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HSB-2 cells stably expressing LDB1 or mutant LDB1 proteins

ABSTRACT: 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

ORGANISM(S): Homo sapiens

SUBMITTER: Yan Guo

PROVIDER: E-GEOD-75839 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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HSB-2 cells stably expressing LDB1 or mutant LDB1 proteins

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

Ldb1 is required for Lmo2 oncogene-induced thymocyte self-renewal and T-cell Acute Lymphoblastic Leukemia

Project description:Prolonged or enhanced expression of the proto-oncogene Lmo2 is associated with a severe form of T-cell Acute Lymphoblastic Leukemia (T-ALL), designated Early T-progenitor ALL (ETP-ALL), that is characterized by the aberrant self-renewal and subsequent oncogenic transformation of immature thymocytes. Recent data suggest that Lmo2 may exert these effects by functioning as component of a multi-subunit transcription complex that includes the ubiquitous adapter Ldb1 along with b-HLH and/or GATA family transcription factors. In this study, we investigated the importance of Ldb1 for Lmo2-induced T-ALL by conditional deletion of Ldb1 in thymocytes in a Lmo2 transgenic mouse model of T-ALL. Our results identify a critical requirement for Ldb1 in the induction of thymocyte self-renewal, thymocyte radio-resistance and transition to T-ALL in Lmo2 transgenic mice. Ldb1 was also required for acquisition of the pre-leukemic ETP gene expression signature in immature Lmo2 transgenic thymocytes. Together, these results support a model where Lmo2-induced T-ALL results from failure to down-regulate Ldb/Lmo2 nucleated transcription complexes that normally function to enforce self-renewal in bone marrow hematopoietic progenitors

2020-03-19 | GSE129244 | GEO

The role of Ldb1 in hemangioblast development: genome-wide analysis shows that Ldb1 controls essential hematopoietic genes/pathways in mouse early development and reveals novel players in hematopoiesis (sequencing)

Project description:The first site exhibiting hematopoietic activity in mammalian development is the yolk sac blood island, which originates from the hemangioblast. Here we performed differentiation assays, as well as genome-wide molecular and functional studies in BL-CFCs to gain insight into the function of the essential Ldb1 factor in early primitive hematopoietic development. We show that the previously reported lack of yolk sac hematopoiesis and vascular development in Ldb1-/- mouse result from a decreased number of hemangioblasts and a block in their ability to differentiate into erythroid and endothelial progenitor cells. Transcriptome analysis and correlation with the genome wide binding pattern of Ldb1 in hemangioblasts revealed a number of direct target genes and pathways misregulated in the absence of Ldb1. The regulation of essential developmental factors by Ldb1 defines it as an upstream transcriptional regulator of hematopoietic/endothelial development. We show the complex interplay that exists between transcription factors and signaling pathways during the very early stages of hematopoietic/endothelial development and the specific signalling occurring in hemangioblasts in contrast to more advanced hematopoietic developmental stages. Finally, by revealing novel genes and pathways, not previously associated with early development, our study provides novel candidate targets to manipulate the differentiation of hematopoietic and/or endothelial cells. Examination of endogenous Ldb1 genome-wide binding sites comparsion between ChIP and Control on Flk1+ BL-CFCs RNA was isolated from Ldb1+/+ and Ldb1-/- Flk1+ cells with the QIAGEN RNeasy Mini Kit and integrity was checked on the Agilent 2100 Bioanalyzer. RNA sequencing was performed on Illumina HiSeq 2000 platform according to the manufacturer instructions.

2013-02-09 | E-GEOD-43041 | biostudies-arrayexpress

Project description:The first site exhibiting hematopoietic activity in mammalian development is the yolk sac blood island, which originates from the hemangioblast. Here we performed differentiation assays, as well as genome-wide molecular and functional studies in BL-CFCs to gain insight into the function of the essential Ldb1 factor in early primitive hematopoietic development. We show that the previously reported lack of yolk sac hematopoiesis and vascular development in Ldb1-/- mouse result from a decreased number of hemangioblasts and a block in their ability to differentiate into erythroid and endothelial progenitor cells. Transcriptome analysis and correlation with the genome wide binding pattern of Ldb1 in hemangioblasts revealed a number of direct target genes and pathways misregulated in the absence of Ldb1. The regulation of essential developmental factors by Ldb1 defines it as an upstream transcriptional regulator of hematopoietic/endothelial development. We show the complex interplay that exists between transcription factors and signaling pathways during the very early stages of hematopoietic/endothelial development and the specific signalling occurring in hemangioblasts in contrast to more advanced hematopoietic developmental stages. Finally, by revealing novel genes and pathways, not previously associated with early development, our study provides novel candidate targets to manipulate the differentiation of hematopoietic and/or endothelial cells. We used microarrays to detail the global programme of gene expression underlying the Ldb1+/+ and Ldb1-/- in Flk1+ cells. RNA was isolated from Ldb1+/+ and Ldb1-/- Flk1+ cells with the QIAGEN RNeasy Mini Kit and integrity was checked on the Agilent 2100 Bioanalyzer. RNA was converted to biotin-labelled cRNA, hybridised on the Mouse Genome 430 2.0 Array and analyzed with the Affymetrix GeneChipÂ® Scanner 3000 according to the manufacturer protocol.

2013-02-09 | E-GEOD-43042 | biostudies-arrayexpress

Project description:The first site exhibiting hematopoietic activity in mammalian development is the yolk sac blood island, which originates from the hemangioblast. Here we performed differentiation assays, as well as genome-wide molecular and functional studies in BL-CFCs to gain insight into the function of the essential Ldb1 factor in early primitive hematopoietic development. We show that the previously reported lack of yolk sac hematopoiesis and vascular development in Ldb1-/- mouse result from a decreased number of hemangioblasts and a block in their ability to differentiate into erythroid and endothelial progenitor cells. Transcriptome analysis and correlation with the genome wide binding pattern of Ldb1 in hemangioblasts revealed a number of direct target genes and pathways misregulated in the absence of Ldb1. The regulation of essential developmental factors by Ldb1 defines it as an upstream transcriptional regulator of hematopoietic/endothelial development. We show the complex interplay that exists between transcription factors and signaling pathways during the very early stages of hematopoietic/endothelial development and the specific signalling occurring in hemangioblasts in contrast to more advanced hematopoietic developmental stages. Finally, by revealing novel genes and pathways, not previously associated with early development, our study provides novel candidate targets to manipulate the differentiation of hematopoietic and/or endothelial cells. We used microarrays to detail the global programme of gene expression underlying the Ldb1+/+ and Ldb1-/- in Flk1+ cells.

2013-02-09 | GSE43042 | GEO

2013-02-09 | GSE43041 | GEO

Role of LDB1 in the transition from chromatin looping to transcription activation

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

LIM domain binding protein 1 regulates a transcriptional program essential for hematopoietic stem cell maintenance

Project description:We used ChIP-Seq to map Ldb1, Scl and Gata2 binding sites in mouse hematopoietic progenitor cells (HPCs). Together with functional studies using conventional and conditional Ldb1 deficient mouse models and bioinformatics analysis, we systematically determined a transcriptional program controlled by Ldb1 complexes in HSC maintenance. To evaluate the role of Ldb1 in hematopoietic stem cell maintenance.

2011-01-04 | E-GEOD-26031 | biostudies-arrayexpress

Expression changes in MEL cells upon differentiation and Ldb1 knockdown

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

A Stable Transcription Factor Complex Nucleated by Dimeric AML1-ETO Controls Leukaemogenesis

Project description:AML1-ETO, a fusion protein generated by the t(8;21) translocation in acute myeloid leukemia, is a transcription factor implicated in both gene repression and activation. We now show that, in leukemic cells, AML1-ETO resides in and functions through a stable protein complex (AETFC) that contains several hematopoietic transcription factors and cofactors. In conjunction with biochemical and leukemia pathological studies, the ChIP-seq and RNA-seq analyses of the AETFC components in leukemic cells reveal that these components stabilize the complex through multivalent interactions, provide multiple DNA-binding domains for diverse target genes, colocalize genome-wide, cooperatively regulate gene expression, and contribute to leukemogenesis. RNA-seq analyses gene expression upon knockdown of each AETFC component, including AML1-ETO, HEB, E2A, LYL1, LDB1 and LMO2, and double-knockdown of HEB and E2A, in Kasumi-1 cells. ChIP-seq analyses of four AETFC components, namely AML1-ETO, HEB, E2A and LMO2, in Kasumi-1 cells.

2013-06-20 | E-GEOD-43834 | biostudies-arrayexpress

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