Chromatin Structure, Epigenetic and Developmental Regulation of Mammalian Gene

National Institute Of Diabetes And Digestive And Kidney Diseases

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Abstract

The eukaryotic genome is organized at varying levels into chromosome territories, transcriptional compartments and topologically associating domains (TADs), which are architectural features largely shared between different cell types and across species. In contrast, within TADs, chromatin loops connect enhancers and their target genes to establish unique transcriptomes that distinguish cells and tissues from each other and underlie development and differentiation. We have been studying the widely expressed Lim domain binding 1 protein, LDB1, which we have shown plays a critical role in connecting enhancers and genes during hematopoiesis by forming a complex with erythroid cell-type specificity. LDB1 is a widely expressed and highly conserved protein from worms to humans. GATA1, TAL1 and LMO2 are erythroid transcription factors that contribute to the chromatin looping and transcription activation properties of the LDB1 complex in erythroid cells. In fact, LDB1 enhancers are the predominate activators of erythroid genes. However, the role of enhancer looping protein LDB1 in stem cells has not been explored. We generated Ldb1(-/-) embryonic stem cells (ESC) using CRISPR/Cas9 editing and observed a reduction in key stem cell factors SOX2 and KLF4 upon LDB1 loss. Embryoid bodies (EB) derived from Ldb1(-/-) ESC displayed reduced expression of lineage-specific markers and impaired ability to undergo terminal differentiation to erythroblasts. Differential gene expression, including of the Lin28-mediated self-renewal pathway genes, was observed between WT and Ldb1(-/-) ESC and EB but was most pronounced after differentiation to erythroblasts. Importantly, LDB1 occupied super enhancers, including those of pluripotency genes, in ESC together with pluripotency factors. Conditional LDB1-deficient mice displayed reduced hematopoietic stem cell markers on bone marrow cells, and dysregulation of the Lin28 pathway. Thus, LDB1 function is critical for ESC and EB development and becomes progressively more important during differentiation to erythroblasts. We had previously observed that an enhancer RNA (eRNA), BGLT3, encoded within the human beta-globin locus, contributes to LDB1 long range chromatin looping between the locus control region enhancer and the globin genes to activate transcription. Enhancer RNAs (eRNAs) function in diverse modes and increasing studies have shown that they play important roles in normal development and disease. However, their full role in erythropoiesis is not well understood. We analyzed published RNA-seq and Promoter Capture Hi-C data from mouse E14.5 fetal liver cells to identify enhancer RNAs in erythroid cells with long-range interactions. We discovered an erythroid-specific enhancer RNA (CpoxeRNA) transcribed from an enhancer region upstream of Cpox, an enzyme important for heme synthesis. CpoxeRNA is important for erythropoiesis, as knockdown of CpoxeRNA by shRNA results in impaired enucleation and cell proliferation during terminal differentiation. CpoxeRNA interacts with cohesin and acts both in cis and in trans to regulate erythroid genes. CpoxeRNA emerges as a potent regulator of terminal erythropoiesis. Super enhancers are important regulators of gene expression that often overlap with protein-coding genes. However, it is unclear whether the overlapping protein-coding genes and the RNA derived from them contribute to enhancer activity. Using an erythroid-specific super enhancer that overlaps the Cpox gene as a model, we found that Cpox pre-mRNA has a non-coding function in regulating neighboring protein-coding genes, CpoxeRNA expression and TAD interactions. Depletion of Cpox pre-mRNA leads to accumulation of H3K27me3 and release of p300 from the Cpox locus, activating an intra-TAD enhancer and gene expression. Additionally, a head-to-tail interaction between the TAD boundary genes Cpox and Dcbld2 was identified, facilitated by a novel type of repressive loop anchored by p300 and PRC2/H3K27me3. These results uncover a regulatory role for pre-mRNA transcribed within a super enhancer context and provide insight into head-to-tail inter-gene interaction in the regulation of gene expression and oncogene activation.