Profiling replication timing in WT, MLL3KO and MLL3/4dKO cells during naive to formative ESC differentiation [Bio-Repli-seq]
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PROVIDER: PRJNA893680 | ENA |
REPOSITORIES: ENA
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Project description:We performed BioRepli-seq for WT, MLL3KO and MLL3/4dKO ESCs the naive to formative ESC differentiation.
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Project description:H3K4me1 methyltransferases MLL3 (KMT2C) and MLL4 (KMT2D) are critical for enhancer activation, cell differentiation and development. However, roles of MLL3/4 enzymatic activities and MLL3/4-mediated enhancer H3K4me1 in these processes remain unclear. Here, we report that constitutive elimination of both MLL3 and MLL4 enzymatic activities prevents initiation of gastrulation and leads to early embryonic lethality in mice. However, selective elimination of MLL3/4 enzymatic activities in embryonic, but not extraembryonic, lineages leaves gastrulation largely intact. Consistently, embryonic stem cells (ESCs) lacking MLL3/4 enzymatic activities can differentiate towards the three embryonic germ layers but show aberrant differentiation to extraembryonic endoderm and trophectoderm. The failure in extraembryonic endoderm differentiation can be attributed to markedly reduced enhancer-binding of the lineage-determining transcription factor GATA6. Furthermore, we show that MLL3/4-catalyzed H3K4me1 is largely dispensable for enhancer activation during ESC differentiation. Together, our findings suggest a lineage-selective, but enhancer activation-independent, role of MLL3/4 methyltransferase activities in early embryonic development and ESC differentiation.
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Project description:Profiling of chromatin features during naive to formative ESC differentiation [CUT&Tag]
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Project description:The gene regulatory network in naïve mouse embryonic stem cells (ESC) is reconfigured to enable lineage commitment. Tcf3 sanctions rewiring to formative pluripotency by suppressing components of the ESC transcription factor circuitry. However, Tcf3 depletion only delays, and does not prevent transition. Here we delineate major contributions of Ets-family transcription factor Etv5 and the repressor Rbpj. ERK signalling triggers genome relocation of Etv5 to commission formative pluripotency enhancers. Concomitant up-regulation of Rbpj prevents reversion by repressing potent naïve factors, Nanog and Tbx3. Triple deletion of Etv5, Rbpj and Tcf3 disables naïve ESC, such that they remain undifferentiated and locked in self-renewal even in the presence of differentiation stimuli. Thus, pluripotency dynamics are driven by combined action of two repressors that respectively dissolve and extinguish the naive network, and an activator that initiates transcription of formative network genes. Similar tripartite modality might be a general requirement for robust cell state transitions.
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Project description:FOXA1 is a pioneer factor that is important in hormone dependent cancer cells to stabilise nuclear receptors, such as estrogen receptor (ER) to chromatin. FOXA1 binds to enhancers regions that are enriched in H3K4mono- and dimethylation (H3K4me1, H3K4me2) histone marks and evidence suggests that these marks are requisite events for FOXA1 to associate with enhancers to initate subsequent gene expression events. However, exogenous expression of FOXA1 has been shown to induce H3K4me1 and H3K4me2 signal at enhancer elements and the order of events and the functional importance of these events is not clear. We performed a FOXA1 Rapid Immunoprecipitation Mass Spectrometry of Endogenous Proteins (RIME) screen in ERα-positive MCF-7 breast cancer cells in order to identify FOXA1 interacting partners and we found histone-lysine N-methyltransferase (MLL3) as the top FOXA1 interacting protein. MLL3 is typically thought to induce H3K4me3 at promoter regions, but recent findings suggest it may contribute to H3K4me1 deposition, in line with our observation that MLL3 associates with an enhancer specific protein. We performed MLL3 ChIP-seq in breast cancer cells and unexpectedly found that MLL3 binds mostly at non-promoter regions enhancers, in contrast to the prevailing hypothesis. MLL3 was shown to occupy regions marked by FOXA1 occupancy and as expected, H3K4me1 and H3K4me2. MLL3 binding was dependent on FOXA1, indicating that FOXA1 recruits MLL3 to chromatin. Motif analysis and subsequent genomic mapping revealed a role for Grainy head like protein-2 (GRHL2) which was shown to co-occupy regions of the chromatin with MLL3. Regions occupied by all three factors, namely FOXA1, MLL3 and GRHL2, were most enriched in H3K4me1. MLL3 silencing decreased H3K4me1 at enhancer elements, but had no appreciable impact on H3K4me3 at enhancer elements. We identify a complex relationship between FOXA1, MLL3 and H3K4me1 at enhancers in breast cancer and propose a mechanism whereby the pioneer factor FOXA1 can interact with a chromatin modifier MLL3, recruiting it to chromatin to facilitate the deposition of H3K4me1 histone marks, subsequently demarcating active enhancer elements.
2016-12-12 | PXD005253 | Pride
Project description:Enhancers are essential in defining cell fates through the control of cell type specific gene expression. Enhancer activation is a multi-step process involving chromatin remodelers and modifiers. One of these steps is monomethylation of histone H3 lysine 4 (H3K4m1) by MLL3 (KMT2C) and MLL4 (KMT2D) is thought to be critical for enhancer activation and cognate gene expression including through the recruitment of acetyltransferases targeting H3K27. Here we test this model by evaluating the impact of MLL3/4 loss on chromatin states and gene expression during early embryonic stem cell (ESC) differentiation. We find that MLL3/4 activity is required at most if not all sites that normally gain or lose H3K4m1, but infrequently at sites that remain unchanged during this transition. This requirement extends to H3K27 acetylation (H3K27a) at most transitional sites. Unexpectedly, many sites gain H3K27a independent of MLL3/4 or H3K4m1 including enhancers regulating key factors in early differentiation. Furthermore, despite the failure to gain active histone marks at thousands of enhancers, transcriptional activation of nearby genes is largely unaffected, thus uncoupling enhancer regulation from transcription during this transition. These data challenge current models of enhancer activation and imply distinct mechanisms between stable and dynamically changing enhancers. Moreover, the data suggest limited roles for enhancers in transcriptional regulation in early ESC differentiation. Collectively, our study highlights gaps in knowledge about the steps and epistatic relationships of enzymes necessary for enhancer activation and cognate gene transcription.
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Project description:The gene regulatory network in naïve mouse embryonic stem cells (ESCs) must be reconfigured for lineage competence. Tcf3 enables rewiring to formative pluripotency by repressing components of the ESC transcription factor circuitry. However, elimination of Tcf3 only delays, and does not prevent, state transition. Here we delineate distinct contributions of the Ets-family transcription factor Etv5 and the repressor Rbpj. Downstream of Erk1/2 signalling, Etv5 activates enhancers for formative pluripotency. Concomitant up-regulation of Rbpj ensures irreversible exit from the naïve state by extinguishing reversal factors, Nanog and Tbx3. Triple deletion of Etv5, Rbpj and Tcf3 incapacitates ESCs, such that they remain undifferentiated and locked in self-renewal even in the presence of differentiation stimuli. Thus, pluripotency progression is driven hierarchically by two repressors, that respectively dissolve and extinguish the naive network, and an initiator that commissions the formative network. Similar tripartite action may be a general mechanism for efficient cell transitions.
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Project description:MLL3 inactivation mutations occurs frequently in human breast cancer. To understand the function of MLL3 inactivation, we compared the gene expression profiles of the vector control (WT) and Mll3-knockout MCF7 cells generated by CRISPR-CAS9. Affymetrix human Gene 2.0ST arrays were used for microarray.
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