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A Synthetic Lethality Screen Reveals ING5 as a Genetic Dependency of Catalytically Dead Set1A/COMPASS in Mouse Embryonic Stem Cells

ABSTRACT: Embryonic stem cells (ESCs) are defined by their ability to self-renew and the potential to differentiate into all tissues of the developing organism. We previously demonstrated that deleting the catalytic SET domain of the Set1A/COMPASS histone methyltransferase in mouse ESCs does not impair their viability or ability to self-renew, however, leads to defects in differentiation. The precise mechanisms by which Set1A executes these functions remain to be elucidated. In this study, we demonstrate that mice lacking the SET domain of Set1A are embryonic lethal at a stage that is unique from null alleles. To gain insight into Set1A function in regulating pluripotency, we conducted a CRISPR/Cas9-mediated dropout screen and identified the MOZ/MORF and HBO1 acetyltransferase complex member ING5 as a synthetic perturbation to Set1A. The loss of Ing5 in Set1AΔSET mESCs decreases the fitness of these cells; and the simultaneous loss of ING5 and in Set1AΔSET leads to upregulation of differentiation-associated genes. Taken together, our results point towards Set1A and ING5 as potential co-regulators of the self-renewal and differentiation status of ESCs.

ORGANISM(S): Mus musculus

PROVIDER: GSE196946 | GEO | 2022/03/28

REPOSITORIES: GEO

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Histone H3K4 methylation-dependent and independent functions of Set1A/COMPASS in embryonic stem cell self-renewal and differentiation

Project description:Of the six members of the COMPASS (complex of proteins associated with Set1) family of histone H3 Lys4 (H3K4) methyltransferases identified in mammals, Set1A has been shown to be essential for early embryonic development and the maintenance of embryonic stem cell (ESC) self-renewal. Like its familial relatives, Set1A possesses a catalytic SET domain responsible for histone H3K4 methylation. Whether H3K4 methylation by Set1A/COMPASS is required for ESC maintenance and during differentiation has not yet been addressed. Here, we generated ESCs harboring the deletion of the SET domain of Set1A (Set1AΔSET); surprisingly, the Set1A SET domain is dispensable for ESC proliferation and self-renewal. The removal of the Set1A SET domain does not diminish bulk H3K4 methylation in ESCs; instead, only a subset of genomic loci exhibited reduction in H3K4me3 in Set1AΔSET cells, suggesting a role for Set1A independent of its catalytic domain in ESC self-renewal. However, Set1AΔSET ESCs are unable to undergo normal differentiation, indicating the importance of Set1A-dependent H3K4 methylation during differentiation. Our data also indicate that during differentiation, Set1A but not Mll2 functions as the H3K4 methylase on bivalent genes and is required for their expression, supporting a model for transcriptional switch between Mll2 and Set1A during the self-renewing-to-differentiation transition. Together, our study implicates a critical role for Set1A catalytic methyltransferase activity in regulating ESC differentiation but not self-renewal and suggests the existence of context-specific H3K4 methylation that regulates transcriptional outputs during ESC pluripotency.

2017-09-26 | GSE98988 | GEO

Gene Expression Profile of ING5-knockdown Brain Tumor Initiating Cell Lines

Project description:Transcriptome analysis on ING5-knockdown brain tumor stem cell lines Stem cell-like brain tumor initiating cells (BTICs) cause recurrence of glioblastomas, with BTIC "stemness" affected by epigenetic mechanisms. The ING family of epigenetic regulators (ING1-5) function by targeting histone acetyltransferase (HAT) or histone deacetylase (HDAC) complexes to the H3K4me3 mark to alter histone acetylation and subsequently, gene expression. Here we find that ectopic expression of ING5, the targeting subunit of HBO1, MOZ and MORF HAT complexes increases expression of the Oct4, Olig2 and Nestin stem cell markers, promotes self-renewal, prevents lineage differentiation and increases stem cell pools in BTIC populations. This activity requires the plant homeodomain region of ING5 that interacts specifically with the H3K4me3 mark. ING5 also enhances PI3K/AKT and MEK/ERK activity to sustain self-renewal of BTICs over serial passage of stem cell-like spheres. ING5 exerts these effects by activating transcription of calcium channel and follicle stimulating hormone (FSH) pathway genes. In silico analyses of The Cancer Genome Atlas (TCGA) data suggest that ING5 is a positive regulator of BTIC stemness, whose expression negatively correlates with patient prognosis, especially in the Proneural and Classical subtypes, and in tumors with low SOX2 expression. These data suggest that altering histone acetylation status and signaling pathways induced by ING5 may provide useful clinical strategies to target tumor resistance and recurrence in glioblastoma.

2017-06-24 | GSE100398 | GEO

A forward genetic screen for modifiers of transgene silencing in the mouse identifies Inhibitor of Growth Family Member 5 (Ing5)

Project description:Here we report the identification of a mutant allele of the Ing5 gene, also known as Inhibitor of Growth Family Member 5 and a member of the MOZ/MORF histone acetyltransferase complex. The mutation likely alters functionality of the Ing5 protein by inhibiting its ability to bind to its histone substrate, H3K4me3. We find that Ing5 acts as a suppressor of a multi-copy transgene reporter suggesting a complex role for Ing5 in regulating gene expression and show that Ing5 is required for normal embryonic development.

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Diverse epigenetic strategies interact to control epidermal differentiation [ChIP-Seq]

Project description:It is becoming clear that interconnected functional gene networks, rather than single genes in isolation, govern stem cell self-renewal and differentiation. To identify potential epigenetic networks that impact on human epidermal stem cells we performed siRNA based genetic screens for 332 chromatin modifiers. We developed a Bayesian mixture model to predict putative functional interactions between those epigenetic modifiers that regulated differentiation. This allowed us to discover a network of genetic interactions involving EZH2, UHRF1 (both known to regulate epidermal self-renewal), ING5 (a MORF complex component), BPTF and SMARCA5 (NURF complex components). Genome-wide localisation and global mRNA expression analysis revealed that these factors impact two distinct but functionally related gene sets, including integrin extracellular matrix receptors that mediate anchorage of epidermal stem cells to their niche. Using a competitive epidermal reconstitution assay we confirmed that ING5, BPTF, SMARCA5, EZH2 and UHRF1 control differentiation under physiological conditions. Thus, regulation of distinct gene expression programs through the interplay between diverse epigenetic strategies protects epidermal stem cells from differentiation. Examination of genome-wide localisation of ING5 in primary human keratinocytes

2012-06-23 | E-GEOD-34556 | biostudies-arrayexpress

A Synthetic Lethality Screen Reveals ING5 as a Genetic Dependency of Catalytically Dead Set1A/COMPASS in Mouse Embryonic Stem Cells

Project description:A Synthetic Lethality Screen Reveals ING5 as a Genetic Dependency of Catalytically Dead Set1A/COMPASS in Mouse Embryonic Stem Cells

| PRJNA807952 | ENA

Diverse epigenetic strategies interact to control epidermal differentiation [Illumina bead array]

2012-06-24 | GSE34557 | GEO

Diverse epigenetic strategies interact to control epidermal differentiation [ChIP-Seq]

2012-06-24 | GSE34556 | GEO

Diverse epigenetic strategies interact to control epidermal differentiation [Illumina bead array]

Project description:It is becoming clear that interconnected functional gene networks, rather than single genes in isolation, govern stem cell self-renewal and differentiation. To identify potential epigenetic networks that impact on human epidermal stem cells we performed siRNA based genetic screens for 332 chromatin modifiers. We developed a Bayesian mixture model to predict putative functional interactions between those epigenetic modifiers that regulated differentiation. This allowed us to discover a network of genetic interactions involving EZH2, UHRF1 (both known to regulate epidermal self-renewal), ING5 (a MORF complex component), BPTF and SMARCA5 (NURF complex components). Genome-wide localisation and global mRNA expression analysis revealed that these factors impact two distinct but functionally related gene sets, including integrin extracellular matrix receptors that mediate anchorage of epidermal stem cells to their niche. Using a competitive epidermal reconstitution assay we confirmed that ING5, BPTF, SMARCA5, EZH2 and UHRF1 control differentiation under physiological conditions. Thus, regulation of distinct gene expression programs through the interplay between diverse epigenetic strategies protects epidermal stem cells from differentiation. Primary human keratinocytes (line Lka, passage 2) were cultured in Keratinocyte Serum Free Medium (KSFM) to 70% confluency in 10 cm dishes. Cells were treated with: vehicle (0.2% DMSO), the EGFR inhibitor AG1478 (10 uM), recombinant human BMP2/7 heterodimer (200 ng/ml) or a combination of AG1478+BMP2/7 (10 uM and 200 ng/ml, respectively) for 48 hours. RNA was extracted directly using a Qiagen RNeasy kit

2012-06-23 | E-GEOD-34557 | biostudies-arrayexpress

RNA sequencing data of Ing4;Ing5 compound mutant embryonic mouse hearts (ING4 and ING5 are essential for histone H3 lysine 14 acetylation and epicardial cell lineage development)

Project description:Inhibitor of growth 4 and 5 (ING4 and ING5) are chromatin-binding proteins in the KAT6A, KAT6B and KAT7 histone acetyltransferase protein complexes. Heterozygous mutations in the KAT6A or KAT6B gene cause human disorders with cardiac defects, but the contribution of their chromatin adaptor proteins to development is unknown. We found that Ing5-/- mice had isolated cardiac ventricular septal defects. ING4 and ING5 are structurally similar proteins, suggesting a degree of redundancy. Combined loss of ING4 and ING5 caused developmental arrest at embryonic day E8.5, loss of histone H3 lysine 14 acetylation (H3K14ac) and a reduction in H3K23ac levels. Ing4+/-Ing5-/- hearts showed a paucity of epicardial cells and epicardium-derived cells, failure of myocardium compaction and coronary vasculature defects, accompanied by a reduction in the expression of epicardium genes. Our findings suggest that ING4 and ING5 are essential for heart development and promote epicardium and epicardium-derived cell fates and mutation of the human ING5 gene as a possible cause of isolated ventricular septal defects.

2024-02-01 | GSE246403 | GEO

RNA sequencing data of Ing4;Ing5 double knock-out embryonic mouse hearts

Project description:Inhibitor of growth 4 and 5 (ING4 and ING5) are chromatin-binding proteins in the KAT6A, KAT6B and KAT7 histone acetyltransferase protein complexes. Heterozygous mutations in the KAT6A or KAT6B gene cause human disorders with cardiac defects, but the contribution of their chromatin adaptor proteins to development is unknown. We found that Ing5–/– mice had isolated cardiac ventricular septal defects. ING4 and ING5 are structurally similar proteins, suggesting a degree of redundancy. Combined loss of ING4 and ING5 caused developmental arrest at embryonic day E8.5, loss of histone H3 lysine 14 acetylation (H3K14ac), a reduction in H3K23ac levels and disruption of developmental gene expression in Ing4–/–Ing5–/– compared to control embryos. E12.5 Ing4+/–Ing5–/– hearts showed a paucity of epicardial cells and epicardium-derived cells, failure of myocardium compaction and coronary vasculature defects, accompanied by a reduction in the expression of epicardium genes compared to control hearts. In addition, a reduction in the expression of genes required for cell adhesion, both in whole E8.75 Ing4–/–Ing5–/– embryos and in E10.5 Ing4+/–Ing5–/– hearts compared to controls was observed. In vitro assessment of fibroblast and proepicardium explants revealed cell spreading and outgrowth defects. Our findings suggest that ING4 and ING5 are essential for heart development and promote epicardium and epicardium-derived cell fates and mutation of the human ING5 gene as a possible cause of isolated ventricular septal defects.

2024-02-01 | GSE252338 | GEO

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