An embryonic stem cell-specific heterochromatin state allows core histone exchange in the absence of DNA accessibility

Nucleosome turnover concomitant with incorporation of the replication-independent histone variant H3.3 is a hallmark of regulatory regions in the animal genome. In our current understanding, nucleosome turnover is universally linked to DNA accessibility and histone acetylation. In mouse embryonic stem cells, H3.3 is also highly enriched at interstitial heterochromatin, most prominently intracisternal-A particle endogenous retroviral elements. Interstitial heterochromatin is established over confined domains by the TRIM28/SETDB1 corepressor complex and has stereotypical features of repressive chromatin, such as H3K9me3 and recruitment of all HP1 isoforms. Here, we demonstrate that fast histone turnover and H3.3 incorporation is compatible with these hallmarks of heterochromatin. Further, we find that histone H3.3 is required to maintain minimal DNA accessibility in this surprisingly dynamic heterochromatin state. Loss of H3.3 in mouse embryonic stem cells elicits a highly specific opening of interstitial heterochromatin with minimal effects on other silent or active regions of the genome.


INTRODUCTION
Histone H3.3 is an evolutionary conserved variant of the canonical histone H3 proteins, termed H3.1 and H3.2 in mammalian cells 1 . First described to associate with regions of active transcription in Drosophila 2 , H3.3 has been recognized as the sole substrate for replication-independent chromatin assembly in mammalian cells 3 . While H3.1/2 expression and replication-dependent incorporation is limited to S phase, H3.3 is expressed through the cell cycle and incorporated at sites of dynamic nucleosome turnover. A multitude of genome-wide studies have identified such dynamic chromatin at promoters, enhancers, gene bodies and origins of replication [4][5][6][7][8][9][10][11][12][13] . In these instances, nucleosome turnover is thought to be a consequence of chromatin remodeling, transcriptional activity, nucleosome-destabilizing DNA sequences, competition with other chromatin binding factors, or a combination thereof. While histone H3.3 itself has been proposed to destabilize the nucleosome, it is unclear if H3.3 has a causal role in promoting nucleosome turnover [14][15][16] . Histone H3.3 has been shown to be enriched in histone posttranslational modifications (PTMs) characteristic of euchromatin, i.e.
H3K4me3, H3K9ac, H3K27ac 17,18 . Accumulation of active PTMs on histone H3.3 is sufficiently explained by their co-occurrence at sites of active enhancers or transcription, and H3.3 appears not to be required for maintaining those active PTMs. However, a notable exception represents phosphorylation of Ser31, a H3.3-specific residue. H3.3Ser31 phosphorylation facilitates rapid activation of genes 19,20 , thus providing a mechanistic link between H3.3 incorporation and gene activation. In summary, histone H3.3 is a well-established component of euchromatin, acting as a replacement histone for a range of dynamic processes. While H3.3 has to be considered predominantly neutral to the underlying dynamic process, it can -as exemplified in the specific instance above -partake mechanistically in establishing open chromatin.
Interstitial heterochromatin is highly CpG-methylated, enriched in linker histone and bound by HP1 family proteins, the stereotypical H3K9me3 readers [28][29][30] . HP1 proteins have been shown to bridge nucleosomes and compact chromatin into phase-separated liquid condensates 31,32 . HP1 and linker histone render DNA in an inaccessible state 32 , and underlying genes/repeats are refractory to activating factors 33 .

H3.3 is a pluripotent stem cell-specific component of interstitial heterochromatin.
Several studies have assessed genome-wide H3.3 dynamics in ESC, either measuring incorporation or turnover of ectopically tagged H3.3 5,8,9,34 , albeit exclusively looking at dynamics related to enhancers and transcription. A recent study assessed nucleosome turnover using a method termed 'time-ChIP' in mouse embryonic stem cells as well as neural stem cells (NSC) 9 , reporting high nucleosome turnover at enhancers correlating with DNA accessibility. For the time-ChIP method, SNAP-tagged H3.3 was expressed from a Tet-controlled transgene and biotin-labeled using the SNAP-tag at the onset of a time course (0h). The biotin-labeled fraction then was observed over a time range of 3-12 hours (Figure 1a, b). Dynamic regions are marked by H3.3 at 0h and gradually decay towards the genome-wide average, implying replication-independent turnover of H3.3 9 .
We reanalyzed the time-ChIP datasets with respect to the known heterochromatic enrichment of  Figure 1b). Thus, as previously concluded 21 , co-enrichment of H3.3 and H3K9me3 is a feature of pluripotent stem cells which appears to be lost early upon differentiation. Interestingly, H3K9me3 and H3.3 has also been studied genome-wide during reprogramming of mouse fibroblasts to induced-pluripotent cells 35 ; while H3K9me3 becomes enriched over IAP ERV in pre-iPSC, H3.3 is absent at this stage (Extended Data Figure 1c). Pre-iPSC are stable late-stage reprogramming intermediates that can be converted to the pluripotent state through MEK inhibition. Thus, these data corroborate a link between pluripotency and the acquisition of H3.3 at interstitial heterochromatin, where H3K9me3 has already been established during reprogramming. H3K27ac, H3K36me3 and RNAP2 37 , in line with the fact that H3K9me3 appears mutually exclusive with these signatures of active chromatin. Binning the genome into 5kb windows, we found a bimodal H3.3 distribution, with regions of very high and very low levels of H3K9me3 coinciding with the highest H3.3 enrichment (Figure 1e). Overlaying the 5kb bins with repeat annotation, we confirmed that the strongest H3.3+H3K9me3 co-enrichment was linked to the presence of an IAP element (Figure 1e, Extended Data Figure 3). Further, ETn and MusD elements also defined a subset of H3.3+H3K9me3 co-enriched regions (Extended Data Figure   3). It is known that a small fraction of ETn/MusD elements are active and, in fact, highly transcribed. This is evident from elongating RNA Polymerase II running into the 3' flanking region of few elements, and the complete absence of H3K9me3 around these elements (Extended Data Figure 4a,b). Since we cannot unambiguously attribute ChIP-Seq reads within the highly conserved ETn/MusD internal sequence to specific instances, we are not able to discern how much H3.3 incorporation can be attributed to the small fraction of active versus majority of repressed copies. Therefore, we did not further focus on ETn/MusD elements.
Amongst the IAP repeat family, none of the annotated instances showed similar evidence of transcriptional activity (Extended Data Figure 4c,d), and RNA-Seq transcript levels were 1-2 orders of magnitude lower than those of ETn (Extended Data Figure 4e).
Rapid histone turnover at interstitial heterochromatin in the absence of DNA accessibility provide complementary evidence for nucleosome turnover at interstitial heterochromatin. While rapid H3.3 turnover has been attributed to 'hyperdynamic' promoter and enhancer nucleosomes 8,9 , our analysis suggests that similar dynamics also apply to H3.3+H3K9me3 peaks.
We next compared H3.3 dynamics across 15 ChromHMM states 37 and H3.3+H3K9me3 heterochromatic regions and found that highest histone H3.3 turnover is observed at active promoters and enhancers followed by H3.3+H3K9me3 regions (Figure 2d, ExtendedData Figure   5b). Analysing individual repeat families using UCSC RepeatMasker annotation, the~340bp IAPLTR1/1a long terminal repeats showed comparable turnover to active promoters and enhancers, followed by IAPLTR2 and internal IAP regions (Extended Data Figure 6). Examining H3.3 dynamics across full-length IAPLTR1 elements, it was interesting to note that histone turnover was not confined to individual positioned nucleosome but broadly occurred over LTRs and adjacent~2kb of internal region ( Figure 2e). Remarkably, regions of highest turnover were also highly enriched for H3K9me3, concomitant with HP1 binding (Figure 2f-g). Prior analysis suggested that, as stereotypical for heterochromatin, DNA accessibility at ERVs is generally low 29 , and analysis of recent ATAC-Seq data in mESC 20 confirmed that IAPLTRs neither show defined nucleosome-free regions nor broader domains of accessible DNA (Figure 2h). In summary, we find that histone turnover does not require or induce an increase in DNA accessibility at IAP ERVs (Figure 2f), Instead, it appears that histone H3.3-H4 is swapped into chromatin directly replacing an existing H3-H4 histone dimer or tetramer, hinting at the possibility of a concerted mechanism.
Interstitial heterochromatin becomes accessible in the absence of histone H3.3.
To achieve an exchange of core histones without rendering DNA at least transiently accessible, a tightly regulated supply of H3.3 would appear necessary. We thus wondered how a lack of H3.3 would affect DNA accessibility at H3.3+H3K9me3 regions. ATAC-Seq profiles of wildtype and H3.3 knockout cells have previously been compared 20 . The authors note that DNA accessibility is largely unchanged across the genome in the absence of H3.3, including sites of rapid histone exchange, such as promoters and enhancers 20 Figure 9). In addition to full-length or truncated IAP ERVs, the mouse genome contains many single LTRs (predominantly of the IAPLTR2 family). Using only uniquely mappable read pairs, we found that the 651 H3.3-enriched orphan LTRs also gained DNA accessibility in the absence of H3.3 ( Figure 3g).
In summary, we find that interstitial heterochromatin maintains underlying DNA in an inaccessible state, a key characteristic of all heterochromatin. Despite this, interstitial heterochromatin is permissive to nucleosome turnover and histone H3.3 incorporation, providing a mechanistic explanation for the co-occurence of H3.3 and H3K9me3. According to the collective evidence from the data analyzed here, nucleosome eviction and assembly of new histone H3.3 nucleosomes must be accomplished through a mechanism that does not allow the underlying DNA to become accessible in the process. Loss of histone H3.3 may uncouple eviction of existing nucleosomes from immediate reassembly of a new nucleosome and consequently lead to the observed broad gain in DNA accessibility.

DISCUSSION
In the current literature, heterochromatin is invariably associated with an inert chromatin packaging 33 . Dynamic histone exchange is inherently viewed as a mechanism that opposes heterochromatin formation 40 . Collectively, our analyses shed first light into a dynamic heterochromatin state that defies these stereotypical distinction of euchromatin and heterochromatin. While the purpose and mechanism remain only poorly understood, our study provides a novel hypothesis and functional insights for future mechanistic studies. Histone H3.3 is known to be deposited at interstitial heterochromatin through the histone chaperone DAXX 21,[41][42][43] . DAXX has been proposed to contribute to ERV silencing through its histone chaperone activity and, independently, recruitment of histone deacetylases to ERVs 21,39 . Loss of H3.3 also reduces global DAXX levels in mouse ESC, but H3.3+H3K9me3 regions did not gain histone H3 acetylation in the absence of H3.3 (Extended Data Figure 8). This suggests that impaired nucleosome assembly in the absence of H3.3 was the cause of the The chromatin remodeler Smarcad1 has recently been linked to IAP ERVs in mouse ESC 47 .
Smarcad1 has further been shown to have nucleosomes sliding and eviction activity, thus representing another candidate for mediating H3.3 turnover at ERVs 48,49 . Structural studies of the HP1-bound dinucleosome corroborate the view that heterochromatin can be accessible for nucleosome remodeling 32 . We show that supply of H3.3 is necessary for maintaining chromatin structure at IAP ERVs, implying that existing nucleosome eviction is directly linked to DAXX-dependent deposition of new H3.3. A major question remains why ATP-dependent turnover of old and incorporation of new histone is required as part of a repressive chromatin domain. Transient eviction of nucleosomes could allow KRAB zinc-finger proteins to sample the DNA more effectively and thereby facilitate recruitment of TRIM28 corepressor complex.
Alternative models could be proposed where nucleosome eviction represents a mere side effect of a more widespread chromatin remodeling activity in embryonic stem cells, imposed by the need for a highly dynamic chromatin structure to maintain pluripotency 50 . Irrespective of the purpose of remodeling, nucleosome turnover necessitates sustained SETDB1 methyltransferase activity for maintaining high H3K9me3 levels at ERVs. This is because newly synthesized H3.3 is devoid of H3K9 methylation, and SETDB1 enzyme is known to establish H3K9me3 only on nucleosomal substrates. Histone H3K9me3 levels decrease in the absence of H3.3 21 , showing that histone H3.3 incorporation promotes rather than counteracts establishment of H3K9me3 and the formation of interstitial heterochromatin in mESC.
Collectively, our analyses shed first light into a dynamic heterochromatin state that defies the stereotypical distinction of euchromatin and heterochromatin. While the purpose and mechanism remain only poorly understood, our study provides a novel hypothesis and functional insights for future mechanistic studies.

ACKNOWLEDGEMENTS
Bioinformatics analyses were performed on resources provided by the Swedish National Infrastructure for Computing (SNIC) at Uppmax server (projects SNIC 2020/15-9, SNIC 2020/6-3, uppstore2018208, SNIC 2018/3-669, sllstore2017057, SNIC 2017/1-508).          replicates were combined to obtain a reliable set of peaks. A peak is considered if it is called in at least two out of the three replicates. This was performed for both ESC and NSC at initial timepoint (0h). The region considered as a reliable peak is then the result of merging the overlapping called peak regions.

FIGURES
Bin-based analyses . For the genome-wide coverage analyses in 5kb bins, mean coverage bin files were generated with deepTools multiBigWigSummary bins tool from the final bigwig files using a bin size of 5kb. Where available, input datasets were used to normalize the ChIP-Seq coverage by calculating the log2 of the ChIP/input ratio.
Repeat element annotation . To filter the many instances of short and fragmented repeat occurrences in the genome, we curated repeat elements from UCSC RepeatMasker as follows: mm9 RepeatMasker track was clustered to merge LTRs and internal sequence into an single continuous interval. For this, adjacent elements (allowing for no gaps) were clustered into single elements, keeping the name of the largest fragment for the cluster (typically the internal sequence, e.g. IAPEz-int, that defines the repeat family). Any single or clustered element shorter than 2kb was removed, clusters larger than 2kb were retained as continuous intervals and used as repeat annotation.

Curated set of shared IAPEz elements across datasets from different mouse strains.
A mm9 reference IAP ERV annotation was obtained from UCSC RepMasker track, merging internal IAPEz-int and flanking LTR intervals and retaining elements longer than 2kb as described above (resulting in 3046 full-length and truncated IAP ERVs). In each available paired-end ChIP-Seq and/or Input dataset, read pairs overlapping with the reference IAP ERVs were retained if at least one of the two reads was unambiguously assigned to one genomic match (thus, the pair could be niquely mapped with bowtie2 mapping quality MQ > 10). One or more such anchoring read was used as evidence that the IAP ERV insertion is present in the respective mouse strain. Evidence from all ChIP-Seq and matched inputs were collectively used to define the IAP ERVs present in a given cell line/mouse background. Finally, the annotation of 2640 shared IAP ERVs was obtained by retaining only those IAP ERVs present in all the cell lines (see Extended Data Figure 10) .

Annotated bins.
Genome-wide 5kb bins were intersected with our repeat annotation using bedtools (v2.27.1) intersect 57 . Bins were assigned the repetitive element group of the element that overlapped the most with each bin, in the unusual case where the same bin overlaps more than one repetitive element.
Annotated peaks. A peak list from endogenous H3.3 ChIP-seq 21 was intersected with peaks called from 9 as described above. Any amount of overlap > 0 was considered. Other loci of interest were also intersected with these peaks in the same fashion: IAPEz and ChromHMM15 regions. Since ChromHMM15 is a partition of the genome, a peak is expected to fall in at least one category. For the cases where a peak overlapped with more than one ChromHMM annotation, the largest overlap was reported. Statistics . Plot statistics were calculated using R package ggpubr. Size effect estimations were calculated using R package effsize.

Genome-wide enrichment over 5kb bins for ES and NS cells, timepoints 0h, 3h, 6h and
12h . Bins were generated using deepTools multiBigWigSummary (see Bins). Each bin value was divided by its corresponding input value. Bins with zero values were excluded from this analysis. Additionally, outliers were removed using Tukey's 1.5 IQR rule for the violin plots.
Venn and Euler diagrams . Venn diagrams were calculated and visualized using Intervene 58 .
Proportional representations of these as Euler diagrams were visualized with R package eulerr.
Profile plots . Average plots over genomic regions were generated with ngsplot 59 , including a modification to produce plots normalized to 1x Genome Coverage. Since ngsplot considers the coverage of actual fragments defined by paired-end reads for more realistic occupancy profiles, the y axis reports average Fragments Per Genome Coverage (FPKM) rather than RPKM.