Trends in Genetics

Spotlight

De novo DNA
Methylation: Who’s
Your DADdy?
Marla E. Tharp1,2
and Alex Bortvin1,3,*,@DNA methylation regulates the organization and function of the genome.
Yamanaka et al. now report that denovo methylation of male germ cellsof mice involves the transient openingof heterochromatin at megabase-sizedifferentiallyaccessible
domains(DADs). This chromatin remodelinglikely facilitates de novo methylationof the germ cell genome.

Throughout a man’s lifetime, theodds of any individual sperm cell heproduces influencing the next generation is worse than winning the lottery.
However, each of his gametes, fruitfulor wasted, represents an epic featof chromatin reorganization and proper
spatiotemporalgeneexpression.
From the start, primordial germ cells(PGCs) are given the colossal task of
epigenetic reprogramming involving
genome-wide erasure of repressive
5-methylcytosine marks, removing
parental imprints, and creating a naı̈ve
potency state to prime sex-specificcell differentiation [1]. Following theirmigration to the embryonic testis,
male PGCs (now termed ‘gonocytes’)undergo mitotic arrest and rapidly reestablish DNA methylation de novo,
an essential task for differentiation
into spermatogonia, the source ofsperm. Indeed, absence of de novo
methylation in mouse male germ cells,such as in DNA methyltransferase(DNMT) 3 family mutants Dnmt3a–/–and Dnmt3l–/–, results in failure tomethylate paternal imprinting control
regions, activation of transposable elements (TEs), and impaired spermatogenesis [2,3].

Two waves of de novo DNA methylation have been reported in mouse
male gonocytes between embryonicday (E) 13.5 and postnatal day (P) 2,transitioning around E16.5 [4]. The firstwave encompasses de novo methylation of most of the genome, while thesecond uses small Piwi-interacting (pi)RNAs to guide methylation of TEs [4].
One plausible feature distinguishingthese waves is the accessibility of
DNMTs to the genome to deposit
DNA methylation marks. In dividing
cells, DNMT recruitment is tightly
coupled to DNA replication, taking
advantage of locally unwound DNA at
replication forks during S phase togain occupancy of the genome inboth euchromatic and heterochromatic
regions [5]. However, in cell cyclearrested gonocytes, the players andstrategies for DNMT accessibility andsubsequent de novo methylation, especially at inaccessible heterochromatic
regions, become ambiguous.

Transient Accessibility of
Gonocyte Chromatin Reveals
DADs
To understand where and when denovo DNMTs might be recruited to
the genome, Yamanaka et al. usedpure populations of germ cells to probe
the dynamics of genome-wide chromatin accessibility [6]. By performing
assay for transposase-accessible chromatin using sequencing (ATAC-seq) toprobe chromatin accessibility andNanoCAGE-seq to determine transcriptional start site and transcriptabundance, the authors identified specific chromatin regions that acquire
greater accessibility between E13.5
and E17.5, and subsequently close by
P2. Such timing implicates these gonocyte-specific DADs in the second wave
of de novo DNA methylation. Using
the stringent 1-Mb minimal size cutoff, Yamanaka et al. identified a totalof 143 DADs on all chromosomes,

except the Y chromosome, accountingfor 14% of the mouse genome.

Genomic Features of DADs
Highlight TEs
The authors next interrogated thedefining features of DADs. Ultimately,
110 of the 143 DADs were found ingene-poor regions of the genome that
encode fewer than ten genes per megabase and are instead enriched in TEsequences (70% of E17.5-specific peaks
on TEs). Interestingly, a few DADs also
included gene clusters, such as thosebelonging to olfactory receptor orV(D)J recombination, origins of which
are based in TEs [7]. Deeper analysisof TE subfamilies and structural elements that overlapped with DADs revealed that 50 regions of sequences
belonging to evolutionarily ‘young’LINE-1 subfamilies become accessible,
features of full-length elements stillwith retrotransposition activity. Indeed,
upregulation of TE transcripts wasobserved at E17.5, in correlation with
their transiently accessible regions.
Given that TE expression is a prerequisite for piRNA production, these data
corroborate findings that methylationof young LINE-1 elements during thesecond wave of de novo methylation ispiRNA dependent [4].

Epigenetic Regulation of DADs
and Vice Versa
To understand the players and dynamics of DAD formation that are independent of DNA methylation and DNA
replication, the authors interrogatedanother type of epigenetic regulation,
namely, methylation of specific lysine
residues in the amino-terminal tail of histone H3. Indeed, DADs correspondedwith the reprogramming of active histone mark H3K4me3 and repressive histone marks H3K27me3 and H3K9me3
measured by ChIP-seq. Acquisition of
H3K4me3 and complementary loss of
H3K27me and H3K9me3 in DADs at

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1Trends in Genetics

E17.5 resulted in the opening of chromatin and a more euchromatic state,permissive for DNMT binding. DADs
gradually returned to a compacted stateby P2 with a decrease in H3K4me4 andincrease in H3K27me3 and H3K9me3.
The plasticity of histone marks exemplified in DADs defined these regions as
facultative heterochromatin that canreorganize in response to environmental cues, as opposed to static
constitutive heterochromatin regions
found at centromeres and telomeres.

P2

Secondwave
DNAme
Transposon mRNA
H3K4me3P0

H3K9me3
H3K27me3
DNMT
E17.5

The observed histone dynamics werereflected by the dynamic reorganization of chromatin 3D architecture
measured by Hi-C. Indeed, the frequencies of long-range intrachromosomal interactions present at E13.5
decreased between E13.5 and E17.5
during DAD formation, signifying decompaction of chromatin structure. By
P2, these interactions returned. This
momentary decompaction of higherorder chromatin structure represents atransition state responding to DAD
formation. Interaction patterns within
the transition state, particularly between E17.5 and P0, were significantly
rearranged compared with other time
points. Specifically, switching between
open and closed chromosome conformations based on A/B compartmentloci was observed in some DADs [6].
Cumulatively, Yamanaka et al. describe
DADs as novel features of facultative
heterochromatin that assume a transient relaxed state to permit DNMT
recruitment and subsequent depositionof 5-methylcytosine marks for de novo
methylation (Figure 1). However, thedependency of de novo methylation
on DADs remains unexplored, forexample, upon DNMT overexpression
or perturbation of H3K4me3 methyltransferase activity. The mechanisms
driving histone remodeling for DAD
organization are also unsolved. A tanta2DNAmeFirstwave
DNAme

E13.5
Gonocyte
stageAccessible
chromatin

Heterochromatic DADs
Trends in Genetics

Figure 1. De novo DNA Methylation of Heterochromatic Differentially Accessible Domains(DADs).
De novo DNA methylation (DNAme) in male gonocytes occurs in two waves. The first wave
targets accessible chromatin regions. The second wave, beginning around embryonic day (E)17.5,
targets inaccessible chromatin regions, including gene deserts, gene clusters, and transposons. De
novo methylation of inaccessible regions is marked by the transient formation of 1–12 Mb DADs,
which is accomplished by rewriting active (H3K4me3) and repressive (H3K27me3 and H3K9me3)
histone marks. Transient DAD formation permits a burst of transposon expression and allows DNA
methyltransferase (DNMT) enzymes access to DNA for de novo methylation. Ultimately, DADs
resume their compacted chromatin state at the end of gonocyte development. Abbreviation: P,postnatal day.

lizing candidate is the piRNA pathway
due to direct relationships with theremoval of H3K4me2 and establishment of de novo methylation andH3K9me3 patterns on TE sequences[8,9]. The authors speculate that denovo methylation related to DADs islikely not sequence specific due to thepromiscuous nature of DNMTs, butgiven the requirement for piRNAs in
de novo methylation of LINE-1 sequences, further analysis of their overall
contribution to DAD organization isTrends in Genetics, Month 2019, Vol. xx, No. xx

warranted [4,6]. Finally, since female
germ cells regain methylation postnatally and only after enduring massive
LINE-1-driven oocyte death [10], it will
be intriguing to determine whether oocytes use the same chromatin-remodeling strategy elucidated by Yamanaka
et al. in males [6].

1Department of Embryology, Carnegie Institution

for Science, Baltimore, MD 21218, USA
2Department of Biology, Johns Hopkins

University, Baltimore, MD 21218, USATrends in Genetics

3Web: https://alexbortvinlab.org@Twitter: @alexbortvinlab*Correspondence:
bortvin@carnegiescience.edu
https://doi.org/10.1016/j.tig.2019.09.001ª 2019 Elsevier Ltd. All rights reserved.

References
1.. Hill, P.W.S. et al. (2018) Epigenetic
reprogramming enables the transition fromprimordial germ cell to gonocyte. Nature555, 392–396

2.. Bourc’his, D. and Bestor, T.H. (2004) Meiotic
catastrophe and retrotransposon
reactivation in male germ cells lacking
Dnmt3L. Nature 431, 96–99
3.. Kaneda, M. et al. (2004) Essential role for denovo DNA methyltransferase Dnmt3a inpaternal and maternal imprinting. Nature429, 900–903
4.. Molaro, A. et al. (2014) Two waves ofde novo methylation during mouse germ
cell development. Genes Dev. 28, 1544–1549
5.. Hervouet, E. et al. (2018) Specific or not
specific recruitment of DNMTs for DNA
methylation, an epigenetic dilemma. Clin.
Epigenetics 10, 17
6.. Yamanaka, S. et al. (2019) Broad
heterochromatic domains open in gonocyte
development prior to de novo DNAmethylation. Dev. Cell. Published online
August 29, 2019. https://doi.org/10.1016/j.
devcel.2019.07.023.
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8.. Nagamori, I. et al. (2018) Relationshipbetween PIWIL4-mediated H3K4me2
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9.. Pezic, D. et al. (2014) piRNA pathway targets
active LINE1 elements to establish therepressive H3K9me3 mark in germ cells.
Genes Dev. 28, 1410–1428
10.. Malki, S. et al. (2014) A role forretrotransposon LINE-1 in fetal oocyte
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