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Active and repressive chromatin in the mammalian genome intersperse without spreading in imprinted gene clusters

ABSTRACT: The Igf2r imprinted cluster is an epigenetic model of cis-acting silencing in which expression of a ncRNA silences multiple genes. Here, we map chromatin profiles on the maternal and paternal chromosome in a 250 kb region. We show that histone modifications associated with expressed and silent genes are mutually exclusive and localize to discrete regions. Expressed genes are modified in two ways; at promoter regions by H3K4me3+H3K4me2+H3K9Ac and on putative regulatory elements by H3K4me2+H3K9Ac. Repressed genes displayed two types of non-overlapping profiles. Type 1 localized to short regions and contained H3K9me3+H4K20me3 and sometimes HP1. Type 2 spanned a large domain containing multiple tissue-specific silent genes and contained H3K27me3 alone. These data identify different forms of repressive chromatin on chromosome arms that resemble constitutive and facultative heterochromatin but are restricted to short regions and interspersed with euchromatin. Keywords: ChIP-Chip, chromatin profile, genomic imprinting, noncoding RNA

ORGANISM(S): Mus musculus

PROVIDER: GSE5834 | GEO | 2007/07/01

SECONDARY ACCESSION(S): PRJNA97231

REPOSITORIES: GEO

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Project description:The Igf2r imprinted cluster is an epigenetic model of cis-acting silencing in which expression of a ncRNA silences multiple genes. Here, we map chromatin profiles on the maternal and paternal chromosome in a 250 kb region. We show that histone modifications associated with expressed and silent genes are mutually exclusive and localize to discrete regions. Expressed genes are modified in two ways; at promoter regions by H3K4me3+H3K4me2+H3K9Ac and on putative regulatory elements by H3K4me2+H3K9Ac. Repressed genes displayed two types of non-overlapping profiles. Type 1 localized to short regions and contained H3K9me3+H4K20me3 and sometimes HP1. Type 2 spanned a large domain containing multiple tissue-specific silent genes and contained H3K27me3 alone. These data identify different forms of repressive chromatin on chromosome arms that resemble constitutive and facultative heterochromatin but are restricted to short regions and interspersed with euchromatin. Keywords: ChIP-Chip, chromatin profile, genomic imprinting, noncoding RNA Chromatin immunoprecipitaion (ChIP) was combined with a custom PCR tiling array (Chip) to determine the profile of histone modifications on the maternal and paternal allele of the imprinted Igf2r cluster on mouse chromosome 17. ChIP-Chip experiments were carried out on mouse embryo fibroblast (13.5dpc) cell lines. The main steps in the array design were (i) design of primers to amplify single copy DNA, (ii) spotting each PCR fragment 8 times on the array.The main steps in the ChIP were: (i) preparation of chromatin by micrococcal nuclease (MNase) digestion, (ii) use of specific antibodies, (iii) amplification of input and ChIP enriched chromatin using two rounds of T7 in vitro transcription, (iv) conversion of amplified RNA to cDNA and labelling with Alexa dyes, (v) hybridisation, washing, scanning. Two biological replicates were performed for each antibody in each cell line. A dye swap was also incorporated in the second hybridisation. A novel method to test gene expression was also employed. PCR fragments lying within the exons of expressed genes gave a significant signal. This method also allowed for the detection of non-coding RNAs. A total of forty hybridizations were performed in the study.

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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Active and repressive chromatin in the mammalian genome intersperse without spreading in imprinted gene clusters

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