RESUMEN
ABSTRACT: Long noncoding RNAs (lncRNAs) are extensively expressed in eukaryotic cells and have been revealed to be important for regulating cell differentiation. Many lncRNAs have been found to regulate erythroid differentiation in the mouse. However, given the low sequence conservation of lncRNAs between mouse and human, our understanding of lncRNAs in human erythroid differentiation remains incomplete. lncRNAs are often transcribed opposite to protein coding genes and regulate their expression. Here, we characterized a human erythrocyte-expressed lncRNA, GATA2AS, which is transcribed opposite to erythroid transcription regulator GATA2. GATA2AS is a 2080-bp long, primarily nucleus-localized noncoding RNA that is expressed in erythroid progenitor cells and decreases during differentiation. Knockout of GATA2AS in human HUDEP2 erythroid progenitor cells using CRISPR-Cas9 genome editing to remove the transcription start site accelerated erythroid differentiation and dysregulated erythroblast gene expression. We identified GATA2AS as a novel GATA2 and HBG activator. Chromatin isolation by RNA purification showed that GATA2AS binds to thousands of genomic sites and colocalizes at a subset of sites with erythroid transcription factors including LRF and KLF1. RNA pulldown and RNA immunoprecipitation confirmed interaction between GATA2AS and LRF and KLF1. Chromatin immunoprecipitation sequencing (ChIP-seq) showed that knockout of GATA2AS reduces binding of these transcription factors genome wide. Assay for transposase-accessible chromatin sequencing (ATAC-seq) and H3K27ac ChIP-seq showed that GATA2AS is essential to maintain the chromatin regulatory landscape during erythroid differentiation. Knockdown of GATA2AS in human primary CD34+ cells mimicked results in HUDEP2 cells. Overall, our results implicate human-specific lncRNA GATA2AS as a regulator of erythroid differentiation by influencing erythroid transcription factor binding and the chromatin regulatory landscape.
Asunto(s)
Cromatina , Eritropoyesis , Factor de Transcripción GATA2 , ARN Largo no Codificante , Humanos , Eritropoyesis/genética , ARN Largo no Codificante/genética , Cromatina/metabolismo , Cromatina/genética , Factor de Transcripción GATA2/genética , Factor de Transcripción GATA2/metabolismo , Diferenciación Celular/genética , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Células Precursoras Eritroides/metabolismo , Células Precursoras Eritroides/citologíaRESUMEN
The Myb proto-oncogene encodes the transcription factor c-MYB, which is critical for hematopoiesis. Distant enhancers of Myb form a hub of interactions with the Myb promoter. We identified a long non-coding RNA (Myrlin) originating from the -81-kb murine Myb enhancer. Myrlin and Myb are coordinately regulated during erythroid differentiation. Myrlin TSS deletion using CRISPR-Cas9 reduced Myrlin and Myb expression and LDB1 complex occupancy at the Myb enhancers, compromising enhancer contacts and reducing RNA Pol II occupancy in the locus. In contrast, CRISPRi silencing of Myrlin left LDB1 and the Myb enhancer hub unperturbed, although Myrlin and Myb expressions were downregulated, decoupling transcription and chromatin looping. Myrlin interacts with the KMT2A/MLL1 complex. Myrlin CRISPRi compromised KMT2A occupancy in the Myb locus, decreasing CDK9 and RNA Pol II binding and resulting in Pol II pausing in the Myb first exon/intron. Thus, Myrlin directly participates in activating Myb transcription by recruiting KMT2A.
Asunto(s)
Elementos de Facilitación Genéticos , N-Metiltransferasa de Histona-Lisina , Proteína de la Leucemia Mieloide-Linfoide , Proteínas Proto-Oncogénicas c-myb , Transcripción Genética , Proteínas Proto-Oncogénicas c-myb/metabolismo , Proteínas Proto-Oncogénicas c-myb/genética , Animales , Proteína de la Leucemia Mieloide-Linfoide/metabolismo , Proteína de la Leucemia Mieloide-Linfoide/genética , Ratones , N-Metiltransferasa de Histona-Lisina/metabolismo , N-Metiltransferasa de Histona-Lisina/genética , Elementos de Facilitación Genéticos/genética , ARN Polimerasa II/metabolismo , ARN Largo no Codificante/genética , ARN Largo no Codificante/metabolismo , Humanos , Quinasa 9 Dependiente de la Ciclina/metabolismo , Quinasa 9 Dependiente de la Ciclina/genética , Proto-Oncogenes Mas , Unión Proteica , Diferenciación Celular/genética , ARN PotenciadoresRESUMEN
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 mRNA 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 mRNA has a non-coding function in regulating neighboring protein-coding genes, eRNA expression and TAD interactions. Depletion of Cpox mRNA leads to accumulation of H3K27me3 and release of p300 from the Cpox locus, activating an intra-TAD enhancer and gene expression. Additionally, we identified a head-to-tail interaction between the TAD boundary genes Cpox and Dcbld2 that is facilitated by a novel type of repressive loop anchored by p300 and PRC2/H3K27me3. Our results uncover a regulatory role for 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.