Project description:Haematopoietic stem cells (HSCs) have long been the focus of developmental and regenerative studies, yet our understanding of the signalling events regulating their specification remains incomplete. We demonstrate that supt16h, a component of the FAcilitates Chromatin Transcription (FACT) complex, is required for HSC formation. Zebrafish supt16h mutants express reduced levels of Notch signalling components, genes essential for HSC development, due to abrogated transcription. Classically, Supt16h regulates transcription and nucleosome reorganization. Whereas global chromatin accessibility in supt16h mutants is unaffected, we observe a specific increase in accessibility at the p53 locus, causing an accumulation of p53 mRNA and protein. We further demonstrate that P53 levels directly influence expression of the Polycomb Group protein, phc1, which functions as a transcriptional repressor of Notch genes. Suppression of phc1 or its upstream regulator, p53, rescues both loss of Notch and loss of HSC phenotypes in supt16h mutants. Taken together, our results highlight a previously uncharacterized relationship between supt16h, p53, and phc1 to specify HSCs via modulation of Notch signalling.
Project description:Hematopoietic stem cells (HSCs) have long been the focus of developmental and regenerative studies, yet our understanding of the signaling events regulating their specification remains incomplete. We demonstrate that supt16h, a component of the FAcilitates Chromatin Transcription (FACT) complex, is required for HSC formation. Zebrafish supt16h mutants express reduced levels of Notch signaling components, genes essential for HSC development, due to abrogated transcription. Classically, Supt16h regulates transcription and nucleosome reorganization. Whereas global chromatin accessibility in supt16h mutants is unaffected, we observe a specific increase in accessibility at the p53 locus, causing an accumulation of p53 mRNA and protein. We further demonstrate that P53 levels directly influence expression of the Polycomb Group protein, phc1, which functions as a transcriptional repressor of Notch genes. Suppression of phc1 or its upstream regulator, p53, rescues both loss of Notch and loss of HSC phenotypes in supt16h mutants. Taken together, our results highlight a previously uncharacterized relationship between supt16h, p53, and phc1 to specify HSCs via modulation of Notch signaling.
Project description:Chromatin organization and accessibility are fundamental to how genes are transcriptionally controlled. We identify the first vertebrate mutant for supt16h, a component of the FACT (FAcilitates Chromatin Transcription) complex along with Ssrp1 known to reorganize nucleosomes and assist in transcriptional elongation. We demonstrate its importance in hematopoietic stem cell (HSC) specification by regulating the elongation of Notch genes. Unexpectedly, Assay for Transposase Accessible Chromatin (ATAC) sequencing revealed that loss of supt16h does not affect histone accessibility on a Notch-specific or global level. Although the majority of genes are unaffected, loss of supt16h alters chromatin accessibility significantly at the p53 locus, leading to its overexpression in mutants. Upon downregulation of p53, both loss of Notch and loss of HSC phenotypes are rescued. Notably, ssrp1 mutants possessed normal elongation of Notch genes, levels of P53, and specification of HSCs. Our results highlight the discrete effects of Supt16h and Ssrp1 during HSC specification. Additionally, we demonstrate the relationship between supt16h and p53 during transcriptional elongation to specify HSC fate via modulation of Notch signaling.
Project description:Chromatin organization and accessibility are fundamental to how genes are transcriptionally controlled. We identify the first vertebrate mutant for supt16h, a component of the FACT (FAcilitates Chromatin Transcription) complex along with Ssrp1 known to reorganize nucleosomes and assist in transcriptional elongation. We demonstrate its importance in hematopoietic stem cell (HSC) specification by regulating the elongation of Notch genes. Unexpectedly, Assay for Transposase Accessible Chromatin (ATAC) sequencing revealed that loss of supt16h does not affect histone accessibility on a Notch-specific or global level. Although the majority of genes are unaffected, loss of supt16h alters chromatin accessibility significantly at the p53 locus, leading to its overexpression in mutants. Upon downregulation of p53, both loss of Notch and loss of HSC phenotypes are rescued. Notably, ssrp1 mutants possessed normal elongation of Notch genes, levels of P53, and specification of HSCs. Our results highlight the discrete effects of Supt16h and Ssrp1 during HSC specification. Additionally, we demonstrate the relationship between supt16h and p53 during transcriptional elongation to specify HSC fate via modulation of Notch signaling.
Project description:To assess the mechanisms by which FACT depletion leads to increased sensitivity of cells to be reprogrammed, we measured the chromatin accessibility landscape using ATAC-seq following mock treatment, SSRP1 knockdown, or SUPT16H knockdown in human fibroblasts and mock, hmg-3 or hmg-4 knockdown in whole worms, and differential gene expression in hmg-3 knockout mutants or following mock, hmg-4, or spt-16 knockdown by RNAseq.
Project description:We used a combination of genetic and proteomic approaches to characterize tmRNA (ssrA) activity in the genome-reduced bacterium Mycoplasma pneumoniae. For this, we generated tmRNA mutants encoding a tag resistant to proteolysis. Endogenous protein tagging by the mutant tmRNA gene (ssrAmk) was then examined by immunoprecipitation (IP) enrichment followed by LC-MS/MS analysis. Additionally, RNA-seq differential expression analysis of the mutants compared to the wild-type strain was assessed.