الفهرس 
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Abstract
Congenital heart disease is a health, social and economic burden in Egypt and worldwide. It results from the disturbance in the molecular process which takes place during heart development. Heart development is a complex process which requires tightly controlled sequential gene expression to generate myocardium sequentially from cardiac precursors, primitive cardiomyocytes and differentiated cardiomyocytes.
Epigenetic regulation of gene expression plays a critical role in normal development and cell functions. Any disturbance in the epigenetic regulation causes serious human illnesses like congenital anomalies and cancers. One of the important epigenetic changes studied is chromatin structure and its modifications. The dynamics of chromatin structure are tightly regulated through multiple mechanisms including histone modification, chromatin remodelling and histone variant incorporation.Histone variants differ from canonical histones in having a distinct amino acid sequence that can influence both the physical properties of the nucleosome, making DNA more accessible for transcription. H3.3 is a histone variant that differs from H3.1 and H3.2 in 4 and 5 amino acids respectively. The incorporation of the histone variant H3.3 into chromatin plays an important role in activating transcription.
Histone chaperone is a protein that escorts histones or histone variants. All histone chaperones share the ability to transfer histones onto naked DNA to reconstitute nucleosomes.
HIRA is a histone chaperone which deposits the histone variants H3.3 in the gene bodies of highly active genes, some active enhancers and regulatory elements. Functional attenuation of Hira in chick cardiac neural crest results in patent truncus arteriosus. Hira-null mouse embryos showed abnormal heart looping and pericardial oedema at embryonic day (E) 10.5. Conditional knockout of Hira in the cardiogenic mesoderm resulted in embryonic lethality with atrial and ventricular septal defects at E15.5.However, the molecular mechanisms through which HIRA affects the early stages of cardiomyocytes differentiation has not yet been studied. Differentiation of mESCs towards cardiomyocytes mimics the early stages of heart development. The aim of this study was to characterize the role of HIRA in embryonic stem cells differentiating toward a cardiac mesoderm, looking at gene expression changes, epigenetics changes, and the different pathways where HIRA may be involved.This study was performed on mouse embryonic stem cells. Two clones were used; wild type (W9.5) and Hira- null (Clone 104) mESCs. HIRA antibodies were produced from hybridoma cell lines. These cell lines were cultured in the laboratory under special conditions.In this study, both WT and Hira-null mESCs were differentiated towards a cardiomyocytes lineage using embryoid bodies (EBs) cultured by hanging DROP method (HD). EBs can be generated using various culture methods, the most commonly used methods are aggregation in HD) and aggregation in suspension culture (mass culture). In the HD method, a defined number of cells are cultured as drops of cells in culture medium, hanging from the underside of the lid of a petridish. In the suspension culture method, a known number of cells are dropped as aggregates directly into differentiation medium. Both methods were compared microscopically and by qRT-PCR. HD method was found to be more reproducible, thus it was used in this study.Phenotypically, there was no microscopic difference between WT and Hira-null cells during differentiation. A change in the gene expression of key stage specific genes was observed. Brachyury and Nkx2-5 were significantly upregulated in Hira-null compared to WT on day3 and day 8 respectively. On the contrary Mesp1 expression was downregulated in Hira-null compared to WT on day 5.Using co-immunoprecipitation (Co-IP) as a technique to detect protein interactions, HIRA was found to interact with BRG1, a chromatin remodelling protein, and with the histone methyltransferase WHSC1 only on day 15 of differentiated cardiomyocytes. It is suggested that absence of HIRA leads to disruption of these interactions and consequently to heart defects similar to the defects discovered in Brg1 and Whsc1 knockout embryonic mouse hearts.Chromatin immunoprecipitation (ChIP) is a technique used to identify the DNA binding site of a certain protein. qChIP technique was used in this study and it was shown that HIRA was enriched at the cardiac enhancer locus Tte only in differentiated cardiomyocytes. Tte enhancer locus was shown previously to be a common enhancer site for Tnni2 and Tnnt3. H3.3 deposition at this locus was found to be HIRA dependent, as there was no H3.3 deposition in Hira-null differentiated cardiomyocytes. These results show that HIRA plays a role in cardiac development/mESCs differentiation through binding to a common enhancer which suppresses Tnni2 and Tnnt3.Next generation sequencing (NGS) caused a revolution in genomic research by providing unlimited data related to genome, transcriptome and epigenome. ChIP-seq is a technique which combines ChIP and NGS. It allows the determination of where a protein binds the genome. This protein can be a transcription factor, histone, histone variant or histone chaperone.To test for the effect of HIRA on the genome wide enrichment of H3.3 in differentiated cardiomyocytes, H3.3-HA ChIP-seq was performed, on both WT and Hira-null differentiated cardiomyocytes. In WT cardiomyocytes, H3.3 was deposited mainly in the intronic followed by distal intergenic region, with 9.2% in the promotor region. In the absence of HIRA, more H3.3 was deposited at the distal intergenic region compared to the intronic region, with a 3% deposition in promotor region. Motif discovery showed that H3.3 significantly binds to the DNA sequence TGTGTG.
In WT differentiated cardiomyocytes, 31.6% of HIRA-dependent H3.3 enrichment co-localised with enhancer marks, Suggesting that HIRA influences transcription of genes through deposition of H3.3 at genes’ specific enhancer loci.RNA sequencing (RNA-Seq) is a transcriptome sequencing approach which can identify novel sequences. RNA-Seq is a powerful tool for accurate quantification of expression levels and for variant detection.RNA-seq was performed on day 15 in both WT and Hira-null cells. Analysis of differentially expressed genes showed that genes related to the development of the cardiac septum, chamber and outflow tract were downregulated in Hira-null differentiated cardiomyocytes compared to WT. Calcium signalling pathway was upregulated while vascular smooth muscle contraction pathway was downregulated.Co-relating ChIP-seq with RNA-seq data, three transcription factors were found to be directly influenced by HIRA dependent H3.3 deposition; Meis1, Gata6 and Tbx2. Meis1 was downregulated in Hira-null cardiomyocytes. There was co-localization of HIRA-dependent H3.3 deposition with NKX2.5 binding site at a previously identified enhancer locus in the gene body of Meis1.Gata6 expression was downregulated in Hira-null cardiomyocytes. In addition, HIRA-dependent H3.3 co-localized with NKX2-5 at a previously identified enhancer locus upstream transcription start sitein WT differentiated cells.Tbx2 was downregulated in Hira-null differentiated cardiomyocytes. This downregulation was associated with HIRA-dependent H3.3 deposition at the promotor of Tbx2 in WT differentiated cells. This locus co-localizes with H3K4me3, NKX2-5 and TBX5. This result suggests that HIRA may play a role in AVC and OFT development by influencing the transcription of Tbx2 through H3.3 deposition at its promotor.This study introduces new sets of genes, affected by the absence of HIRA, involved in DNA repair, development of the immune and nervous systems. Several studies link HIRA-dependent H3.3 deposition to the induction of stress responsive associated genes following interferons and heat-shock stimulations. Absence of HIRA was shown previously to increased neuronal differentiation. Appearance of gene sets related to nervous system development can be due to using EB method for differentiation, which can produce undesired mesodermal cell lineages.
This study provides new information that can be clinically applicable in the fields of cell replacement therapy and personalized medicine. The optimized ESCs differentiation protocol can be implemented in human ESCs differentiation into cardiomyocytes. It can be useful in cardiac regeneration following myocardial infarction. In addition, screening for the candidate genes in patients with DiGeorge syndrome can show similar changes in gene expression or point mutations which can be corrected by genetic manipulations.This study shows new molecular mechanisms through which HIRA influences heart development and offers new sets of genes that can be further investigated. However the limitations of this study include the inability to validate the expression of Meis1, Gata6 and Tbx2 at a protein level. In addition, we performed RNA-seq and ChIP-seq analysis in duplicates. However, we validated the main hits found by both RNA-seq and ChIP-seq using reverse RT-qPCR and qChIP respectively, on three biological replicates