![Figure 5. Loss of Setd1a in HSPCs results in deregulated expression of genes encoding for DNA damage sensing and repair. (A) Experimental outline. (B,C) Plots show biological processes that are enriched in genes downregulated (B) or upregulated (C) in Setd1a-deficient KSL in comparison with wild-type controls. Analysis was performed using the gene ontology (GO)/biological process (BP) database of DAVID. Enrichment scores (log transformation of the DAVID Expression Analysis Systematic Explorer [EASE] score) were calculated to determine overrepresentation of particular biological processes and are indicated on the x-axis. Terms included in the GO pathways are listed in supplemental Table 1. (D) Normalized enrichment scores of the GO categories based on gene sets associated with DNA repair. Negative enrichment scores indicate global downregulation of the genes within the gene set. Terms included in GSEA gene sets are listed in supplemental Table 3. (E) ChIP-qPCR analysis specific for H3K4me3 at promoter regions. Lin− cells from R.26-CreERT2+;Setd1aF/F or control mice were used, and target genes were selected from RNA sequence data. (F) Photographs from colonies from 600 LT-HSCs and ST-HSCs from R.26-CreERT2+;Setd1aF/F and control mice that were kept in culture for 8 days. The first 4 days, TAM was added to deplete SETD1A expression. Scale bars, 500 μm. (G) Recombination efficiency of the Setd1aF allele in indicated cells. (H) Absolute cell numbers per well after 8 days of culture. Data from 3 independent experiments are shown. (I) Frequency of annexin V+ cells per culture. Data from 2 independent experiments are shown. (J) Frequencies of TUNEL positive cells of cultivated LT-HSCs and ST-HSCs are shown. Data from 2 independent experiments are pooled. (K) MFI of Cell Rox Deep Red to detect ROS levels in each culture. Data from 2 independent experiments are shown. BER, base excision repair; ctrls, controls; DSB, double-strand break; dUTP, deoxyuridine triphosphate; HR, homologous recombination; ncRNA, noncoding RNA; NER, nucleotide excision repair; tRNA, transfer RNA.](https://ash.silverchair-cdn.com/ash/content_public/journal/blood/131/12/10.1182_blood-2017-09-806844/4/blood806844f5.jpeg?Expires=1743021837&Signature=nGJgw5UFYPfvCEURrZSoel5fwAVT2RBeZWZT00XvwKUjq8t0dp4XJmFmXLdGJ~YmPwNwDCgw1YweXScbBLcQ7JDHZR55xZg2zmd8bHB2NjsXNFwLJHVppFYxoEbuttkIwG10RBnxhsD4xeoG6SU8xGLJVxo~rxhAGghLiNKqZVqMq66lNH3XbeCqIA3R8RFLqhQYQOHQTILXmQONQhlU96dFnWWuE8v0LOfv2tzuzHS~DRCBcYBvSOYkGep61WLTNA1fshGflf9rX1kBVCyhElezJZcpvWvDjO1d3kZPXdxJ8rfkXh-zrJ2IuaCkUgtmKBOfnFnpjEJPUjiLSGFyNQ__&Key-Pair-Id=APKAIE5G5CRDK6RD3PGA)
Loss of Setd1a in HSPCs results in deregulated expression of genes encoding for DNA damage sensing and repair. (A) Experimental outline. (B,C) Plots show biological processes that are enriched in genes downregulated (B) or upregulated (C) in Setd1a-deficient KSL in comparison with wild-type controls. Analysis was performed using the gene ontology (GO)/biological process (BP) database of DAVID. Enrichment scores (log transformation of the DAVID Expression Analysis Systematic Explorer [EASE] score) were calculated to determine overrepresentation of particular biological processes and are indicated on the x-axis. Terms included in the GO pathways are listed in supplemental Table 1. (D) Normalized enrichment scores of the GO categories based on gene sets associated with DNA repair. Negative enrichment scores indicate global downregulation of the genes within the gene set. Terms included in GSEA gene sets are listed in supplemental Table 3. (E) ChIP-qPCR analysis specific for H3K4me3 at promoter regions. Lin− cells from R.26-CreERT2+;Setd1aF/F or control mice were used, and target genes were selected from RNA sequence data. (F) Photographs from colonies from 600 LT-HSCs and ST-HSCs from R.26-CreERT2+;Setd1aF/F and control mice that were kept in culture for 8 days. The first 4 days, TAM was added to deplete SETD1A expression. Scale bars, 500 μm. (G) Recombination efficiency of the Setd1aF allele in indicated cells. (H) Absolute cell numbers per well after 8 days of culture. Data from 3 independent experiments are shown. (I) Frequency of annexin V+ cells per culture. Data from 2 independent experiments are shown. (J) Frequencies of TUNEL positive cells of cultivated LT-HSCs and ST-HSCs are shown. Data from 2 independent experiments are pooled. (K) MFI of Cell Rox Deep Red to detect ROS levels in each culture. Data from 2 independent experiments are shown. BER, base excision repair; ctrls, controls; DSB, double-strand break; dUTP, deoxyuridine triphosphate; HR, homologous recombination; ncRNA, noncoding RNA; NER, nucleotide excision repair; tRNA, transfer RNA.
