Enhancer-associated H3K4 methylation safeguards in vitro germline competence

Tore Bleckwehl, Giuliano Crispatzu, Kaitlin Schaaf, Patricia Respuela, Michaela Bartusel, Laura Benson, Stephen J. Clark, Kristel M. Dorighi, Antonio Barral, Magdalena Laugsch, Wilfred F. J. van IJcken, Miguel Manzanares, Joanna Wysocka, Wolf Reik, Álvaro Rada-Iglesias

Posted on: 13 May 2021 , updated on: 17 May 2021

Preprint posted on 20 January 2021

Article now published in Nature Communications at http://dx.doi.org/10.1038/s41467-021-26065-6

H3K4me1 keeps the enhancer doors open during mammalian development for reactivation?

Selected by Sree Rama Chaitanya

Categories: cell biology, developmental biology, genomics, molecular biology

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Enhancers are crucial cis-regulating elements that synchronize spatiotemporal gene expression during development by relaying cellular signals. Enhancers are epigenetically coded by H3K4me1 (histone 3 lysine 4 mono-methylation) and H3K27ac (histone 3 lysine 27 acetylation) which are often associated with binding of transcription factors (TF), RNA polymerase II activity (RNAPII), and active gene expression.

However, like any other life process, enhancer regulation is not black and white. For example, the lack of H3K4me1 at enhancers does not seem to impact RNAPII occupancy and gene expression, or H3K27ac levels. Hence, H3K4me1 and H3K27ac may be dispensable for enhancer function and gene expression, albeit useful as epigenetic markers to identify enhancers. Also, some decommissioned enhancers (scored by suboptimal levels of H3K27ac, TF, and RNAPII) in differentiated macrophages still retain H3K4me1, possibly primed for reactivation at the onset of specific signals. Hence, enhancer decommissioning may be partial during development. Building on this, the authors of the current preprint investigated the role of H3K4me1 and enhancer-mediated regulation of gene expression during mammalian development.

Key findings

In this work, the authors used a well-established in vitro primordial germ cell (PGC) differentiation system to unravel the role of H3K4me1 and enhancers during development. Using defined culture conditions, they differentiated embryonic stem cells (ESCs, naïve) to germline competent epiblast like-cells (EpiLC), PGC like-cells (PGCLC), and germline incompetent epiblast stem cells (EpiSC) (Fig 1a). While EpiLC can further differentiate to PGCLC in embryonic bodies (EB), EpiSC cannot. Measuring gene expression (using single-cell and cell pool RNA-seq), they defined stage-specific gene expression profiles corroborating with previously published datasets. They found that ESCs and PGCLCs manifested similar gene expression profiles, which were silent in EpiLC and EpiSC (Fig 1b). Thus, in multiple stages of PGCLC differentiation, genes that are silenced in EpiLC could be reactivated in PGCLC.
Figure 1: (a) Schematic representation of the in vitro PGCLC differentiation system and in vivo location of PGCs at the proximal-posterior end of a mouse embryo. (b) single-cell RNA-seq data collected at different stages of in vitro PGCLC differentiation. Heatmaps showing the genome-wide distribution of H3K27ac (c), H3K4me1 (d), and mCpG (e) at different stages of in vitro PGCLC differentiation.
By evaluating H3K27ac peaks, they identified that PGCLC-enhancers are also active in ESCs, but not in EpiLC and EpiSC (Fig 1c). They also experimentally validated the impact of some of the PGCLC enhancers on gene expression in both PGCLCs and ESCs by deleting a handful of PGCLC enhancers using CRISPR/Cas9 technology. Hence, they suggest that some PGCLC enhancers could be relevant for PGCLC induction. Also, a subset of PGCLC enhancers seems to be active in ESCs (i.e., H3K27ac, TF binding) and slowly become partially decommissioned in EpiLC and fully silenced in EpiSC. They also found that PGCLC enhancers retained H3K4me1 (Fig 1d) in EpiLC, while the establishment of heterochromatin marks (CpG methylation) was delayed (Fig 1e) in comparison to EpiSC. Additionally, they also computed the same active and heterochromatin marks at the PGCLC-enhancers in different stages of mouse embryos (i.e., E4.5, E5.5, E6.5) in vivo using published datasets. Intriguingly, they found that the enhancer decommissioning is heterogeneous in vivo. Altogether, they suggest that PGCLC enhancers could be going through partial decommissioning retaining the chromatin accessibility for transcription factor responsiveness during embryonic development.
Figure 2: (a) Schematic representation of catalytically dead MLL3/4 mutants used in this study. (b) PGCLC quantification (CD15+ CD61+ cells) in wild-type and MLL3/4 mutant cells using FACS analysis. Heatmaps showing the genome-wide distribution of H3K4me1 (c) and H3K27ac (d) in MLL3/4 mutant cells.
To address the impact of enhancer decommissioning on PGCLC induction, the authors used mESC expressing wild-type and catalytic dead mutants of mixed-lineage leukemia 3 and 4 (MLL3/4, the enzyme that catalyzes H3K4me1/2, Fig 2a). They found that the PGCLC induction is challenged in MLL3/4 double mutants (dCD and dCT), but not in the single mutant, suggesting the redundant function of MLL3/4 (Fig 2b). Further, they report significant loss of H3K4me1 at PGCLC enhancers in all stages (Fig 2c), with the concomitant acquisition of heterochromatin marks (i.e., mCpG). However, they only found marginal loss of the other active enhancer mark H3K27ac at the PGCLC enhancers in the mutant cells (Fig 2d). Interestingly, the genes associated with PGCLC enhancers did not show significant changes in their expression in ESC, EpiLC, or EpiSC. However, upon PGCLC differentiation, the H3K27ac distribution and associated gene expression were impeded in the mutant cells compared to their wild-type counterparts. Thus, they report that H3K4me1 facilitates in vitro germline competence, although H3K4me1 seems dispensable for the enhancer maintenance and upon differentiation into EpiLC and EpiSC.

Conclusion and perspective

The authors propose a model where H3K4me1 may not be necessary for enhancer activity but provides a chromatin platform that facilitates stage and cell-type-specific gene expression. They suggest that H3K4me1 could ward off heterochromatinization at enhancer elements and promote (re)activation of transcription at the onset of relevant cellular signals (here during PGCLC-development). This could explain the partial decommissioning of enhancers during differentiation in mammalian embryonic development. How a combination of active and inactive epigenetic marks at enhancer impacts different mammalian developmental stages remains open for future research.

Figure 3: Model proposed in this preprint illustrating the partial decommissioning of PGCLC enhancers during PGCLC differentiation and its relevance for germline competence.

Acknowledgments

Thanks to Tore Bleckwehl and Álvaro Rada-Iglesias for their insightful comments on this preLight. Thanks to all the authors of this preprint for their support.

All the figures used in this preLight are taken directly from Bleckwehl T et. al., 2021 under a CC BY-NC-ND 4.0 international license.

References

Tags: development, enhancers, epigenetics

doi: https://doi.org/10.1242/prelights.28840

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Author's response

Tore Bleckwehl (TB) and Álvaro Rada-Iglesias (ARI) shared

1. The authors assessed enhancer marks in NANOG and PRDM14 over-expressed cells. Did the authors evaluate H3K4me1 and gene expression changes in these cells?

TB and ARI: PGCLC enhancers are strongly bound by NANOG and PRDM14 in germline competent EpiLC but not EpiSC. This indicates that the PGCLC enhancers are more accessible in EpiLC. When we performed the same experiments for the active histone modifications H3K4me2 and H3K27ac, we found an increase in both modifications. Hence, we concluded that the PGCLC enhancers are also more responsive in EpiLC. However, we did not evaluate H3K4me1 in the over-expression experiments, partly because this histone modification was already higher in EpiLC than in EpiSC under normal conditions.

2. Do the authors intend to use RNA Polymerase II ChIP-seq profiles to correlate with enhancer activity?

TB and ARI: We have analyzed public RNA Polymerase II ChIP-seq profiles for the differentiation and found a positive correlation with enhancer activity that resembled the dynamics of H3K27ac during the differentiation of ESC into EpiLC and EpiSC. However, considering the amount of genomic data already, we decided not to include it in the manuscript.

3. It is very interesting that MLL3/4 mutant cells do not manifest transcription changes, even though the H3K4me1 patterns are severely challenged. How do the authors envisage that H3K4me1 impacts the differentiation process despite the lack of transcriptional changes? It would be great to hear the authors’ perspective on this.

TB and ARI: This is indeed very interesting. We showed that in ESC and upon differentiation to EpiLC and EpiSC the absence of H3K4me1 seems to have a rather minor effect on gene expression. On the other side, we showed that the loss of H3K4me1 impaired the proper induction of PGCLC as measured by FACS and single-cell RNA-seq and it also reduced the induction of genes associated with PGCLC enhancers. Based on our current data, we propose that H3K4me1 might facilitate the reactivation of previously active enhancers by rendering them more accessible and responsive to transcriptional activators during the germline competent state. Further research is needed to investigate the importance of H3K4me1 in different cellular contexts and to explore whether H3K4me1 might contribute to enhancer function through additional mechanisms (e.g. 3D chromatin architecture).

4. What do the authors think about the heterogenous enhancer decommissioning in the in vivo data? Do the authors predict if this is because of a plethora of cellular signals present in vivo?

TB and ARI: That is another interesting question. Work from other labs showed that the asynchronous differentiation from pluripotent cells and/or genome-wide oscillations in CpG methylation levels can contribute to the heterogeneity in CpG methylation, which could be related to the multiple and opposing signals that the embryo faces upon implantation. Assuming that CpG methylation within enhancers is negatively correlated with germline competence, we could envision that the epigenetic heterogeneity within enhancers might facilitate the diversity of cellular transitions. For instance, this heterogeneity might ensure that during the germline competence phase some epiblast cells might display lower CpG methylation within PGCLC enhancers and might be particularly responsive to the germline inductive signals. Thereby, this heterogeneity might ensure that only a limited number of epiblast cells give rise to PGCs.

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Enhancer-associated H3K4 methylation safeguards in vitro germline competence

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Key findings

Conclusion and perspective

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