Mammalian erythropoiesis is coordinated by a series of sequential events and precise regulation of a gene expression program. The Ldb1 complex, consisting of transcription factors Tal1 and Gata1, the Lim domain protein Lmo2, and bridging protein Ldb1, regulates expression of key genes during erythropoiesis. The transcriptional co-repressor Eto2 has been shown to associate with the Ldb1 complex to inhibit transcription of target genes. Mice lacking Eto2 exhibit decreased proliferation of hematopoietic stem cells and defective murine T-cell development. However, the role of Eto2 in erythropoiesis is unclear. We find an expansion of immature erythroid cells from the murine fetal liver at the expense of mature erythrocytes in Eto2 null animals. Using RNA-seq and ChIP-seq, we identified genes functioning downstream of Eto2 at different developmental stages in mice and in an immortalized human cell line depleted of Eto2 by RNAi. Using these approaches, we are defining the role of Eto2 in normal erythropoiesis in murine and human cells. Recent genome-wide studies suggest that long non-coding RNAs (nc-RNAs) have potential as regulators of gene expression. BGL3, a non-coding RNA transcript located 2,000 bp downstream of the A-gamma-globin gene, harbors an Ldb1 transcription-factor binding site that is involved in locus control region (LCR) looping. It has been shown that the BGL3 gene interacts with the gamma-globin gene promoter and the LCR region located upstream of the beta globin genes. The expression of BGL3 parallels that of the gamma-globin genes in various erythroid cell models although at a much lower level. We mapped the 5' end of the BGL3 gene by both 5' RACE and RT-PCR in erythroid leukemia K562 cells. In addition, we knocked down and over-expressed BGL3. We found that gamma-globin expression increased when BGL3 is transiently overexpressed and decreased when BGL3 was reduced using anti-sense oligonucleotides. We further confirmed the requirement of gamma-globin transcription for BGL3 by deletion of the BGL3 gene using CRISPR/Cas9 genome editing. These analyses support the idea that long non-coding RNA BGL3 is involved in the regulation of the gamma-globin genes. To extend these results, we are knocking out different regions of the BGL3 gene using CRISPR/Cas9. Once we generate the mutants, we will perform RNA-seq experiments to investigate how BGL3 influences the expression of gamma-globin and potentially other genes in erythroid cells. Our recent findings demonstrated that the H3K9 methyltransferase G9a establishes epigenetic conditions preventing activation of gamma-globin genes during differentiation of adult erythroid progenitor cells. In this view, manipulation of G9a represents a promising epigenetic approach for treatment of -hemoglobinopathies. However, the full repertoire of activities modifying histones in the beta-globin locus during development remains unclear. Moreover, the influence of histone modification on enhancer-promoter long range interactions to activate gene expression is unknown. To address these issues, we have undertaken a genome wide CRISPR/Cas9-mediated knock out screen for epigenetic modifications essential for mediating enhancer-promoter communication. This approach should yield results with implications for understanding enhancer mechanisms and the function of overall genome folding in gene regulation.
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