RESUMO
Distal enhancers commonly contact target promoters via chromatin looping. In erythroid cells, the locus control region (LCR) contacts ß-type globin genes in a developmental stage-specific manner to stimulate transcription. Previously, we induced LCR-promoter looping by tethering the self-association domain (SA) of Ldb1 to the ß-globin promoter via artificial zinc fingers. Here, we show that targeting the SA to a developmentally silenced embryonic globin gene in adult murine erythroblasts triggers its transcriptional reactivation. This activity depends on the LCR, consistent with an LCR-promoter looping mechanism. Strikingly, targeting the SA to the fetal γ-globin promoter in primary adult human erythroblasts increases γ-globin promoter-LCR contacts, stimulating transcription to approximately 85% of total ß-globin synthesis, with a reciprocal reduction in adult ß-globin expression. Our findings demonstrate that forced chromatin looping can override a stringent developmental gene expression program and suggest a novel approach to control the balance of globin gene transcription for therapeutic applications.
Assuntos
Cromatina/metabolismo , Hemoglobina Fetal/genética , Técnicas Genéticas , Região de Controle de Locus Gênico , Ativação Transcricional , Globinas beta/genética , Animais , Antígenos CD34/metabolismo , Cromatina/química , Embrião de Mamíferos/metabolismo , Eritroblastos/metabolismo , Hemoglobinopatias/genética , Hemoglobinopatias/terapia , Humanos , Camundongos , Cultura Primária de CélulasRESUMO
During mitosis, RNA polymerase II (Pol II) and many transcription factors dissociate from chromatin, and transcription ceases globally. Transcription is known to restart in bulk by telophase, but whether de novo transcription at the mitosis-G1 transition is in any way distinct from later in interphase remains unknown. We tracked Pol II occupancy genome-wide in mammalian cells progressing from mitosis through late G1. Unexpectedly, during the earliest rounds of transcription at the mitosis-G1 transition, â¼50% of active genes and distal enhancers exhibit a spike in transcription, exceeding levels observed later in G1 phase. Enhancer-promoter chromatin contacts are depleted during mitosis and restored rapidly upon G1 entry but do not spike. Of the chromatin-associated features examined, histone H3 Lys27 acetylation levels at individual loci in mitosis best predict the mitosis-G1 transcriptional spike. Single-molecule RNA imaging supports that the mitosis-G1 transcriptional spike can constitute the maximum transcriptional activity per DNA copy throughout the cell division cycle. The transcriptional spike occurs heterogeneously and propagates to cell-to-cell differences in mature mRNA expression. Our results raise the possibility that passage through the mitosis-G1 transition might predispose cells to diverge in gene expression states.
Assuntos
Ciclo Celular/genética , Fase G1/genética , Genoma/genética , Mitose/genética , Ativação Transcricional/genética , Animais , Linhagem Celular , Células Cultivadas , Cromatina/metabolismo , DNA Intergênico/genética , Elementos Facilitadores Genéticos/genética , Eritroblastos/citologia , Camundongos , Regiões Promotoras Genéticas/genética , Regulação para CimaRESUMO
Inhibitors of bromodomain and extraterminal motif proteins (BETs) are being evaluated for the treatment of cancer and other diseases, yet much remains to be learned about how BET proteins function during normal physiology. We used genomic and genetic approaches to examine BET function in a hematopoietic maturation system driven by GATA1, an acetylated transcription factor previously shown to interact with BETs. We found that BRD2, BRD3, and BRD4 were variably recruited to GATA1-regulated genes, with BRD3 binding the greatest number of GATA1-occupied sites. Pharmacologic BET inhibition impaired GATA1-mediated transcriptional activation, but not repression, genome-wide. Mechanistically, BETs promoted chromatin occupancy of GATA1 and subsequently supported transcriptional activation. Using a combination of CRISPR-Cas9-mediated genomic engineering and shRNA approaches, we observed that depletion of either BRD2 or BRD4 alone blunted erythroid gene activation. Surprisingly, depletion of BRD3 only affected erythroid transcription in the context of BRD2 deficiency. Consistent with functional overlap among BET proteins, forced BRD3 expression substantially rescued defects caused by BRD2 deficiency. These results suggest that pharmacologic BET inhibition should be interpreted in the context of distinct steps in transcriptional activation and overlapping functions among BET family members.
Assuntos
Células Eritroides/metabolismo , Regulação da Expressão Gênica , Hematopoese/genética , Proteínas Serina-Treonina Quinases/fisiologia , Proteínas de Ligação a RNA/fisiologia , Animais , Células Cultivadas , Fator de Transcrição GATA1/genética , Técnicas de Silenciamento de Genes , Humanos , Camundongos , Proteínas Serina-Treonina Quinases/química , Estrutura Terciária de Proteína , Proteínas de Ligação a RNA/químicaRESUMO
Background On October 1, 2015, the Center for Medicare and Medicaid Services transitioned from the International Classification of Diseases, Ninth Revision ( ICD-9) to the ICD, Tenth Revision ( ICD-10) compendium of codes for diagnosis and billing in health care, but translation between the two is often inexact. Here we describe a validated crosswalk to translate ICD-9 codes into ICD-10 codes, with a focus on complications after carotid revascularization and endovascular aortic aneurysm repair. Methods and Results We devised an 8-step process to derive and validate ICD-10 codes from existing ICD-9 codes. We used publicly available sources, including the General Equivalence Mapping database, to translate ICD-9 codes used in prior work to ICD-10 codes. We defined ICD-10 codes as validated if they were concordant with the initial ICD-9 codes after manual comparison by two physicians. Our primary validation measure was the percent of valid ICD-10 codes out of the total ICD-10 codes obtained during translation. We began with 126 ICD-9 diagnosis codes used for complication identification after carotid revascularization procedures, and 97 ICD-9 codes for complications after endovascular aortic aneurysm procedures. Translation generated 143 ICD-10 codes for carotid revascularization, a 14% increase from the initial 126 codes. Manual comparison demonstrated 98% concordance, with 99% agreement between the reviewers. Similarly, we identified 108 ICD-10 codes for endovascular aortic aneurysm repair, an 11% increase from the initial 97 ICD-9 codes. We again noted excellent concordance and agreement (98% and 100%, respectively). Manual review identified 4 ICD-10 codes incorrectly translated from ICD-9 codes for carotid revascularization, and 3 codes incorrectly translated for endovascular aortic aneurysm repair. Conclusions Algorithms to crosswalk lists of ICD-9 codes to ICD-10 codes can leverage electronic resources to minimize the burden of code translation. However, manual review for code validation may be necessary, with collaboration across institutions for researchers to share their efforts.
Assuntos
Algoritmos , Angioplastia/efeitos adversos , Aneurisma Aórtico/cirurgia , Doenças das Artérias Carótidas/terapia , Mineração de Dados/métodos , Endarterectomia das Carótidas/efeitos adversos , Procedimentos Endovasculares/efeitos adversos , Pesquisa sobre Serviços de Saúde/métodos , Classificação Internacional de Doenças , Avaliação de Resultados em Cuidados de Saúde/métodos , Complicações Pós-Operatórias/etiologia , Angioplastia/instrumentação , Aneurisma Aórtico/diagnóstico , Doenças das Artérias Carótidas/diagnóstico , Bases de Dados Factuais , Humanos , Complicações Pós-Operatórias/classificação , Complicações Pós-Operatórias/diagnóstico , Reprodutibilidade dos Testes , Medição de Risco , Fatores de Risco , Stents , Resultado do TratamentoRESUMO
Polyribosomes, mRNA, and other elements of translational machinery have been reported in peripheral nerves and in elongating injured axons of sensory neurons in vitro, primarily in growth cones. Evidence for involvement of local protein synthesis in regenerating central nervous system (CNS) axons is less extensive. We monitored regeneration of back-labeled lamprey spinal axons after spinal cord transection and detected mRNA in axon tips by in situ hybridization and microaspiration of their axoplasm. Poly(A)+mRNA was present in the axon tips, and was more abundant in actively regenerating tips than in static or retracting ones. Target-specific polymerase chain reaction (PCR) and in situ hybridization revealed plentiful mRNA for the low molecular neurofilament subunit and ß-tubulin, but very little for ß-actin, consistent with the morphology of their tips, which lack filopodia and lamellipodia. Electron microscopy showed ribosomes/polyribosomes in the distal parts of axon tips and in association with vesicle-like membranes, primarily in the tip. In one instance, there were structures with the appearance of rough endoplasmic reticulum. Immunohistochemistry showed patches of ribosomal protein S6 positivity in a similar distribution. The results suggest that local protein synthesis might be involved in the mechanism of axon regeneration in the lamprey spinal cord. J. Comp. Neurol. 524:3614-3640, 2016. © 2016 Wiley Periodicals, Inc.