Atrx promotes heterochromatin formation at retrotransposons

Identification of a short heterochromatin inducing sequence (SHIN)

To monitor the silencing potential of specific DNA sequence elements of mouse IAP retrotransposons, we developed a lentiviral hEF1α-EGFP reporter system (Fig1A). When we combined the reporter with the well-characterized repressor binding site (RBS) of the MLV retrovirus ^9,37, strong silencing of EGFP expression was observed 2 days post-transduction (Fig1A). Similar effects were observed with the GAG region of IAP retrotransposons (Fig1A). In order to more systematically test silencing potential of IAP sequence elements, we analyzed different IAP regions in our reporter assay (Fig1B). A previously characterized region containing the UTR of IAP retrotransposons ¹⁰ displayed reporter silencing; however, the strongest effect was exerted by the ∼2,000-bp-long IAP-GAG region (Fig1B). As de novo silencing of IAP retrotransposons is restricted to ES cells ³⁸, we did not detect silencing in mouse embryonic fibroblasts (MEF). To refine potential silencing initiating elements within the GAG region, we further dissected this element into 400-bp fragments (Supplementary Fig S1A) and could finally determine a region of 160 bp which was sufficient to induce strong reporter silencing (Fig1C). This sequence could not be further shortened without compromising silencing potential (Fig1C) and is highly conserved in more than 600 IAP elements, predominantly of the IAP-Ez subclass (Supplementary Fig S1C, Supplementary Table S1). As will be outlined below, this short sequence element is sufficient to trigger heterochromatin formation; thus, we termed this region SHIN for short heterochromatin inducing sequence. Notably, SHIN silencing is not restricted to the hEF1α promoter (Supplementary Fig S1B) and is orientation independent (Supplementary Fig S1B), suggesting that silencing is initiated by sequence-specific DNA binding factors.

The SHIN sequence induces heterochromatin

In ES cells, IAP silencing is mediated by heterochromatin that depends on Trim28 which, in turn, recruits the H3K9me3 methyltransferase Setdb1 ^6,10,39. Similar to pericentric heterochromatin, the H3K9me3-rich domains across IAP elements coincide with H4K20me3 and DNA methylation. To test if SHIN silencing is dependent on any of these chromatin modifications, we assessed the extent of SHIN repression in ES cell lines that are depleted for the major silencing machineries. Here, we measured the number of EGFP-positive cells relative to the control reporter that does not contain the SHIN sequence. Interestingly, cells deficient for Suv39h, Suv4-20h, Dnmt1, or Dnmt3ab can still effectively silence the SHIN reporter (Fig2A). In contrast, Setdb1 knockout cells are severely compromised in SHIN silencing (Fig2A). Also, lentiviral knockdown of Trim28, which did not even completely abolish Trim28 expression (Supplementary Fig S1D and E), resulted in strong silencing defects. These data demonstrate that SHIN silencing requires the major players that also regulate silencing of endogenous IAP elements.

Next, we asked if the SHIN sequence can directly trigger Trim28-/Setdb1-dependent heterochromatin formation. To address this question, we used a well-established recombinase-mediated cassette exchange (RMCE) cell line to integrate the SHIN sequence into a defined position of the ES cell genome (Supplementary Fig VSports - S1F) ^40,41. We combined the SHIN sequence with a Tet-inducible EGFP-T2A-zeocin reporter which is not transcribed in the absence of doxycycline (Fig2B). We also generated a control reporter lacking the SHIN sequence (Fig2B). RMCE of the two reporter constructs resulted in the HA36::SHIN and HA36::control cell lines (Fig2B). ChIP-qPCR analysis revealed high levels of H3K9me3 across the RMCE locus in HA36::SHIN cells, comparable to endogenous IAP elements (Fig2B). In contrast, only residual occupancy for H3K9me3 was detected in HA36::control cells (Fig2B). To test if H3K9me3 across the SHIN reporter depends on Setdb1, we performed CrispR/Cas knockout of Setdb1 in HA36::SHIN cells. Prolonged knockout of Setdb1 is lethal for ES cells. Thus, we could not select for Setdb1 knockout cells and used a mixed population of Setdb1-deficient/Setdb-proficient cells for ChIP analyses 4 days post-sgRNA transduction (Fig2C, Supplementary Fig S1G). H3K9me3 levels at major satellite repeats are dependent on Suv39h enzymes and therefore remained almost unchanged upon Setdb1 depletion (Fig2C). Levels of H3K9me3 at endogenous IAP sequences were only mildly affected, indicating that this modification may have a low turnover on these sequences. Importantly, H3K9me3 was clearly reduced on a unique Setdb1 target region (pos control) and across the RMCE locus containing the SHIN sequence (Fig2C). In summary, our data show that the SHIN sequence represents an initiation site for heterochromatin formation from which H3K9me3 can spread over the entire locus.

SHIN silencing requires the Snf2-type chromatin remodeler Atrx

Heterochromatin formation across SHIN strictly depends on the Trim28/Setdb1 pathway and recapitulates silencing of endogenous IAP retrotransposons. Thus, we utilized our reporter assay to screen for additional factors that regulate retrotransposon silencing using a genome-wide shRNA screen (Fig3A). We transduced wild-type ES cells with a pooled genome-wide shRNA library, selected shRNA expressing cells with puromycin and then transduced the SHIN reporter. Cells that escaped SHIN silencing were isolated based on their EGFP fluorescence, and shRNA sequences from these escaper cells were cloned into a plasmid library (Fig3A). We tested 71 individual shRNA plasmids in a secondary screen. One shRNA which most strongly inhibited SHIN repression was found to target the SNF2-type chromatin remodeler Atrx (Fig3B, Supplementary Table S2). This shRNA clearly reduces Atrx mRNA and protein levels in mouse ES cells (Supplementary Fig S2A and B). However, to rule out off-target effects, we generated four independent knockout ES cell lines (Supplementary Fig S2C) with loss of Atrx protein (Fig3C). Atrx knockout (ko) cells grow normally and show no obvious alterations in their cell cycle profile (Supplementary Fig S2D). Importantly, SHIN silencing was defective in all Atrx knockout ES cell lines (Fig3D), which confirmed the shRNA screening result.

We then asked if Atrx is important for the Trim28/Setdb1 pathway and performed SHIN silencing assays in wild-type versus Atrx ko cells, depleted for Setdb1 and Trim28, respectively (Supplementary Fig S2E and F). Although Setdb1 could be depleted by around 70% (Supplementary Fig S2E), SHIN silencing was only mildly impaired (Fig3E). Setdb1 depletion in Atrx ko cells, in contrast, resulted in strong silencing defects (Fig3E). This suggests that Atrx enhances the efficiency of Setdb1-dependent repression, although we cannot exclude the possibility that Atrx may act independently of Setdb1 in repressing the SHIN reporter, leading to synergistic effects when both Setdb1 and Atrx are depleted. Similar effects were observed for shRNA knockdown of Trim28 (Fig3E).

To test if Atrx is required for Trim28/Setdb1-dependent repression outside of IAP retrotransposons, we performed silencing assays using a heterologous reporter system. The well-established repressor binding site of MLV retrotransposons (RBS) is recognized by the KRAB zinc finger protein Zfp809, which in turn recruits Trim28 and Setdb1 to induce silencing in a variety of cell types. In wild-type cells, the RBS-EGFP reporter was efficiently silenced (Fig3F, Supplementary Fig S2G). However, RBS silencing was compromised upon knockdown of Atrx in MEFs and in Atrx ko ES cells (Fig3F, Supplementary Fig S2G). Taken together, our data demonstrate Atrx as a crucial component of the Setdb1/Trim28 silencing pathway.

SHIN silencing is independent of Daxx-mediated H3.3 deposition

On telomeres and pericentric heterochromatin, Atrx recruits Daxx to mediate H3.3 deposition ^28,29,30. In order to test if the Daxx/H3.3 pathway plays a role in SHIN repression, we generated Daxx ko cells (Supplementary Fig S3A). We found that SHIN silencing is defective in Daxx ko cells (Supplementary Fig S3B), suggesting that Daxx is an important component for retrotransposon silencing. To address the question whether H3.3 deposition by Daxx is critical for SHIN silencing, we generated rescue cell lines expressing wild-type and mutant Daxx proteins (Supplementary Fig S3C). Re-expression of full-length Daxx protein in Daxx ko cells leads to a rescue in SHIN silencing (Supplementary Fig S3D). However, the expression of Daxx-mutant proteins with relaxed binding specificity for histone H3.3 (E231A) or impaired H3.3 interaction (R257A) also resulted in rescued SHIN silencing (Supplementary Fig S3D and E). In contrast, the expression of Daxx with a deletion of the C-terminal repressor domain ⁴² does not rescue SHIN silencing (Supplementary Fig S3D). These data point toward a H3.3-independent function of Daxx in SHIN repression. To confirm that H3.3 is really dispensable for retrotransposon silencing, we generated H3.3 ko ES cell lines (Supplementary Fig S4A). Histone H3.3 is mainly transcribed from two loci in the mouse genome, H3f3a and H3f3b. Consequently, H3f3a/H3f3b double knockout cells have essentially lost H3.3 protein (Supplementary Fig S4B). Notably, SHIN silencing is completely intact in H3.3 ko cells (Supplementary Fig S4C). Due to H3f3a duplications in the mouse genome that could eventually give rise to functional H3.3 transcripts (see blat search and alignment report in Supplementary Table S3), we performed H3f3a knockdown in H3f3b ko cells. These cells have lost H3.3 (Supplementary Fig S4D) but display robust SHIN silencing (Supplementary Fig S4E). Finally, we re-analyzed published H3.3 ChIP-seq datasets in control and Atrx ko ES cells ³⁰. We found only low enrichment of H3.3 in control ES cells (Supplementary Fig S4F). Surprisingly, upon deletion of Atrx, H3.3 becomes strongly enriched at IAP elements (Supplementary Fig S4F), suggesting that Atrx does not promote H3.3 incorporation at endogenous IAP elements, but rather inhibits excessive H3.3 deposition. Alternatively, higher chromatin turnover at IAP elements in the absence of Atrx may indirectly lead to more H3.3 incorporation. Together with our finding that H3.3 interaction mutants of Daxx can repress retrotransposon sequences and H3.3-depleted cells are not impaired in SHIN repression, our data demonstrate that SHIN silencing is independent of Daxx-mediated histone H3.3 incorporation.

Atrx is required for efficient heterochromatin formation

Next, we sought to investigate how Atrx mediates SHIN silencing on the molecular level. One possibility was that Atrx could mediate efficient heterochromatin formation across the SHIN locus. To test this hypothesis, we performed a re-silencing assay in HA36::SHIN ES cells in which we depleted Atrx by shRNA-mediated knockdown (Fig4A). Strong induction of the Tet-inducible promoter using doxycycline alleviates SHIN silencing in a small population of cells, and constant selection pressure with zeocin allowed us to isolate a homogeneous population of cells with strong EGFP expression. Lack of promoter stimulation by doxycycline removal resulted in the rapid loss of EGFP expression within 4 days in both control (shScr) and Atrx-depleted (shAtrx) cells (Fig4B, left panel). Loss of EGFP expression coincided with presence of H3K9me3 across the RMCE locus in shScr cells (Fig4C, left panel, Supplementary Fig S5A). H3K9me3 could also be established in shAtrx cells; however, abundance of H3K9me3 appeared reduced in the test region 1 most distal to the SHIN sequence (Fig4C, left panel), indicating reduced heterochromatin spreading. Surprisingly, re-silencing was very efficient in shScr cells despite continuous doxycycline induction (Fig4B, right panel, Supplementary Fig S5A). A large proportion of these cells displayed strongly reduced EGFP expression and the establishment of H3K9me3 across the RMCE locus (Fig4B and C, right panels). In contrast, shAtrx cells are completely unable to induce heterochromatin formation across the RMCE locus in continuous presence of doxycycline (Fig4B and C, right panels). In summary, these data indicate that Atrx is necessary for efficient establishment of heterochromatin. On transcriptionally inactive loci, heterochromatin can be formed, but spreading appears reduced. In addition, Atrx is strictly required when heterochromatin formation is challenged by transcriptional activity.

Atrx is important for heterochromatin maintenance

Thus far, out data showed that Atrx is necessary for efficient de novo establishment of heterochromatin. However, in embryonic stem cells endogenous IAP elements are already covered by a heterochromatic structure which needs to be maintained. We thus wondered if Atrx is important for heterochromatin maintenance. When we analyzed published ChIP-seq datasets for Atrx, Setdb1, Trim28, and H3K9me3 in ES cells ^43,44,45, we found strong enrichment of Atrx across IAP retrotransposons with highest occupancy over the SHIN region (Fig5A). These data suggest that Atrx has a permanent role on these elements. ChIP-qPCR analyses for Atrx could confirm the enrichment on IAP elements and a non-repetitive control region (Fig5B, top panel). However, heterochromatin marks, such as H3K9me3 and DNA methylation, were not altered in Atrx ko cells (Fig5B, Supplementary Fig S5B and C).

The strong enrichment of Atrx, H3K9me3, Trim28, and Setdb1 on IAP sequences close to the SHIN region (Fig5A) suggests that Atrx is recruited by H3K9me3 and/or Trim28/Setdb1. Thus, we tested if Atrx is de novo recruited to a Setdb1-dependent heterochromatin locus by analyzing HA36::SHIN cells. Enrichment of Atrx at the SHIN region within the reporter locus demonstrates that the recruitment of Atrx occurs in the context of Setdb1 and Trim28 (Fig5C). Thus, we investigated whether Atrx generally associates with Setdb1/Trim28 target region in repetitive and non-repetitive regions of the genome. The overlap between Atrx, Trim28, and Setdb1 in non-repetitive regions is relatively small (Supplementary Fig S7A). Prominent enrichment was mainly observed on imprinted genes and few intragenic loci (Supplementary Fig S7B–D, Supplementary Tables S5 and S6). In contrast, our analyses revealed that Atrx co-occupies many ERV classes which are also enriched for Setdb1, Trim28, and H3K9me3, for example, IAP-Ez, MusD/ETn, and MMERVK10 elements (Fig5D, Supplementary Fig S6, Supplementary Table S4). Thus, we sought to investigate if some of these ERV sequences become de-repressed in Atrx ko cells. Out of the three tested ERV classes with the enrichment of Setdb1/Trim28/Atrx, we detected the de-repression of MusD/ETn retrotransposons (Fig5E), suggesting that Atrx function is rate limiting for the silencing of distinct ERV classes.

Challenges like DNA replication or transcription through intronic IAP repeats require heterochromatin to be constantly re-established. Based on our finding that Atrx is crucial for efficient establishment of heterochromatin, we wondered if heterochromatin maintenance is perturbed in Atrx ko cells when the Setdb1/Trim28 pathway has reduced activity. Moderate knockdown of Trim28 in wild-type cells resulted in slight up-regulation of endogenous IAP elements, demonstrating that heterochromatin maintenance is not fully ensured. However, when we performed Trim28 knockdown in Atrx ko cells, we observed severe derepression of IAP retrotransposons (Fig5F). These findings indicate that Atrx is required and rate limiting for the repression of endogenous IAP elements when heterochromatin maintenance is challenged.

Another way of challenging heterochromatin is to provoke transcription across a heterochromatinized locus. In HA36::SHIN cells, the EGFP reporter gene is not transcribed and covered with H3K9me3 (Fig2C). However, transcription can be induced by doxycycline that allows the recruitment of the reverse tetracycline transactivator (rtTA) to the Tet-responsive promoter of the reporter gene. Due to the heterochromatic state of the reporter gene in control cells (shScr + dox), doxycycline induction leads to a very low percentage of EGFP-positive cells (Fig5G, Supplementary Fig S5D). In contrast, doxycycline induction upon Atrx knockdown (shAtrx + dox) results in a much higher percentage of EGFP-expressing cells (Fig5G, Supplementary Fig S5D). These data demonstrate that heterochromatin is more vulnerable in Atrx ko cells.

Atrx regulates heterochromatin accessibility (VSports在线直播)

Heterochromatin is generally characterized by a high compaction grade which makes it largely inaccessible to transcriptional activators and refractory to challenges. Our finding that transcription of a heterochromatic locus can be more easily stimulated in the absence of Atrx suggests that structural properties of heterochromatin like local nucleosome density or higher order folding are compromised when Atrx is lost. To test this hypothesis, we performed MNase accessibility assays in wild-type versus Atrx ko ES cells. Atrx is only binding to a small fraction of the mouse genome, and thus, we did not detect global alterations in MNase accessibility ⁴³ (Fig6A and B). To investigate Atrx target regions, we employed a qPCR strategy to measure the MNase digestion degree on specific genomic loci with increasing MNase concentrations (Fig6C). Curve fitting through these data points allows statistical assessment of the digestion degree between wild-type and Atrx-mutant cells (Supplementary Fig S7E). Further, we derive a chromatin accessibility score which represents the digestion rate at which 50% of the target locus is digested (Supplementary Fig S7E). MNase accessibility at endogenous IAP retrotransposons is significantly increased, whereas control regions, such as promoters of active and inactive genes, show no alterations (Fig6D and E, Supplementary Fig S7F). Interestingly, chromatin accessibility was also increased on non-repetitive Setdb1/Trim28 targets (Fig6E, Supplementary Fig S7E). These data demonstrate that Atrx renders Setdb1-dependent heterochromatin more inaccessible.

Reporter gene silencing assay (V体育2025版)

Reporter constructs were stably integrated into cells by lentiviral transduction, and the percentage of EGFP⁺ cells was measured by FACS after 2–4 days. Lentiviral particles were generated using standard protocols, and virus titers were determined by titration in HeLa cells. Mouse ES cells were transduced on gelatinized multi-well dishes using spinoculation at a low multiplicity of infection to ensure a linear relationship between virus titer and transduction rate. The ratio of the percentage of EGFP⁺ cells generated by the reporter relative to the percentage of EGFP⁺ cells generated by a control EGFP vector of the same virus titer was used to quantify reporter silencing (relative % EGFP⁺ cells). In Fig1 and Supplementary Fig S1, the reciprocal ratio (fold repression) is plotted.

shRNA screen (V体育官网)

Feeder-independent ES cells were transduced with a genome-wide pooled shRNA library (Lentiplex, Sigma-Aldrich) at a low MOI, selected for shRNA expression using 0.5 μg/ml puromycin, and then transduced with the SHIN reporter. Cells that escaped SHIN silencing were isolated by FACS sorting based on their high EGFP fluorescence. shRNA sequences of the EGFP⁺ cells were amplified by PCR and cloned into the pLKO1 backbone. For validation of hits, 71 random shRNA sequences of the primary screen were separately transduced into ES cells and the SHIN reporter assay was performed as indicated above. The number of EGFP-positive cells was compared to a non-silencing scrambled control. Details are given in the Supplementary Materials and Methods.

Resilencing assay

T86 cells (HA36::SHIN cells harboring a reverse Tet transactivator) were treated with 2 μg/ml doxycycline for 2 days, and the re-expression of the silenced reporter locus was selected with 50 μg/ml zeocin and 2 μg/ml doxycycline for at least 2 weeks. Reactivated reporter cells were then transduced with a lentiviral shRNA targeting either Atrx or a scrambled control sequence, and knockdown cells were selected by the addition of 1 μg/ml puromycin 2 days after transduction. After additional 3 days, zeocin selection was released and resilencing of the EGFP reporter was monitored daily in the presence or absence of doxycyline by FACS. The median EGFP fluorescence was calculated using FlowJo™ (TreeStar). For ChIP experiments, cells were harvested 4 days after zeocin release. Details are given in the Supplementary Materials and Methods.