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

ABSTRACT: 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.

ORGANISM(S): Mus musculus

PROVIDER: GSE54549 | GEO | 2014/07/02

SECONDARY ACCESSION(S): PRJNA237032

REPOSITORIES: GEO

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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

Distinct Ldb1/NLI complexes orchestrate γ-globin repression and re-activation through ETO2 in human adult erythroid cells.

Project description:The Ldb1/GATA-1/TAL1/LMO2 complex mediates long range interaction between the β-globin locus control region (LCR) and gene in adult mouse erythroid cells but whether this complex mediates chromatin interactions at other developmental stages or in human cells is unknown. We investigated human NLI (Ldb1 homologue) complex occupancy and chromatin conformation of the β-globin locus in human erythroid cells. In addition to the LCR, we find robust NLI complex occupancy at a site downstream of the Aγ-globin gene, within sequences of BGL3, an intergenic RNA transcript. In cells primarily transcribing β-globin, BGL3 is not transcribed and BGL3 sequences are occupied by NLI core complex members together with co-repressor ETO2 and γ-globin repressor BCL11A. The LCR and β-globin gene establish proximity in these cells. In contrast, when γ-globin transcription is re-activated by cytokines in these cells, ETO2 participation in the NLI complex at BGL3 is diminished, as is BCL11A occupancy, and both BGL3 and γ-globin are transcribed. In these cells, proximity between the BGL3/γ-globin region and the LCR is established. Thus, alternative NLI complexes mediate γ-globin transcription or silencing through long range LCR interactions involving an intergenic site of non-coding RNA transcription and ETO2 is critical to this process. ChIP-chip of GATA-1, NLI, and pol II in cell lines Comparison of GATA-1, NLI, and pol II binding on chromatin. 2 replicates for each protein; ChIP and input DNA for each replicate

2012-01-05 | E-GEOD-30047 | biostudies-arrayexpress

Distinct Ldb1/NLI complexes orchestrate γ-globin repression and re-activation through ETO2 in human adult erythroid cells.

2012-01-05 | GSE30047 | GEO

Forced enhancer-promoter rewiring to alter gene expression in animal models

Project description:Transcriptional enhancers can be in physical proximity with their target genes via chromatin looping. The enhancer at the beta-globin locus (LCR) contacts the fetal (HBG) and adult (HBB) type beta-globin genes during corresponding developmental stages. We previously demonstrated that forcing proximity between the LCR and HBG genes in cultured adult-stage erythroid cells can activate HBG transcription. Activation of HBG expression in erythroid cells is of benefit to patients with sickle cell disease. Here, using the beta-globin locus as a model we provide proof-of-concept at the organismal level that forced enhancer re-wiring might present a strategy to alter gene expression for therapeutic purposes. Hematopoietic stem and progenitor cells (HSPC) from mice bearing human beta-globin genes were transduced with lentiviral vectors expressing a synthetic transcription factor (ZF-Ldb1) that fosters LCR-HBG contacts. When engrafted into host animals, HSPCs gave rise to adult-type erythroid cells with elevated HBG expression. Vectors containing ZF-Ldb1 were optimized for activity in cultured human and rhesus erythroid cells. Upon transplantation into rhesus macaques, erythroid cells from HSPCs expressing ZF-Ldb1 displayed elevated HBG production. These findings in two animal models suggest that forced redirection of gene regulatory elements may be used to alter gene expression to treat disease.

2023-02-08 | GSE221182 | GEO

Epigenetic modifications and chromosome conformations of the beta globin locus throughout development

Project description:Human embryonic stem cells provide an alternative to using human embryos for studying developmentally regulated gene expression. The co-expression of high levels of embryonic epsilon and fetal gamma globin by the hESC-derived erythroblasts allows the interrogation of epsilon globin regulation at the transcriptional and epigenetic level which could only be attained previously by studying cell lines or transgenic mice. In this study, we compared the histone modifications across the beta globin locus of the undifferentiated hESCs and hESC-, FL-, and mobilized PB CD34+ cells-derived erythroblasts, which have distinct globin expression patterns corresponding to their developmental stages. We demonstrated that the histone codes employed by the beta globin locus are conserved throughout development. Furthermore, in spite of the close proximity of the epsilon globin promoter, as compared to the gamma or beta globin promoter, with the LCR, a chromatin loop was also formed between the LCR and the active epsilon globin promoter, similar to the loop that forms between the gamma or beta globin promoters and the LCR, in contrary to the previously proposed tracking mechanism. Human embryonic stem cells, hESC-, FL-, and PB derived erythroblasts were studied. The enrichment of H3K4me3 and AcH3 acrossed the beta globin locus was studied using ChIP-seq.

2015-01-26 | E-GEOD-35375 | biostudies-arrayexpress

Micro-Capture-C to determine the long-range interaction of HBG1/2 promoter with the locus control region (LCR), a powerful upstream enhancer in -175 A>G mutantant HUDEP-2 cells.

Project description:Naturally occurring mutations in the γ-globin promoters can result in hereditary persistence of fetal hemoglobin (HPFH), a benign condition that ameliorates the severity of β-hemoglobinopathies through increased post-natal fetal hemoglobin (HbF) expression. Base editing to recreate the HPFH mutations is a promising approach to induce HbF. Compared with Cas9-generated indels, base-editing approaches were more potent, with the −175 A>G conversion resulting in the strongest induction of HbF by creating a new TAL1 binding motif that stimulates long-range interaction with the locus control region (LCR), a powerful upstream enhancer. Micro-Capture-C analysis showed that introducing the −175 A>G variant stimulated long-range interaction between the γ-globin promoters and the LCR in erythroid progenitor cell line. Together, these findings show that the −175 A>G variant creates a bipartite GATA–TAL1 motif, resulting in the assembly of a GATA1/TAL1/LMO2/LDB1 complex, which activates γ-globin gene transcription by enhancing its physical association with the LCR.

2023-04-28 | GSE228570 | GEO

Epigenetic modifications and chromosome conformations of the beta globin locus throughout development

2015-01-26 | GSE35375 | 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

Editing the core region in HPFH deletions alters fetal and adult globin expression for treatment of β-hemoglobinopathies [4C]

Project description:We have identified putative repressor region (PRR) to exon-1 of β-globin (βE1) in the β-globin cluster as a core region in alll the HPFH deletions that activates fetal hemoglobin and silences adult hemoglobin. To test the impact of PRR-βE1 gene editing in β-globin cluster configuration, we employed Circular chromosome conformation capture (4C) analysis to assess the LCR interaction to the gamma-globin promoter. Using HBG2 promoter as viewpoint,4C analysis showed an increased interaction of HBG promoter to the hypersensitive (HS) sites of the LCR in HUDEP-2 clones harbouring PRR-βE1 biallelic deletions compared to control HUDEP-2 cells. Link for 4C output files Mendeley - Venkatesan, Vigneshwaran (2022), “4C data set ”, Mendeley Data, V1, doi: 10.17632/cz3thnr9kf.1

2022-04-26 | GSE201388 | GEO

Expression changes in MEL cells upon differentiation and Ldb1 knockdown

2012-12-11 | GSE42839 | GEO

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