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Cytokine-sensing primary enhancer cluster launches secondary enhancers to activate multi-gene locus [RNA-seq]

ABSTRACT: Enhancer clusters are platforms occupied at high-density by the transcription machinery to activate lineage-specific genes. However, it is not clear yet whether they are merely a collection of conventional enhancers or represent unique and new types of regulatory elements. Specifically, the question remains whether they activate additional enhancer programs in multigene loci. We addressed this issue in the mouse mammary gland, where genes are highly activated during pregnancy. We identify a 300kb multi-gene locus with twenty-one regulatory elements, including a cytokine-dependent primary enhancer cluster (PEC). Using genome editing in combination with ChIP-seq, RNA-seq and 3C-technologies, we show that the PEC initiates the activation of lineage-specific genes through contacting and activating secondary enhancers. This new framework of enhancer clusters indicates a hitherto unrecognized hierarchy of regulation by which a PEC activates genes via regulating secondary enhancers.

ORGANISM(S): Mus musculus

PROVIDER: GSE127140 | GEO | 2021/02/12

REPOSITORIES: GEO

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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.

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Cytokine-sensing primary enhancer cluster launches secondary enhancers to activate multi-gene locus [RNA-seq]

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