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:Plant development is characterised by the capacity to reprogram differentiated cells to initiate asexual reproduction and to produce new organs such as lateral roots. To test whether HIRA could be involved in aspects of developmental reprogramming we studied the effect of HIRA during de-differentiation Arabidopsis root tissue was digested with enzyme to generate protoplast. This process would induce plant cell dedifferentiation. Experiments were performed in WT and hira background to understand how hira affect plant dedifferentiation process.
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:Purpose: The goals of this study are to compare transcriptome profiling (RNA-seq) resulting from the knockout of Hira in undifferentiated mouse embryonic stem cells (mESCs) and in day 15 differentiated cardiomyocytes.Methods: RNA extraction was done in duplicate from WT and Hira-null mESCs at day0 and day15 using TRIzol reagent. RNAseq was done onIllumina Nextseq500 and processed by the ICH genomics facility, reads were aligned and normalised using BOWTIE and DEseq R2 package. Gene lists were filtered using adjusted p-value ≤ 0.05 and absolute fold change ≥ 2. Results:We identified 1680 transcripts changed in the absence of HIRA in day 15 differentiated cardiomyocytes. GO term cardiovascular system development was the most downregulated gene set(p-value ≤ 0.01 and FDR ≤0.1. Conclusion: this study analysis the role of HIRA in early cardiac mesoderm development usinf an invitro mESCs model.
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
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:Hi-C experiment was performed to map and compare potential evolutionary changes in chromatin structural organisation of human, chimpanzee and macaque iAstrocytes.
Project description:Hira has been implicated in replication-independent chromatin assembly. To determine the role of Hira in the regulation of embryonic stem (ES) cell gene expression, we used microarrays to analyze the global program of gene expression in Hira null versus WT undifferentiated ES cells.