A Synthetic Lethality Screen Reveals ING5 as a Genetic Dependency of Catalytically Dead Set1A/COMPASS in Mouse Embryonic Stem Cells
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ABSTRACT:
PROVIDER: PRJNA807952 | ENA |
REPOSITORIES: ENA
Dataset's files
| Action | DRS | |||
|---|---|---|---|---|
| SRR18058100.fastq.gz | Fastqsanger.gz | |||
| SRR18058101.fastq.gz | Fastqsanger.gz | |||
| SRR18058102.fastq.gz | Fastqsanger.gz | |||
| SRR18058103.fastq.gz | Fastqsanger.gz | |||
| SRR18058104.fastq.gz | Fastqsanger.gz |
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Project description: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.
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Project description:Histone H3 lysine 4 trimethylation (H3K4me3) facilitates recruitment of transcription factors and epigenetic effectors to promote transcriptional activation and ensure cellular identity. This conserved histone mark is implemented by the COMPASS (COMplex of Proteins ASsociated with Set1) family of H3K4 methyltransferases. COMPASS members Set1A and Set1B have been accredited as primary depositors of global H3K4me3 in mammalian cells. Our previous studies in mouse embryonic stem cells (ESCs) demonstrated that deleting the enzymatic SET domain of Set1A does not perturb bulk H3K4me3, indicating possible compensatory roles played by other COMPASS methyltransferases. Here, we generated a series of ESC lines harboring compounding mutations of the COMPASS enzymes. We find that Set1B is functionally redundant to Set1A in implementing H3K4me3 at highly expressed genes, while Mll2 deposits H3K4me3 at less transcriptionally active promoters. Furthermore, Set1A and Set1B are responsible for broad H3K4me3 peaks, whereas Mll2 establishes H3K4me3 with narrow breadth. Most importantly, Mll2 helps preserve global H3K4me3 levels and peak breadth. Our results illustrate the biological flexibility of such enzymes in regulating transcription in a context-dependent manner to maintain stem cell identity, which could assist our understanding of their disease liability.
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Project description:MUC1-C activates the COMPASS SET1A/WDR5 complex in integrating the induction of genes that promote chronic inflammation, pluripotency and the cancer stem cell state
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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.
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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
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.
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Project description:In this study, we performed chromatin immunoprecipitation-coupled massive parallel DNA sequencing (ChIP-seq) to analyze the genome-wide transcriptional profile of ING5 in hepatocytes. ChIP was performed first in HepG2 cells with antibodies against ING5. Following ChIP, ING5-associated DNAs were amplified using non-biased conditions, labeled, and sequenced with HiSeq 2500. The raw sequencing image data were examined by the Illumina analysis pipeline, aligned to the unmasked human reference genome (GRCh37, hg19) using Bowtie2, and further analyzed by MACS (Model-based Analysis for ChIP-Seq).
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Project description:To explore the functional significance of JFK-mediated ING5 degradation, we performed genome-wide analysis of the transcriptome in hepatocytes that is regulated by the JFK-ING5 axis using RNA-based deep sequencing (RNA-seq). The data obtained from RNA-seq experiments were cross-analyzed with ChIP-seq data of ING5 from HepG2 cells.
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