ll

Leading Edge

Review
3D genome, on repeat:
Higher-order folding principles
of the heterochromatinized repetitive genome
Spencer A. Haws,1,2,3,4 Zoltan Simandi,1,2,3,4 R. Jordan Barnett,1,2,3 and Jennifer E. Phillips-Cremins1,2,3,*
1Department of Bioengineering, University of Pennsylvania, Philadelphia, PA, USA
2Epigenetics Institute, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, USA
3Department of Genetics, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, USA
4These authors contributed equally

*Correspondence: jcremins@seas.upenn.edu
https://doi.org/10.1016/j.cell.2022.06.052

SUMMARY

Nearly half of the human genome is comprised of diverse repetitive sequences ranging from satellite repeats
to retrotransposable elements. Such sequences are susceptible to stepwise expansions, duplications, inver-
sions, and recombination events which can compromise genome function. In this review, we discuss the
higher-order folding mechanisms of compartmentalization and loop extrusion and how they shape, and
are shaped by, heterochromatin. Using primarily mammalian model systems, we contrast mechanisms gov-
erning H3K9me3-mediated heterochromatinization of the repetitive genome and highlight emerging links be-
tween repetitive elements and chromatin folding.

HETEROCHROMATIN: A HISTORICAL PERSPECTIVE ON
PROPERTIES, MECHANISMS, AND FUNCTIONS
Eukaryotic genomes are broadly partitioned into heterochromat-
in and euchromatin. The existence of heterochromatin was first
reported by Emil Heitz in 1928 as high-intensity staining of
DNA dye throughout the cell cycle in specific genome fragments
(Heitz, 1928). In the nearly 100 years following Heitz’s initial
discovery, the molecular properties and biological roles of het-
erochromatin have been elucidated in detail (Figure 1, top). Het-
erochromatin is relatively compact, replicates at the end of S
phase, and is refractory to both enzymatic digestion and sonicat-
ion (Becker et al., 2017; Lima-de-Faria and Jaworska, 1968;
Wallrath and Elgin, 1995). A leading theory is that heterochro-
matin is relatively inaccessible to transcription factors due to
its condensed state, and, therefore, the genes it encompasses
are transcriptionally silent (Becker et al., 2016). By contrast,
euchromatin is gene rich, transcriptionally active, and accessible
to DNA binding factors that promote gene expression. Therefore,
Heintz’s original observation of heterochromatin as a distinct nu-
clear feature has stood the test of time over nearly 100 years of
imaging, genetics, genome engineering, and sequencing
studies.
Significant advances have been made in our understanding of
the classes of heterochromatin and their interplay with genome
function. The causal effect of heterochromatin on gene silencing
was demonstrated with classic genetic screens or transgene
studies in Drosophila. As early as the 1930s, the phenomenon
of position-effect variegation (PEV) was reported in which active
genes are stochastically silenced upon ectopic placement in
juxtaposition to heterochromatin (Elgin and Reuter, 2013; Muller
and Altenburg, 1930). Moreover, in 1949, the inactive X chromo-
some was observed as a heterochromatinized Barr body to
achieve dosage compensation of X-linked genes in females
(Barr and Bertram, 1949). These classic studies, among many
others, provided early insight which suggested that heterochro-
matin is generally associated with a repressive role on gene
expression.
Heterochromatin also plays a chief role in the protection of
genome stability by repressing repetitive DNA elements (Peters
et al., 2001). Repetitive DNA elements constitute
54% of the
human genome (Hoyt et al., 2022). Since the initial discovery of
mobile transposable elements (TEs) in maize (McClintock,
1950), several other repetitive elements has been identified in eu-
karyotic genomes, including short and long interspersed nuclear
elements (SINEs and LINEs, respectively) (Kit, 1961; Schmid and
Deininger, 1975), pericentromeric and centromeric satellite re-
peats (Tyler-Smith and Brown, 1987), telomeric repeats (Black-
burn and Gall, 1978), retroviral sequences (Martin et al., 1981),
and short tandem repeats (STRs) (La Spada et al., 1991; Oberlé
et al., 1991; Verkerk et al., 1991; Yu et al., 1991) (Figure 1, bot-
tom). Targeted repression of repetitive elements is critical to
counter their propensity for instability events (e.g., stepwise ex-
pansions, duplications, inversions, and recombination). Thus,
heterochromatin has a critical role in controlling genome function
at the interface between gene expression and repeat stability.
Heterochromatin can be earmarked by multiple known chro-
matin modifications. Repetitive DNA is packaged into what is
canonically referred to as ‘‘constitutive heterochromatin,’’ a tran-
scriptionally silent chromatin state characterized by the crosstalk

2690 Cell 185, July 21, 2022 ª 2022 Published by Elsevier Inc.
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Review

Figure 1. History of classic heterochromatin and repetitive genome discoveries

Key principles of heterochromatin acquisition, maintenance, and function since the initial discovery in 1928 by Emil Heitz (top). In parallel, insights into the
abundance, regulation, and function of repetitive elements have also come to light, including Barbara McClintock’s 1950 foundational discovery of mobile genetic
elements in maize and recent reports of the first gapless human genome (bottom). Crosstalk between heterochromatin and the repetitive genome has emerged as
understanding has accumulated in both disciplines.

between H3 Lys-9 tri-methylation (H3K9me3) and DNA methyl-
ation (Millán-Zambrano et al., 2022; Saksouk et al., 2015). Consti-
tutive heterochromatin at telomeres and pericentromeres con-
denses vulnerable repetitive regions in an invariant manner
across developmental lineages and phases of the cell cycle.
H3K9me3 can also be developmentally regulated, as genome-
wide changes in occupancy patterns have been linked to gene
expression changes during mammalian differentiation and re-
programming (Magklara et al., 2011; Nicetto et al., 2019; Soufi
et al., 2012). Moreover, cell-type-specific heterochromatin
marked by the histone modification H3 Lys-27 tri-methylation
(H3K27me3) plays a critical role in developmentally regulated
gene expression (Millán-Zambrano et al., 2022). Together, these
data highlight that distinct heterochromatin signatures can exhibit
context-specific constitutive and developmentally regulated pat-
terns of acquisition and maintenance in the mammalian genome.
An open question at the forefront of the last several decades
has been the mechanisms governing context-specific deposition
and maintenance of heterochromatin modifications. In the case of
H3K9me3, genetic screens in Drosophila and S. Pombe identified

Cell 185, July 21, 2022 2691

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Review

Figure 2. Interplay between higher-order chromatin organization and heterochromatin

Mammalian genomes are folded into (A) A/B compartments, (B) chromatin-associated subnuclear bodies, (C) topologically associating domains (TADs),
(D) subTADs, and (E) long-range looping interactions.
(F) TADs/subTADs formed by cohesin-mediated loop extrusion manifest as corner-dot TADs/subTADs in ensemble Hi-C maps, whereas dot-less TADs/subTADs
have weaker boundaries or are formed by cohesin-independent mechanisms.
(G) Loop extrusion is refractory to B compartment and heterochromatin domain formation (based on Haarhuis et al. [2022]).

more than fifty candidate genetic loci—termed Su(Var) group
genes—as suppressors of PEV (Allshire et al., 1995; Reuter and
Spierer, 1992). Two such genes—SUV39H1 and SUV39H2—
encode histone methyltransferases (HMTs) which selectively
methylate H3K9 in mammals. Gain- and loss-of-function studies
for SUV39H1/2 have demonstrated their causal role in methylating
H3K9 in mammalian cell lines (Firestein et al., 2000; Melcher et al.,
2000; Rea et al., 2000). Another HMT, SET domain bifurcated his-
tone lysine methyltransferase 1 (SETDB1), also possesses H3K9-
specific tri-methylation activity (Schultz et al., 2002). In addition to
HMTs, eukaryotes employ histone demethylases (HDMs) for tar-
geted methyl-group removal, and the mammalian Jumonji
domain-containing protein 2 (JMJD2/KDM4) isoforms A–D were
identified as H3K9me3 HDM enzymes (reviewed in Kooistra and
Helin [2012]). Similarly, H3K27me3 is also deposited in a cell-
type-specific manner by the HMT activity of specific subunits of
the Polycomb-repressive complex 2 (PRC2) (Yu et al., 2019),
and can be removed by HDM KDM6 isoforms A–B (Kooistra
and Helin, 2012). The existence of evolutionarily conserved
H3K9me3 and H3K27me3 writers and erasers highlights the likely
importance of precise spatiotemporal regulation of heterochro-
matin during mammalian development and adulthood.
In this review, we highlight the contrasting higher-order
folding patterns of compartmentalization and loop extrusion
and their interplay with repressive heterochromatin in the
three-dimensional (3D) nucleus. In light of the first gapless,
complete genome assembly created by the Telomere-to-
Telomere (T2T) consortium (Hoyt et al., 2022), we summarize
the diversity of repetitive DNA sequences and mobile genetic
elements in the human genome. Using primarily mammalian
model systems, we compare and contrast the mechanisms of
H3K9me3-mediated heterochromatinization of repetitive ele-
ments and highlight emerging evidence suggesting a link be-
tween genome folding and the repetitive genome.
HIGHER-ORDER FOLDING PATTERNS OF REPRESSIVE
HETEROCHROMATIN
Technological advances in imaging, sequencing, and computa-
tional biology over the last two decades have revealed that het-
erochromatin not only functions in a linear fashion as described
above, but also in concert with higher-order genome folding
structures. As imaging and sequencing technologies enabled
the creation of genome-wide, ultra-high-kilobase-resolution
maps of chromatin folding in single cells, we now have the abil-
ity to detect genome-wide locations of A/B compartments,
subnuclear bodies, topologically associating domains (TADs),
subTADs, and loops across many mammalian cell types
(Figure 2). Here we discuss whether and how each distinct
genome folding pattern is shaped by and informs H3K9me3-
mediated heterochromatin—and the mechanistic principles un-
derlying the establishment and maintenance of each folding
feature.
B compartments can be established via HP1a
Compartments were originally identified in ensemble Hi-C maps
binned at 1 Megabase resolution as a chromosome-wide plaid
pattern of ultra-long-range intra-chromosomal and inter-

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Review
chromosomal contacts (Lieberman-Aiden et al., 2009; Rao et al.,
2014) (Figure 2A). The plaid pattern has been interpreted to sug-
gest genome partitioning into A compartments of euchromatin
and B compartments enriched for heterochromatin (Lieber-
man-Aiden et al., 2009). As Hi-C data has increased in read
depth and quality over the last decade, it has enabled the addi-
tional partitioning of 1 kilobase-binned mammalian genomes into
several subtypes of smaller sub-compartments marked by
signature combinations of active and repressive chromatin mod-
ifications (Liu et al., 2021; Rao et al., 2014; Spracklin et al., 2021).
Super-resolution and widefield imaging experiments with Oligo-
paints DNA fluorescence in situ hybridization (FISH) probes veri-
fied compartments independently via direct visualization in situ
(Bintu et al., 2018; Mateo et al., 2019; Takei et al., 2021; Nir
et al., 2018). Compartmentalization has also been observed in
C. elegans and Drosophila (Bian et al., 2020; Rowley et al.,
2017), suggesting that it may be an essential feature of genome
folding across mammalian and non-mammalian eukaryotic or-
ganisms.
Following their initial stratification into A and B states (Lieber-
man-Aiden et al., 2009), compartments were further classified
into multiple sub-compartments (e.g., A1, A2, B1, B2, B3) based
on their combinations of chromatin modifications, DNA replica-
tion timing, and association with subnuclear structures (i.e., nu-
clear lamina and nucleolus) (Liu et al., 2021; Rao et al., 2014). B
compartments are generally heterochromatic and gene poor and
have since been stratified into sub-B compartments marked by
(1) H3K9me3, HP1a, and HP1b; (2) H3K9me2 (but not
H3K9me3) and the H2A.Z histone variant; or (3) H3K27me3
and PRC 1 and 2 (Spracklin et al., 2021). By contrast, A compart-
ments are euchromatic and encompass accessible, gene-dense
DNA enriched for histone modifications linked to transcriptional
activation (e.g. H3K27ac and H3K4me3). Although the full iden-
tification of sub-compartment subtypes in each cell type is
ongoing, these data indicate that multiple different heterochro-
matin mechanisms exist within the generalized B compartment,
and thus they should be sub-stratified by signatures of chromatin
marks when ascertaining function.
The mechanisms underlying B compartment formation are an
active area of investigation. The adaptor protein heterochromat-
in protein 1 homolog alpha (HP1a) binds to H3K9me3 to maintain
heterochromatin via the direct recruitment of additional effectors
across the cell cycle and in development (Bannister et al., 2001;
Lachner et al., 2001; Nakayama et al., 2001). HP1a has been
directly implicated in the establishment of B compartments dur-
ing the early developmental stage of zygotic genome activation
(ZGA) in the Drosophila embryo (Zenk et al., 2021). Depletion of
HP1a during ZGA causes a 20% decrease in B compartment
strength and disrupted trans interactions between pericentro-
meric regions (Zenk et al., 2021). However, its knockdown
does not perturb genome folding in somatic Drosophila S2 cells,
thus suggesting that it is required for establishment and not
maintenance of B compartments. Computational modeling of
HP1a-H3K9me3-dependent chromatin compaction is only
partially capable of reproducing the distinctive alternating plaid
pattern exhibited by A/B compartments on Hi-C maps, suggest-
ing additional organizing mechanisms may be involved (Mac-
Pherson et al., 2018). The apparent discrepancy might in part
ll
be explained by the ability of HP1a to nucleate into spherical
droplets exhibiting the physical properties of liquid-like, mem-
braneless condensates (Larson et al., 2017; Strom et al., 2017).
Liquid-like droplets of HP1a can condense a stretched fragment
of linear DNA into a dense focal point in vitro, thus raising the
possibility that B compartments could form in principle through
phase separation. Biochemical studies indicate that the ability
of human HP1a to form liquid-like droplets is dependent on
binding to DNA via its intrinsically disordered hinge region and
N-terminal HP1a phosphorylation (Larson et al., 2017). While ob-
servations from these and other studies are suggestive of a role
for HP1a in the establishment of B compartments via oligomer-
ization or phase separation mechanisms, more perturbative
gain and loss of function studies will ascertain the extent of
HP1a0 s role in B compartment establishment in mouse and hu-
man systems across developmental stages.
H3K9me2/3 is necessary and sufficient for formation
of LADs
The association of genomic loci with the nuclear lamina in lam-
ina-associated domains (LADs) was reported using a DNA
adenine methyltransferase-based assay (DamID) targeting
B-type lamin proteins first in Drosophila Kc cells (Pickersgill
et al., 2006) (Figure 2B). DamID was subsequently modified to
target lamin B1 in Tig3 human lung fibroblasts, revealing that
LADs are approximately 100 kb–10 Mb in size and span up to
40% of the human genome (Guelen et al., 2008). It is well estab-
lished that LADs correlate with repressed genes and B compart-
ments, and localization to the nuclear periphery has long been
discussed as a model for gene silencing (van Steensel and Bel-
mont, 2017).
The mechanisms by which LADs are established and main-
tained are of significant importance for understanding
H3K9me2/3 deposition. LADs are enriched for H3K9me2/3 and
reduction of H3K9me2/3 via depletion or inhibition of H3K9
HMTs results in disruption of chromatin contacts with the nuclear
periphery across model organisms (Bian et al., 2013, 2020; Harr
et al., 2015; Towbin et al., 2012). Moreover, dCas9 targeting of
the HMT G9afor genomic locus-specific H3K9 di-methylation
promotes lamina interactions in a methyltransferase-activity-
dependent manner (See et al., 2020). HP1a is a candidate in
the establishment of the interface between H3K9me2/3 and
the lamina, as it binds directly to H3K9me2/3 through its chromo-
domain and can also bind directly to lamina-associated proteins
(Holla et al., 2020; Ye et al., 1997). Together, these collective ob-
servations suggest a possible model where H3K9me2/3 is suffi-
cient for the establishment and necessary for the maintenance of
chromatin-lamina associations.
The functional relationship between gene expression and LAD
localization remains an open question. In mammalian systems,
thousands of genes are localized within LADs, and the majority
are actively repressed or transcriptionally inactive (Leemans
et al., 2019). Artificial tethering to LADs results in silencing of spe-
cific genes, which supports the argument that positioning at the
periphery instructs gene expression (Finlan et al., 2008; Reddy
et al., 2008). However, it is not yet fully clear whether specific
lamina proteins or H3K9me2/3 drive silencing given the
complexity of experiments decoupling periphery localization
Cell 185, July 21, 2022 2693
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with the factors involved. For example, depletion of lamina com-
ponents does not typically lead to significant changes in gene
expression (Amendola and van Steensel, 2015; Solovei et al.,
2013). Moreover, at least 10% of genes in LADs retain their
expression, and many such ‘‘escaper’’ genes are locally de-
tached from the lamina and depleted of H3K9me2/3 signal, sug-
gesting that mechanisms also exist for transcription to overcome
the conventionally repressive environment at the nuclear periph-
ery (Guelen et al., 2008; Leemans et al., 2019). Together, these
studies suggest that tethering to the nuclear lamina can be caus-
ally linked to gene expression silencing but that there are
escapee genes which are exceptions to this general rule.
An emerging diversity of subnuclear bodies exhibits
distinct heterochromatin and genome-function
signatures
In addition to compartments and LADs, chromatin loci can
spatially co-localize with proteinaceous sub-nuclear bodies
including nuclear pores, nuclear speckles, stress granules, and
nucleoli-associated proteins (Figure 2B). For example, nucle-
olus-associated domains (NADs) were discovered by high-
throughput sequencing of genomic DNA associated with purified
nucleoli (Németh et al., 2010; van Koningsbruggen et al., 2010).
NADs can encompass as much as
4% of the human genome
and are enriched for repetitive ribosomal RNA genes, pericentro-
meric and centromeric satellite repeats, and specific telomere
arms (Németh et al., 2010; Su et al., 2020). NADs can overlay
with the heterochromatin marks of H3K9me3 (Type I) or
H3K27me3 (Type II) (Bersaglieri et al., 2022; Vertii et al., 2019).
Type I NADs have been hypothesized to facilitate repression
and stabilization of repetitive DNA elements, whereas Type II
NADs have been implicated in developmentally regulated gene
expression (Vertii et al., 2019). The nucleolus might also exhibit
the biophysical properties of liquid-like droplets (Frottin et al.,
2019; Lafontaine et al., 2021), consistent with the possibility
that repetitive elements may associate in the nucleolus via phase
separation mechanisms. The roles and mechanisms by which
nucleolar associated DNA can be heterochromatinized and
silenced remains and exciting area for future investigation.
Recently, the development of new genomics and computa-
tional technologies brought to light that repressive sub-compart-
ments could be further refined beyond the foundational B1, B2,
and B3 convention (Liu et al., 2021; Quinodoz et al., 2018;
Wang et al., 2021). Tyramide signal amplification sequencing
(TSA-seq) directly measures the cytological distance of chro-
matin fragments to nuclear bodies such as speckles or stress
granules (Chen et al., 2018). The spatial position inference of
the nuclear genome (SPIN) computational method integrates
data from TSA-seq for speckles and the nuclear lamina, Hi-C,
and DamID for the lamina and nucleolus and has successfully
identified at least 4 active and 6 repressive subtypes of nuclear
bodies (Wang et al., 2021) (Figure 2B). These observations open
up new questions as to whether and how specific subnuclear
bodies functionally contribute to or might be shaped by gene
expression and heterochromatinization. The coupling of TSA-
seq with synthetic biology technologies such as CRISPR-
genome organization (CRISPR-GO) (Wang et al., 2018b) or
light-activated-dynamic-looping (LADL) (Kim et al., 2019) to
2694 Cell 185, July 21, 2022
Review
tether specific genomic segments to the nuclear periphery and
subnuclear bodies will be critical for addressing these underex-
plored concepts. As genomics and computational technologies
continue to advance, the granularity with which repressive sub-
compartments are classified will improve, allowing mechanistic
and functional perturbative studies to precisely dissect the ge-
nome’s structure-function relationship.
TADs and subTADs formed via loop extrusion
antagonize B compartments and heterochromatin
Within larger A/B compartments and subnuclear bodies, the
mammalian genome folds into TADs and subTADs (Dixon
et al., 2012; Nora et al., 2012; Phillips-Cremins et al., 2013;
Sexton et al., 2012). As defined algorithmically in low-resolution
Hi-C data, TADs are Mb-scale domains in which genomic frag-
ments have a higher interaction frequency with themselves
compared to loci outside of the domains (Figure 2C). TADs and
their nested subMb-sized counterparts subTADs (Figure 2D)
are demarcated by boundaries—which are molecularly distinct
regions of the genome refractory to permitting chromatin inter-
actions between up and downstream genomic loci (Beagan
and Phillips-Cremins, 2020; Norton and Phillips-Cremins, 2017;
Phillips-Cremins, 2014) (orange segments, Figures 2C and 2D).
Although TADs were initially reported as conserved across cell
types, evidence is mounting that TAD and subTAD boundaries
can vary significantly in strength across cell types and in disease
(Beagan et al., 2016, 2017, 2020; Norton et al., 2018; Sun
et al., 2018).
Over the last decade, major progress has been made toward
understanding the molecular mechanisms that regulate TAD/
subTAD formation (reviewed in detail in Beagan and Phillips-Cre-
mins [2020]). Many TADs and subTADs are formed by the cohe-
sin-based mechanism of loop extrusion in which the subunits of
the structural maintenance of chromosomes (SMC) proteins
form a ring-like structure which progressively moves in an
ATP-dependent manner along the genome and reels flanking
loci through the inner diameter of its ring (Figure 2E) (Banigan
and Mirny, 2020; Davidson and Peters, 2021). Long-range loop-
ing interactions form when the cohesin complex stalls at TAD/
subTAD boundaries bound by the architectural protein
CCCTC-binding factor (CTCF) (Figure 2E) (Davidson et al.,
2016; Fudenberg et al., 2016; Sanborn et al., 2015). Extrusion-
based long-range looping interactions manifest structurally in
ensemble Hi-C maps as dots, and, therefore, TADs/subTADs
formed by extrusion exhibit corner-dots (Figure 2F). Due to the
transient nature of loop extrusion, a corner dot feature is not al-
ways readily detectable in TADs formed by extrusion at a popu-
lation level (Beagan and Phillips-Cremins, 2020; Emerson et al.,
2022; Rao et al., 2017). The majority of dot-like structure-indica-
tive loops are lost upon short-term knockdown of CTCF and co-
hesin protein levels, thus reinforcing that these factors are
essential for loop establishment and maintenance (Nora et al.,
2017; Rao et al., 2017; Schwarzer et al., 2017). Hereinafter, we
refer to TADs/subTADS as all domain-like structures in Hi-C
and single-cell imaging data which form mechanistically via
loop-extrusion mechanisms—but we note that CTCF/cohesin-
independent mechanisms of TAD, subTAD, and loop formation
are an active area of intense investigation.
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Review

Figure 3. Types and distribution of repetitive DNA elements across the human genome
(A) The human genome consists of
54% repetitive sequences. Class I repeats comprise retrotransposable elements, including long interspersed nuclear el-
ements (LINEs, grey), short interspersed nuclear elements (SINEs, dark orange), and long terminal repeat (LTR) endogenous retroviruses (ERVs, light orange). The
human genome has primarily one abundant LINE, LINE-1, that is
6 kb long and encodes its own requisite reverse transcription and transposition machinery. The
most common class of SINE elements in the human genome are the
280-bp-long Alu-SINEs which are non-autonomous and rely on LINE-1 encoded machinery
to facilitate their transposition. ERV remnants are present in the human genome in fragments (HERVs) and in some cases can be actively transcribed. Tandem
repeats are non-mobile DNA sequences in which the copies of one or more nucleotides are repeated in a head-to-tail manner. They are distributed throughout the
genome and are most prevalent as satellite repeats at pericentromeric and centromeric chromatin regions.
(B) The recent gapless telomere-to-telomere (T2T) human genome assembly consists of assemblies for all 22 human autosomes and chromosome X and
comprises 3.05 gigabase pairs (Gbp) of nuclear DNA (CHM13v1) (adapted from Nurk et al. [2022]). Bar plot, recent T2T estimates of the percentage of the human
genome made up of each repeat class.
TSD, target site duplication; ORF, open reading frame; EN, endonuclease; RT, reverse transcriptase; A and B, split Pol III RNA promoter; LTR, long terminal
repeats; Gag, group antigens; Prt, protease; Pol2, reverse transcriptase; Env, envelope protein.

It is established that the loss of cohesin-based loop extrusion
leads to gained compartments in the mammalian genome (Nora
et al., 2017; Rao et al., 2017; Schwarzer et al., 2017). Knockdown
of subunits of both the Mediator and cohesin complex leads to
lost loops, increased genome compartmentalization, and in-
creases in heterochromatin domain formation (Figure 2G) (Haar-
huis et al., 2022). Knockdown of the Setdb1 H3K9 HMT in
mammalian neurons results in lost H3K9me3 and gained ectopic
binding of CTCF (Jiang et al., 2017). Moreover, stabilization of
chromatin loops by knock-down of the cohesin-unloading factor
wings apart-like protein homolog (WAPL) can result in ablation of
heterochromatin domains and a decrease in compartmentaliza-
tion (Figure 2G) (Haarhuis et al., 2017, 2022). Altogether, these ob-
servations raise the possibility that stabilization of chromatin
loops can counteract the formation of B compartments and sug-
gest that mechanisms driving the loss or gain of chromatin loops
could actively shape heterochromatin.
LINEAR HETEROCHROMATINIZATION OF THE
REPETITIVE GENOME
Repeat composition of the complete gapless human
genome assembly
The recent T2T-CHM13 human genome assembly released by
the Telomere-to-Telomere (T2T) consortium offers the most ac-
curate estimation to date regarding the composition of the repet-
itive genome (Figure 3). Repetitive sequences comprise
54%
of the T2T-CHM13v1 assembly (Figure 3A-B) (Hoyt et al., 2022;
Nurk et al., 2022). Transposable elements (TEs) are mobile
DNA sequences capable of integrating into new genomic loci

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and are the largest class of repetitive DNA elements. Class I TEs,
or retrotransposable elements (RTEs) including long inter-
spersed nuclear elements (LINEs), short interspersed nuclear el-
ements (SINEs), and long terminal repeat (LTR) endogenous ret-
roviruses (ERVs), are dominant in the CHM13v1 assembly due to
their ‘‘copy-and-paste’’ mechanism where an RNA intermediate
is reverse transcribed to cDNA prior to its integration into a new
genomic locus (Chuong et al., 2017). By contrast, class II TEs, or
DNA transposons, leverage a ‘‘cut-and-paste’’ mechanism
where terminal internal repeats are bound by the self-encoded
transposase, facilitating TE excision and reintegration into a
new genomic locus (Chuong et al., 2017). Non-mobile repetitive
DNA sequences, including pericentric and centromeric satellite
repeats, short tandem repeats (STRs), and variable number of
tandem repeats (VNTRs), also occur at high frequency in the hu-
man genome. We anticipate that as our knowledge of the
complexity and diversity of repetitive sequences among individ-
uals increases, it will transform our understanding of the func-
tions and roles for the repetitive genome in phenotypic variation
in the coming years.
Repetitive elements threaten genome stability due to their sus-
ceptibility to stepwise expansions, duplications, inversions, and
recombination events (Lupski and Stankiewicz, 2005). A prin-
cipal role for H3K9me3 is the repression of repetitive DNA se-
quences and mobile genetic elements. Such mechanisms are
a double-edged sword, however, because some repetitive ele-
ments have evolved to regulate their host’s genomes (reviewed
in detail in Hermant and Torres-Padilla [2021] and Sundaram
and Wysocka [2020]). Here, we discuss the distinguishing fea-
tures of satellite repeats, STRs and VNTRs, and RTEs, as well
as the linear H3K9-related heterochromatic mechanisms regu-
lating their accessibility and expression.
Pericentromeric satellite repeat tracts: Crosstalk
between H3K9me3 and DNA methylation facilitates
silencing
In both humans and mice, pericentromeric and centromeric DNA
is enriched for repetitive satellite repeat sequences (reviewed in
Thakur et al. [2021]). Human centromeric DNA is enriched for re-
petitive a-satellite repeat tracts consisting of 171-bp monomer
subunits. In their most simple form, a-satellite repeat units are di-
mers, but they can also be found in greater repeating arrays
known as higher-order repeats. Mouse centromeric DNA con-
tains primarily minor satellite (MiSat) tracts consisting of
repeating 120-bp subunits. In human pericentromeres, there
are 6 unique classes of satellite sequences: satellites I (17- and
25-bp), satellites II (10- to 80-bp), satellites III (5- and 10-bp),
a-satellites (171-bp), b-satellites (68-bp), and g-satellites (220-
bp). Mouse pericentromeres are predominantly populated by
major satellite (MajSat) tracts comprised of a repeating 234-bp
subunit. As long-read sequencing and alignment technologies
continue to improve, the further characterization of satellite
repeat tract diversity and structure represents an exciting area
for future scientific exploration.
Although centromeric repeats are actively transcribed and
generally remain accessible to facilitate kinetochore assembly
(McKinley and Cheeseman, 2016), the satellite repeats within
pericentromeres are constitutively heterochromatinized by
2696 Cell 185, July 21, 2022
Review
H3K9me2/3 and 5-methylcytosine (5mC) DNA methylation (re-
viewed in Thakur et al. [2021]). In mammals, H3K9me3 is depos-
ited at pericentromeres by SUV39H1/2 HMTs (Peters et al.,
2001; Maison et al., 2011; Rice et al., 2003) and can be recog-
nized by the chromodomain of HP1a (Bannister et al., 2001;
Lachner et al., 2001; Nakayama et al., 2001) (Figure 4A). HP1a
can co-immunoprecipitate with the H3K9me3 HMTs SUV39H1/
2 and SETDB1, as well as the DNA methyltransferase DNMT3b
in mammalian cell lines (Lehnertz et al., 2003; Maeda and Tachi-
bana, 2022; Smallwood et al., 2007) (Figure 4A). Double
knockout of SUV39H1 and SUV39H2 in HeLa cells results in
the depletion of H3K9me3 and HP1a on chromatin (Johnson et
al., 2017). Once localized to pericentromeres, HP1a functions
as an adaptor protein to facilitate heterochromatin spreading
by acting as a scaffold for the recruitment of additional
heterochromatin effectors (Lechner et al., 2005; Smothers and
Henikoff, 2000). The mechanisms by which HMTs, DNMTs,
HP1a, and other chromatin effectors interplay to heterochroma-
tinize mammalian pericentromeres remain an exciting open
question.
It is noteworthy that an early study knocking out the mouse en-
zymes for DNA methylation maintenance (i.e., Dnmt1) or de novo
deposition (i.e., Dnmt3a/Dnmt3b double knockout) do not show
decreased H3K9me3 at pericentromeres (Lehnertz et al., 2003).
By contrast, knock-down of both Suv39h1 and Suv39h2 reduces
pericentromeric DNA methylation and de-represses MajSat
repeat expression (Lehnertz et al., 2003). Although still under
active investigation, this early work suggests that DNA methyl-
ation at pericentromeres may occur downstream of H3K9me3.
Together, these data suggest a working model of heterochro-
matin at pericentromeres similar to the non-repetitive genome.
In this working model, HP1a associates with H3K9me3 and sta-
bilizes—either directly or indirectly—local H3K9 HMT and DNMT
occupancy, resulting in a self-reinforcing cycle of H3K9me3 and
DNA methylation (Figure 4A).
It is worth pointing out that crosstalk between H3K9me2/3
and 5mC DNA methylation might also occur at pericentromeres
independently from HP1a during S phase of the cell cycle via
the recruitment of ubiquitin-like PHD and RING finger
domain-containing protein 1 (UHRF1). UHRF1 (previously
known as NP95 in mice and ICBP90 in humans) co-localizes
with 40 ,6-diamidino-2-phenylindole (DAPI)-dense pericentro-
meric foci (Fang et al., 2016) and recognizes both H3K9me3
and hemi-5mC DNA generated during replication (Rothbart
et al., 2012). Heterochromatin-bound UHRF1 can recruit
DNMT1 to facilitate local DNA methylation through two distinct
molecular models (Figure 4B). In one leading model, hemi-5mC
DNA provides UHRF1 binding specificity, and H3K9me2/3 im-
proves binding affinity (Rothbart et al., 2012). Upon stable bind-
ing to existing H3K9me2/3 and hemi-5mC DNA, UHRF1 can re-
cruit DNMT1 via a direct interaction to further mediate DNA
methylation maintenance (Achour et al., 2008) (Model A, Figure
4B. Alternatively, it has also been suggested that UHRF1 can
ubiquitinate local H3 residues which may then directly recruit
DNMT1 to guide DNA methylation maintenance (Model B,
Figure 4B) (Ishiyama et al., 2017). In addition to DNMT1, the
HMT SUV39H1 can directly associate with UHRF1 in human
cells, and UHRF1 knockdown can reduce both SUV39H1 and
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Review

Figure 4. Distinct repeat elements require unique heterochromatinization mechanisms

Linear heterochromatin mechanisms play an integral role in repressing both the accessibility and expression of repetitive DNA elements.
(A) At pericentric chromatin, H3K9me3 and DNA 5mC cooperate to silence satellite repeats. DNMT3B and the HMT SUV39H1 can be recruited to pericentromeric
repeats and co-immunoprecipitation (co-IP) with HP1a .
(B) The interplay between H3K9me3 and DNA 5mC can occur via two distinct models in a DNA-replication-dependent manner. In model A, UHRF1 is located to
DNAP-replicating loci through an interaction with PCNA. At replicating loci, UHRF1 binds hemi-5mC DNA and H3K9me3 where it can recruit DNMT1 via a direct
protein interaction for 5mC maintenance. In model B, UHRF1 ubiquitinates histone 3 residues which are recognized by DNMT1, leading to its local recruitment for
5mC maintenance.
(C) Transcription of pericentromeric satellite repeats generates ncRNAs that can recruit the HMT SUV39H1 in cis for H3K9me3 deposition.
(D) At telomeric short tandem repeats (STRs), TERRA ncRNA can recruit heterochromatin machinery to the TRF2 subunit of the shelterin complex.
(E) ERVs are primarily targeted for repression by KRAB-containing zinc finger proteins (KRAB-ZFPs) which interact with the corepressor KRAB-associated protein
1 (KAP1) to facilitate recruitment of additional heterochromatin effector proteins.
(F) Actively transcribed young LINE-1s are silenced by the HUSH complex, which recruits the H3K9me3 HMT SETDB1 to chromatin. Prematurely terminated
transcripts can be degraded via the HUSH-associated NEXT complex.
(G) SINEs can be targeted for H3K9me3-dependent repression by SUV39H1 as well as HP1a.
SUV39H1, suppressor of variegation 3-9 homolog 1; DNMT3B, DNA methyltransferase 3B; HP1a, heterochromatin protein 1a; UHRF1, ubiquitin-like PHD and
RING finger domain-containing protein 1; DNMT1, DNA methyltransferase 1; PCNA, proliferating cell nuclear antigen; DNAP, DNA polymerase; RNAPI, RNA po-
lymerase I; ncRNA, non-coding RNA; TERRA, telomeric repeat-containing RNA; TRF2, telomeric repeat-binding factor 2; ORC1, origin recognition complex sub-
unit 1; ERV, endogenous retroviruses; KRAB, Krüppel-associated box domain containing-zinc-finger proteins; C2H2, Cys-2 His-2 zinc finger; SETDB1, SET
domain bifurcated 1; RNAPIII, RNA polymerase II; HUSH, human silencing hub; Transgene activation suppressor; MPP8, M-phase phosphoprotein 8;
PPHLN, periphilin; NEXT, nuclear exosome targeting; RNAPIII, RNA polymerase III.

H3K9me3 signal at specific target genes (Babbio et al., 2012). tion, these data raise a potential working model for HP1a-inde-
Although the functional role for SUV39H1 recruitment by pendent maintenance of pericentromeric heterochromatin dur-
UHRF1 specifically at pericentromeres requires further explora- ing S phase via UHRF1.

Cell 185, July 21, 2022 2697

 ll
Noncoding RNA-mediated H3K9me3 deposition at
pericentromeric repeats
Although pericentromeric heterochromatin is targeted for consti-
tutive repression, it is not transcriptionally inert. Some of its
derived transcripts, such as MajSat non-coding RNA (ncRNA),
can remain largely chromatin associated through RNA:DNA hy-
brids (Velazquez Camacho et al., 2017). Biochemical studies
suggest that the N-terminal basic domain of mouse SUV39H2
contains a single-stranded RNA (ssRNAs) recognition module
that selectively binds MajSat ncRNA. N-terminal truncation of
the ssRNA recognition domain reduces pericentromeric repeat
association with SUV39H2 and H3K9me3, suggesting a critical
role for MajSat ncRNA in pericentromere heterochromatinization
(Velazquez Camacho et al., 2017). Biochemical studies have also
shown that human SUV39H1 can interact with a-satellite ncRNA
in vitro (Johnson et al., 2017). In contrast to mice, human
SUV39H1 binds ssRNA through its chromodomain, as it does
not possess an N-terminal RNA binding domain (Johnson
et al., 2017). Non-specific RNase A digestion or mutation of the
SUV39H1 ssRNA binding domain reduced SUV39H1 enrichment
at pericentromeres in HeLa cells, suggesting these ncRNAs may
also be critical for heterochromatinization in humans (Figure 4C).
Together, these findings support the existence of satellite repeat
ncRNA-facilitated heterochromatinization of pericentromeres.
An exciting avenue for future inquiry is to dissect and further un-
derstand the seemingly discordant relationship between satellite
repeat transcription and heterochromatin acquisition and main-
tenance at mammalian pericentromeres.
Local heterochromatin silencing of genes containing
unstable STR tracts in a subset of repeat expansion
disorders
Over 1 million STR tracts, also known as microsatellites, are
distributed across the human genome in introns, exons, and in-
tergenic regions (Hannan, 2018). STRs consist of 1–6 bp motifs
arranged in tandem (Hannan, 2018). Recent genome-wide
studies have revealed that thousands of STRs are polymorphic
across individuals and serve as key regulators of gene expres-
sion (Gymrek et al., 2016). STR instability occurs at significantly
higher rates than single nucleotide mutations, and small changes
in tract lengths contribute to the diversity of healthy human
phenotypic traits (Ellegren, 2000). Thus, STRs are abundant in
the human genome and play important functional roles in devel-
opment and phenotypic diversity.
By a process that is poorly understood, a small number of spe-
cific normal-length STRs undergo somatic or germline expan-
sion and transition to long-normal, intermediate, pre-mutation,
and mutation STR tract lengths. Unstable expansion of STRs
was first reported simultaneously by four independent groups
and serves as the mechanistic basis for a growing list of more
than 40 inherited repeat expansion disorders, including fragile
X syndrome (FXS), Huntington’s disease, amyotrophic lateral
sclerosis, and Friedreich’s ataxia (La Spada et al., 1991; Oberlé
et al., 1991; Verkerk et al., 1991; Yu et al., 1991). Patients with
repeat expansion diseases typically suffer from a complex set
of neuropsychiatric symptoms such as anxiety, hyperactivity,
low IQ, social deficits, and seizures (Orr and Zoghbi, 2007). Un-
derstanding the mechanisms governing STR instability is of
2698 Cell 185, July 21, 2022
Review
immense importance for human health and also an unsolved
pathophysiological question.
Emerging evidence suggests that heterochromatin acquisition
at unstable STRs might stabilize disease-related expansion
events. In the prototypic example of FXS, expansion of a CGG
STR tract in the 5’UTR of FMR1 expands to mutation length of
200 triplets or more to cause disease (Oberlé et al., 1991; Verkerk
et al., 1991; Yu et al., 1991). STR expansion from pre-mutation to
mutation length causes transcriptional inhibition of FMR1 and
consequent severe reduction in levels of the Fragile X Mental
Retardation Protein (FMRP) it encodes. Upon STR expansion
above 200 CGGs to full-mutation length, classic models assert
that the unstable STR tract undergoes local DNA hypermethyla-
tion and heterochromatinization by H3K9me3. Heterochromati-
nization is thought to stabilize the STR tract, but it also spreads
upstream to the FMR1 promoter to cause transcriptional
silencing (Oberle et al., 1991; Sutcliffe et al., 1992; Kumari and
Usdin, 2010). The role for heterochromatin in stabilizing dis-
ease-expanding STRs and the downstream consequences in
disease is an exciting area for future inquiry, with crosscutting
impact across multiple repeat expansion disorders already
linked to DNA methylation and H3K9me3 (reviewed in Dion and
Wilson [2009]).
Finally, VNTRs, also known as minisatellites, are distin-
guished from STRs as their repeating monomer units are
greater than six bps in length (Course et al., 2021). Although
more than 55,000 VNTRs are present in the human genome,
relatively little is known regarding their mechanisms of hetero-
chromatinization and variation across patient cohorts. Limited
knowledge of STRs is largely a consequence of their greater
size relative to STRs, which has prevented their accurate
mapping to human reference genomes and repeat length quan-
tification due to limitations associated with short-read seq-
uencing. The recent advent of long-read sequencing may
address these limitations, as evidenced by the identification
of VNTRs in the long-read generated T2T CHM13 genome
(Hoyt et al., 2022). Long-read sequencing has recently shown
promise in identifying disease-associated VNTRs (daVNTRs),
such as the amyotrophic lateral sclerosis (ALS)-associated
WDR7 daVNTR (Course et al., 2020).
A hexanucleotide STR tract facilitates the association
between heterochromatin effectors and shelterin to
maintain telomere length
Human telomeres contain an internal hexanucleotide 50 -TTA
GGG-30 STR tract that recruits the protein complex shelterin as
a critical mechanism supporting telomere stability (de Lange,
2018). Telomeres are exceptionally unstable as their unpro-
tected ends can stimulate DNA damage repair pathways and
undergo progressive shortening with each cell division in non-
cancerous, somatic cells (Shay and Wright, 2019). To protect
telomere ends from initiating DNA damage response pathways,
shelterin facilitates telomere loop (t-loop) formation in which the
single-stranded 30 DNA overhang at telomere ends invades the
double-stranded telomere DNA to create a protective loop struc-
ture (de Lange, 2018). Shelterin also recruits telomerase in germ-
line and embryonic tissues to maintain telomere length (Hocke-
meyer and Collins, 2015). Together, these studies highlight the

Review
functional role for telomere hexanucleotide STR tracts in promot-
ing telomere stability via the recruitment of the shelterin protein
complex.
Telomeres and heterochromatin were first linked upon discov-
ery of the telomere position effect (TPE), a phenomenon
conserved from yeast to humans where reporter genes incorpo-
rated near telomeres become silenced due to the spread of
nearby heterochromatin (Blasco, 2007). Follow-up studies
have begun deciphering the specific mechanisms supporting
heterochromatin acquisition, maintenance, and function at telo-
meres. For example, in cells derived from a Suv39h1 and
Suv39h2 double-knockout mouse, telomeres are depleted for
H3K9me2/3 and HP1 isoforms concomitant with aberrant telo-
mere lengthening (Garcı́a-Cao et al., 2004). Additionally, the
long ncRNA termed ‘‘telomeric repeat-containing RNA’’
(TERRA) has been reported to promote telomere heterochroma-
tinization by recruiting HP1a to shelterin in human colorectal
cancer HCT116 cells (Figure 4D) (Deng et al., 2009). TERRA
depletion leads to an increase in telomere-free ends, telomere
doublets, and chromatid duplications, thus supporting a role
for telomere heterochromatinization in promoting telomere sta-
bility (Deng et al., 2009). It is important to acknowledge that these
studies, while exciting and thought provoking, still represent an
area of active investigation and conflicting observations have
been reported (Barral and Déjardin, 2020). Future research into
the interplay between telomeric heterochromatinization, tran-
scription, and stability are needed to dissect the TERRA-depen-
dent and -independent mechanisms of heterochromatinization
of telomeres.
Long terminal repeat retrotransposons: SETDB1-
dependent H3K9 methylation is essential for silencing
of ERVs
One of three major subclasses of class I TEs (termed ‘‘RTEs’’)
are the long terminal repeat (LTR) ERVs comprising
8–9%
of the human genome (Hoyt et al., 2022). ERVs were originally
integrated into the germline via an exogenous provirus infec-
tion, after which they utilized a virus-like mechanism leveraging
self-encoded Gag, Pol, and Env proteins driven by an internal
LTR Pol II promoter to infect a new host (Chuong et al.,
2017). This once primary role of ERVs has been lost due in
part to Env truncations. Of the thirty or more ERV subfamilies
known to be present within the human genome (HERVs),
none are thought to contain all the necessary components to
support their mobilization (Goodier, 2016).
ERVs are targeted by distinct molecular machinery to facilitate
their heterochromatinization and repression. In early embryonic
development, when global DNA demethylation promotes a tran-
sient burst in ERV expression (Fu et al., 2019; Reik and Surani,
2015), ERV silencing is primarily mediated by Krüppel-associ-
ated box-domain-containing-zinc-finger proteins (KRAB-ZFPs).
Mammalian KRAB-ZFPs bind to motifs internal to the ERV
DNA sequence via their C2H2 zinc fingers (Figure 4E) (Geis
and Goff, 2020). The KRAB domain facilitates direct interactions
with KRAB-associated protein 1 (KAP1/TRIM28), which in turn
recruits heterochromatin effectors including HP1a and the
H3K9 HMT SETDB1 (Geis and Goff, 2020; Schultz et al.,
2002)(Figure 4E). Thus, ERVs appear to be targeted by
ll
SETDB1 rather than SUV39H1 or SUV39H2 as the predominant
HMT mediating its heterochromatinization.
The necessity for SETDB1-mediated ERV repression has been
illustrated using primordial germ cells from Setdb1 conditional
knockout mice. In this model system, primordial germ cells
exhibit an ectopic increase in ERV expression as well as severe
postnatal defects, suggesting that SETDB1-mediated ERV
silencing is critical for proper germ cell development (Liu et al.,
2014). The requirement of SETDB1-mediated KRAB-ZFP
silencing of ERVs has also been demonstrated in somatic mouse
cells and tissues, supporting a pervasive role for SETDB1-
dependent heterochromatin in maintaining ERV repression
beyond development (Ecco et al., 2016).
Non-LTR retrotransposons: DNA methylation and
H3K9me3 cooperate to repress LINE-1 expression in
early development
Non-LTR RTEs are the most prevalent repetitive DNA sequences
present within the human genome (Hoyt et al., 2022) and primar-
ily include LINEs and SINEs (Figure 3). Within the autonomous
LINE subdivision of RTEs, only LINE-1s have retained their
genomic mobility, with
100 active LINE-1s thought to currently
reside in an individual human’s genome (Brouha et al., 2003).
LINE-1 retrotransposition is facilitated by an internal 50 UTR Pol
II promoter that transcribes two open reading frames (i.e.,
ORF1 and ORF2) ending with a 30 UTR polyadenylation signal.
ORF1 encodes a protein possessing RNA binding and nucleic
acid chaperone abilities while ORF2 encodes a protein with
endonuclease and reverse transcriptase properties that are
fundamental to the RTE copy-and-paste mechanism (Beck
et al., 2011).
Roughly 100 cases of sporadic human disease have been
attributed to LINE-1 or SINE retrotransposition into gene bodies
(Hancks and Kazazian, 2016). LINE-1 exon insertions, such as
those first detected in two hemophilia A patients as early as
1988 (Kazazian et al., 1988), represent the most direct mecha-
nism for disrupting protein function by altering the coding
sequence of the target gene. Intron integration of non-LTR
RTEs can also contribute to human disease by altering RNA
splicing (Burns and Boeke, 2012; Payer et al., 2019), high-
lighting the diverse mechanisms by which gene body retro-
transposition events can impair a downstream protein’s
function.
LINE-1 elements are expressed shortly after human embryo
fertilization and then are silenced by the 16-cell stage or after.
Recent reports have linked LINE-1 expression to the establish-
ment of chromatin accessibility during early embryogenesis
(Guo et al., 2014; Jachowicz et al., 2017). LINE-1 expression co-
incides, but does not directly overlap, with the wave of global
DNA demethylation which occurs in pre-implantation embryos
(Seisenberger et al., 2012), suggesting that DNA methylation is
a critical regulator of LINE-1 expression during early develop-
ment. H3K9me3-mediated silencing is also crucial in the repres-
sion of LINE-1s, as gain of H3K9me3 occurs during the 16-cell
stage in early embryogenesis in parallel with gained DNA methyl-
ation (Wang et al., 2018a). Together these studies suggest that
both H3K9me3 and DNA methylation regulate the silencing
and heterochromatinization of LINE-1 expression.
Cell 185, July 21, 2022 2699
 ll
The mechanisms governing LINE-1 heterochromatinization in
mammalian systems are an active area of investigation.
SETDB1 facilitates H3K9me2/3 deposition in both early develop-
ment and in somatic cells in LINE-1 elements via its association
with the human silencing hub (HUSH) complex (Figure 4F) (Tcha-
sovnikarova et al., 2015; Tunbak et al., 2020). The HUSH com-
plex localizes to intronless DNA, a characteristic feature of evolu-
tionarily young LINE-1s, where it interacts with actively
transcribed LINE-1 RNA to promote H3K9me3 deposition
(Figure 4F) (Seczynska et al., 2022). HUSH produces prema-
turely terminated transcripts which may then be locally degraded
via the nuclear exosome targeting (NEXT) complex (Figure 4F)
(Garland et al., 2022). Together, these studies highlight that
DNA methylation as well as RNA-mediated deposition of
H3K9me3 via the HUSH complex can silence LINE-1 elements
in mammalian development and somatic cells.
Non-LTR retrotransposons: SUV39H1 represses SINE
transcription via local H3K9me3
By contrast to LINEs, SINE RTEs are not known to encode for
proteins and thus rely on LINE-1 encoded proteins to facilitate
their non-autonomous retrotransposition. In total, there are

850k full-length SINEs across three primary lineages (i.e.,
AluJ, AluS, and AluY) within the human genome. Although all
full-length AluJ elements (i.e.,
160k) are believed to be non-
functional, a significant portion of full-length AluS (i.e.,
550k)
and nearly all full-length AluY (i.e.,
130k) elements are sug-
gested to have retained their retrotransposition abilities based
on in vitro mobilization assays (Bennett et al., 2008). Thus, along
with LINE-1s, SINEs maintain a significant threat to genome
instability.
SINE DNA is enriched for 5mC DNA methylation (Arand et al.,
2012). However, multiple studies argue against DNA methylation
as sufficient to repress SINE expression. Both 5-Azacytidine, a
DNMT small-molecule inhibitor, and DNMT1 repression are un-
able to induce SINE transcriptional activation (Varshney et al.,
2015). The heterochromatin effectors HP1a and SUV39H1 as
well as H3K9me3 are enriched at SINEs even in the absence of
DNA methylation (Varshney et al., 2015). Small molecule inhibi-
tion of SUV39H1 results in significant decreases in H3K9me3
abundance, along with increased RNA polymerase III occupancy
leading to elevated SINE transcription (Varshney et al., 2015).
These data suggest that SUV39H1-mediated H3K9me3 can at
least in part govern SINE transcription repression (Figure 4G).
INTERPLAY AMONG 3D GENOME FOLDING,
HETEROCHROMATIN, AND REPETITIVE DNA
ELEMENTS
While the literature provides support for a link between hetero-
chromatin and higher-order chromatin organization, and a role
for heterochromatin in silencing the repetitive genome, the
higher-order folding principles of the repetitive genome have
only recently begun to be explored. We highlight some early
case studies exploring the relationship among heterochromatin,
higher-order chromatin folding patterns, and the repetitive
genome.
2700 Cell 185, July 21, 2022
Review
Spatial positioning of genomic loci with double strand
breaks outside of chromocenters promotes high-fidelity
repair of pericentromeric repeats in mice
A notable example for how the higher-order folding of hetero-
chromatin interplays with the repetitive genome comes from
studies exploring the activation of DNA repair pathways within
clustered pericentromeric repeats. The high concentration of re-
petitive satellite DNA sequences in pericentromeric chromatin
pose a significant challenge for double strand break (DSB) repair
by homologous recombination (HR). Although HR is typically an
exceptionally high-fidelity DSB repair pathway (Verma and
Greenberg, 2021), the presence of repetitive sequences in-
creases the possibility for improper recombination in cis (i.e., be-
tween similar satellites present at distinct genomic loci on
homologous chromosomes) or in trans (i.e., between similar sat-
ellites present on non-homologous chromosomes). In mouse
model systems, the heterochromatinization and condensation
of pericentromeric satellite repeat clusters into chromocenters
can establish a barrier to prevent improper access of HR ma-
chinery (Mitrentsi et al., 2022). It has been suggested that this
mechanism likely works cooperatively with the relocation of peri-
centromeric DSBs towards the periphery to improve the fidelity
of HR DSB repair (Caridi et al., 2018; Chiolo et al., 2011; Tsour-
oula et al., 2016). Notably, in NIH 3T3 mouse fibroblasts, enrich-
ment of H3K9me3 at CRISPR-induced pericentromeric DSBs re-
mains unchanged following its relocation to the periphery, which
may provide continued additional protection against nonspecific
HR (Tsouroula et al., 2016). It is interesting that these mecha-
nisms have been shown to be absent in human cell model sys-
tems (Mitrentsi et al., 2022), which is somewhat unexpected
given the conservation of spatial mechanisms governing peri-
centromeric DSB repair from yeast to mice. Therefore, future in-
vestigations into how both spatial genome organization and het-
erochromatinization influence the fidelity of pericentromeric DSB
repair in humans will be critical for addressing this intriguing
discrepancy.
STR expansion disrupts TAD boundaries in fragile X
syndrome
Although repeat expansion disorders share the commonality of
unstable STR expansion, the repeat tracts themselves are quite
diverse in their specific properties across diseases. For
example, the repeat unit sequence, as well as the repeat unit
length, can be highly variable. Each disorder has a different
premutation and mutation threshold, and the tracts are evenly
distributed across the genome in exons and introns and the
50 UTR, 30 UTR, and intergenic regions. We recently set out to
ascertain if chromatin folding patterns could shed light on
why some regions of the genome grow unstable in disease,
whereas hundreds of thousands of sequence- and transcrip-
tion-matched STR tracts remain stable. Using Hi-C maps
across multiple cell types, Sun et al. reported that nearly all
STRs which are known to grow unstable in disease co-localize
with the boundaries of TADs/subTADs (Sun et al., 2018)
(Figure 5A). On the basis of this observation, it was hypothe-
sized that a subset of genetically encoded boundaries would
serve as hotspots in the human genome for vulnerability to
instability events. Using fibroblasts, B cells, and post-mortem
 ll
Review

Figure 5. STR expansion and HERV-H transcription influence TAD boundary integrity
(A) A CGG trinucleotide short tandem repeat (STR) tract residing in the 50 UTR of FMR1 is associated with fragile X syndrome.
(B) A study by (Sun et al., 2018) reports that in healthy individuals, the CGG STR (i.e., 6–40 repeating monomer subunits) is present at a TAD boundary, where
FMR1 is actively transcribed.
(C) As the CGG STR expands to full mutation length of 200 triplets or more, CTCF is displaced and local TAD/subTAD boundaries within 1 Mb around FMR1 are
disrupted.
(D) A study by Zhang et al. (2019) shows that transcription of HERV-H by the RNA Polymerase II complex presents a physical barrier that interferes with cohesin-
mediated loop extrusion, leading to the creation of a local CTCF-independent TAD boundary.
(E) De novo HERV-H insertion is sufficient for the formation of a new TAD boundary.
(F) Transcriptional repression of an endogenous HERV-H locus can dissolve a pre-existing TAD boundary.

brain tissue from male FXS patients compared to healthy
normal-length individuals, Sun et al. observed severe boundary
disruption around the FMR1 gene upon long mutation-length
CGG expansion (Sun et al., 2018) (Figures 5B and 5C). Tran-
scriptional silencing of FMR1 correlated with severe boundary
disruption, suggesting a link between structural boundary
integrity and proper gene expression. Together, these data
suggest that STR instability events may be linked to the molec-
ular and structural features at a subset of TAD boundaries.
Although mutation-length STR expansion events coincide with
boundary disruption, it is not yet known whether instability
drives genome misfolding or vice versa. A recent BioRxiv pre-
print reports the deposition of Megabase-scale H3K9me3 do-
mains along with widespread genome misfolding and trans in-
teractions between autosomes and the X chromosome in FXS
(Zhou et al., 2021), suggesting an interplay between
H3K9me3, genome folding, and repeat instability.
Transcription of HERV-H is sufficient to create a TAD/
subTAD boundary
Transcriptionally active RTEs can actively shape chromatin ar-
chitecture and genome function independent of whether they

Cell 185, July 21, 2022 2701

 ll
mobilize or destroy architectural protein motifs (Zhang et al.,
2019). Human ES cells are hypomethylated with respect to differ-
entiated cell lines, which can lead to the de-repression of LTR
and non-LTR RTEs. In human embryonic stem cells, the RTE
HERV-H (Figure 5D) is transcribed, and its transcripts account
for 2% of the total poly A RNA pool (Santoni et al., 2012).
Random integration of an active HERV-H element into a new
genomic locus is sufficient to form a de novo TAD boundary in
a transcription-dependent manner (Figure 5E). Moreover,
repression of HERV-H expression via differentiation of human
ES cells to cardiomyocytes or dCas9-KRAB mediated silencing
attenuates the ability of the HERV-H sequence to serve as a
boundary element (Figure 5F). In the development of the mouse
2 cell stage embryo, transcription of murine endogenous retro-
virus element (MERV) family elements is also sufficient to form
TAD boundaries (Kruse et al., 2019). Together, these studies
highlight that specific subclasses of ERV repeat elements can
form TAD/subTAD boundaries in a transcription-dependent
manner.
SINEs and LINEs may shape human genome folding via
redistribution CTCF binding sites
Finally, it has been hypothesized that SINEs and LINEs shape
the higher-order folding of the genome by harboring binding
sites for the architectural protein CTCF within their own se-
quences (Choudhary et al., 2022; Diehl et al., 2020). Activation
of retroelements during evolution can lead to species-specific
expansion of CTCF binding sites. Over 95% of mammalian
CTCF sites are derived from SINEs, LINEs, and LTRs (Choudh-
ary et al., 2020)., and SINEs in mice are enriched for CTCF-
bound motifs (Schmidt et al., 2012). It is possible that the mobi-
lization of SINE sequences during evolution could provide
significant alterations to genome folding patterns in a CTCF-
dependent manner. To minimize this risk, the CTCF motifs in
SINEs are targeted by the ChAHP complex, which competes
with CTCF for motif binding to prevent novel SINE B2 integra-
tions from disrupting the 3D genome (Kaaij et al., 2019). Addi-
tionally, SETDB1 facilitates the heterochromatinization of SINE
B2s in mouse macrophages, thus preserving the regulation of
lipopolysaccharide-inducible genes by preventing inappro-
priate CTCF binding and ectopic chromatin looping events
(Gualdrini et al., 2022). Comparatively, in human lymphoblas-
toid cells, only a small fraction of CTCF-bound loop anchors
are present in SINE elements, as LTR RTEs contained the
greatest percentage of CTCF-bound loop anchors (Choudhary
et al., 2020). Thus, CTCF binding sites in RTEs can form loops
and mechanistically contribute to the formation of TAD
boundaries.
CONCLUSIONS
Contrasting mechanisms of compartmentalization and loop
extrusion shape the higher-order folding of eukaryotic genomes
in ways that directly influence heterochromatin acquisition and
maintenance. While the heterochromatic mechanisms associ-
ated with repetitive DNA element biology have been well studied
in the context of the linear chromatin fiber, emerging evidence
reveals critical crosstalk between higher-order folding patterns
2702 Cell 185, July 21, 2022
Review
and heterochromatin acquisition. Looking ahead, technological
innovations including high-resolution microscopy, spatial geno-
mics, single-cell technologies, and systematic perturbative
studies will be well positioned to catalyze a wave of fresh insights
into how the genome’s structure-function relationship intricately
governs stability of the repetitive genome across space and time
in development and human disease.
ACKNOWLEDGMENTS
We regret that many important works of our colleagues are not cited due to the
large scope of the topic and journal space limits. S.A.H. is a New York Stem
Cell Foundation – Druckenmiller Fellow. This research was supported by the
New York Stem Cell Foundation (to S.A.H.); NIH National Institute of Mental
Health (1R01MH120269; 1DP1MH129957) (to J.E.P.-C.); NIH National Institute
of Neural Disorders and Stroke (1R01-NS114226) (to J.E.P.-C.); 4D Nucleome
Common Fund grants (1U01DK127405, 1U01DA052715) (to J.E.P.-C.); NSF
CAREER Award (CBE-1943945) (to J.E.P.-C.); and Chan Zuckerberg Initiative
Neurodegenerative Disease Pairs Award (2020-221479-5022) (to J.E.P.-C.).
DECLARATION OF INTERESTS
The authors declare no competing interests.
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