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LDB1 regulates gene expression and chromatin structure in pluripotency and lineage differentiation [ATAC-seq]

ABSTRACT: Chromatin organization is a pivotal factor in stem cell pluripotency and differentiation. However, despite considerable study, the role of enhancer looping protein LDB1 in these processes has not been explored. Here, we generated Ldb1(-/-) embryonic stem cells (ESC) using CRISPR/Cas9 editing to understand the significance of LDB1 during development. Ldb1(-/-) ESC exhibited a reduction in key stem cell factors SOX2 and KLF4. Embryoid bodies (EB) derived from Ldb1(-/-) ESC displayed reduced expression of lineage-specific markers and impaired ability to undergo terminal differentiation to erythroblasts. Transcriptome analysis revealed differential gene expression between WT and Ldb1(-/-) ESC and EB but altered gene expression was most pronounced after differentiation to erythroblasts. The Lin28-mediated stem cell self-renewal pathway was dysregulated in Ldb1(-/-) cells. Our data reveal that LDB1 occupies super enhancers of pluripotency genes in ESC together with pluripotency factors. LDB1 loss resulted in a global decrease in chromatin accessibility in ESC and EB. Conditional LDB1-deficient mice showed reduced hematopoietic stem cell markers on bone marrow cells, and dysregulation of the Lin28/Let-7/Hmga2 pathway. We conclude LDB1 function is critical for ESC and EB development and becomes progressively more important during differentiation to erythroblasts.

ORGANISM(S): Mus musculus

PROVIDER: GSE282375 | GEO | 2026/06/12

REPOSITORIES: GEO

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LDB1 regulates gene expression and chromatin structure in pluripotency and lineage differentiation [ChIPmentation]

Project description:Chromatin organization is a pivotal factor in stem cell pluripotency and differentiation. However, despite considerable study, the role of enhancer looping protein LDB1 in these processes has not been explored. Here, we generated Ldb1(-/-) embryonic stem cells (ESC) using CRISPR/Cas9 editing to understand the significance of LDB1 during development. Ldb1(-/-) ESC exhibited a reduction in key stem cell factors SOX2 and KLF4. Embryoid bodies (EB) derived from Ldb1(-/-) ESC displayed reduced expression of lineage-specific markers and impaired ability to undergo terminal differentiation to erythroblasts. Transcriptome analysis revealed differential gene expression between WT and Ldb1(-/-) ESC and EB but altered gene expression was most pronounced after differentiation to erythroblasts. The Lin28-mediated stem cell self-renewal pathway was dysregulated in Ldb1(-/-) cells. Our data reveal that LDB1 occupies super enhancers of pluripotency genes in ESC together with pluripotency factors. LDB1 loss resulted in a global decrease in chromatin accessibility in ESC and EB. Conditional LDB1-deficient mice showed reduced hematopoietic stem cell markers on bone marrow cells, and dysregulation of the Lin28/Let-7/Hmga2 pathway. We conclude LDB1 function is critical for ESC and EB development and becomes progressively more important during differentiation to erythroblasts.

2026-06-12 | GSE282373 | GEO

LDB1 regulates gene expression and chromatin structure in pluripotency and lineage differentiation [RNA-seq]

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LDB1 regulates gene expression and chromatin structure in pluripotency and lineage differentiation [Cut&Tag]

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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. RNAseq were performed on HSB cell lines to examine their expression patterns

2015-12-15 | E-GEOD-75839 | biostudies-arrayexpress

HSB-2 cells stably expressing LDB1 or mutant LDB1 proteins

2015-12-15 | GSE75839 | GEO

Reevaluation by CRISPR-Cas9 approach reveals multiple lincRNAs dispensable for pluripotency maintenance and SNORA73 essential for pluripotency exit (scRNA-Seq)

Project description:Many lincRNAs have been identified as essential regulators for ESC pluripotency through siRNA-based screening. However, the biological and molecular functions of the large intergenic noncoding RNAs (lincRNAs) remain unclear. Here, we employed CRISPR/Cas9-mediated knockout technology to explore the functions of 8 previously annotated lincRNAs in ESCs. Unexpectedly, it was found that most lincRNAs are dispensable for pluripotency maintenance, cloning formation ability, proliferation and apoptosis in ESCs when we disrupted these lincRNAs individually or in combinations. Single-cell transcriptomic analysis of a set of lncRNA knockout ESCs showed consistent results, indicating that previously observed functions of lincRNAs on ESC pluripotency maintenance may be due to off-targeted effects. Interestingly, deletion and transcriptional repression of linc1343 disrupts embryoid body (EB) differentiation in ESC. Notably, GM17300 and two small RNAs (Snora73a, Snora73b) derived from the lnc1343 locus are highly expressed in ESCs. Further overexpression of Snora73a and Snora73b in linc1343 knockout cells rescue the phenotype, suggesting that snoRNAs derived from linc1343 are the responders for EB differentiation. Taken together, our results demonstrate the divergent effects of lincRNAs on regulating ESC pluripotency and EB differentiation.

2024-08-07 | GSE225238 | GEO

Reevaluation by CRISPR-Cas9 approach reveals multiple lincRNAs dispensable for pluripotency maintenance and SNORA73 essential for pluripotency exit (RNA-Seq)

2024-08-07 | GSE225237 | GEO

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

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 (Affymetrix)

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. 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

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