Characterization of human erythroid chromatin in a two-phase liquid differentiation system
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ABSTRACT:
ORGANISM(S): Homo sapiens
PROVIDER: GSE125753 | GEO | 2020/06/29
REPOSITORIES: GEO
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Project description:SCL/TAL1, a tissue-specific transcription factor of the basic helix-loop-helix (bHLH) family, and c-Kit, a tyrosine kinase receptor, control hematopoietic stem cell survival and quiescence. Here we report that SCL and c-Kit signaling control a common gene expression signature, of which 19 genes are associated with apoptosis. In vivo, SCL levels are limiting for the clonal expansion of Kit+ multipotent and erythroid progenitors. In addition, increased SCL expression specifically enhances the sensitivity of multipotent and megakaryocyte/erythroid progenitors to Steel factor (KIT ligand), whilst a DNA binding mutant antagonizes KIT function and induces apoptosis in progenitors. We conclude that Scl operates downstream of Kit to support the survival of megakaryocyte/erythroid progenitors. Finally, higher SCL expression upregulates Kit in normal bone marrow cells and increases chimerism after bone marrow transplantation, indicating that Scl is also upstream of Kit. We conclude that Scl and Kit establish a positive feedback loop in multipotent and megakaryocyte/erythroid progenitors.
2013-07-06 | GSE48557 | GEO
Project description:SCL/TAL1, a tissue-specific transcription factor of the basic helix-loop-helix (bHLH) family, and c-Kit, a tyrosine kinase receptor, control hematopoietic stem cell survival and quiescence. Here we report that SCL and c-Kit signaling control a common gene expression signature, of which 19 genes are associated with apoptosis. In vivo, SCL levels are limiting for the clonal expansion of Kit+ multipotent and erythroid progenitors. In addition, increased SCL expression specifically enhances the sensitivity of multipotent and megakaryocyte/erythroid progenitors to Steel factor (KIT ligand), whilst a DNA binding mutant antagonizes KIT function and induces apoptosis in progenitors. We conclude that Scl operates downstream of Kit to support the survival of megakaryocyte/erythroid progenitors. Finally, higher SCL expression upregulates Kit in normal bone marrow cells and increases chimerism after bone marrow transplantation, indicating that Scl is also upstream of Kit. We conclude that Scl and Kit establish a positive feedback loop in multipotent and megakaryocyte/erythroid progenitors. c-Kit regulated genes were extrapolated from gene expression profiles of TF-1 erythroid progenitor cells (empty MSCV vector) stimulated with SF (Kit ligand), Epo or GM-CSF. Second, SCL-regulated genes were obtained by expressing a DNA binding-defective SCL mutant (DbSCL) and selecting genes that were differentially expressed in M-oM-^AM-^DbSCL cells versus control cells (MSCV) stimulated with the same cytokines.
2013-07-06 | E-GEOD-48557 | biostudies-arrayexpress
Project description:We have previously proposed two distinct molecular mechanisms by which SCL binds its targets in hematopoiesis; either by direct contact with specific DNA sequences or by indirect recruitment through interaction with other proteins. We have established that direct DNA binding is the major non-redundant mechanism SCL exerts in red cells. A DNA-binding mutant form of SCL (SCLRER) had detrimental effect on erythropoiesis in vivo. To extend these data to a molecular and mechanistic level, we have set out to identify the genomic sequences bound by SCL in vivo in erythroid precursors; we performed anti-SCL ChIP assays on immature, Ter119- erythroid cell populations isolated from day E12.5 wild-type (SCLWT/WT) fetal livers followed by ultra-throughput sequencing (ChIP-SEQ). To compare SCL’s direct versus indirect DNA-binding activities and, thus, gain insight into its mechanisms of action, we also analysed material isolated from SCLRER/RER fetal livers. anti-SCL ChIP-enriched DNA from mouse fetal liver erythroblast chromatin was analysed by Solexa sequencing. Four samples were processed: chromatin from SCL wildtype erythroblasts (WT-SCL) and SCL mutant erythroblasts (RER-SCL) were ChIPed by anti-SCL antibody and sequenced with their respective 'no antibody' controls.
2010-05-18 | E-GEOD-18720 | biostudies-arrayexpress
Project description:ChIP-Seq experiments using an Scl/TAL1 antibody were carried out in primary erythroid cells cultured from two individuals of Caucasian origin. ChIP-Seq libraries were prepared from two biological replicates for each individual. ChIP DNA was processed for Illumina High-throughput sequencing according to Illumina protocol. By obtaining over four billion bases of sequence from chromatin immunoprecipitated DNA from each experiments, we generated a high-resolution map of Scl/TAL1 genomic targets in human erythroid cells. We find that WGATAR and the combinatorial CTG(n9)GATA are the most significant motifs responsible for the chromatin occupancy by Scl/TAL1. In addition, other motifs are also significantly enriched at the Scl/TAL1 targets. Amongst these were binding sites for known TFs (Sp/XKLF, RUNX1, NFE2). We next investigated how many of these targets varied between these two individuals. We find that 0.8% of Scl/TAL1 binding regions differ significantly between the two individuals.
2014-11-08 | GSE42390 | GEO
Project description:Coordination of cellular processes through the establishment of tissue-specific gene expression programmes is essential for lineage maturation. The basic helix-loop-helix haemopoietic transcriptional regulator SCL/Tal1 is required for terminal differentiation of red blood cells. To gain insight into SCL function and mechanisms of action in erythropoiesis, we performed ChIP-sequencing and gene expression analyses from primary fetal liver erythroid cells. We show that SCL coordinates expression of genes in most known red cell-specific processes. The majority of SCLâs genomic targets require direct DNA-binding activity. However, one fifth of SCLâs target sequences, mainly amongst those showing high affinity for SCL, can recruit the factor independently of its DNA binding activity. An unbiased DNA motif search of sequences bound by SCL identified CAGNTG as SCL-preferred E-box motif in erythroid cells. Novel motifs were also characterised that may help distinguish activated from repressed genes and suggest a new mechanism by which SCL may be recruited to DNA. Finally, analysis of recruitment of GATA1, a protein partner of SCL, to sequences occupied by SCL suggests that SCLâs binding is necessary prior or simultaneous to that of GATA1. This work provides the framework to study regulatory networks leading to erythroid terminal maturation and to model mechanisms of action of tissue-specific transcription factors. Total RNA extracted from wild-type (WT) day E12.5 fetal liver Ter119- erythroid progenitor cells was compared to total RNA extracted from E12.5 fetal liver Ter119- cells expressing a DNA-binding mutant form of SCL (SclRER/RER).
2010-05-27 | E-GEOD-21877 | biostudies-arrayexpress
Project description:Coordination of cellular processes through the establishment of tissue-specific gene expression programmes is essential for lineage maturation. The basic helix-loop-helix haemopoietic transcriptional regulator SCL/Tal1 is required for terminal differentiation of red blood cells. To gain insight into SCL function and mechanisms of action in erythropoiesis, we performed ChIP-sequencing and gene expression analyses from primary fetal liver erythroid cells. We show that SCL coordinates expression of genes in most known red cell-specific processes. The majority of SCL’s genomic targets require direct DNA-binding activity. However, one fifth of SCL’s target sequences, mainly amongst those showing high affinity for SCL, can recruit the factor independently of its DNA binding activity. An unbiased DNA motif search of sequences bound by SCL identified CAGNTG as SCL-preferred E-box motif in erythroid cells. Novel motifs were also characterised that may help distinguish activated from repressed genes and suggest a new mechanism by which SCL may be recruited to DNA. Finally, analysis of recruitment of GATA1, a protein partner of SCL, to sequences occupied by SCL suggests that SCL’s binding is necessary prior or simultaneous to that of GATA1. This work provides the framework to study regulatory networks leading to erythroid terminal maturation and to model mechanisms of action of tissue-specific transcription factors.
2010-05-18 | GSE21877 | GEO
Project description:We have previously proposed two distinct molecular mechanisms by which SCL binds its targets in hematopoiesis; either by direct contact with specific DNA sequences or by indirect recruitment through interaction with other proteins. We have established that direct DNA binding is the major non-redundant mechanism SCL exerts in red cells. A DNA-binding mutant form of SCL (SCLRER) had detrimental effect on erythropoiesis in vivo. To extend these data to a molecular and mechanistic level, we have set out to identify the genomic sequences bound by SCL in vivo in erythroid precursors; we performed anti-SCL ChIP assays on immature, Ter119- erythroid cell populations isolated from day E12.5 wild-type (SCLWT/WT) fetal livers followed by ultra-throughput sequencing (ChIP-SEQ). To compare SCL’s direct versus indirect DNA-binding activities and, thus, gain insight into its mechanisms of action, we also analysed material isolated from SCLRER/RER fetal livers.
2010-05-18 | GSE18720 | GEO
Project description:Identification of cell-type specific enhancers is important for understanding the regulation of programs controlling cellular development and differentiation. Enhancers are typically marked by the co-transcriptional activator protein p300 or by groups of cell-expressed transcription factors. We hypothesized that a unique set of enhancers regulates gene expression in human erythroid cells, a highly specialized cell type evolved to provide adequate amounts of oxygen throughout the body. Using chromatin immunoprecipitation followed by massively parallel sequencing, genome-wide maps of candidate enhancers were constructed for p300 and four transcription factors, GATA1, NF-E2, KLF1, and SCL, using primary human erythroid cells. These data were combined with gene expression analyses and candidate enhancers identified. Consistent with their predicted function as candidate enhancers, there was statistically significant enrichment of p300 and combinations of co-localizing erythroid transcription factors within 1-50 kb of the TSS of genes highly expressed in erythroid cells. Candidate enhancers were also enriched near genes with known erythroid cell function or erythroid cell phenotypes. Candidate enhancers exhibited only moderate conservation with mouse and minimal conservation with nonplacental vertebrates. Candidate enhancers were mapped to a data set of erythroid-associated, biologically relevant, SNPs from the GWAS catalog of the NHGRI. Fourteen candidate enhancers, representing 10 genetic loci, mapped to sites associated with biologically relevant erythroid traits. Fragments from these loci directed statistically significant expression in reporter gene assays. Identification of enhancers in human erythroid cells will allow a better understanding of erythroid cell development, differentiation, structure, and function, and provide insights into inherited and acquired hematologic disease.
2013-01-28 | GSE43625 | GEO
Project description:Identification of cell-type specific enhancers is important for understanding the regulation of programs controlling cellular development and differentiation. Enhancers are typically marked by the co-transcriptional activator protein p300 or by groups of cell-expressed transcription factors. We hypothesized that a unique set of enhancers regulates gene expression in human erythroid cells, a highly specialized cell type evolved to provide adequate amounts of oxygen throughout the body. Using chromatin immunoprecipitation followed by massively parallel sequencing, genome-wide maps of candidate enhancers were constructed for p300 and four transcription factors, GATA1, NF-E2, KLF1, and SCL, using primary human erythroid cells. These data were combined with gene expression analyses and candidate enhancers identified. Consistent with their predicted function as candidate enhancers, there was statistically significant enrichment of p300 and combinations of co-localizing erythroid transcription factors within 1-50 kb of the TSS of genes highly expressed in erythroid cells. Candidate enhancers were also enriched near genes with known erythroid cell function or erythroid cell phenotypes. Candidate enhancers exhibited only moderate conservation with mouse and minimal conservation with nonplacental vertebrates. Candidate enhancers were mapped to a data set of erythroid-associated, biologically relevant, SNPs from the GWAS catalog of the NHGRI. Fourteen candidate enhancers, representing 10 genetic loci, mapped to sites associated with biologically relevant erythroid traits. Fragments from these loci directed statistically significant expression in reporter gene assays. Identification of enhancers in human erythroid cells will allow a better understanding of erythroid cell development, differentiation, structure, and function, and provide insights into inherited and acquired hematologic disease.
2013-01-28 | GSE43624 | GEO
Project description:Identification of cell-type specific enhancers is important for understanding the regulation of programs controlling cellular development and differentiation. Enhancers are typically marked by the co-transcriptional activator protein p300 or by groups of cell-expressed transcription factors. We hypothesized that a unique set of enhancers regulates gene expression in human erythroid cells, a highly specialized cell type evolved to provide adequate amounts of oxygen throughout the body. Using chromatin immunoprecipitation followed by massively parallel sequencing, genome-wide maps of candidate enhancers were constructed for p300 and four transcription factors, GATA1, NF-E2, KLF1, and SCL, using primary human erythroid cells. These data were combined with gene expression analyses and candidate enhancers identified. Consistent with their predicted function as candidate enhancers, there was statistically significant enrichment of p300 and combinations of co-localizing erythroid transcription factors within 1-50 kb of the TSS of genes highly expressed in erythroid cells. Candidate enhancers were also enriched near genes with known erythroid cell function or erythroid cell phenotypes. Candidate enhancers exhibited only moderate conservation with mouse and minimal conservation with nonplacental vertebrates. Candidate enhancers were mapped to a data set of erythroid-associated, biologically relevant, SNPs from the GWAS catalog of the NHGRI. Fourteen candidate enhancers, representing 10 genetic loci, mapped to sites associated with biologically relevant erythroid traits. Fragments from these loci directed statistically significant expression in reporter gene assays. Identification of enhancers in human erythroid cells will allow a better understanding of erythroid cell development, differentiation, structure, and function, and provide insights into inherited and acquired hematologic disease. CD34+-selected stem and progenitor cells were expanded for three days in the absence of EPO, and total RNA was isolated from a portion of the cells. The cells were further cultured in the presence of EPO, and RNA was isolated after cells differentiated into R3/R4 nucleated erythroid cells. There were 3 replicates of CD34 cells and 9 replicates of R3/R4 erythroid cells.
2013-01-28 | E-GEOD-43624 | biostudies-arrayexpress