Ldb1-nucleated transcription complexes function as primary mediators of erythroid gene activation
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ABSTRACT:
ORGANISM(S): Mus musculus
PROVIDER: GSE42843 | GEO | 2013/04/29
SECONDARY ACCESSION(S): PRJNA183626
REPOSITORIES: GEO
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Project description:We used ChIP-Seq to map Ldb1, Scl and Gata1 binding sites in mouse total bone marrow cells. Together with functional studies comparing gene expression in Murine Erythroleukemia (MEL) cells expressing Ldb1 shRNA or control shRNA and bioinformatics analysis, we systematically determined the transcriptional program controlled by Ldb1 complexes in erythropoiesis. This represents the ChIP-Seq component of the study only To evaluate the role of Ldb1complexes in erythroid gene activation
2013-04-29 | E-GEOD-42843 | biostudies-arrayexpress
Project description:Carbonic anhydrase 1 (Car1), an early specific marker of the erythroid differentiation, has been used to distinguish fetal and adult erythroid cells since its production closely follows the γ- to β-globin transition, but the molecular mechanism underlying transcriptional regulation of Car1 is unclear. Here, we show that Car1 mRNA decreases significantly when erythroid differentiation is induced in MEL cells. The Ldb1 protein complex including GATA1/SCL/LMO2 binds to the Car1 promoter in uninduced cells and reduced enrichment of the complex during differentiation correlates with loss of Car1 expression. Knockdown of Ldb1 results in a reduction of Ser2 phosphorylated RNA Pol II and Cdk9 at the Car1 promoter region, suggesting that Ldb1 is required for recruitment of Pol II as well as the transcription regulator P-TEFb to enhance elongation of Car1 transcripts. Taken together, these data show that Ldb1 forms a regulatory complex to maintain Car1 expression in erythroid cells. Expression analysis of induced and uninduced MEL cells after control or Ldb1 shRNA knockdown.
2012-12-11 | E-GEOD-42839 | biostudies-arrayexpress
Project description:Carbonic anhydrase 1 (Car1), an early specific marker of the erythroid differentiation, has been used to distinguish fetal and adult erythroid cells since its production closely follows the γ- to β-globin transition, but the molecular mechanism underlying transcriptional regulation of Car1 is unclear. Here, we show that Car1 mRNA decreases significantly when erythroid differentiation is induced in MEL cells. The Ldb1 protein complex including GATA1/SCL/LMO2 binds to the Car1 promoter in uninduced cells and reduced enrichment of the complex during differentiation correlates with loss of Car1 expression. Knockdown of Ldb1 results in a reduction of Ser2 phosphorylated RNA Pol II and Cdk9 at the Car1 promoter region, suggesting that Ldb1 is required for recruitment of Pol II as well as the transcription regulator P-TEFb to enhance elongation of Car1 transcripts. Taken together, these data show that Ldb1 forms a regulatory complex to maintain Car1 expression in erythroid cells.
2012-12-11 | GSE42839 | GEO
Project description:Stem Cell Leukemia (Scl or Tal1) and Lymphoblastic Leukemia 1 (Lyl1) are highly related members of the basic helix-loop-helix (bHLH) family of transcription factors that are co- expressed in hematopoietic stem cells and the erythro-megakaryocytic lineages. Previous studies suggest that Scl is essential for hematopoietic development including primitive erythropoiesis. However, analysis of single-cell RNA-sequencing data of early embryos showed that primitive erythroid cells express both Scl and Lyl1. Therefore, to determine whether Lyl1 has a functional role in erythropoiesis, we crossed conditional Scl mice with transgenic mice expressing a Cre recombinase under the control of the Epo receptor, active in erythroid progenitors. Surprisingly, embryos with markedly reduced expression of Scl from E9.5 survived to adulthood. In contrast, mice with reduced expression of Scl and absence of Lyl1 (double knockout; DKO) died at E10.5 due to progressive loss of erythropoiesis. Consistent with a phenocopy of Gata1-null mice, gene expression profiling of DKO yolk sacs prior to the loss of erythrocytes (E9.5) revealed loss of Gata1 and many of the known target genes of the SCL-GATA1 complex. ChIP-seq analyses showed that LYL1 exclusively bound a small subset of SCL targets including GATA1. Together, these data show for the first time that Scl and Lyl1 share functional roles in primitive erythropoiesis.
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Project description:Many questions remain about how close association of genes and distant enhancers occurs and how this is linked to transcription activation. In erythroid cells, LDB1 is recruited to the β-globin locus via LMO2 and is required for looping of the β-globin locus control region (LCR) to the active β-globin promoter. We show that the LDB1 dimerization domain (DD) is necessary and, when fused to LMO2 is sufficient, to completely restore LCR-promoter looping and transcription in LDB1 depleted cells. The looping function of the DD is unique and irreplaceable by heterologous dimerization domains. Dissection of the DD revealed distinct functional properties of conserved subdomains. Notably, a conserved helical region (DD4/5) is dispensable for LDB1 dimerization and chromatin looping but essential for transcriptional activation. DD4/5 is required for the recruitment of the co-regulators FOG1 and NuRD complex. Lack of DD4/5 alters histone acetylation and RNA polymerase II recruitment and results in failure of the locus to migrate to the nuclear interior as normally occurs during erythroid maturation. These results uncouple enhancer-promoter looping from nuclear migration and transcription activation and reveal new roles for LDB1in these processes. RNA-seq in LDB1 knockdown, LDB1 delta4/5 construct, LDB1 full-length construct, and control in induced MEL cells; three replicates each.
2014-07-02 | E-GEOD-54549 | biostudies-arrayexpress
Project description:MEL clone 745 cells that are blocked at the pro-erythroblast stage serve as a model for terminal erythroid differentiation when treated with DMSO. A stable MEL cell line expressing (Dox)-dependent shRNA sequence targeting different regions of G9a mRNA was established and screened as previously described in (Demers, 2007 PMID17707229). Knock down was induced by adding 5 micrograms/ml final of Dox in the culture medium. For a total of six samples, 10 micro grams of RNA was extracted from each three biological replicates of mouse erythroleukemia (MEL) cells and their corresponding G9a knock downs. The RNA was extracted and purified using the RNeasy Mini Kit (Qiagen) including the on-column DNAse I digestion step. Experiment Overall Design: Mouse erythroleukemia cells: wild type vs G9 knock down. For each microarray hybridization, data analysis was performed using MAS and GCRMA.
2009-11-09 | E-GEOD-15620 | biostudies-arrayexpress
Project description:LMO2 is a component of multisubunit DNA-binding transcription factor complexes that regulate gene expression in hematopoietic stem and progenitor cell development. Enforced expression of LMO2 causes leukemia by inducing hematopoietic stem cell-like features in T-cell progenitor cells, but the biochemical mechanisms of LMO2 function have not been fully elucidated. In this study we systematically dissected the LMO2/LDB1 binding interface to investigate the role of this interaction in T-cell leukemia. Alanine scanning mutagenesis of the LIM interaction domain of LDB1 revealed a discrete motif R320LITR required for LMO2 binding. Most strikingly, co-expression of full length, wild type LDB1 increased LMO2 steady state abundance, whereas co-expression of mutant proteins deficient in LMO2 binding compromised LMO2 stability. These mutant LDB1 proteins also exerted dominant negative effects on growth and transcription in diverse leukemic cell lines. Raw gene expression data on HSB-2 cells is presented here.
2015-12-15 | GSE75839 | GEO
Project description:LMO2 is a component of multisubunit DNA-binding transcription factor complexes that regulate gene expression in hematopoietic stem and progenitor cell development. Enforced expression of LMO2 causes leukemia by inducing hematopoietic stem cell-like features in T-cell progenitor cells, but the biochemical mechanisms of LMO2 function have not been fully elucidated. In this study we systematically dissected the LMO2/LDB1 binding interface to investigate the role of this interaction in T-cell leukemia. Alanine scanning mutagenesis of the LIM interaction domain of LDB1 revealed a discrete motif R320LITR required for LMO2 binding. Most strikingly, co-expression of full length, wild type LDB1 increased LMO2 steady state abundance, whereas co-expression of mutant proteins deficient in LMO2 binding compromised LMO2 stability. These mutant LDB1 proteins also exerted dominant negative effects on growth and transcription in diverse leukemic cell lines. Raw gene expression data on HSB-2 cells is presented here. RNAseq were performed on HSB cell lines to examine their expression patterns
2015-12-15 | E-GEOD-75839 | 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.
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