The long non-coding RNA LINC00941 modulates MTA2/NuRD occupancy to suppress premature human epidermal differentiation

  1. Eva Morgenstern
  2. Uwe Schwartz
  3. Johannes Graf
  4. Astrid Bruckmann
  5. Markus Kretz

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Abstract

Numerous long non-coding RNAs (lncRNAs) were shown to have functional impact on cellular processes such as human epidermal homeostasis. However, the mechanism of action for many lncRNAs remains unclear to date. Here, we report that lncRNA LINC00941 regulates keratinocyte differentiation on an epigenetic level through association with the NuRD complex, one of the major chromatin remodelers in cells. We find that LINC00941 interacts with NuRD-associated MTA2 in human primary keratinocytes. LINC00941 perturbation changes MTA2/NuRD occupancy at bivalent chromatin domains in close proximity to transcriptional regulator genes, including the EGR3 gene coding for a transcription factor regulating epidermal differentiation. Notably, LINC00941 depletion resulted in reduced NuRD occupancy at the EGR3 gene locus, increased EGR3 expression in human primary keratinocytes, as well as increased abundance of EGR3-regulated epidermal differentiation genes in cells and human organotypic epidermal tissue. Our results therefore indicate a role for LINC00941/NuRD in repressing EGR3 expression in non-differentiated keratinocytes, consequentially preventing premature differentiation of human epidermal tissue.

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  1. Review Commons

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    Referee #3

    Evidence, reproducibility and clarity

    In the manuscript entitled "The long non-coding RNA LINC00941 modulates MTA2/NuRD occupancy to suppress premature human epidermal differentiation", Morgenstern and colleagues describe a novel mechanism by which Nur complex interacting with a ncRNA LINC00947 represses expression of a late differentiation transcription factor EGR3 in the skin. These findings are novel and will be of interest in the field. Of note, the authors use a plethora of biochemical and cell biology techniques such as chromatin occupancy, transcriptomics and organotypic skin culture. Both the Introduction and Discussion outline a necessary background in the field of ncRNAs and skin biology as well as clearly state and discuss the scientific problem. The Methods are comprehensive and accurate. Importantly, any possible ethical issues are discussed. The Results are clear and support the data shown. Also the Figures are well organised and easy to follow. Overall, the manuscript is well prepared and will be of interest for broad readership. Before publication, however, the authors should address some points to further enhance their work:

    Major points:

    1. It is peculiar that there is no evident K10 and FLG staining in the organotypic skin from siControl cells in Fig 2E but it is present on the Fig 5C. The authors should explain these inconsistences. Ideally the authors should provide a western blot analysis of these proteins which would help to give a more quantitative picture of the phenomenon.
    2. There iare no evidences at protein level of an efficiency of MTA2 and EGR3 knock-down. The authors use an MTA2 for western blot, so it should not be difficult to confirm. For EGR3, it is essential, as EFR3 is expected to increase only during late differentiation, while experiments from Fig 5B are performed only at 3 days of differentiation. Is it enough to induce EGR3 expression? Is the knock-down efficient at protein level at that time point? This is important as the work by Kim et al 2019 shows a detectable expression of EGR3 only after 7 days of differentiation.
    3. The authors should demonstrate an increase of EGR3 at protein level after LINC00947 knock-down (by western blot in vitro and/or in epidermal organotypic tissue).
    4. A key experiment to confirm the proposed model should be a double knock-down of both LINC00941 and EGF3. Will it rescue the observed increase of pro-differentiation genes?

    Minor points:

    1. 4E - EGR3 or LINC00941 knock-down?
    2. 4E - The RNA seq label states "RNA seq (siLINC00941 d3)" but apparently shows both scr and siLINC00941
    3. Please add the recipe of the lysis buffer used for western blot analysis of keratinocytes lysates.
    4. Page 10 - Fig 4D description - is it "chromatin conformation state" or just "chromatin state"?

    Significance

    These findings are novel and will be of interest in the field.

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    Referee #2

    Evidence, reproducibility and clarity

    In this manuscript, the authors investigate the role of LINC00941 epidermal differentiation. Specifically the authors show interaction with MTA2 and other NuRD subunits. Next, the authors show that LINC00941 and MTA2 restricts premature keratinocyte differentiation, where KD of either results in increased differentiation marker expression. To understand molecular impacts, the authors perform ChIPseq of MTA2 in control and LINC00941 depletion. Curiously, MTA2 binds in a trend differing from other cell types with predominant binding over active promoters. Upon LINC00941 KD, MTA2 binding is changed at 33 locations, where the majority show reduced binding. Overlapping binding changes with gene expression changes, the authors identify EGR3 as the only direct candidate upregulated upon LINC00941 KD and upregulated during differentiation. KD of EGR3 results in opposite trends of LINC00941 KD, suggesting the proposed mechanism of LINC00941 repressing EGR3 until appropriate time in differentiation. I have the following suggestions for this work:

    1. While data support MTA2 acting in NuRD, beyond Fig 1, the authors exclusively use MTA2 as a proxy for NuRD. Of course there are some subunits that are within other complexes and should not be used, others are options. While I do not expect the authors to perform all experiments on an additional subunit of NuRD, I do think there are a few things the authors should consider:
      • a. Be more precise with language to point out only MTA2 rather than say NuRD complex throughout many aspects of the paper, and only assume the complex in limited settings and when it is clear it is speculative
      • b. Perform a subset of experiments on another subunit. For example, the Mass Spec in Fig 1A/B shows an interaction with other subunits, but the verification was only done for MTA2 (Fig 1C/D). This could easily be blotted (or another Western performed) and/or primers for other subunits for the qPCR for a couple additional subunits. Similarly straightforward, looking at MTA2 RNA expression changes during differentiation (Fig 2A): if additional primers were used to other subunits, these additional subunits could be used to verify.
    2. Related to the above comment, does MTA2 KD (or LINC00941 KD for that matter) result in loss of NuRD complex formation? If so, this would be sufficient to address point 1.
    3. Finally, in relation to NuRD complex here, it is important to note that mutually exclusive NuRD complexes (MBD2/NuRD and MBD3/NuRD) have been documented. Because the Mass Spec did not show interaction to MBD2 or MBD3, it is not clear if this is limited to one of these complexes. Related to this, the authors show by Mass spec that LINC00941 interacts with CHD4, but not CHD3. Is this because Chd3 is not expressed in these cells, or because there is some mutual exclusivity to CHD4 and LINC00941 is acting through this subcomplex?
    4. Immunofluorescence images showing increased Keratin 10 and Filaggrin in LINC00941 or MTA2 KD (Fig 2E) and decreased Keratin 10 and Filaggrin in EGR3 KD (Fig 5C) are curious as the control look very different. In 2E, the control shows barely detectable levels, whereas in 5C the levels look similar to what is seen in Fig 2E KDs. Is this variability? If so, more representative images as well as quantification to the changes are necessary to make these two points.
    5. In Figure 4, the authors present ChIPseq data for MTA2 in LINC00941 KD. One interesting trend is that the KD alters binding of MTA2 at mostly bivalent/repressed locations, rather than at active locations which is the majority of MTA2 binding (from Fig 3). It would be nice to show then these data rather than only stating it. The authors include a browser track for 2 genes (Fig 4D and S4C), but for the other 31 locations, a heatmap or something to show the level of K27me3 vs K27ac/K4me3 would be helpful to support this claim. Notably saying "Most of the differential MTA2/NuRD occupied sites were marked by repressive histone modification H3K27me3..." is the point that doesn't seem to be shown, and also a precise number should be included. a. Related to this, I believe the authors performed K27me3 ChIPseq in the KD, and if so, it would be nice to see more genome wide effects here.
    6. This is perhaps beyond the scope of the paper, but the obvious question to me is if EGR3 is relocalized in LINC00941 KD. Specifically, we would anticipate that EGR3 localization in the KD would mimic that of a more differentiated cell (binding to differentiated genes). A quick ChIPqPCR experiment for a few locations would be sufficient to support this model.

    Minor points:

    1. Importantly, CHD5 can also be incorporated in NuRD, in place of CHD3 or CHD4.
    2. The authors use heatmaps and metaplots in Fig S3 to show reproducibility of the ChIPseq datasets. Importantly, the PCA does show some variation. XY scatterplots for replicates vs one another would be a more robust QC.
    3. In figure 4D, the authors present nice data showing changes in histone mods during differentiation, but it is very hard to see the color changes and the tracks as presented. (same point for Fig S4C)
    4. it is unclear from the methods or the figure legend if RTqPCR data are biological or technical replicates.

    Significance

    In this manuscript, the authors present a molecular function for LINC00941 in epidermal differentiation, where it interacts directly with NuRD subunit MTA2. LINC00941 has been previously described but this activity was not described. LINC00941 seems to specifically help target or maintain MTA2 localization to EGR3 to promote repression of this gene. Then, the model suggests that during differentiation, LINC00941 and MTA2 levels decrease, permitting activation of EGR3 during epidermal differentiation and subsequent activation of appropriate genes. These findings will be of interest to individuals interested in NuRD function, lncRNA activity and/or epidermal cell fate.

    My expertise is in chromatin biology, chromatin remodelers, epigenomics, and cell identity.

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    Referee #1

    Evidence, reproducibility and clarity

    In this manuscript, Morgenstern et al investigated the molecular mechanism by which LINC00941 regulates keratinocyte differentiation. They found the LINC00941 interacts with the NuRD chromatin remodeling complex in human primary keratinocytes. Furthermore, LINC00941 silencing by RNAi results in changes in the genomic occupancy of MTA2, a core NuRD subunit, especially near a number of bivalent genes. In particular, they showed that LINC00941 depletion resulted in reduced MTA2 occupancy at the EGR3 gene, increased EGR3 expression, and increased expression of EGR3-regulated epidermal differentiation genes. Together, they propose that LINC00941 prevents premature differentiation of human epidermal tissue by repressing EGR3 expression in non-differentiated keratinocytes via NuRD. The interaction between LINC00941 and NuRD is a novel finding and will likely provide new insights for the function of LINC00941, which has been implicated in keratinocytes, tissue homeostasis and cancer. It will also shed light on the role of lncRNAs in epigenetic gene regulation and cell fate transition in general. The conclusion of this study can be much strengthened if the authors can identify LINC00941-occupied genomic regions by ChIRP (PMID: 21963238) or RAP (PMID: 23828888). In addition, the authors are also encouraged to address the following questions and comments to further improve the manuscript.

    Fig 1C: Since multiple NuRD subunits were identified in the LINC00941 pull-down (Fig 1B), can the authors validate at least one other subunit? CHD4 is also a NuRD-specific subunit and appears to be a strong hit based on supplemental Fig 1.

    Fig 1D: Can the authors also try RNA-IP on MTA2 and endogenous LINC00941?

    Fig 2B: It seems that MTA2 protein level still remains reasonably high at day-4.

    Fig 3C: How many bivalent promoters are there in keratinocytes? How many of those are bound by MTA2?

    Fig S3A: Can the authors examine MTA2 occupancy at TSS and bivalent TSS in control vs. siLINC00941 cells (by meta-gene analysis)? This will show whether LINC00941 KD affects MTA2 occupancy at bivalent TSS in general.

    Fig 4B: Does LINC00941 KD only affect 33 out of the 3613 MTA2 peaks? If yes, can the authors comment on why only such a small fraction of MTA2 occupied regions are affected?

    Fig 4C: The authors only examined a small number of MTA2-associated genes. To provide a more complete view of the potential involvement of LINC00941-regulated genes in keratinocytes differentiation, can the authors provide the total number of differentially expressed genes (DEGs) in LINC00941 KD, the total number of DEGs during keratinocytes differentiation, and the overlap between the two (maybe using a venn diagram)? In addition, among all the overlapping DEGs from above, how many of them have MTA2 peaks nearby? Finally, in the overlapping DEGs occupied by MTA2, can the authors compare MTA2 occupancy at up- vs. down-regulated DEGs caused by LINC00941 KD, to see whether reduced MTA2 occupancy associates with increased expression after LINC00941 KD?

    Fig 4D: Can the authors add the H3K4me3 track to the figure? Can the authors provide ChIP-qPCR result to validate the changes in MTA2 occupancy near EGR3 after LINC00941 KD?

    Fig 5A: Some of the EGR3 target genes (eg., GJB4, SERTAD1) appear to be expressed before EGR3 up-regulation in siCtrl, and some of them (eg. HMOX1, ESYT3, SMPD3) appear to show stronger up-regulation than EGR3 in siLINC00941. This is not entirely consistent with the idea that they are regulated by LINC00941 via EGR3.

    Significance

    The interaction between LINC00941 and NuRD is a novel finding and will likely provide new insights for the function of LINC00941, which has been implicated in keratinocytes, tissue homeostasis and cancer. It will also shed light on the role of lncRNAs in epigenetic gene regulation and cell fate transition in general. The conclusion of this study can be much strengthened if the authors can identify LINC00941-occupied genomic regions by ChIRP (PMID: 21963238) or RAP (PMID: 23828888). In addition, the authors are also encouraged to address the above-mentioned questions and comments to further improve the manuscript.

  5. Version published to 10.1101/2023.07.25.549662v1 on bioRxiv