GATA‐1‐dependent histone H3K27 acetylation mediates erythroid cell‐specific chromatin interaction between CTCF sites

  1. Yea Woon Kim
  2. Yujin Kang
  3. Jin Kang
  4. AeRi Kim

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  1. Version published to 10.1096/fj.202001526r
  2. eLife

    ###Reviewer #3:

    Kim et al. studied the roles of GATA1 binding and H3K27ac on chromatin interactions associated with binding of the architectural factor CTCF. The authors focused on a TAD and sub-TAD surrounding the human beta-globin locus. The sub-TAD that is flanked by tissue-invariant CTCF sites that form contacts with each other only in erythroid cells. Loss of GATA1 binding at the beta-globin enhancer (LCR) and perturbation of H3K27ac affects some CTCF associated contacts, both involving the sub-TAD as well as the (tissue-invariant) TAD boundaries.

    The mechanisms by which tissue specific contacts are established are of great interest in the field. However, while this report provides some correlative data to support a link between H3K27ac and CTCF contacts, the study in its current form is too preliminary and failed to address alternative explanations.

    1. A major concern for this study: the MEL/chr11 cell line might not be a suitable model system. The CTCF/Rad21 binding profiles in the MEL/chr11 seem quite different from normal human erythroid cells. Specifically, the signals of CTCF binding at C4, C5 and C6 are almost at the baseline (Fig2A/B and Fig3D/E) in MEL/chr11. However, those sites should have comparable CTCF binding strength with C3 and C7, (e.g. Fig2E). The diminished CTCF binding at the C4-C6 sites might affect the sub-TAD structures in MEL/chr11 cells.

    2. Loss of GATA1 binding at HS2 or HS3 affects GATA1 binding at other sites as well. It is also possible that loss of GATA1 binding impairs binding by other nuclear factors that might be involved in sub-TAD formation. The same holds for histone modifications other than H3K27ac. This should be discussed.

    3. Fig.2: the correlation between H3K27ac and CTCF contacts does not hold at all examined sites. For example, H3K27ac is reduced also at C7 and C3 that maintain contacts. Same in Fig.3 where increases in H3K27ac is also increased near CTCF sites whose contacts don't increase.

    4. Fig.2: total H3 is reduced at C5 to the same extent as asH3. How was H3K27ac normalized?

    5. Why were p300 inhibition experiments done in K562 cells? In Fig2E and Fig4D it seems that the H3K27ac signals are not concordant between MEL/chr11 and K562 cells. For example, in Fig2E, the H3K27ac at C3 site is very strong in the MEL/chr11; However, in Fig4D, the H3k27ac signal at C3 site is near baseline. The H3K27ac signals also suggest that the MEL/chr11 cell line is different from normal human erythroid cells and also the leukemia cell line K562. The different H3K27ac profiles at CTCF binding sites confound the interpretation.

    6. Fig1E, the interactions between HS5-3'HS1 (C4-C5) were not significantly changed in the HS3/HS2 mutated MEL/chr11 cells. Actually this well known interaction between HS5-3'HS1 was not even detected by 3C in the WT control MEL/chr11 cells, contrary to previous studies. Again, this suggests that the MEL/chr11 cell line is not an ideal system for this study. The reduced interactions between beta-globin and C5/HS5 could be the result of loss of GATA1 binding but not CTCF. For clarity it would be better to plot the 3C data scale to the actual genomic distance.

    7. The authors should make clearer distinctions throughout whether they are considering supposedly tissue-invariant CTCF contacts near the TAD boundaries or the tissue-specific sub-TAD. It appears that both can change upon their perturbations.

    8. Data mining: "acetylation level was decreased remarkably in the boundaries of longer interactions (over than 100 Kb)" why is this remarkable? How does contact distance inform the role of H3K27ac? What is the correlation genome wide between H3K27ac and CTCF ChIA PET? This could be done using data sets from a variety of cell types.

    9. Fig.6A: GATA1 knock down seems to affect H3K27 broadly and not any more extensively (or even less) at CTCF sites.

  3. eLife

    ###Reviewer #2:

    The authors analyzed long and short range chromatin interactions in erythroid cell lines after perturbation of globin associated enhancers, histone acetylation, GATA1 expression, or CBP/p300 expression. The data show that CTCF binding sites are associated with high levels of H3K27 acetylation in the context of short range interactions. Deletion of GATA1 binding sites in the human LCR in hypersensitive sites 2 or 3 caused a reduction in H3K27ac at nearby CTCF sites and impaired the interaction of those sites, without interfering with the binding of CTCF or the cohesin subunit Rad21. Treatment of cells with TSA or inhibition of CBP/p300 or GATA1 expression had a similar effect.

    The manuscript provides interesting and novel observations with respect to GATA1 binding and H3K27ac at nearby CTCF sites. However, the mechanism(s) by which GATA1 modulates H3K27ac levels at CTCF sites remain(s) unknown.

    Specific comments.

    1. One possible interpretation of the results, which has not been addressed by the authors, is that the reduction in histone acetylation levels may modulate the stiffness of chromatin, which may particularly affect short-range interactions.

    2. Figure 3: TSA treatment also affects other histone acetylation events. This should be mentioned. Furthermore, the data in Figure 3D are not consistent with data in Figure 1E with respect to the control cells. In Figure 1E it is shown that the anchor C5 interacts with beta and C6, Figure 3C shows no interactions between these elements.

    3. Figure 4: There is no description of how the CBP/p300 depleted cells were generated. Was this a single shRNA? If so, it would be important to repeat the experiment using a second shRNA targeting a different region of the RNA to avoid off-target effects. Furthermore, it would be important to show the CBP/p300 binding pattern across the globin locus and the CTCF sites. Does H3K27ac at the CTCF sites correlate with CBP/p300 binding? These data should be available from K562 cells.

    4. Figure 6: there is no description of how the GATA1 depleted cells were generated. Again, was this a single shRNA?

  4. eLife

    ###Reviewer #1:

    In this study, Kim et al described the GATA1-dependent histone H3K27ac on a subset of CTCF binding sites and CTCF-mediated chromatin interactions in erythroid cells. The authors generated a GATA1 binding site deletion in MEL cells and observed impaired CTCF-mediated interactions around the beta-globin gene cluster. They further modulated H3K27ac by TSA treatment of CBP/p300 knockdown, and noted that altered H3K27ac affected interactions between a subset of CTCF sites. The authors further performed global correlation analysis between public ChIA-PET and H3K27ac in K562 cells with or without GATA1 depletion, and presented evidence supporting a modest effect of H3K27ac at some CTCF sites upon depletion of GATA1 in K562 cells. Based on these findings, the authors concluded that GATA1-dependent H3K27ac mediates erythroid-specific chromatin interactions between CTCF sites.

    Overall, this manuscript provides molecular details of the role of GATA1-mediated transcriptional programs in regulating chromatin interactions between CTCF sites. The experiments related to GATA1 binding site mutations were well designed and executed. The authors also make use of existing public datasets to extend the analysis to global scales. However, the current study falls short in providing strong evidence to support several conclusions due to a combination of insufficient data and suboptimal data quality. The overall conclusions that GATA1 regulates chromatin interactions through H3K27ac-mediated effects do not significantly extend our current understanding based on previous studies in the authors' group (Kang et al., 2017 BBA 1860:416) and others (Hsu et al., 2017 Mol Cell 66:102). The lack of replicate experiments for several ChIP-seq studies limited the robustness of relevant conclusions.

    In summary, although the current study provides additional insights into the role of GATA1-dependent transcriptional programs in regulating CTCF-mediated chromatin interactions, the overall findings were not developed in sufficient depth in light of previous studies and some conclusions were not sufficiently supported by the evidence.

    Major points:

    1. The authors previously showed that interactions between CTCF sites associated with sub-TADs are dependent on the binding of GATA1 to LCR enhancers (Kang et al., 2017 BBA 1860:416). In this study, the authors provided further details of the relationship between GATA1 binding, H3K27ac, and CTCF-mediated interactions. These data provide additional insights into the molecular mechanisms of GATA1-mediated effect on chromatin interactions, although they do not add significantly to previous studies from these authors and others (e.g. Hsu et al., 2017 Mol Cell 66:102).

    2. To determine the effect of GATA1 binding site KO on CTCF-mediated interactions, the authors performed 3C-qPCR analysis (Fig. 1E). Given the higher resolution and ability to quantitatively contextualize the data obtained from next-gen sequencing based 3C methods, I recommend these be done in the future. Similar for the studies in Figs. 3B-E and 4E.

    3. Page 5, line 115-117. It is stated that 'when it is compared to the results from knocking down GATA-1...'; however, no results from GATA-1 knockdown were included for the comparison. These studies would be helpful to determine whether the effect is directly caused by loss of GATA1 binding at the LCR enhancers.

    4. It is unclear whether GATA1 site KO also affected H3K27ac signals at other CTCF-bound chromatin regions besides the b-globin gene cluster (C3 to C7). For example, in Fig. 2E, it would be helpful to include C2 and C8 regions in the ChIP-seq track as controls. Additional analyses of global changes of H3K27ac and CTCF binding also will be helpful to determine whether the effect is limited to the b-globin gene cluster.

    5. In Fig. 3, to determine the effect of H3K27ac on chromatin interactions, the authors treated the cells with the HDAC inhibitor TSA. A major caveat of these studies is related to the pleiotropic effects of TSA on global H3K27ac and gene expression, thus the indirect effects on chromatin interactions due to altered gene regulation and/or erythroid maturation. This limitation should be discussed.

    6. In Fig. 5, it would be important to include analyses by separating CTCF binding sites depending on whether or not they are associated with TAD boundaries. Is there correlation between the range of CTCF-mediated interactions and TAD boundary? These studies will provide a global assessment of the findings based on the beta-globin gene cluster in Figs. 1-4.

    7. The effects of GATA1 knockdown on H3K27ac signals at CTCF binding sites at the global or selected regions are marginal (Fig. 6A, C,D, noting the scales of y-axis not started with 0 in Fig. 6C,D). It is unclear whether this is due to suboptimal data quality (see below) or limited effects at a global scale. These results raise questions about the extent to which the GATA1-mediated effect on H3K27ac at CTCF sites and CTCF-mediated interactions in erythroid cells.

    8. There are concerns about the quality of several genomic datasets including H3K27ac ChIP-seq that may limit the robustness of relevant analyses. Specifically, the quality of H3K27ac ChIP-seq in K562 cells is suboptimal and no apparent H3K27ac enrichment is noted at the CTCF-binding sites (C3, C4, C6 and C7). Comparing Fig. 2E with Figs. 4D and 6A, the signal-to-noise ratio is highly variable in different experiments. No replicate experiment or quality control analysis was provided.

  5. eLife

    ##Preprint Review

    This preprint was reviewed using eLife’s Preprint Review service, which provides public peer reviews of manuscripts posted on bioRxiv for the benefit of the authors, readers, potential readers, and others interested in our assessment of the work. This review applies only to version 1 of the manuscript.

    ###Summary

    This study investigates the relationship between the binding of the transcription factor GATA-1 and histone acetylation on CTCF sites in the same genomic region. Although the reviewers find this work interesting, they raise concerns about the strength of the novel conclusions that can be drawn at this stage. Specifically, the reviewers are concerned about the strength of the data supporting a specific role of GATA1 binding in regulating CTCF-associated sub-TAD interactions (Reviewer #1 and #3), the lack of technical details on several key experiments (Reviewer #1, #2 and #3), the lack of consideration of alternative interpretations on GATA1- and/or H3K27ac-mediated chromatin regulation (Reviewer #2 and #3), and the use of MEL/chr11 cell line as the experimental model system (Reviewer #1 and #3).

  6. Version published to 10.1101/2020.05.14.095547v1 on bioRxiv