ChAHP2 and ChAHP control diverse retrotransposons by complementary activities

  1. Josip Ahel
  2. Aparna Pandey
  3. Michaela Schwaiger
  4. Fabio Mohn
  5. Anja Basters
  6. Georg Kempf
  7. Aude Andriollo
  8. Lucas Kaaij
  9. Daniel Hess
  10. Marc Bühler

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Abstract

Retrotransposon control in mammals is an intricate process that is effectuated by a broad network of chromatin regulatory pathways. We previously discovered ChAHP, a protein complex with repressive activity against SINE retrotransposons, composed of the transcription factor ADNP, chromatin remodeler CHD4, and HP1 proteins. Here we identify ChAHP2, a protein complex homologous to ChAHP, wherein ADNP is replaced by ADNP2. ChAHP2 is predominantly targeted to ERVs and LINEs, via HP1β-mediated binding of H3K9 trimethylated histones. We further demonstrate that ChAHP also binds these elements in a mechanistically equivalent manner to ChAHP2, and distinct from DNA sequence-specific recruitment at SINEs. Genetic ablation of ADNP2 alleviates ERV and LINE1 repression, which is synthetically exacerbated by additional depletion of ADNP. Together, our results reveal that the ChAHP and ChAHP2 complexes function to control both non-autonomous and autonomous retrotransposons by complementary activities, further adding to the complexity of mammalian transposon control.

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    Referee #3

    Evidence, reproducibility and clarity

    Here, Ahel et al characterize complex composiiton, chromatin binding, and respressive function of ADNP2. First they show that ADNP2 forms a complex with CHD4 and HP1b, very similar to, although biochemically distinct from, the ChAHP complex formed by ADNP. This complex is prevalently bound at repeats, and in particular at LTR retrotranspoons. Recruitment of ADNP2 is mostly mediated by HP1b binding to H3K9 and a PxVxL mutant that cannot bind to HP1 does not localize properly to chromatin. While ADNP has its own specific targets in SINE elements, it also binds some ADNP2 targets in an HP1-dependent manner. Depletion of ADNP or ADNP2 results in upregulation of some shared and some distinct transposons, indicating distinct roles but also partial redundancy.

    This is a solid study that adds to our knowledge of these repressive complexes and their transposable element targets. I feel that the data could be analyzed a bit more in depth, especially in the comparisons of ChIP-seq vs. RNA-seq.

    Major points

    • Fig. 5: it would be interesting to analyze differences between repeats that are only under ADNP2 control vs. those that are sensitive to additional loss of ADNP. Presumably they both have K9me3 so why some are also repressed by ADNP?
    • Fig. S5B, S7A: degrons are known to destabilize some proteins even before induction of degradation. These western blots should include a WT line to compare abundance of the endogenous protein with the tagged version.
    • FIg. 5: it would be good to show a bit more overlap between RNA-seq and ChIP-seq. How many of the bound transposons are derepressed? Can co-occupancy by ADNP and ADNP2 explain which transposons will display synergistic effets from removal of both proteins?

    Minor points

    • Fig. 2B: Upset plot seems like a strange choice for visualization. I would show as stacked bar plot compared to genome distribution. I.e. are peaks enriched on repeats or is it just that a larger genomic space is occupied by repeats compared to TSSs?
    • Fig. 3A: if ADNP2 only binds to a subset of K9me+ retrotransposons, why arent' there regions of K9me+ ADNP2- in this plot?
    • Check figure calls for 3B and 3C, some of them seem off.
    • Fig. S7B: the text says several dozen genes but I only see 21 up and 15 down upon ADNP with ADNP2 present.

    Significance

    This is a solid study that adds to our knowledge of these repressive complexes and their transposable element targets. I feel that the data could be analyzed a bit more in depth, especially in the comparisons of ChIP-seq vs. RNA-seq.

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    Referee #2

    Evidence, reproducibility and clarity

    In this work, Ahel, Pandey and Schwaiger et al. examine the function of ChAHP complexes in transposon silencing in mouse embryonic stem cells. This works builds on a proteomics approach previously developed in the Buhler lab, which identified ADNP1-ChAHP as a repressor complex of SINE retrotransposons. In their previous work they also identified the zinc-finger protein ADNP2 as an interactor of the ChAHP component CHD4. Here, the authors test the hypothesis of whether ADNP2 is part of an alternative ChAHP, which they termed ChAHP2. They first establish, through proteomics/biochemistry experiments, that the ChAHP2 complex can form independently of ChAHP. By ChIP-sequencing they then show that the ChAHP2 member ADNP2 predominantly occupies retrotransposons of the LTR families (e.g., ERV elements). They further show that recognition of target transposable elements (TEs) is conferred by the ChAHP2 complex member HP1 and its affinity for H3K9me3. Using RNA-seq they then addressed the repressive activities of ChAHP and ChAHP2 on TEs through degradation of ADNP, ADNP2 or both. The experimental approach is very well thought out and experiments are done with extensive controls.

    I have the following comments/suggestions:

    • Regarding the ChIP-seq experiments, it is not entirely clear from the manuscript text and also not from the Material and Methods whether ChIP-seq was done with a tagged-version of ADNP2, which is what I assume, or an endogenous ADNP2 antibody. Could you please state this clearly?
    • The authors map short read sequencing data to individual TE insertions and conclude that ADNP2 binds retrotransposons both internally and at their terminal sequences. Particularly for the internal parts, how sure can the authors be that their approach, mapping to repeat consensus sequences, is not confounded by e.g., genetic differences between their ESC lines and the references they map to, or simply by the limitations of short read data?
    • Page 7, the first sentence starting with "Contrary to the expected behavior of a TF, ...". Could the authors please elaborate on what they mean with "the expected behavior of a TF"?
    • In Figure S4B, could the authors please clarify if HP1, CHD4 and Tubulin are high/low exposure?
    • SETDB1 is the H3K9me3 methyltransferase responsible for LTR/ERV silencing in mESCs and its disruption leads to a pronounced upregulation of TEs (Karimi et al., 2011). Could the authors comment on the effect of their degron-mediated SETDB1 depletion on de-repression of LTR/ERV elements? At least for the elements shown to lose H3K9me3 upon SETDB1 depletion (Figure S5D - strong depletion replicate)? Could you please also provide information about morphological/pluripotency characteristics of the 2HA-FKBPSETDB1 ESC lines before and after the 48h treatment with 500nM dTAG13?
    • Maybe I missed this, but are H3K9me3 levels affected in the ADNP and/or ADNP2 degron lines where transcriptional dysregulation of TEs is observed (Figure 5)? If not H3K9me3, is TE de-repression accompanied by reduction in repressive histone marks in ADNP2 degraded lines?
    • When looking at TE de-repression, the upregulation seems very modest (Figure 5) and Log2FoldChanges >0.9 (FDR<0.05) were considered as statistically significant. Is this the result of using mESCs that "approximate a constitutive KO situation through inducible ADNP2 degradation" and 14 days of treatment with 250nM dTAG13? As mention above, could you please also provide information about morphological/pluripotency characteristics?

    Significance

    The findings are interesting and expand on our understanding of how complexes of chromatin bound proteins control epigenetic silencing of individual retrotransposon classes/families in mouse embryonic stem cells. More specifically, this study adds a new player, ChAHP2, a ChAHP alternative that associates with ERV and LINE1 retrotransposons via HP1-mediated binding of H3K9me3 and is required for transcriptional repression of LTR class retrotransposons. Although, in my opinion, it remains somewhat uncertain how depletion of ADNP2 results in TE upregulation.

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    Referee #1

    Evidence, reproducibility and clarity

    The Buhler group previously identified the ChAHP complex and showed it to be made up of the transcription factor ADNP, chromatin remodeler CHD4, and HP1 proteins. They found that genetic removal of ADNP leads to increased expression of SINE B2 elements in mouse ES cells (mESCs), and an increase in chromatin accessibility and CTCF binding.

    In the current manuscript they describe a related protein complex they name ChAHP2. ChAHP2 has a vertebrate paralogue of ADNP, a transcription factor they call ADNP2 and the same chromatin remodeler CHD4, and HP1 proteins. By investigating chromatin occupancy they find that ChAHP2 chromatin binding specificity is distinct from ChAHP, and predominantly associates with ERV and LINE1 retrotransposons via HP1beta-mediated binding of H3K9 trimethylated histones. The ChAHP and ChAHP2 complexes control a wide variety of molecularly disparate retrotransposons, including SINEs, LINEs, and ERVs.

    The findings are of significance as the ChAHP2 complex is novel - it has not previously been identified or characterized and its description is therefore worthy of publication. Overall this study was well-performed and nicely-presented. In general, the text is clear, the flow of experiments seemed logical, easy to follow and supported by the presented evidence without the findings being overstated.

    The data is mainly descriptive and it was at times hard to identify what were the clear conclusions from their study. Having identified the new ChAHP2 complex they show that its chromatin binding characteristics are distinct from ChAHP, which predominantly binds SINE elements. In contrast, ChAHP2 binds different classes of retrotransposons and recruitment to H3K9me3 heterochromatin is dependent on HP1-Beta. This was all relatively clear. It became a little more confusing trying to unravel the functional consequences of the loss of ChAHP2 - either alone, or in combination with ChAHP.

    Although they see changes - both up- and down-regulation of a large number of genes, they say that: 'None of the changing gene categories showed strong and significant patterns either in terms of GO term enrichment (FigureS7C), distance between the promoter and the nearest ADNP/ADNP2 peaks'- does this mean that none of the upregulated genes following ADNP1/2 deletion show ADNP/ADNP2 peaks over the specific gene in question - i.e. do they think that the majority of effects here are indirect?

    When it came to the 'repeat' families of genes - the situation was also not entirely clear.

    They say: All these differentially expressed repeats belonged to the LTR class of retrotransposons, including families identified as ADNP2-bound in ChIP sequencing - it would be helpful to know which familes are bound and maybe show snapshots of ADNP2 binding. Are these differentially expressed repeats directly regulated by ADNP2? - they seem like a fairly heterogenous group of repeat elements? - do they have any shared features? They say that expression of two LINE1 subclasses were significantly upregulated - do these L1s (L1Mdas) have common features - are they old or young - it would be helpful if these different issues could be clarified.

    In the discussion they are upfront about their inability to identify any specific sequence motifs required for ADNP2 occupancy. Since the repression is not obviously H3K9Me3 dependent, then what is the role of H3K9me3 in repressing here? They surmise that repression is likely to be regulated through chromatin remodeling by CHD4 - would ATAC-seq help clarify this suggestion? - particularly as they suggest that having CHD4 as an integral component of the repressor complex is a unique feature of ChAHP1/2?

    In this context, do they not consider MORC2 to be an essential component of HUSH-dependent silencing?

    Other points

    The language in the introduction somewhat confusing and could be improved: They jumble ZNFs/TRIM28/HUSH/HP1 as if there is no specificity here - there clearly is - needs a more balanced and informative description.

    What is firstly referring to? Firstly, histone deacetylation and the removal of activating histone methylations disfavor transcription13,14. Secondly, the underlying DNA is extensively methylated, further repressing the locus

    They state: Like ERVs, LINE1 retrotransposons are autonomous- would help if they could clarify exactly what is meant here.

    Significance

    The findings are of significance as the ChAHP2 complex is novel - it has not previously been identified or characterized and its description is therefore worthy of publication. Overall this study was well-performed and nicely-presented. In general, the text is clear, the flow of experiments seemed logical, easy to follow and supported by the presented evidence without the findings being overstated.

    The findings will be of broad interest to chromatin biologists - particularly those interested in the regulation of retroelements and repeat regions

  5. Version published to 10.1101/2024.02.05.578923v1 on bioRxiv