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•REVIEW• November 2022 Vol.65 No.11: 2162–2190

https://doi.org/10.1007/s11427-022-2120-1

Mechanisms of chromatin-based epigenetic inheritance

Wenlong Du

1†

, Guojun Shi

2†

, Chun-Min Shan

3†

, Zhiming Li

4†

, Bing Zhu

1,5*

, Songtao Jia

Qing Li

& Zhiguo Zhang

National Laboratory of Biomacromolecules, CAS Center for Excellence in Biomacromolecules, Institute of Biophysics, Chinese Academy of

Sciences, Beijing 100101, China;

State Key Laboratory of Protein and Plant Gene Research, School of Life Sciences and Peking-Tsinghua Center for Life Sciences,

Peking University, Beijing 100871, China;

State Key Laboratory of Plant Genomics, Institute of Microbiology, Chinese Academy of Sciences, Beijing 100101, China;

Institutes of Cancer Genetics, Herbert Irving Comprehensive Cancer Center, Columbia University Medical Center, New York, NY 10032, USA;

College of Life Sciences, University of Chinese Academy of Sciences, Beijing 100049, China;

Department of Biological Sciences, Columbia University, New York, NY 10027, USA

Received February 9, 2022; accepted April 27, 2022; published online June 30, 2022

Multi-cellular organisms such as humans contain hundreds of cell types that share the same genetic information (DNA se-

quences), and yet have different cellular traits and functions. While how genetic information is passed through generations has

been extensively characterized, it remains largely obscure how epigenetic information encoded by chromatin regulates the

passage of certain traits, gene expression states and cell identity during mitotic cell divisions, and even through meiosis. In this

review, we will summarize the recent advances on molecular mechanisms of epigenetic inheritance, discuss the potential impacts

of epigenetic inheritance during normal development and in some disease conditions, and outline future research directions for

this challenging, but exciting field.

epigenetic inheritance, histone modification, DNA methylation, histone deposition, DNA replication

Citation: Du, W., Shi, G., Shan, C.M., Li, Z., Zhu, B., Jia, S., Li, Q., and Zhang, Z. (2022). Mechanisms of chromatin-based epigenetic inheritance. Sci China

Life Sci 65, 2162–2190. https://doi.org/10.1007/s11427-022-2120-1

Introduction

Epigenetics was coined by Waddington in 1942 as a frame-

work for the generation of distinct phenotypes in multi-cel-

lular organisms (Waddington, 1942). At the time, DNA was

not discovered as the carrier of genetic information that

governs the transmission of genetic traits from generation to

generation. Since then, it has been increasingly clear that

epigenetic regulation plays a critical role in the development

of multicellular organisms including human, and mis-reg-

ulations of the epigenetic network are the drivers for many

forms of diseases including cancer and aging (Margueron

and Reinberg, 2010; Benayoun et al., 2015; Allis and Jenu-

wein, 2016; Jones et al., 2016). In this review, we will focus

on discussing the molecular mechanisms underlying how

epigenetic information is inherited into daughter cells during

mitotic cell divisions. While we will mention several ex-

amples on the trans-generational epigenetic inheritance, we

will concentrate our discussion on epigenetic inheritance

during mitosis, and refer the readers to other reviews dis-

cussing the mechanisms and the impacts of trans-genera-

tional epigenetic inheritance (Heard and Martienssen, 2014;

Horsthemke, 2018).

In early days of epigenetic research, scientists described

SCIENCE CHINA

Life Sciences

†Contributed equally to this work

*Corresponding authors (Qing Li, email: [email protected]; Songtao Jia, email:

[email protected]; Bing Zhu, email: [email protected]; Zhiguo Zhang, email:

[email protected])

and studied biological phenomena that cannot be explained

by genetic information alone. These examples include po-

sition effect variegation observed in Drosophila, X chro-

mosome inactivation in female mammals, genome

imprinting in mammals, and para-mutations observed in

plants. Position effect variegation is a phenomenon in which

the white gene in Drosophila eye is expressed in some cells

but silenced in others when the white gene translocates closer

to heterochromatin region, a highly condensed chromatin

domain that is transcriptionally silent (Tartof et al., 1984).

That the expression of a gene was based on its location on the

chromosome, but not the gene itself, was also observed in

budding yeast when a gene was inserted closer to telomeres

(telomere position effects) (Gottschling et al., 1990). X-

chromosome inactivation in female mammals is a mechan-

ism whereby one of two X-chromosomes is inactivated in

female mammals during early embryogenesis to balance the

expression of genes on X-chromosomes between male and

female. Moreover, once silenced, the inactivated X-chro-

mosome remains silent during subsequent cell divisions

(Plath et al., 2002). Genome imprinting is a phenomenon in

which the maternal or paternal allele of a gene is expressed,

while the other allele is silenced (Ferguson-Smith and

Bourc’his, 2018). These examples remain the best to illus-

trate the modern definition of epigenetics, heritable changes

in gene expression/phenotypes without alterations at the

underlying DNA sequences (Allis et al., 2007; Margueron

and Reinberg, 2010). While not all inheritable epigenetic

information is encoded by the chromatin, such as prions, in

this review, we will focus on discussion of inheritance of

epigenetic information encoded by chromatin in eukaryotes.

In eukaryotic cells, the genetic material forms a highly

ordered structure, chromatin, consisting of proteins, DNA

and RNA. The basic repeat unit of chromatin is the nucleo-

some, consisting of 147 bp of DNA wrapped around a his-

tone octamer composed of one H3-H4 tetramer and two

H2A-H2B dimers (Zhou et al., 2019; Talbert and Henikoff,

2021). Chromatin is further organized into distinct domains

such as heterochromatin and euchromatin, which tradition-

ally represent chromatin regions with inactive and active

gene transcription, respectively. For in-depth discussion,

please see recent reviews on insights of three-dimensional

chromatin structures (Dekker and Mirny, 2016; Yu and Ren,

2017; Li et al., 2020a). Furthermore, heterochromatin and

euchromatin are marked by different posttranslational mod-

ifications on histones (Figure 1). For instance, di- and tri-

methylation of histone H3 lysine 9 (H3K9me2/me3) mark

constitutive heterochromatic regions, such as repetitive DNA

sequences including endogenous retroviral elements (ERVs),

pericentric heterochromatin regions and telomeric hetero-

chromatin (Grewal and Moazed, 2003). On the other hand,

tri-methylation of histone H3 lysine 27 (H3K27me3) plays

an important role in the repression of gene transcription

during development (Margueron and Reinberg, 2011). Be-

sides these repressive marks, other histone modifications are

associated with active gene transcription. Tri-methylation of

H3 lysine 4 (H3K4me3) is highly enriched at promoters of

actively transcribed genes (Shilatifard, 2012), whereas

H3K36me3 marks the gene bodies of actively transcribed

genes (Wagner and Carpenter, 2012). In addition to histone

modifications, histone variants, a group of proteins that

adopt similar fold as core histones, reside in specific chro-

matin regions and are also important for the establishment

and maintenance of chromatin states (Loyola and Almouzni,

2007; Talbert and Henikoff, 2010). For instance, histone H3

variant CenH3 proteins occupy centromeric heterochromatin

regions and are critical for the establishment of a functional

kinetochore for chromosome segregation during mitosis.

Histone variant H3.3, which differs from canonical H3.1/

H3.2 by 4 or 5 amino acids, marks actively transcribed re-

gions, whereas canonical H3.1/H3.2 are enriched at hetero-

chromatin. Moreover, DNA cytosine can be methylated

Figure 1 A cartoon depicts representative chromatin states.

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(5mC) or hydroxymethylated (5hmC), which are distributed

on chromatin differently. At constitutive heterochromatin

regions, 5mC co-localizes with H3K9me2/me3 (see detailed

discussion below). In contrast, 5hmC in general is found at

promoters and enhancers of actively transcribed genes. Fi-

nally, non-coding RNAs also play a role in forming distinct

chromatin states (Zaratiegui et al., 2007). In summary,

chromatin is demarcated by histone modifications, histone

variants, DNA methylation and non-coding RNA (not dis-

cussed in this review). Together, they play an important role

in the establishment and maintenance of chromatin struc-

tures, gene expression and cell identity.

During DNA replication, chromatin structures are tran-

siently disassembled to allow DNA replication machinery

to access replicating DNA. Following DNA replication,

distinct chromatin states, marked by different histone

modifications, histone variants, DNA methylation and non-

coding RNA must be restored to maintain chromatin

structures and gene expression states (Moazed, 2011; Ma-

cAlpine and Almouzni, 2013; Serra-Cardona and Zhang,

2017). How distinct chromatin states are inherited follow-

ing DNA replication lies in the heart of epigenetics. In this

review, we will first discuss how nucleosomes, the basic

repeat units of chromatin, are assembled following DNA

replication, and outline the general principles in the passage

of histone modifications into daughter cells. As an example,

we will discuss in depth on how H3K9 methylation in S.

pombe is inherited during mitotic cell division. Further-

more, we will discuss how DNA methylation is inherited,

and highlight the potential interplay between DNA me-

thylation and histone modifications to maintain chromatin

states. Finally, we will discuss the potential impact of

dysregulation of epigenetic inheritance in development and

human diseases and outline future research directions for

this challenging, but exciting field.

DNA replication-coupled nucleosome assembly

A brief overview of DNA replication in eukaryotic cells

During S phase of the cell cycle, DNA sequence must be

faithfully replicated to maintain genome integrity. DNA re-

plication initiates stochastically from DNA replication ori-

gins (MacAlpine, 2021). While replication origins are well-

defined and contain consensus sequence motifs in Sacchar-

omyces cerevisiae, DNA replication origins in higher eu-

karyotic cells are specified and influenced by local chromatin

structures (Hu et al., 2020; Long et al., 2020). The initial step

in the initiation of DNA replication is the assembly of pre-

replication complex (pre-RC) at a replication origin. During

this process, a group of proteins are orderly assembled into a

large complex at G1 phase at replication origins (Bell and

Dutta, 2002; Burgers and Kunkel, 2017). First, origin re-

cognition complex (ORC), which is composed of six sub-

units (Orc1–6), recognizes replication origins (Bell and

Stillman, 1992), and together with CDC6 and CDC10-de-

pendent transcript 1 (CDT1), loads the hexameric mini-

chromosome maintenance (MCM) complex, consisting of

MCM2–7, at replication origins to form the pre-RC complex

(Donovan et al., 1997; Tanaka et al., 1997). The loaded

MCM complexes at this stage are head-to-head inactive

double hexamers and encircle double-stranded (ds) DNA.

Phosphorylation of the MCM complex by DDK (DBF4-de-

pendent kinase) and CDKs and subsequent binding of

CDC45 and the DNA replication complex GINS (go-ichi-ni-

san) lead to formation of two active replicative helicases, the

CMG helicase (Cdc45-MCM-GINS) (Ilves et al., 2010). The

CMG complex unwinds dsDNA into ssDNA, which is

coated with ssDNA binding protein, replication protein A

(RPA). Two short RNA-DNA primers are then synthesized

by the primase-DNA polymerase alpha (Polα) complex,

which are used by DNA polymerase epsilon (Polε) to syn-

thesize the leading strands continuously and DNA poly-

merase delta (Polδ) to synthesize the lagging strands as

Okazaki fragments. Finally, Ctf4 (AND1 in mammalian

cells) connects the CMG helicase with Polα primase, which

likely coordinates leading and lagging DNA synthesis as

well as nucleosome assembly of parental histones (See

Discussion below). Together, the multi-component protein

machinery, namely the replisome, replicates DNA in a highly

regulated manner.

An overview of DNA replication-coupled nucleosome

assembly

In general, nucleosomes limit the accessibility of protein

machinery involved in various DNA transactions such as

DNA replication, repair and gene transcription to the nu-

cleosomal DNA. Therefore, during DNA replication, 1-2

nucleosomes ahead of DNA replication forks are temporarily

disassembled to allow the replisome to access DNA. Fol-

lowing the passage of DNA replication forks, replicated

DNA is reassembled into nucleosomes using both parental

histones and newly synthesized histones in a process tightly

coupled to on-going DNA replication (DNA replication-

coupled nucleosome assembly) (McKnight and Miller, 1977;

Stillman, 1986; Li et al., 2013) (Figure 2). Moreover, par-

ental (H3.1-H4)

tetramers remain intact and generally do

not split during DNA replication (Xu et al., 2010). Mean-

while, newly synthesized H3.1-H4 are deposited onto re-

plicating DNA in tetramer forms mediated by histone

chaperones (Fazly et al., 2012; Liu et al., 2012b; Su et al.,

2012). Therefore, parental and newly synthesized (H3.1-

H4)

tetramers form distinct nucleosomes following DNA

replication. On the contrary, newly synthesized H2A-H2B

could be found in nucleosomes containing parental H3-H4

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tetramers in one cell cycle, consistent with the idea that

nucleosomal H2A-H2B can exchange relatively freely with

parental H2A-H2B following DNA replication. Further-

more, deposition of H3-H4 tetramers is the rate-limiting step

of nucleosome formation (Smith and Stillman, 1991).

Therefore, we will focus on the discussion of replication-

coupled nucleosome assembly into three parts, dis-assembly

of preexisting nucleosomes (or parental nucleosomes) lo-

cated ahead of the replication fork, recycling of parental

histone H3-H4 tetramers, and deposition of newly synthe-

sized H3-H4 tetramers to form nucleosomes de novo.

Disassembly of parental nucleosomes

Previous studies reveal that approximately 300 bp of naked

DNA resides ahead of the replication forks, suggesting that

1-2 nucleosomes ahead of DNA replication forks are tem-

porarily disrupted (Lucchini et al., 2001). In Xenopus egg

extracts, using single-molecule imaging, it was reported that

nucleosome ahead of the replication fork is evicted and

parental histones are recycled (Gruszka et al., 2020). To-

gether, these studies support the idea that nucleosomes ahead

of DNA replication forks are disassembled temporarily.

Several factors are likely involved in the disassembly of

nucleosomes ahead of DNA replication forks. First, ATP-

dependent chromatin remodeling complexes, which utilize

the energy of ATP hydrolysis to alter the position of nu-

cleosomes along the DNA and to evict nucleosomal histones,

are likely involved in this process. Supporting this idea,

several chromatin remodeling complexes including INO80,

SWR1, ISW1 and ISW2 in budding yeast and their mam-

malian counterparts also participate in the DNA replication

process (Papamichos-Chronakis and Peterson, 2008; Vincent

et al., 2008; Morrison and Shen, 2009; Kurat et al., 2017).

However, to what extent that these chromatin remodeling

complexes remodel parental nucleosomes ahead of DNA

replication forks remains elusive. Second, the FACT (facil-

itates chromatin transactions) complex has been implicated

in remodeling nucleosomes ahead of DNA replication forks.

FACT, consisting of two subunits, Spt16 and Pob3 (SSRP1

in mammals), is a histone chaperone that binds to both H3-

H4 tetramers and H2A-H2B dimers (Belotserkovskaya and

Reinberg, 2004; Formosa and Winston, 2020). It has been

shown that FACT is essential for transcription on chromatin

template in vitro proposedly through removing H2A-H2B

from nucleosomes (LeRoy et al., 1998; Orphanides et al.,

1998). Recent studies using purified proteins in reconstituted

DNA replication system indicate that FACT is also essential

for DNA replication through chromatin template (Kurat et

al., 2017). Thus, FACT plays an important role in both DNA

replication and gene transcription through chromatin. In vi-

tro, FACT can alter the contacts between histones and DNA

without ATP hydrolysis. However, FACT itself could not

disassemble nucleosomes in vitro (Chen et al., 2018; Wang et

al., 2018b). Based on the Cryo-EM structures, FACT re-

cognizes partially unwrapped nucleosome structures (Liu et

al., 2020). In cells, FACT co-purifies with MCM2-7 complex

in both yeast and mammalian cells. FACT can also promote

DNA unwinding by MCMs in vitro (Gambus et al., 2006;

Tan et al., 2006). Together, these studies suggest that after

nucleosome disassembly, FACT may work with MCM he-

licase complex to facilitate nucleosome reassembly during

DNA replication (Figure 2). However, whether and how

FACT functions in parental nucleosome disassembly and

subsequent transfer of parental histones onto replicated DNA

remain unclear. Finally, Asf1, another histone chaperone

proposed to be involved in parental nucleosome disassembly,

is best known for its role in shuttling newly synthesized H3-

H4 in the process of de novo nucleosome assembly. It has

been shown that Asf1 co-purifies with MCM2–7 complex in

mammalian cells, and this interaction is bridged by histone

H3-H4 in the nucleus (Groth et al., 2007). A mutation on

Asf1-V94R, which disrupts Asf1 binding to H3-H4, also

compromise the Asf1-MCM interactions. Structure analysis

of the Asf1-H3-H4-MCM2 complex indicates that MCM2

N-terminus can bind to the H3-H4 tetramer and hijack the H3

interface involved in tetramer formation (Clément and Al-

mouzni, 2015; Huang et al., 2015). In cells, it has been

shown that histone chaperone Asf1 can facilitate nucleosome

disassembly at promoter region or gene body during tran-

scription (Adkins et al., 2004; Adkins and Tyler, 2004; Gao

et al., 2018). However, Asf1 cannot disassemble nucleo-

somes in vitro, indicating that other factors collaborate with

Asf1 to accomplish parental histone eviction in vivo (Don-

ham et al., 2011). Together, these studies suggest that mul-

tiple factors including chromatin remodeling complexes and

histone chaperones are likely involved in the disassembly of

nucleosomes ahead of DNA replication forks. However, to

what extent these factors function in nucleosome dis-

assembly and subsequent parental histone transfer remains to

be determined.

Parental histone transfer at the replication forks

Once parental nucleosomes ahead of replication forks are

disassembled, parental histones with modifications must be

transferred onto replicating DNA strands for the formation of

nucleosomes. This parental histone transfer and/or recycling

process is critical for the inheritance of histone modifica-

tions, but remains elusive for over 4 decades. For instance,

based on metabolic labeling of DNA and proteins during S

phase, it was proposed that parental histones are randomly

and equally distributed onto replicated DNA strands (Seale,

1976). Recent studies indicate that parental H3-H4 tetramers

likely remember their position along the DNA. These studies

are made possible with the development of novel techniques.

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For instance, by monitoring parental H3-H4 on a plasmid in

two different in vitro DNA replication systems, it has been

shown that parental H3-H4 are transferred locally in the

Xenopus DNA replication system, but are dispersed in SV40

DNA replication system (Madamba et al., 2017). The major

distinction between these two systems is that different heli-

cases are used in the DNA replication. In Xenopus extract,

CMG is the replicative helicase, whereas large T antigen is

the replicative helicase in the SV40 DNA replication system.

More recently, two studies show that parental nucleosomes

form positional memory following DNA replication (Esco-

bar et al., 2019; Schlissel and Rine, 2019). Both studies

started with labeling parental nucleosomes at a particular

locus covalently with biotin, and then tracked the fate of

labeled nucleosomes through DNA replication. In budding

yeast, it has been shown that labeled histone H3 can re-

member its positions along the DNA following replication

and gene transcription (Schlissel and Rine, 2019). In mouse

embryonic stem (ES) cells, by monitoring parental nucleo-

somal H3.1 that is enriched at silent chromatin regions, it has

been shown that parental H3.1 is transferred locally at re-

pressive regions, but is dispersed at actively transcribed re-

gions (Escobar et al., 2019). Of note, H3.3, but not H3.1, is

enriched at actively transcribed regions (Loyola and Al-

mouzni, 2007; Talbert and Henikoff, 2010). Therefore, it

would be interesting to determine whether parental H3.3 is

also transferred locally or dispersed at actively transcribed

regions.

Recent studies have discovered specific protein factors

involved in the transfer of parental H3-H4 onto replicating

DNA (Figure 2). First, it has been shown in both yeast and

mouse ES cells, mutations at the histone binding motif of

MCM2, a subunit of the CMG helicase, result in defects in

the transfer of parental H3-H4 to lagging strands of DNA

replication forks (Gan et al., 2018; Petryk et al., 2018). Early

studies indicate that human MCM complex binds to H3 and

H4 in HeLa cell extracts, and the N-terminus of mouse

MCM2 is required for the histone binding activity (Ishimi et

al., 1998; Ishimi et al., 2001). Similarly, the N-terminal

histone binding motif (HBM) of yeast Mcm2 was reported to

interact with all four histones released from chromatin

(Foltman et al., 2013). Interestingly, mouse MCM2 can bind

to H3-H4 and assemble a nucleosome-like structure in vitro,

supporting the idea that MCM2 histone binding domain also

possesses histone chaperone activity. Using the eSPAN

(enrichment and sequencing of protein-associated nascent

DNA) that measures the relative amount of parental and

newly synthesized histones at the leading and lagging strands

of DNA replication forks, it has been shown that parental H3

marked with H3K4me3 are transferred almost equally to

leading and lagging strands, with a slight preference for

lagging strands (Yu et al., 2018a). In contrast, new histones

marked by H3K56ac (acetylation on H3 lysine 56) showed

an opposite pattern. In cells with mcm2-3A mutation that

disrupts the interaction between Mcm2 and H3-H4, parental

H3K4me3 are enriched at leading strands due to defects in

the transfer of parental histones to lagging strands (Gan et al.,

2018). Similarly, using SCAR-seq (sister chromatids after

replication by DNA sequencing) in mouse ES cells with

mutations disrupting MCM2 binding to histones, marks on

Figure 2 DNA replication coupled nucleosome assembly pathways with key factors involved in nucleosome assembly indicated.

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parental histone show asymmetric distribution (Petryk et al.,

2018). These results show that the histone binding ability of

MCM2 is critical for parental histone transfer to lagging

strands of DNA replication forks.

The CMG helicase interacts with leading strand poly-

merase Polε and travels along with leading strand template

(Fu et al., 2011; Burgers and Kunkel, 2017). How does

MCM2, traveling along the leading strands, facilitates the

transfer of parental histones to the lagging strands of DNA

replication forks? To answer this question, it should be noted

that the CMG helicase interacts with Ctf4, which forms a

trimer that also interacts with Pol1, the catalytic subunit of

Polα primase enriched at lagging strands (Simon et al.,

2014). Studies from budding yeast show that mutations at

Ctf4 that cannot bridge the CMG-Pol1 interaction or Pol1

mutants that cannot bind to Ctf4 display similar defects in

parental histone transfer to lagging strands (Gan et al., 2018).

Finally, like Mcm2, Pol1 also contains a conserved histone

binding motif (Evrin et al., 2018). Both yeast and mouse Pol1

bind to H3-H4 preferentially over H2A-H2B. Mutations at

the histone binding motif of Pol1 also result in defects in

parental histone transfer in a manner similar to Mcm2 mutant

defective in histone binding (Li et al., 2020b). Together,

these studies indicate that Mcm2-Ctf4-Polα axis regulates

the transfer of parental histone H3-H4 to lagging strands of

DNA replication forks.

In budding yeast and mouse ES cells, using eSPAN ana-

lysis, it has been shown that deletion of Dpb3 (POLE4 in

mammals) or Dpb4 (POLE3 in mammals) leads to the dra-

matic reduction of the transfer of parental histones to leading

strands of DNA replication forks (Yu et al., 2018a). Dpb3

and Dpb4 are two subunits of leading strand DNA poly-

merase, Polε. However, Dpb3 and Dpb4 are not required for

enzymatic activity of Polε. Dpb3 and Dpb4 in fission yeast

form a dimer with the structure similar to H2A-H2B (He et

al., 2017). Moreover, Dpb3-Dpb4 co-purify with all four

core histones (Tackett et al., 2005) and interact with H3-H4

preferentially over H2A-H2B in vitro (Yu et al., 2018a).

Similarly, POLE3-POLE4 formed a stable dimer and could

bind to histone H3-H4 directly but not H2A-H2B (Bellelli et

al., 2018). Together, these studies indicate that Dpb3 and

Dpb4 serve as histone chaperones to promote the transfer of

parental histones to leading strands of DNA replication

forks.

Budding yeast cells with mcm2-3A mutation showed mild

defects in the loss of transcriptional silencing at hetero-

chromatin loci. Similar effects were also observed for cells

lacking Dpb3 and Dpb4 in both budding and fission yeast

(He et al., 2017; Yu et al., 2018a). Moreover, mcm2-3A dpb3

double mutant cells show defects in memory of nucleosome

positions following DNA replication (Schlissel and Rine,

2019). In mouse ES cells, the MCM2 and Polα mutants with

impaired parental histone transfer show defects in the re-

pression of ERVs (Li et al., 2020b). Together, these studies

indicate that the precise transfer of parental H3-H4 to re-

plicating DNA strands is important to maintain hetero-

chromatin states. Of note, both yeast and mouse ES cells

lacking these factors involved in parental histone transfer

have largely normal growth, suggesting that additional fac-

tors participate in the transfer of parental histones.

Deposition of newly synthesized histone H3-H4

After DNA duplication, parental histones contribute to only

half of the total histones required for the assembly of re-

plicating DNA into nucleosomes. Therefore, newly synthe-

sized histones are needed to complete the nucleosome

assembly of replicated DNA. Compared with the transfer of

parental histones, de novo deposition of new H3-H4 is re-

latively well studied (Serra-Cardona and Zhang, 2017). As

detailed below, de novo deposition of new H3-H4 requires a

group of histone chaperones that mediate histone folding,

import and deposition onto replicating DNA. Moreover,

modifications on newly synthesized H3-H4 also regulate the

interactions between histones and histone chaperones. Fi-

nally, these histone chaperones interact with components of

replisomes to facilitate the deposition of new H3-H4 onto

replicating DNA strands (Figure 2).

Histone chaperones form a coordination network for

deposition of new H3-H4

Histone chaperones are essential for de novo histone de-

position. These histone chaperones form a coordination

network for the deposition of newly synthesized H3-H4,

which first form a heterodimer. With the aid of other protein

chaperones involved in protein folding, new H3-H4 form a

complex with histone chaperone Asf1, which does not show

nucleosome assembly activity in vitro, indicating that Asf1

may not participate in the assembly event directly (Tyler et

al., 1999; English et al., 2005; English et al., 2006). Con-

sistent with this observation, the structure of Asf1-H3-H4

complex reveals that Asf1 binds to the H3-H4 dimer through

the H3 interface involved in the formation of H3-H4 tetra-

mers, and thus Asf1 blocks the H3-H4 tetramer formation

(English et al., 2006). Therefore, once associated with Asf1,

H3-H4 must be transferred to downstream chaperones in-

cluding chromatin assembly factor 1 (CAF-1) for deposition

onto replicating DNA.

CAF-1 was the first histone chaperone discovered in-

volved in replication coupled nucleosome assembly (Still-

man, 1986; Verreault et al., 1996; Kaufman et al., 1997).

CAF-1 consists of three subunits, Cac1, Cac2 and Cac3 in

yeast, corresponding to p150, p60 and p48 in mammalian

cells. One CAF-1 molecule binds to one H3-H4 dimer and

the dimerization of two CAF-1 complexes triggers the for-

mation of a H3-H4 tetramer (Liu et al., 2016; Mattiroli et al.,

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2017). Asf1 binds to the Cac2 subunit of histone chaperone

CAF-1 and the conformational changes allow the delivery of

H3-H4 dimer from Asf1 to CAF-1, thus providing direct

evidence for coordination between histone chaperones (Tyler

et al., 2001; Mello et al., 2002). In addition to direct inter-

action between Asf1 and CAF-1, previous studies suggest

that ubiquitination of H3K122 will destabilize the interaction

between Asf1 and H3-H4 complex, which in turn facilitates

the transfer of H3-H4 from Asf1 to CAF-1 (Han et al., 2013).

In yeast, yeast cells lacking CAF-1 are viable (Kaufman et

al., 1997), suggesting that other histone chaperones likely

promote deposition of new H3-H4 onto replicating DNA.

Indeed, it has been shown that Rtt106 (Regulator of Ty1

transposon 106) functions in parallel with CAF-1 in de-

position of new H3-H4 (Huang et al., 2005; Huang et al.,

2007). In addition to CAF-1 and Rtt106, using a separation

of functional mutant alleles, FACT has also been shown to

function in the deposition of newly synthesized H3-H4

during replication (Yang et al., 2016). FACT contains mul-

tiple PH (pleckstrin homology) domains and can bind to H3-

H4 with newly synthesized histone marks. Thus, multiple

chaperones function in the deposition of new H3-H4 onto

replicating DNA. Furthermore, in cells, these chaperones co-

purify with each other. For instance, FACT can co-purify

with CAF-1 and Rtt106, and the interaction between them is

bridged by H3K56Ac and peaks during S phase (Yang et al.,

2016). In addition, CAF-1 also co-purifies with Rtt106

(Huang et al., 2005). These physical interactions indicate that

these chaperones form a coordination network for de novo

histone deposition during S phase.

Histone modifications and variant amino acids on histone

proteins regulate the interaction between newly synthesized

histones and histone chaperones

Newly synthesized histones are also modified post-trans-

lationally and most of these modifications are distinct from

modifications on parental histones. For instance, acetyla-

tion of histone H4 lysine 5 and 12 (H4K5,12) by HAT1-

RbAp46 acetyltransferase and acetylation at some lysine

residues on H3 tails (H3K4,9,14,23,27) are marks on newly

synthesized histones across almost all species (Sobel et al.,

1995; Verreault et al., 1996). In fungal species, H3K56ac is

a mark on new H3 (Masumoto et al., 2005; Zhou et al.,

2006). H3K56ac is catalyzed by the Rtt109-Vps75 com-

plex and histone chaperone Asf1 is essential for H3K56

acetylation (Han et al., 2007a; Han et al., 2007b). The

structure of Rtt109 in complex with Asf1-H3-H4 indicates

that while Asf1 has little contact with Rtt109, it positions

H3 lysine 56 for acetylation by Rtt109 (Zhang et al., 2018).

In addition to histone acetylation, mono-methylation of

histone H3K9 (H3K9me1) by SETDB1 is also found on

H3.1 prior to deposition in mammalian cells (Loyola et al.,

2006).

Several functions have been uncovered for the modifica-

tions on newly synthesized H3-H4. First, the acetylation of

H4K5,12 occurs in cytoplasm and promotes the nuclear

import of histone H3-H4 mediated by histone chaperone

Asf1 and the Importin complex (Zhang et al., 2012; An et al.,

2017). Importin Kap123 contains two lysine binding pock-

ets, and acetylation at lysine residues on histone H3 and H4

weakens the interaction of H3-H4 with importin (An et al.,

2017). Second, H3K56 acetylation regulates the interactions

between H3-H4 and CAF-1 and Rtt106 (Chen et al., 2008; Li

et al., 2008). Moreover, acetylation at both H3 and H4 tails

also significantly increases the interaction of CAF-1 and

Rtt106 with new H3-H4 and promotes replication-coupled

nucleosome assembly (Burgess et al., 2010). Rtt106 contains

two tandem PH domains that likely bind to H3K56 acety-

lated H3-H4 (Su et al., 2012). However, how CAF-1 re-

cognizes H3K56ac and acetylates H3 and H4 tails remains to

be determined. Furthermore, it remains unclear whether

H3K56ac, which is present at low abundance in metazoans,

also has a role in replication-coupled nucleosome assembly.

Finally, it has been proposed that H3K9me1 helps the re-

storation of H3K9me2/me3 by serving as a substrate for

H3K9 methyl-transferases that catalyze di- and tri-methyla-

tion (Loyola et al., 2006). For a detailed description of his-

tone modifications’ role in replication-coupled nucleosome

assembly we refer readers to other reviews like “All roads

lead to chromatin”(Li et al., 2013).

In addition to histone modifications, variant amino acids

found on histone H3.1/H3.2 and H3.3 play a key role in

regulating the interaction between H3-H4 and the corre-

sponding histone chaperones. Histone H3.1/H3.2 differ from

H3 variant H3.3 by four or five amino acids, with the three

variant amino acids located at residues 87 to 90 (SAVM in

H3.1/H3.2 vs. AAIG in H3.3). H3.1/H3.2 bind to histone

chaperone CAF-1 and is deposited during S phase of cell

cycle in the replication-coupled nucleosome assembly

pathway (Ahmad and Henikoff, 2002a, 2022b). In contrast,

H3.3 associates with histone chaperones HIRA and DAXX,

and it can be deposited both during and outside of S phase

(Tagami et al., 2004; Drané et al., 2010; Goldberg et al.,

2010). Mutating the three variant amino acids between H3.1/

H3.2 and H3.3 can alter their interactions with CAF-1 and/or

HIRA/DAXX and subsequent deposition onto DNA (Lewis

et al., 2010; Elsässer et al., 2012; Liu et al., 2012a). Finally, it

has been shown that phosphorylation of H4 serine 47 inhibits

the interaction between CAF-1 and H3-H4 and promotes the

interaction between HIRA and H3-H4 (Kang et al., 2011).

Together, these studies indicate that modifications on newly

synthesized H3-H4 and variant amino acids on histone pro-

teins regulate the dynamic interactions between histone and

histone chaperones, thereby providing the supply of other

half of histones for the assembly of newly replicated DNA

into nucleosomes.

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Histone chaperones connect to replication forks via inter-

actions with replisome components

How do histone chaperones deposit newly synthesized H3-

H4 specifically at replicated DNA? The answer to this

question lies at least partially in the physical interactions

between histone chaperones and replisome components.

Early studies showed that CAF-1 interacts with PCNA

(proliferating cell nuclear antigen) (Shibahara and Stillman,

1999). PCNA forms a homotrimer (Pol30 subunits in bud-

ding yeast) and functions as a sliding clamp for both Polδ

and Polε involved in lagging and leading strand DNA

synthesis, respectively (Choe and Moldovan, 2017). De-

pletion of PCNA inhibits CAF-1 mediated chromatin as-

sembly in vitro (Shibahara and Stillman, 1999).

Furthermore, site-specific PCNA mutations that disrupt the

CAF-1-PCNA interaction in budding yeast, while showing

minor effects on cell growth, result in defects in transcrip-

tional silencing, in the same pathway as cells lacking CAF-1

(Zhang et al., 2000). A recent discovery found that in-

troduction of the same PCNA mutations in mouse ES cells

led to defects in differentiation in vitro, and embryonic

lethality during mouse early development (Cheng et al.,

2019). Together, these studies suggest that the PCNA-CAF-

1 interaction is important for the deposition of new H3-H4

and embryonic development.

In addition to PCNA, RPA, the single-stranded DNA

binding protein at the replication forks, can also interact with

multiple histone chaperones. RPA contains three subunits

named as Rfa1, Rfa2 and Rfa3 in budding yeast or RPA70,

RPA32 and RPA14 in humans, respectively. RPA interacts

with histone chaperones FACT, CAF-1 and Rtt106, but not

Asf1 (Liu et al., 2017). Genetic analysis suggests the po-

tential coordination between FACT and RPA during nu-

cleosome assembly (VanDemark et al., 2006). Besides

histone chaperones, RPA also binds to free histone H3-H4

directly but not intact nucleosomes or H2A-H2B (Liu et al.,

2017). Furthermore, histone H3-H4 promotes the interaction

of RPA with those histone chaperones. Moreover, RPA can

also deposit H3-H4 onto adjacent double strand DNA when

bound to ssDNA, indicating a role of RPA in histone de-

position mediated by multiple histone chaperones (Liu et al.,

2017). Finally, it has been shown that FACT co-purifies with

MCM helicases in both yeast and mammalian cells (Gambus

et al., 2006; Tan et al., 2006). Together, these studies indicate

that histone chaperones involved in de novo deposition of

new H3-H4 interact with multiple components of replisomes,

which likely mediate the ability of these histone chaperones

to deposit H3-H4 in the DNA replication-coupled process.

However, the functional significance of several aforemen-

tioned interactions between histone chaperones and repli-

some components in replication-coupled nucleosome

assembly remains to be determined.

General principles for the restoration of histone

modifications following DNA replication

Early studies on X-chromosome inactivation, position effect

variegation in Drosophila, genome imprinting, silent chro-

matin at mating type locus in both budding and fission yeast

strongly support the idea that heterochromatin domains can

be inherited through mitotic cell divisions. These studies

were performed before the discoveries that distinct histone

modifications mark active and repressive chromatin domains

(Grewal and Jia, 2007).

It is well accepted that DNA methylation is heritable,

however, it is clear that not all histone modifications are

heritable for various reasons (Zhu and Reinberg, 2011;

Ptashne, 2013; Reinberg and Vales, 2018). Currently, it is

estimated that over 80–100 posttranslational modifications

on four histone proteins can be identified (Zhao and Garcia,

2015). Some of these histone modifications such as acet-

ylation are quite labile with a half-life less than one cell cycle

(Zee et al., 2010). Therefore, it is unlikely that those labile

histone modifications can be used as templates for the re-

storation of the modification following DNA replication

without the aid of other factors. In addition, it is known that

most nucleosomal H2A-H2B proteins exchange relatively

freely with newly synthesized H2A-H2B within one cell

cycle (Xu et al., 2010). Therefore, it is likely that most

modifications on H2A-H2B might not be heritable. Of note,

it has been recently shown that H2AK119 ubiquitination

located at repressive heterochromatin can be inherited (Zhao

et al., 2020a), suggesting that some H2A-H2B modifications

are heritable. Compared with H2A-H2B, H3-H4 tetramers,

once assembled into nucleosomes, are relatively stable and

do not exchange freely with newly synthesized H3-H4. In-

deed, methylation of H3 and H4, including H3K27me3 and

H3K9me2/m3, are widely accepted as inheritable epigenetic

marks (Margueron et al., 2009; Liu et al., 2015; Coleman and

Struhl, 2017; Laprell et al., 2017) and have a half-life over

one cell cycle. These findings suggest that histone mod-

ifications with longer half-life are more likely to be trans-

mitted following DNA replication. Moreover, it is known

that histone H3.1 at active or repressive chromatin regions

shows distinct patterns following DNA replication (Escobar

et al., 2019), suggesting that the heritability of histone

modifications likely also depends on local chromatin en-

vironment. Therefore, future studies are warranted to explore

the regulatory network that governs the inheritance of dif-

ferent epigenetic modifications.

An early insight into the inheritance of histone modifica-

tions came from studies on H3K27me3, which reported that

EED, a subunit of the PRC2 complex catalyzing H3K27me3

(Margueron and Reinberg, 2011; Holoch and Margueron,

2017), has a chromodomain that recognizes H3K27me3. In

vitro studies indicate that binding of H3K27me3 by EED

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Du, W., et al. Sci China Life Sci November (2022) Vol.65 No.11

stimulates the enzymatic activity of PRC2 to methylate

neighboring nucleosomes without this modification (Mar-

gueron et al., 2009). In mouse ES cells, mutations at EED

chromodomain impairing its binding to H3K27me3 result in

defects in the spreading of H3K27me3 (Oksuz et al., 2018).

Similarly, G9a/GLP, the methyltransferases for H3K9me2,

harbor ankyrin repeat domains, and the association of G9a/

GLP with H3K9me2 also stimulates their enzymatic activ-

ities. Mice with mutations at GLP ankyrin repeat show de-

fects in growth ossification and postnatal lethality (Liu et al.,

2015). Moreover, Suv39h1/h2, the enzymes catalyzing

H3K9me3 in mammalian cells, contain a chromodomain that

recognizes H3K9me3, although the functional significance

of this domain is not well explored. In fission yeast, the

recognition of H3K9me2/me3 by the chromodomain of Clr4,

the sole H3K9me3 writer, is important for inheritance of this

mark (Ragunathan et al., 2015; Wang and Moazed, 2017).

Together, these studies support a positive feedback model

whereby H3K9 or H3K27 enzymes first recognize (read)

their cognate modifications on nucleosomes from parental

histones and then modify (write) nucleosomes containing

newly synthesized histones without this mark following

DNA replication (Figure 3).

It was proposed that repressive marks including H3K9me3

and H3K27me3 are inheritable (see discussion below),

whereas active marks such as H3K4me3 are not (Reinberg

and Vales, 2018). However, in the literature, there are ex-

amples that active chromatin domains are also heritable. For

instance, it has been shown that the active gene state can

persist through 24 cell divisions in the absence of gene

transcription in nuclear transfer experiments and this epi-

genetic memory depends on the incorporation of H3.3, a

histone H3 variant marking actively transcribed genes, as

well as on H3.3 lysine 4 (Ng and Gurdon, 2008; Hörman-

seder et al., 2017). In C. elegans, mutations at H3K4me3

methyltransferases result in increased life span, and this in-

crease can be transmitted into descendants up to three gen-

erations, suggesting that certain chromatin loci marked by

H3K4me3 can be maintained trans-generationally (Greer et

al., 2011). In mouse mature oocytes, a non-canonical form of

H3K4me3 that contains broad H3K4me3 peaks at the pro-

moters and distal loci was discovered. These broad

H3K4me3 domains can be inherited in post-fertilization

embryos, before being erased at two cell embryo stages

(Dahl et al., 2016; Zhang et al., 2016). These studies strongly

suggest that active marks such as H3K4me3 may also be

inherited under certain conditions. Supporting this idea, the

Spp1 (CFP1 in humans), a subunit of the COMPASS com-

plex that catalyzes H3K4me3, also contains a PHD domain

that binds to H3K4me3 (He et al., 2019). Indeed, it has been

recently shown that both gene transcription machinery and

the read of H3K4me3 by Spp1 help recruit the COMPASS

complex for the restoration of H3K4me3 following DNA

replication (Serra-Cardona et al., 2022).

The read-write mechanism is just one part of the puzzles

for the restoration of histone modifications following DNA

replication. In fact, the inheritance of histone modifications

is much more complicated. For instance, it has been shown

that different histone modifications are restored on newly

synthesized histones at different rates following DNA re-

plication. Moreover, while restoration of histone modifica-

tions may start at S phase of the cell cycle, it takes until next

G1 for cells to fully restore most histone modifications (Xu

et al., 2011; Alabert et al., 2015). Furthermore, the cis-reg-

ulatory element called PRE involved in the establishment of

H3K27 methylation in early embryo is needed for the stable

maintenance of this mark, most likely through recruiting

PRC2 along with the read-write mechanism, to methylate

H3K27 in nucleosomes formed with newly synthesized H3-

H4 following DNA replication (Coleman and Struhl, 2017;

Laprell et al., 2017). Moreover, when the PRE is removed,

there is still considerable, residual capacity for copying the

mark, likely due to the function of the read-write mechanism

(Coleman and Struhl, 2017). In S. pombe and as described in

detail in the next section, both cis-regulatory elements and

RNAi machinery play important roles in the inheritance of

H3K9 methylation.

Several factors likely contribute to the complex nature for

the stable inheritance of histone modifications. First, com-

pared to DNA sequences, histone modifications are re-

versible due to the presence of eraser proteins, providing a

balance and competition between writers and erasers for a

particular histone modification. Therefore, in principle, cells

need to increase the local concentration of writers and/or

reduce the concentration of the erasers for the histone

modifications in order to faithfully maintain them during cell

division. Second, there are cross-talks among histone mod-

ifications at different chromatin regions. For instance,

H3K36 methylation, an active mark, can counterbalance

H3K27 methylation, a silent chromatin mark (Yuan et al.,

2011). Therefore, an increase in the concentration of writers/

erasers for H3K36 methylation can in principle influence the

dynamics of H3K27 methylation, or vice versa. Third, some

histone modifications such as H3K4me3 are deposited co-

transcriptionally (Soares et al., 2017; Bae et al., 2020).

Moreover, ongoing transcription can promote active histone

turnovers, i.e., exchange between parental histones and

newly synthesized histones. Therefore, it is proposed that

factors inhibiting histone turnover/exchange likely play an

important role in epigenetic inheritance (Aygün et al., 2013).

Finally, there are cross-talks between histone modifications

and DNA methylation (see detailed discussion below). Be-

cause of these complications, we propose that the restoration

of histone modifications following DNA replication requires

the interplay of histone modifications, cis-regulatory DNA

elements, non-coding RNA and DNA methylation.

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Below, we use the inheritance of H3K9 methylation in S.

pome as the model to discuss these ideas for the following

reasons. First, key factors involved in H3K9 methylation and

heterochromatin assembly are highly conserved in higher

organisms. Second, the genetic power of yeast system allows

precise genetic manipulations. Third, heterochromatin pro-

teins are not essential for cell viability, allowing greater

flexibility for genetic analyses. Fourth, there is usually a

single gene encoding H3K9 heterochromatin regulators,

avoiding complications from multiple proteins with partially

overlapping functions. Finally, fission yeast does not have

DNA methylation. Together, this system makes it possible to

dissect the molecular mechanisms underlying the inheritance

of heterochromatin marked by H3K9 methylation.

Inheritance of H3K9 methylation, a lesson learned

from S. pombe

In fission yeast, large heterochromatin domains are present at

the pericentric region, silent mating-type region, and sub-

telomeres (Grewal and Jia, 2007). These regions all contain

repetitive DNA sequences, and the formation of hetero-

chromatin is critical for suppressing recombination between

repeats to maintain genome stability. Heterochromatin also

silences the transcription of genes within and near it in a

sequence-independent manner to regulate gene expression

programs.

Nucleosomes within these heterochromatic regions are

methylated at histone H3 lysine 9 (H3K9me). H3K9me re-

cruits heterochromatin protein 1 (HP1) family proteins Swi6

and Chp2, which in turn recruit diverse proteins to regulate

different biological processes (Grewal and Jia, 2007). Clr4 is

the sole histone H3K9 methyltransferase critical for hetero-

chromatin formation (Rea et al., 2000; Nakayama et al.,

2001), which contains a SET domain that catalyzes

H3K9me, and a chromodomain that recognizes H3K9me3

(Zhang et al., 2008). Mutations of the chromodomain that

affect Clr4 interaction with H3K9me3 reduced binding of

Clr4 to its target sites, and H3K9me3 domains are no longer

properly inherited. These results support the idea that Clr4

not only “writes” H3K9me3 but also “reads” it, forming a

positive feedback loop. The coupling of “read” and “write”

activities is also critical for restoring H3K9me3 domain after

DNA replication, where parental histones containing

H3K9me3 serve as seeds for the recruitment of Clr4 to

modify newly synthesized histones.

Early studies of heterochromatin at the silent mating-type

locus established that heterochromatin can be epigenetically

inherited, even before the role of histone H3K9 methylation

in heterochromatin assembly has been discovered. Fission

yeast has two different mating types: P (plus) and M (minus).

The mating type of a cell is determined by the gene content

within the mat1 locus, which is actively transcribed (Figure

4). Cells can also switch their mating types using one of the

two donor sequences, mat2P or mat3M, located more than 10

kilobases away from mat1. The donors, as well as the se-

quences between them, are silenced by heterochromatin.

Among the sequences between donors is cenH (centromere

homology), which is homologous to pericentric repeats.

Replacing cenH with a ura4

reporter gene leads to cells with

one of two stably maintained states: “ura4-on” (the reporter

is expressed) and “ura4-off” (the reporter is repressed)

(Grewal and Klar, 1996) (Figure 4A). Due to the low

switching rate from ura4-on to ura4-off, heterochromatin of

ura4-off cells is presumed to be maintained in the absence of

de novo establishment. Genetic analyses demonstrate that

these epigenetic states are inherited through both mitosis and

meiosis, behaving similarly to gene alleles (Grewal and Klar,

1996). Later it was demonstrated that the two epigenetic

alleles are different in their chromatin environments, such as

H3K9 methylation and Swi6 protein levels (Nakayama et al.,

2000; Hall et al., 2002).

The cenH sequence as well as pericentric repeats are later

Figure 3 The read and write mechanism contributes to the restoration of key histone modifications following DNA replication. Please note that the

restoration of histone modifications starts during S phase and may last till G1 phase of the cell cycle.

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Du, W., et al. Sci China Life Sci November (2022) Vol.65 No.11

found to initiate heterochromatin formation through the

RNA interference (RNAi) pathway (Hall et al., 2002; Volpe

et al., 2002). The DNA repeats are transcribed, generating

double-stranded RNAs (dsRNAs) (Volpe et al., 2002), which

are processed by the ribonuclease Dicer (Dcr1) into small

interfering RNAs (siRNAs). The Argonaute protein (Ago1)

within the RNA-induced transcriptional silencing complex

(RITS) binds to siRNAs and directs RITS to nascent RNA

transcripts originated from repeat regions (Motamedi et al.,

2004; Verdel et al., 2004). RITS then recruits the CLRC

complex, which contains the H3K9 methyltransferase Clr4,

to initiate H3K9me3 (Zhang et al., 2008; Bayne et al., 2010;

Gerace et al., 2010). Therefore, cenH is critical for the initial

RNAi-mediated targeting of Clr4 to the mating-type region

to establish heterochromatin. But once formed, this hetero-

chromatin is efficiently inherited by subsequent generations

even in the absence of cenH and RNAi.

However, such a simplified explanation is complicated by

later findings that transcription factors Atf1/Pcr1 recognize

target sequences within the silent mating-type region and

cooperate with RNAi to recruit Clr4 to establish hetero-

chromatin (Jia et al., 2004; Kim et al., 2004; Wang and

Moazed, 2017; Wang et al., 2021). Although Atf1/Pcr1

binding sites cannot independently initiate heterochromatin

formation, it still raises concern that removal of cenH does

not completely abolish heterochromatin establishment. To

precisely measure heterochromatin inheritance in the ab-

sence of de novo establishment, ectopic heterochromatin is

established by recruiting a TetR and Clr4-SET domain

(TetR-Clr4-SET) fusion protein to tetO binding sites, leading

to the silencing of adjacent report genes (Audergon et al.,

2015; Ragunathan et al., 2015) (Figure 4B). Releasing TetR-

Clr4-SET from tetO binding sites by the addition of tetra-

cycline allows the examination of heterochromatin main-

tenance through the self-templated restoration of H3K9me3

by endogenous Clr4. This artificial heterochromatin can

persist after TetR-Clr4-SET release, although only after re-

moving an anti-silencing protein Epe1. Moreover, the in-

heritance of such chromatin structure is dependent on the

ability of the Clr4 chromodomain to recognize H3K9me3

(Audergon et al., 2015; Ragunathan et al., 2015). These re-

sults clearly demonstrate that cells can indeed mediate epi-

genetic inheritance of H3K9me3 marked chromatin by

coupling the “reading” and “writing” of H3K9me3. How-

ever, they also indicate that this mechanism alone is not

sufficient to maintain heterochromatin states because of

other mechanisms that counter the inheritance of H3K9

methylation, such as Epe1.

Epe1 contains a JmjC domain, which typically catalyzes

histone demethylation (Tsukada et al., 2006). However, no

demethylase activity of Epe1 has been demonstrated in vitro,

and the commonly used mutations expected to abolish Epe1

Figure 4 Epigenetic inheritance of H3K9me3 in S. Pombe. A, At the silent mating-type region, the replacement of cenH with a ura4

reporter results in two

metastable epigenetic states: ura4-on and ura4-off. The ura4-off state can be maintained during mitosis and meiosis through the coupling of “read-write”

activities of Clr4. B, The tetO sites recruit TetR-Clr4-SET to establish ectopic heterochromatin. The addition of tetracycline release TetR-Clr4-SET and

heterochromatin is maintained by endogenous Clr4.

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Du, W., et al. Sci China Life Sci November (2022) Vol.65 No.11

demethylase activity actually influence protein-protein in-

teractions (Raiymbek et al., 2020). Moreover, Epe1 is known

to exert its function on heterochromatin independent of its

JmjC domain (Wang et al., 2013; Bao et al., 2019; Sorida et

al., 2019). Therefore, the mechanisms whereby Epe1 coun-

teracts histone-based heterochromatin maintenance remain

unclear.

In addition to Epe1, other mechanisms that counteract

heterochromatin inheritance have also been uncovered. At

pericentric repeats, RNAi is the major pathway to establish

heterochromatin. However, heterochromatin is not properly

maintained in RNAi mutants, consistent with the existence of

mechanisms that counteract heterochromatin inheritance.

Interestingly, mutations of the Mst2 histone acetyltransferase

complex, INO80 chromatin remodeling complex, the Paf1C

complex associated with transcription, or Epe1 bypass RNAi

for heterochromatin inheritance (Trewick et al., 2007; Reddy

et al., 2011; Ragunathan et al., 2015; Sadeghi et al., 2015;

Shan et al., 2020). A common theme is that all of them

promote histone turnover at heterochromatin (Aygün et al.,

2013; Sadeghi et al., 2015; Wang et al., 2015; Shan et al.,

2020). A higher histone turnover rate leads to the loss of the

parental histones containing H3K9me3 at the original loca-

tion after DNA replication, therefore breaking the read-write

cycle for chromatin-based epigenetic inheritance (Shan et al.,

2021). As a result, RNAi is constantly needed to maintain a

high concentration of Clr4 to counteract the loss of parental

histones by histone turnover. Supporting this idea, the cou-

pling of siRNA production and H3K9me positive feedback

loops also promotes the inheritance of ectopic hetero-

chromatin induced by siRNAs (Yu et al., 2018b). Therefore,

faithful inheritance of H3K9me3 marked chromatin in fis-

sion yeast adopts multiple approaches including the read-

write mechanism (Wang et al., 2018a), inhibition of histone

turnover, and an increase in the local concentration of H3K9

methyltransferase via the RNAi machinery and DNA se-

quence-specific binding proteins.

DNA methylation inheritance including de novo

deposition and maintenance of DNA

methyltransferases

In mammals, DNA methylation primarily occurs on the fifth

position of cytosine (5-methylcytosine) in the palindromic

CpG context, and DNA methylation is one of the well-stu-

died epigenetic modifications. DNA methylation plays im-

portant roles in stably silencing the inactivated X

chromosome, repetitive elements, imprinting genes and de-

velopmental genes. Long-term transcription repression effect

of DNA methylation is mediated by recruiting methyl-CpG-

binding protein 2 (MECP2) in complex with histone deace-

tylases (HDACs), which reduce chromatin accessibility and

cause local condensation (Jones et al., 1998; Nan et al., 1998;

Muotri et al., 2010). Alternative silencing strategy of DNA

methylation is to prevent methylation-sensitive transcription

factors (TFs) from binding to their cognate sequences (Tate

and Bird, 1993). Besides, DNA methylation could stably

repress gene expression during mitosis and confer plasticity

upon stimulation, suggesting that DNA methylation can also

serve as an epigenetic marker for regulation of epigenetic

transcriptional memory described below.

DNA methylation is catalyzed by DNA methyltransferases

(DNMTs) in mammalian cells, and cytosines in CpG palin-

drome can be unmethylated, hemi-methylated or fully-me-

thylated at various genomic regions. Based on the preference

of the methylated state of cytosine substrates, DNMTs are

classified into two groups: maintenance DNA methyl-

transferase (DNMT1) that shows selective activity toward

hemi-methylated CpG substrates, and de novo DNA me-

thyltransferases (DNMT3A, DNMT3B and rodent specific

DNMT3C) that exhibit comparable activity on both un-

methylated and hemi-methylated substrates (Figure 5).

In the early studies, DNMT1 was defined as maintenance

DNA methyltransferase based on its preferential activity on

hemi-methylated CpGs (Bestor et al., 1988; Pradhan et al.,

1999). However, recent studies challenged this simple clas-

sification model of DNMTs. Firstly, biochemical results

identified considerable de novo methyl-transfer activity of

DNMT1 on unmethylated substrates (Bestor and Ingram,

1983; Jeltsch and Jurkowska, 2014). Further in vivo studies

confirmed the notable de novo activity of DNMT1 enzyme.

In oocytes depleted of Stella, which is a maternal factor

essential for early development, DNMT1 was aberrantly

accumulated at vast chromatin regions, with significant de

novo methyl-transfer activities (Li et al., 2018b). This ac-

tivity was also observed in “Dnmt1” only oocytes (i.e., oo-

cytes with naturally silenced Dnmt3b and genetically

depleted Dnmt3a) (Li et al., 2018b), further confirming the

de novo activity of DNMT1. Furthermore, a well-designed

hairpin-bisulfite sequencing study identified about 0–5% de

novo activity of DNMT1 during replication-coupled phase

(Ming et al., 2021a). Moreover, although DNMT3s mainly

work as de novo methyltransferase, they lack selectivity to-

ward unmethylated and hemi-methylated CpG dinucleotide

substrates. Hairpin-bisulfite sequencing found significant

fully-methylated CpG sites in Dnmt1 depleted ESCs (Arand

et al., 2012), indicating DNMT3s also contribute to DNA

methylation maintenance in vivo. Finally, DNMT3 enzymes

could fill gaps caused by inefficiency of DNMT1 and

counteract demethylation mediated by ten-eleven transloca-

tion (TET) enzymes (Ramsahoye et al., 2000; Liang et al.,

2002; Jackson et al., 2004). Thus, DNMT1 and DNMT3

enzymes are both responsible for considerable de novo de-

position and maintenance of DNA methylation, at least in

some cellular contexts (Ming et al., 2021b). Consistent with

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this idea, these enzymes show context dependent functions in

mammalian development. It would be interesting for future

studies to dissect the contribution of de novo deposition and

maintenance activity of different DNMTs at different de-

velopmental stages or pathological contexts. We will focus

on discussing latest findings on mechanisms of DNA me-

thylation inheritance, the crosstalk between DNA methyla-

tion and histone modifications, and the role of DNA

methylation in epigenetic transcriptional memory.

Dynamics of DNA methylation inheritance

Globally, genomic DNA methylation exhibits a bimodal

distribution pattern in mammalian cells. Most genomic CpG

sites are hypermethylated, however, a fraction of CpG sites

residing in CG-dense DNA sequences named CpG islands

(CGIs) are generally hypomethylated (Cooper et al., 1983;

Bird et al., 1985). CGIs predominantly localize at regions

nearby the transcription start sites (TSSs) or promoters of

house-keeping genes and developmental genes. In addition,

many regulatory elements used to control gene expression

are largely resistant to CpG methylation (Hon et al., 2013;

Ziller et al., 2013; Rasmussen and Helin, 2016). The bimodal

landscape of mammalian methylome is a result of the dy-

namic balance between DNA methylation and demethylation

activities. During development, bulk genomic DNA methy-

lation pattern is static upon differentiation, and demethyla-

tion only occurs at specific sites in response to certain

cellular signals. In contrast, global DNA demethylation

happens in primordial germ cell (PGC) specification stage

and pre-implantation embryos to reset the methylome pat-

tern. In general, DNA methylation inheritance during mitotic

cell division is sophisticatedly regulated by multiple me-

chanisms: chromatin targeting and activity control of

DNMTs to counterbalance the effects of imperfect main-

tenance efficiency of DNMT1 and demethylation mediated

by TET family enzymes. Moreover, DNA methylation

maintenance is mainly a regional regulatory event (Wang et

al., 2020; Ming et al., 2021a) as local chromatin environment

including histone modifications and neighboring CpG state

(CpG densities and methylation levels) are important for the

dynamic turnover and inheritance of mammalian DNA me-

thylome.

Maintenance of DNA methylation during mitotic cell

division

Recent studies indicated that maintenance methylation oc-

curred quickly in a replication-coupled manner, with ap-

proximately 50% of the CpG sites methylated within minutes

and 80% of the sites within 30 min after DNA replication.

However, restoration of DNA methylation following DNA

replication also occured outside the S phase (replication-

uncoupled phase) (Figure 6) (Charlton et al., 2018; Xu and

Corces, 2018; Ming et al., 2021a). To achieve these, mam-

mals have evolved multiple mechanisms to ensure the fide-

lity and robustness of DNA methylation maintenance. For

instance, during mitotic cell division, the chromatin targeting

activity and protein stability of DNMT1 are regulated to

safeguard DNA methylation maintenance. Several key co-

factors, such as PCNA, LIG1 (DNA ligase 1), UHRF1

(ubiquitin-like with PHD and RING finger domains 1),

PAF15 (PCNA associated factor 15) and LSH (lymphoid-

specific helicase), are required for the maintenance role of

DNMT1. During replication-coupled phase, the highly effi-

cient maintenance activity of DNMT1 relies on its connec-

tion with DNA replication forks mediated by multiple

protein-protein interactions including PCNA-DNMT1,

UHRF1-LIG1 and ubiquitinated H3-DNMT1 interactions.

DNMT1 interacts with PCNA, a DNA clamp tethering DNA

polymerases to DNA replication forks, through PBD domain

(PCNA binding domain) (Chuang et al., 1997; Egger et al.,

2006). PAF15 contains a N-terminal H3-like sequence that

could be ubiquitinated at Lys 15 and Lys 24 by UHRF1 (Karg

et al., 2017), and ubiquitinated PAF15 binds to the replication

focus targeting sequence (RFTS) of DNMT1, which facil-

itates the association of DNMT1 with replisomes (González-

Magaña et al., 2019; Nishiyama et al., 2020). LIG1 is a

component of the replication machinery and is responsible

for ligating Okazaki fragments, of which the Lys 126 (K126)

Figure 5 Dynamic interplays between histone modifications and DNA methylation.

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Du, W., et al. Sci China Life Sci November (2022) Vol.65 No.11

and surrounding residues mimicked histone H3K9 site (Ferry

et al., 2017) that can be methylated by G9a/GLP. UHRF1

could bind to methylated LIG1 K126 to contact with re-

plication fork and promote maintenance of the lagging strand

(Ferry et al., 2017). These interactions together facilitate the

efficient methylation maintenance function of DNMT1 in the

replication-coupled phase (Figure 6A).

UHRF1 participates in DNA methylation maintenance

through both replication-coupled phase and replication-un-

coupled maintenance phase (Figure 6B) (Ming et al., 2021a).

Early studies found that UHRF1 was essential for genomic

DNA methylation inheritance by recruiting DNMT1 to me-

thylated sites (Bostick et al., 2007; Sharif et al., 2007).

UHRF1 contains several chromatin targeting domains,

which function cooperatively to promote proper chromatin

targeting of UHRF1 (Arita et al., 2012; Cheng et al., 2013;

Rothbart et al., 2013; Vaughan et al., 2018). The SET and

RING-associated (SRA) domain shows higher binding se-

lectivity towards hemi-methylated CpG sites (hemi-mCG),

which are established after DNA replication (Avvakumov et

al., 2008; Hashimoto et al., 2008; Qian et al., 2008; Fang et

al., 2016). The tandem tudor (TTD) domain of UHRF1

shows high affinity to H3K9me2/3 modification, and the

plant homeodomain (PHD) domain prefers the H3 N-tail

with unmethylated H3R2 (Rajakumara et al., 2011; Arita et

al., 2012; Cheng et al., 2013). Recognition of H3K9me2/3

modified nucleosome is mediated by cooperative binding of

TTD and PHD (Cheng et al., 2013; Rothbart et al., 2013).

Thus, UHRF1 could be recruited to DNA replication foci at

heterochromatin through the UHRF1-LIG1 interaction, re-

cognition of hemi-mCG by SRA domain and the interaction

between TTD-PHD domain of UHFR1 and H3K9me2/3.

Moreover, the TTD domain of UHRF1 and its recognition of

H3K9me2/3 modification are critical also for replication-

uncoupled DNA maintenance (Ming et al., 2021a). UHRF1

contains a RING-finger E3 ligase domain, which is re-

sponsible for histone H3 ubiquitination (Nishiyama et al.,

2013; Qin et al., 2015), and the hydrophobic patch of the

ubiquitin-like (UBL) domain of UHRF1 is required for ef-

ficient H3 ubiquitination, mainly through stabilizing the E2/

E3/chromatin complex (Foster et al., 2018). Early studies

proposed that UHRF1 facilitates methylation maintenance

activity of DNMT1 through recruiting of DNMT1 to re-

plication forks (Bostick et al., 2007; Sharif et al., 2007).

However, recent works indicated that ubiquitinated histone

by UHRF1 could also promote DNMT1 recruitment and

activate its methyltransferase activity (Nishiyama et al.,

2013; Qin et al., 2015; Ishiyama et al., 2017; Foster et al.,

2018; Li et al., 2018a). After DNA replication, many hemi-

CG sites were hindered by histones and other chromatin

proteins. Research found that chromatin remodeler LSH

might function in remodeling nucleosomal CpG sites to ex-

pose them to DNMT1 (Dennis et al., 2001). LSH could

promote nucleosomal CpG methylation maintenance in re-

plication-uncoupled phase, especially in heterochromatin

regions. Besides, LSH also associates with UHRF1, which

could assist the UHRF1-DNMT1 DNA methylation main-

tenance pathway(Han et al., 2020). Among all co-factors of

DNMT1, UHRF1 appears to be the most important co-factor

for the maintenance of DNA methylation, as Uhrf1 depletion

dramatically damages the pattern and kinetics of the main-

tenance of DNA methylation, comparable to Dnmt1 knock-

out.

Mechanisms for restricting excessive activity of DNMT1

Multiple mechanisms are evolved to restrict the activity of

DNMT1 to avoid accumulation of aberrant DNA methyla-

tion at unmethylated sites through its de novo methylation

activity. First, the protein levels of both DNMT1 and its key

Figure 6 Epigenetic inheritance of DNA methylation.

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Du, W., et al. Sci China Life Sci November (2022) Vol.65 No.11

cofactor UHRF1 are cell-cycle regulated. It was reported that

DNMT1 stability is regulated by a series of integrated post-

translational modifications, including methylation (Estève et

al., 2009; Estève et al., 2011), acetylation (Du et al., 2010),

and phosphorylation (Estève et al., 2011), which co-

ordinately determine DNMT1 ubiquitination levels and

protein stability. Methyltransferase SET7/9 methylates

DNMT1 and triggers poly-ubiquitination and degradation of

DNMT1 (Estève et al., 2009; Estève et al., 2011), whereas

lysine-specific demethylase 1 (LSD1) stabilizes DNMT1

proteins, likely through demethylation (Zhang et al., 2019).

Previous studies reported that AKT1 kinase phosphorylates

DNMT1 Ser143 and interferes with lysine 142 methylation

by SET7/9 (Estève et al., 2011). Therefore, the interplay of

these two modifications affects cellular DNMT1 stability.

Another study identified that cell-cycle regulated methyl-

transferase SET8/PR-Set7 could control the stability of both

DNMT1 and UHRF1 through its methylation activity, fol-

lowed by subsequent poly-ubiquitination mediated protein

degradation (Zhang et al., 2019). SET8/PR-Set7 could down-

regulate UHRF1 in G2/M phase, causing repression of the

activity of DNMT1 on post-replicated DNA (Zhang et al.,

2019). Thus, SET8/PR-Set7 and LSD1 compete to regulate

genomic DNA methylation, most likely through regulation

of UHRF1 protein levels. DNMT1 could be acetylated by

acetyltransferase TIP60 for subsequent ubiquitination and

proteasomal degradation (Du et al., 2010). On the contrary,

histone deacetylase 1 (HDAC1) and deubiquitinase could

stabilize cellular DNMT1 levels (Du et al., 2010; Cheng et

al., 2015). The acidic pocket in ubiquitin-specific protease 7

(USP7) interacts with lysine residues within KG linker of

DNMT1, and this interaction is important for USP7 mediated

DNMT1 stabilization (Cheng et al., 2015). Acetylation of

lysine residues in DNMT1 KG linker interferes its binding to

USP7, thus promoting ubiquitination and degradation of

DNMT1 (Cheng et al., 2015). Besides, multiple-interaction

networks among DNMT1, UHRF1, PCNA, LIG1, PAF15,

LSH and histone H3 ubiquitination not only facilitate the

sophisticated contact of DNMT1 with the replication fork in

the replication-coupled phase, but also promote its targeting

to sites for methylation in the replication-uncoupled phase.

For instance, the specific binding between the SRA domain

of UHRF1 and hemi-methylated CpG ensures DNMT1 to

predominantly function as a maintenance methyltransferase.

The interaction between the TTD-PHD module of UHRF1

and H3K9me2/3 helps to confer some degree of targeting

specificity for DNMT1 (Cheng et al., 2013; Rothbart et al.,

2013). Importantly, DNMT1 mediated methylation main-

tenance heavily relies on H3 ubiquitination, which has a fast

turnover rate and is removed by USP7 after DNMT1 re-

cruitment (Nishiyama et al., 2013; Yamaguchi et al., 2017).

Interestingly, the de novo methylation activity of DNMT1

has to be tightly controlled. For example, during oocyte

maturation, Stella is required to prevent aberrant accumula-

tion of DNA methylation mediated by the de novo methy-

lation activity of DNMT1, via disrupting the chromatin

association of UHRF1 (Li et al., 2018b; Du et al., 2019). It is

interesting to investigate whether the de novo methylation

activity of DNMT1 is also under tight control in other post-

mitotic cells that may allow aberrant methylation accumu-

lation by the weak de novo methylation activity of DNMT1,

especially during aging. Finally, DNA methylation main-

tenance efficiency is affected by the methylation levels of

nearby CpG sites, which ensures the robustness in main-

taining a bistable system that allows faithful maintenance of

highly methylated regions and unmethylated regions, but not

intermediately methylated regions (Ming et al., 2021a).

Crosstalk between DNA methylation and histone

modifications

Somatic DNA methylation is set up de novo at early embryo

development and maintained during subsequent mitotic cell

cycles. Recent studies revealed complicated interplay be-

tween DNA methylation and histone modifications. As de-

tailed below, some histone modifications help to recruit

DNMTs to certain genomic regions and boost their methyl-

transfer activities, while others exclude them from chromatin

with certain genomic features and suppress their catalytic

abilities. Moreover, transitions from histone modification to

DNA methylation were also observed in some processes

including X-chromosome inactivation. For instance, during

early stage of X-chromosome inactivation, H3K27 methy-

lation by PRC2 complex silences one of the two X chro-

mosome in female mammals, and this silencing mechanism

is replaced by promoter DNA methylation during later stage

of this process (Avner and Heard, 2001; Csankovszki et al.,

2001; Sado et al., 2004; Disteche and Berletch, 2015; Pinter

et al., 2012). Interestingly, inactivated X-chromosome was

globally hypomethylated due to reduced H3K36 methylation

caused by transcriptional silencing, except for promoter re-

gions.

H3K4 methylation counters DNA methylation

DNA methylation is largely excluded from H3K4 methyla-

tion marked regions, such as active gene promoters and en-

hancers. In recent years, a series of studies revealed the

underlying mechanism whereby H3K4 methylation repels

the deposition of DNA methylation. DNMT3L is an en-

zymatically inactive homolog of DNMT3A and DNMT3B,

and is required for establishing the DNA methylation land-

scape during gametogenesis (Bourc’his et al., 2001). More-

over, DNMT3L was reported to stimulate the activity of

DNMT3A (Chedin et al., 2002). Structure analysis indicates

that DNMT3L forms a functional heterotetramer with

DNMT3A to promote de novo DNA methylation (Jia et al.,

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Du, W., et al. Sci China Life Sci November (2022) Vol.65 No.11

2007), and this interaction also prevents the oligomerization

of DNMT3A (Jurkowska et al., 2011). DNMT3L binds to the

N-terminal H3 sequence, and this interaction was specifi-

cally suppressed by H3K4 methylation both in vitro and in

vivo (Figure 5A) (Ooi et al., 2007). The DNMT3A-

DNMT3L structural data indicates that DNMT3L binds to

unmethylated H3 tail and promotes de novo DNA methyla-

tion through either enhancing the recruitment or activation of

DNMT3A (Ooi et al., 2007). Another structural study found

that the ATRX-DNMT3-DNMT3L (ADD) domain of

DNMT3A also specifically binds to H3 N-tail without H3K4

methylation (Otani et al., 2009), suggesting ADD might re-

cognize the unmethylated state of H3K4 and help DNMT3A

to target chromatin properly. Methylation analysis using in

vitro reconstituted chromatin showed that full-length

DNMT3A and full-length DNMT3A/3L complexes methy-

late DNA, preferentially at linker DNA regions, of H3K4-

unmethylated chromatin more efficiently than H3K4me3

marked chromatin (Zhang et al., 2010). The authors con-

cluded that the improved activity of DNMT3A on H3K4-

unmethylated chromatin was due to the selective binding

property of ADD domain to H3K4-unmethylated region.

Supporting this idea, the activity of catalytic domain of

DNMT3A was not affected by H3K4me3 per se (Zhang et

al., 2010). However, it is still unclear whether unmethylated

H3 tail could induce allosteric activation of functional

DNMT3A complex. Strikingly, an independent study

showed that the activity of DNMT3A was stimulated by up to

8-fold by H3K4-unmethylated H3 tail (Li et al., 2011).

However, the underlying molecular mechanism of allosteric

regulation remains elusive. The autoinhibitory DNMT3A-

DNMT3L complex and catalytically active DNMT3A-

DNMT3L-H3 complex helped to clarify this issue. In the

autoinhibitory structure, ADD domain of DNMT3A sup-

presses the methylation activity of DNMT3A by binding to

catalytic domain (CD) of DNMT3A and thereby blocking

DNA binding of CD (Guo et al., 2015). Histone H3 tail with

unmethylated state of H3K4 specifically disrupts the ADD-

CD interaction, and therefore release the autoinhibitory ef-

fects of DNMT3A (Guo et al., 2015). These studies provide a

new insight in understanding the mutually exclusive geno-

mic distribution of DNA methylation and H3K4 methyla-

tion. H3K4 methylation at promoters might be a potential

mechanism for excluding DNA methylation at CGIs, how-

ever, mechanisms that maintain the hypomethylated state of

CGIs are still elusive. It is proposed that transcription factors

(Brandeis et al., 1994; Macleod et al., 1994), active de-

methylation by TET enzymes (Williams et al., 2012; Putiri et

al., 2014; Verma et al., 2018) and skewed GC distribution

nearby TSS (Ginno et al., 2012) all contribute to this process.

In contrast to Dnmt3a or Dnmt3b deficient mice, Dnmt3l

knockout mice are viable (Okano et al., 1999; Bourc’his et

al., 2001). The functional requirement of DNMT3L for de

novo methylation in vivo is still not fully answered.

Although DNMT3A is generally depleted at CGIs, it has

been found that mutations at PWWP domain result in re-

distribution of DNMT3A to genomic regions including CGIs

marked by ubiquitinated H2AK119 (Figure 5C) (Remacha et

al., 2018; Heyn et al., 2019; Weinberg et al., 2021). The

amino terminus of DNMT3A1 interacts with H2AK119ub-

marked nucleosomes, serving as another chromatin targeting

strategy for DNMT3A1 (Weinberg et al., 2021). This novel

interaction explains the aberrant genomic distribution of

DNMT3A and hypermethylation at Polycomb-regulated re-

gions in paragangliomas and microcephalic dwarfism con-

taining mutations at the PWWP domain of DNMT3A. In the

future, it would be interesting to determine whether the N-

terminus of DNMT3B and the tissue-specific or cancer-

specific DNMT3A/3B isoforms (Chen et al., 2002; La Salle

and Trasler, 2006; Gopalakrishnan et al., 2009; Duymich et

al., 2016) also utilize similar strategies for chromatin tar-

geting.

Different methylation state of H3K36 dictates chromatin

targeting of DNMT3A and DNMT3B

For many years, the PWWP domain of DNMT3A/3B has

been linked to chromatin targeting. PWWP domain, which

contains a conserved aromatic cage for recognition of me-

thylated lysine residue, is important for protein chromatin

targeting through synergistic binding of histone and DNA

(Wu et al., 2011; Qin and Min, 2014; Dukatz et al., 2019).

Early study demonstrated PWWP domains are important for

targeting DNMT3A/DNMT3B to major satellite regions

(Chen et al., 2004; Ge et al., 2004). Loss of DNA methyla-

tion at satellite sequences was found in genetic diseases

bearing PWWP mutations, such as ICF (immunodeficiency,

centromere instability and facial anomalies) syndrome

(Shirohzu et al., 2002). Together, these reports link the

chromatin recruitment of DNMT3A/3B through the PWWP

domain. Biochemical and structural studies indicate that

PWWP domain of DNMT3A/3B interacts with H3K36me3

(Dhayalan et al., 2010; Qin and Min, 2014; Rondelet et al.,

2016). Genomic binding profile studies found that DNMT3B

is preferentially recruited to transcribed gene bodies, which

are enriched with active H3K36me3 mark mediated by SET

domain containing 2 (SETD2) (Figure 5B) (Sun et al., 2005;

Edmunds et al., 2008; Baubec et al., 2015). SETD2 and

PWWP domain are both required for proper targeting of

DNMT3B to active gene bodies (Baubec et al., 2015),

highlighting the importance of H3K36me3-PWWP interac-

tion for de novo methylation. However, DNMT3A, different

to DNMT3B, could interact with both di- and tri-methylated

state of histone H3K36, and shows a higher binding affinity

towards H3K36me2 (Weinberg et al., 2019). H3K36me2 is

distributed at both intergenic regions and gene bodies, and is

catalyzed by two NSD histone methyltransferase family

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Du, W., et al. Sci China Life Sci November (2022) Vol.65 No.11

enzymes, such as NSD1 and NSD2 (Kuo et al., 2011).

Genome wide analysis demonstrated that DNMT3A is tar-

geted to intergenic regions through DNMT3A-H3K36me2

interaction, which can be mistargeted to H3K36me3 mod-

ified gene bodies in cells depleted of Nsd1 and Nsd2

(Weinberg et al., 2019). Thus, the PWWP domains of

DNMT3A and DNMT3B recognize different methylation

state of H3K36 and target these enzymes for methylation at

different chromatin regions.

Relationship between H3K27me3 and DNA methylation

Mammalian H3K27me3 modification is catalyzed by Poly-

comb repressive complex 2 (PRC2) (Margueron and Rein-

berg, 2011). H3K27me3 is almost exclusively associated

with CGI regions, which is generally hypomethylated in

embryonic stem cells (ESCs) (Cooper et al., 1983; Bird et al.,

1985; Ku et al., 2008), indicating the mutually exclusive

distribution of H3K27me3 and DNA methylation in ESCs.

This might be partially explained by the dependency of DNA

methylation on H3K36me2/3, and the antagonizing dis-

tribution pattern of H3K36 and H3K27 methylation (Papp

and Müller, 2006; Schmitges et al., 2011; Yuan et al., 2011;

Gaydos et al., 2012; Popovic et al., 2014; Lu et al., 2016;

Huang and Zhu, 2018). Further studies focused on the PRC2

accessory proteins uncovered a more direct molecular me-

chanism underlying the mutually exclusive distribution of

H3K27me3 and DNA methylation. PRC2 accessory pro-

teins, such as PHF1, MTF2, JARID2, AEBP2 and PHF19,

are likely involved in the recruitment and/or regulation of

enzymatic activity of PRC2 (Cao et al., 2008; Boulay et al.,

2011; Casanova et al., 2011; Ballaré et al., 2012; Brien et al.,

2012; Hunkapiller et al., 2012; Oksuz et al., 2018; Youmans

et al., 2018; Højfeldt et al., 2019). Structural analysis iden-

tified the N-terminus of PHF1 and MTF2 bind to un-

methylated CpG motif (Li et al., 2017), highlighting a

potential mechanism for the restrictive recruitment of PRC2

to these unmethylated regions. In addition, PRC2 was also

functionally associated with TET1 enzyme (Neri et al.,

2013). Collectively, these studies uncovered the mutually

exclusive distribution of H3K27me3 and DNA methylation,

and the underlying potential molecular mechanisms. How-

ever, H3K27me3 and DNA methylation do overlap at some

genomic regions in certain somatic and cancer cells

(Brinkman et al., 2012; Statham et al., 2012). During dif-

ferentiation and carcinogenesis, H3K27me3-silenced gene

promoters contain DNA methylation in some sites (Ohm et

al., 2007; Schlesinger et al., 2007; Mohn et al., 2008; Rose

and Klose, 2014; Chen et al., 2019; Sendžikaitė et al., 2019).

Moreover, it has been shown that during early phase of X-

chromosome inactivation, expression of Xist RNA recruits

Polycomb complex for gene silencing. Subsequently, the

H3K27me3 silencing mechanism is switched to promoter

DNA methylation for long term silencing (Augui et al., 2011;

Jégu et al., 2017; Galupa and Heard, 2018). However, the

inactivated X-chromosome exhibits a global DNA hypo-

methylated state due to transcription silencing and reduced

H3K36 methylation. Therefore, the relationship and transi-

tion between DNA and H3K27 methylation are complicated,

which requires further mechanistic studies.

Crosstalk between H3K9me2/3 and DNA methylation

There is a direct cross talk between H3K9 methylation and

DNA methylation. H3K9 methylation is required for all

DNA methylation in Neurospora crassa (Tamaru and Selker,

2001; Tamaru et al., 2003). In Arabidopsis thaliana CpNpG

methylation is also dependent on H3K9 methylation (Jack-

son et al., 2002). Albeit lack of a strict link between H3K9me

and DNA methylation deposition in mammals, these two

repressive modifications are co-localized at heterochromatin

regions. Early studies proposed that DNMT3A/3B could

deposit DNA methylation through binding to hetero-

chromatin protein 1 (HP1) that recognizes H3K9me3 (Leh-

nertz et al., 2003). Further, several studies describe direct

interactions between DNMT3A/3B and the H3K9 methyl-

transferases, such as SUV39H1 (Fuks et al., 2003), SETDB1

(Li et al., 2006) and G9a/GLP (Epsztejn-Litman et al., 2008;

Chang et al., 2011). However, the functional significance of

these interactions to promote DNA methylation has not yet

been fully studied.

Compared with de novo DNA methylation, DNA methy-

lation maintenance mediated by DNMT1-UHRF1 machinery

shows a closer connection with H3K9me2/3 modification.

As mentioned above, the TTD and PHD domains of UHRF1

cooperatively bind to H3K9me2/3 modification and show a

preference on trimethylated state of H3K9 (Figure 6A)

(Hashimoto et al., 2009; Rottach et al., 2010). Due to the

technical limitations of knockdown and overexpression ex-

periments, TTD was thought to be essential for UHRF1

chromatin targeting and DNA methylation maintenance in

early studies (Rothbart et al., 2012; Rothbart et al., 2013).

However, genome edited homozygous TTD mutant mice

only shows about 10% reduction of DNA methylation in

various tissues tested (Zhao et al., 2016), indicating limited

role of TTD for DNA methylation maintenance. These works

all focused on the readout of global methylation level, but

little is known about the contribution of TTD in the kinetics

of DNA methylation maintenance. Recent kinetic analysis

demonstrated that TTD and H3K9me2/3 are important for

replication-uncoupled DNA methylation maintenance (Ming

et al., 2021a). Besides, RFTS domain of DNMT1 could di-

rectly recognize H3K9me3 and facilitate DNA methylation

maintenance (Ren et al., 2020). DNMT1 was also reported to

directly interact with G9a to promote its maintenance effi-

ciency during replication (Estève et al., 2006). Together,

these studies highlight the importance of direct crosstalk

between H3K9 methylation and DNA methylation.

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H3 ubiquitination facilitates the recruitment and activation

of DNMT1

Detailed molecular mechanisms of the UHRF1-DNMT1

maintenance apparatus in DNA methylation inheritance were

carefully illustrated until recent years. Histone H3 was

identified as a ubiquitination target of UHRF1 using Xenopus

egg extracts (Nishiyama et al., 2013); and both the PHD and

RING domains are important for efficient ubiquitination of

H3 (Qin et al., 2015). Mass spectrometry analysis identified

that H3 could be ubiquitinated at Lys14 (Ishiyama et al.,

2017), Lys18 (Qin et al., 2015; Ishiyama et al., 2017) and

Lys23 (Nishiyama et al., 2013; Ishiyama et al., 2017), and

these ubiquitinated sites could be recognized by the ubiquitin

interacting motif (UIM) of RFTS domain in DNMT1 protein

(Qin et al., 2015). Crystal structure identified a novel re-

cognition mode of RFTS which simultaneously binds to

double ubiquitinated H3 at K18 and K23 (Ishiyama et al.,

2017). It was also reported that RFTS domain mediates the

homodimerization (Fellinger et al., 2009) and autoinhibition

(Syeda et al., 2011; Takeshita et al., 2011) of DNMT1.

Strikingly, DNMT1 opens its active site upon binding to

H3K18ub/K23ub by the RFTS domain, indicating that ubi-

quitinated histone by the UHRF1 could allosterically activate

the activity of DNMT1. Therefore, these works together

demonstrated the essential role of the ubiquitin-binding

module of DNMT1 in DNA methylation maintenance (Ish-

iyama et al., 2017).

A role of H4K20 methylation in recruiting DNMT1 to

LINE-1 region

Generally, DNA methylation, H3K9me3 and H4K20me3 co-

exist at many heterochromatin regions. These repressive

marks function cooperatively to silence repetitive DNA se-

quences in mammalian genomes. Compared with the well-

known connections between DNA methylation and

H3K9me3, the crosstalk between DNA methylation and

H4K20me3 is less reported. Disturbance of DNA methyla-

tion and H4K20me3 frequently occurs in cancer cells

(Feinberg and Vogelstein, 1983; Eden et al., 2003; Fraga et

al., 2005). Reactivation of repetitive elements, especially

long interspersed nuclear element-1 (LINE-1), is tightly as-

sociated with genome rearrangements in cancers (Rodriguez-

Martin et al., 2020), highlighting the importance of the re-

pression of repetitive LINE-1. DNMT1 contains two bromo-

adjacent homology (BAH) domains (Yarychkivska et al.,

2018), which have been recently shown to specifically re-

cognize methylated H4K20 with a preference for H4K20me3

(Ren et al., 2021). Furthermore, H4K20me3 bound to BAH1

domain could induce an allosteric stimulation of DNMT1

activity (Ren et al., 2021), and the BAH1-H4K20me3

binding module facilitates DNA methylation maintenance

especially for the LINE-1 elements (Ren et al., 2021). This

work provides a direct crosstalk between DNA methylation

and H4K20me3. Thus, RFTS and BAH1 domains of

DNMT1 bind to H3ub/H3K9me3 and H4K20me3, respec-

tively, highlighting multivalent communications among re-

pressive marks and DNA methylation maintenance.

Nevertheless, it remains unclear whether the de novo activity

of DNMT1 participates in DNA methylation at LINE-1 re-

gions. Furthermore, novel crosstalk between histone mod-

ifications and different domains of DNMTs warrants further

investigation.

Roles of DNA methylation in transcriptional

memory

Epigenetic memory of gene transcription was described as a

heritable change in gene expression or behavior that is in-

duced by an experienced stimulus (D’Urso and Brickner,

2014). Epigenetic memory could be set up and maintained by

various epigenetic players, including DNA methylation,

histone modifications, histone variants and chromatin re-

modelers. Epigenetic memory can be divided into cellular

transcriptional memory and transgenerational memory based

on the time scales of memory maintained. Cellular tran-

scriptional memory refers to the mitotically heritable tran-

scriptional state in response to development cues or

environmental stimuli, while the transgenerational memory

describes meiotically heritable transcriptional profile gen-

erated by experiences of previous generations (D’Urso and

Brickner, 2014). Adaptive immunity, chronic inflammation,

and neuronal memory are ideally suitable contexts for

studying the molecular mechanisms underlying the estab-

lishment and maintenance of transcriptional memory.

Transcriptional memory allows certain genes to respond

more rapidly and robustly toward previously experienced

signals (Bergink et al., 1973). During past years, transcrip-

tional memory of the inducible inositol-1-phosphate syn-

thase (INO1) and galactokinase (GAL) genes in

Saccharomyces cerevisiae system are thoroughly studied. It

has been shown that several factors/players including nuclear

periphery retention, intragenic looping, H2A.Z variant de-

position, H3K4 methylation and chromatin remodeler SWI/

SNF are important for transcriptional memory. In addition to

histone related players, transcriptional memory of Tat gene

upon glucocorticoid induction is associated with DNA de-

methylation event (Thomassin et al., 2001), and similar de-

methylation was also reported at IL2 gene locus after T cell

activation (Murayama et al., 2006). These studies imply the

biological significance of DNA demethylation on transcrip-

tional memory establishment and maintenance. Recent

published works further demonstrated the importance of

DNA methylation in transcriptional memory regulation.

Although short-term treatment of tumor necrosis factor α

(TNF-α) could activate DNA methylation silenced IL32 gene

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Du, W., et al. Sci China Life Sci November (2022) Vol.65 No.11

without demethylation step, prolonged TNF-α treatment in-

duces DNA demethylation at both the promoter and CGI

region of IL32 gene which depends on TET and p65 (Zhao et

al., 2019). Strikingly, demethylation-induced transcriptional

activation of IL32 persists for a long time after withdrawing

of TNF-α (Zhao et al., 2019). Moreover, sustained TNF-α

administration uncovers a transcriptional memory induced

by the key proinflammatory cytokine TNF-α (Zhao et al.,

2020b). CALCB gene, the key therapeutic target gene in

migraine, shows the strongest transcriptional memory and

relies on the active demethylation mediated by TET enzymes

(Zhao et al., 2020b). These results suggest that inflammatory

signals and memory consolidation might play a role in the

development of chronic migraine. Collectively, these works

demonstrated that transcriptional memory provoked by TNF-

α is governed by active DNA demethylation by TET en-

zymes. The hypomethylated state of memory gene and re-

lated regulatory region might facilitate the chromatin binding

of subsequent methylation-sensitive transcription factors,

which in turn provoke rapid and robust transcription acti-

vation in the subsequent encounter of inflammatory stimuli.

Notably, it is intriguing whether DNA demethylation medi-

ated transcriptional memory towards certain environmental

and cellular stimuli might also be involved in the develop-

ment of adaptive immunity malfunction, chronic inflamma-

tion, aging and cancer.

Functional impact of epigenetic inheritance

Factors discussed above involved in epigenetic inheritance are

important to maintain chromatin states and cell identity.

Therefore, it is easy to envision the critical roles of epigenetic

inheritance in cell identity during normal development, in

disease evolution and in response to environmental stress/cues.

In reality, it is challenging to directly link a malfunction in

epigenetic inheritance to the occurrence of a particular phe-

notypes/disease at organism levels. Below, we outline several

examples in which alterations of factors involved in epigenetic

inheritance contribute to developmental defects and cancers.

Defects in genomic imprinting

A couple of well-studied examples linking defects in epi-

genetic inheritance to human diseases are the Prader-Willi

syndrome (PWS) and Angelman syndrome (AS). PWS and

AS are distinct human neurological disorders resulting from

defects in genomic imprinting of a gene cluster at 15q11q13

locus. While some genes are only expressed from the ma-

ternal allele, several genes including SNRPN and SnoRNA

are expressed paternally, with the maternal allele silenced

through DNA methylation. PWS is caused by the loss of the

expression of paternally expressed genes, whereas AS is

caused by loss of expression of maternally expressed genes.

It is estimated that 86% patients with PWS and 92% patients

with AS are caused by epimutations without changes at un-

derlying DNA sequence. Of note, about one third of these AS

patients show somatic mosaicism in which cells with im-

printing defects and normal cells co-exist (Horsthemke and

Buiting, 2008). These results indicate that a majority of PWS

and AS cases are caused by sporadic errors during the pro-

cess of establishment, and maintenance of this imprinting

locus. Future studies are needed to understand the molecular

basis for the generation of epimutations at this imprinted

gene cluster.

With the advancement of sequencing-based technologies,

more and more imprinted genes have been identified. Cur-

rently, it is estimated that over 220 genes are imprinted in

human genome (Horsthemke and Buiting, 2008). Moreover,

in addition to DNA methylation based on mechanism of

genomic imprinting, H3K27me3 alone can also imprint

genes during mouse early development (Inoue et al., 2017a;

Inoue et al., 2017b). These advancements will likely provide

additional insights into how alterations in imprinting con-

tribute to human diseases. For more information about

genomic imprinting, we direct readers to two recent reviews

on this topic (Peters, 2014; Monk et al., 2019).

A critical role for CAF-1 in maintaining chromatin states

and cell identity during development and tumorigenesis

CAF-1, the first identified histone chaperone involved in

deposition of new H3-H4 onto replicating DNA, plays an

important role in maintaining chromatin states from yeast to

human. An early study in Arabidopsis found that CAF-1 is

important to maintain cellular and functional organization of

both shoot apical meristem and the root apical meristem

(Kaya et al., 2001), which are responsible for postembryonic

development of plant architecture. Recently, it has been

shown that depletion of CAF-1 in mouse ES cells results in

an increase in 2C-like cells (Ishiuchi et al., 2015). Further-

more, depletion of CAF-1 in mouse embryonic fibroblast

increases in the reprograming efficiency of these cells into

iPSC, likely due to an increase in chromatin accessibility

(Cheloufi et al., 2015). Therefore, CAF-1 is important to

maintain chromatin states and cell identity likely in all cell

types during normal development.

Two recent studies also report that alterations in CAF-1

expression can promote tumorigenesis and drive tumor me-

tastasis. It is known for a long time that CHAF1B, a subunit

of CAF-1, is overexpressed in several solid tumors and acute

megakaryocytic leukemia (AMKL) (Polo et al., 2010; Sy-

karas et al., 2021). However, it was not known whether the

overexpression of CHAF1B has any role in tumorigenesis.

Using mouse models, it has been shown that CAF-1 is es-

sential for normal hematopoiesis. However, overexpression

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Du, W., et al. Sci China Life Sci November (2022) Vol.65 No.11

of CHAF1B interferes with the association of transcription

factors such as CEBPA involved in myeloid differentiation,

which in turn promotes leukemia. The effects of CHAF1B

overexpression on leukemia genesis are linked to the role of

CHAF1B in nucleosome assembly of new H3-H4 (Volk et

al., 2018). On newly replicating chromatin, transcription

factor binding sites are temporarily blocked (Ramachandran

and Henikoff, 2016). As parental H3-H4 can memorize their

positions along DNA following DNA replication (Escobar et

al., 2019), it is likely that the block of transcription factors is

caused by the deposition of new H3-H4 by CAF-1. There-

fore, overexpression of CHAF1B likely exacerbates the

blocking effects of CAF-1, thereby inhibiting myeloid dif-

ferentiation. In the future, it would be interesting to de-

termine whether CAF-1 overexpression in solid tumors also

plays a causal role in tumorigenesis.

While CAF-1 overexpression promotes leukemia, a recent

study indicates that reduced CAF-1 expression contributes to

tumor metastasis (Gomes et al., 2019). Tumor metastasis,

referring to cancer cells migrating from the primary organs

through the blood or lymph systems and forming tumors at

new organs, contributes to the largest fraction of cancer-

induced death (Fares et al., 2020). Genome wide analysis of

several tumors and their matched metastatic ones indicate

that epigenetic changes, but not genetic mutations, are likely

the dominant force in the development of tumor metastasis

(McDonald et al., 2017; Chatterjee et al., 2018). Using car-

cinoma models, it has been shown that metastatic signals

suppress the expression of CAF-1, leading to reduced density

of canonical histone H3.1/H3.2, which are assembled into

nucleosomes by CAF-1. This will trigger an increase in

HIRA mediated nucleosome assembly of H3.3 and the ac-

quisition of more aggressive and metastatic characteristics of

cancer. Depletion of HIRA suppresses the metastatic phe-

notypes (Gomes et al., 2019). Together, these studies high-

light the dynamic regulation of CAF-1-mediated nucleosome

assembly of H3.1/H3.2 and HIRA-mediated nucleosome

assembly of H3.3 in changes of cell identity to a more me-

tastatic one. In the future, it would be interesting to de-

termine to what extent other factors involved in epigenetic

inheritance discussed above may play in promoting cell fate

changes and thereby metastasis.

Summary and future directions

In the last several years, we have witnessed major advances

in the understanding of epigenetic inheritance. Specifically,

studies from various systems have established that repressive

histone modifications can be inherited, at least in part,

through the read-and-write mechanism. Because of the dy-

namic and reversible natures of histone modifications, it is

also clear that non-coding RNAs and DNA sequence specific

binding proteins are needed to recruit histone modifying

enzymes locally for the stable inheritance of a histone

modification. More importantly, we have also witnessed

major advances in our understanding of the recycling of

parental histones, which contain epigenetic modifications,

following DNA replication. Finally, we have also begun to

appreciate the importance of maintenance of chromatin

states and cell identity to prevent diseases including tumors.

These advances have laid a solid foundation for dissecting

molecular mechanisms of epigenetic inheritance during

normal development, and in tumorigenesis. However, many

questions still remain to be answered. How is parental his-

tone transfer/recycling regulated? Are there other factors

involved in parental histone transfer? Is there any co-

ordination between parental histone transfer and de novo

deposition of new H3-H4, and if there is, how do these two

pathways coordinate to promote nucleosome formation?

Considering the extensive cross talk between DNA methy-

lation and histone modifications, do protein machineries

involved in the heritance of DNA methylation also con-

tribute to the inheritance of histone modifications, or vice

versa? Do alterations in epigenetic inheritance contribute to

the establishment of alterative chromatin states that specify

disease evolution such as the transition of cancer cells to

metastatic cells? Future studies to address these and other

questions in epigenetic inheritance will advance our under-

standing of epigenetic inheritance and the contribution of

malfunction of this process to human disease.

Compliance and ethics The author(s) declare that they have no conflict

of interest.

Acknowledgements This work was supported by the National Natural

Science Foundation of China (31725015, 31830048 to Q.L. and 32000417

to W.D.), the Beijing Outstanding Young Scientist Program

(BJJWZYJH01201910001005 to Q.L.), the National Key Research and

Development Project of China (2019YFA0508903 to Q.L.), the China

Postdoctoral Science Foundation (2020M670487 to W. D.), the Chinese

Academy of Sciences (XDB 37010100 and QYZDY-SSW-SMC031 to B.Z.),

the K. C. Wong educational foundation (GJTD-2020-06 to B.Z.) and the

National Institutes of Health (R35 GM126910 to S.J. and R35 GM115018 to

Z.Z.). We apologize that we could not cite all references because of space

limitations.

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