Project description:Heart valve formation initiates when endothelial cells of the heart transform into mesenchyme and populate the cardiac cushions. The transcription factor, SOX9, is highly expressed in the cardiac cushion mesenchyme, and is essential for heart valve development. Loss of Sox9 in mouse cardiac cushion mesenchyme alters cell proliferation, embryonic survival, and disrupts valve formation. Despite this important role, little is known regarding how SOX9 regulates heart valve formation or its transcriptional targets. Therefore, we mapped putative SOX9 binding sites by ChIP-Seq in embryonic day (E) 12.5 heart valves, a stage at which the valve mesenchyme is actively proliferating and initiating differentiation. Embryonic heart valves have been shown to express a high number of genes that are associated with chondrogenesis, including several extracellular matrix proteins and transcription factors that regulate chondrogenesis. Consequently, we compared regions of putative SOX9 DNA-binding between E12.5 heart valves and E12.5 limb buds. We identified context-dependent and contextâindependent SOX9 interacting regions throughout the genome. Analysis of context-independent SOX9 binding suggests an extensive role for SOX9 across tissues in regulating proliferation-associated genes including key components of the AP-1 complex. Integrative analysis of tissue-specific SOX9 interacting regions and gene expression profiles on Sox9-deficient heart valves demonstrated that SOX9 controls the expression of several transcription factors with previously identified roles in heart valve development, including Twist1, Sox4, Mecom/Evi1 and Pitx2. Together, our data identifies SOX9 coordinated transcriptional hierarchies that control cell proliferation and differentiation during valve formation. Examination of SOX9 binding sites in E12.5 atrioventricular canal (AVC) and E12.5 embryonic limb and mRNA expression profiling in E12.5 WT and Sox9 mutant AVCs, in duplicate.
Project description:The HIRA chaperone complex, comprised of HIRA, UBN1 and CABIN1, collaborates with histone-binding protein ASF1a to incorporate histone variant H3.3 into chromatin in a DNA replication-independent manner. To better understand its function and mechanism, we integrated HIRA, UBN1, ASF1a and histone H3.3 ChIP-seq and gene expression analyses. Most HIRA-binding sites co-localize with UBN1, ASF1a and H3.3 at active promoters and active and weak/poised enhancers. At promoters, binding of HIRA/UBN1/ASF1a correlates with the level of gene expression. HIRA is required for deposition of histone H3.3 at its binding sites. There are marked differences in nucleosome and co-regulator composition at different classes of HIRA-bound regulatory site. Underscoring this, we report novel physical interactions between the HIRA complex and transcription factors, a chromatin insulator and an ATP-dependent chromatin-remodelling complex. Our results map the distribution of the HIRA chaperone across the chromatin landscape and point to different interacting partners at functionally distinct regulatory sites. Examination of H3.3 histone modification in HeLA cells with accompanying FAIRE data
Project description:The HIRA chaperone complex, comprised of HIRA, UBN1 and CABIN1, collaborates with histone-binding protein ASF1a to incorporate histone variant H3.3 into chromatin in a DNA replication-independent manner. To better understand its function and mechanism, we integrated HIRA, UBN1, ASF1a and histone H3.3 ChIP-seq and gene expression analyses. Most HIRA-binding sites co-localize with UBN1, ASF1a and H3.3 at active promoters and active and weak/poised enhancers. At promoters, binding of HIRA/UBN1/ASF1a correlates with the level of gene expression. HIRA is required for deposition of histone H3.3 at its binding sites. There are marked differences in nucleosome and co-regulator composition at different classes of HIRA-bound regulatory site. Underscoring this, we report novel physical interactions between the HIRA complex and transcription factors, a chromatin insulator and an ATP-dependent chromatin-remodelling complex. Our results map the distribution of the HIRA chaperone across the chromatin landscape and point to different interacting partners at functionally distinct regulatory sites. Examination of 3 histone chaperone proteins in HeLa cells
Project description:Purpose: The goals of this study are to compare transcriptome profiling (RNA-seq) resulting from a Mesp1Cre driven ablation of Hira in the heart at E11.5 and E12.5. Methods: RNA extraction was done in triplicate from Mesp1Cre;Hira-/fl and Mesp1Cre;Hira+/fl embryonic hearts at E11.5 and E12.5 using the QIAGEN RNeasy mini kit. RNAseq was processed by the ICH genomics facility, reads were aligned and normalised using BOWTIE and DEseq R package. Strand NGS 2.5 software was used to re-analyse the aligned files (.bam). By applying the Mann Whitney unpaired test, Benjamini Hochberg False discovery rate (FDR) and filtering the genes using adjusted p-value ≤ 0.05 and absolute fold change ≥ 1.5, 95 % of the results were identical to the DEseq package used by the UCL Genomics facility. Results: We identified 360 coding transcripts changed in the mutant hearts (Mann Whitney unpaired test, Benjamini Hochberg FDR, p ≤ 0.05, FC ≥ 1.5) with no trend towards up- or down-regulation of global transcription (48.8% down vs 51.2% up) at E12.5. Conclusions: This work analyses the role of HIRA in mouse cardiac development. It was found that Tnni2 is the most upregulated gene in the absence of Hira. qRT-PCR validation of 25 targets. Little (<5%) or no variation of fold change between RNAseq and qRT-PCR data were observed.
Project description:The HIRA chaperone complex, comprised of HIRA, UBN1 and CABIN1, collaborates with histone-binding protein ASF1a to incorporate histone variant H3.3 into chromatin in a DNA replication-independent manner. To better understand its function and mechanism, we integrated HIRA, UBN1, ASF1a and histone H3.3 ChIP-seq and gene expression analyses. Most HIRA-binding sites co-localize with UBN1, ASF1a and H3.3 at active promoters and active and weak/poised enhancers. At promoters, binding of HIRA/UBN1/ASF1a correlates with the level of gene expression. HIRA is required for deposition of histone H3.3 at its binding sites. There are marked differences in nucleosome and co-regulator composition at different classes of HIRA-bound regulatory site. Underscoring this, we report novel physical interactions between the HIRA complex and transcription factors, a chromatin insulator and an ATP-dependent chromatin-remodelling complex. Our results map the distribution of the HIRA chaperone across the chromatin landscape and point to different interacting partners at functionally distinct regulatory sites. We used microarrays to detail the global programme of gene expression after knockdown of HIRA HeLa cells were nucleofacted with Dharmacon control siRNA and siRNA to HIRA and RNA was isolated 72 hours after transfection in four biological replicates
Project description:The HIRA chaperone complex, comprised of HIRA, UBN1 and CABIN1, collaborates with histone-binding protein ASF1a to incorporate histone variant H3.3 into chromatin in a DNA replication-independent manner. To better understand its function and mechanism, we integrated HIRA, UBN1, ASF1a and histone H3.3 ChIP-seq and gene expression analyses. Most HIRA-binding sites co-localize with UBN1, ASF1a and H3.3 at active promoters and active and weak/poised enhancers. At promoters, binding of HIRA/UBN1/ASF1a correlates with the level of gene expression. HIRA is required for deposition of histone H3.3 at its binding sites. There are marked differences in nucleosome and co-regulator composition at different classes of HIRA-bound regulatory site. Underscoring this, we report novel physical interactions between the HIRA complex and transcription factors, a chromatin insulator and an ATP-dependent chromatin-remodelling complex. Our results map the distribution of the HIRA chaperone across the chromatin landscape and point to different interacting partners at functionally distinct regulatory sites.
Project description:The HIRA chaperone complex, comprised of HIRA, UBN1 and CABIN1, collaborates with histone-binding protein ASF1a to incorporate histone variant H3.3 into chromatin in a DNA replication-independent manner. To better understand its function and mechanism, we integrated HIRA, UBN1, ASF1a and histone H3.3 ChIP-seq and gene expression analyses. Most HIRA-binding sites co-localize with UBN1, ASF1a and H3.3 at active promoters and active and weak/poised enhancers. At promoters, binding of HIRA/UBN1/ASF1a correlates with the level of gene expression. HIRA is required for deposition of histone H3.3 at its binding sites. There are marked differences in nucleosome and co-regulator composition at different classes of HIRA-bound regulatory site. Underscoring this, we report novel physical interactions between the HIRA complex and transcription factors, a chromatin insulator and an ATP-dependent chromatin-remodelling complex. Our results map the distribution of the HIRA chaperone across the chromatin landscape and point to different interacting partners at functionally distinct regulatory sites.