RESUMO
Precise Hox gene expression is crucial for embryonic patterning. Intra-Hox transcription factor binding and distal enhancer elements have emerged as the major regulatory modules controlling Hox gene expression. However, quantifying their relative contributions has remained elusive. Here, we introduce "synthetic regulatory reconstitution," a conceptual framework for studying gene regulation, and apply it to the HoxA cluster. We synthesized and delivered variant rat HoxA clusters (130 to 170 kilobases) to an ectopic location in the mouse genome. We found that a minimal HoxA cluster recapitulated correct patterns of chromatin remodeling and transcription in response to patterning signals, whereas the addition of distal enhancers was needed for full transcriptional output. Synthetic regulatory reconstitution could provide a generalizable strategy for deciphering the regulatory logic of gene expression in complex genomes.
Assuntos
Padronização Corporal , Regulação da Expressão Gênica no Desenvolvimento , Genes Homeobox , Proteínas de Homeodomínio , Animais , Padronização Corporal/genética , Elementos Facilitadores Genéticos , Genoma , Proteínas de Homeodomínio/genética , Camundongos , Ratos , Transcrição GênicaRESUMO
Ectopic expression of master regulatory transcription factors can reprogram the identity of specific cell types. The effectiveness of such induced cellular reprogramming is generally greatly reduced if the cellular substrates are fully differentiated cells. For example, in the nematode C. elegans, the ectopic expression of a neuronal identity-inducing transcription factor, CHE-1, can effectively induce CHE-1 target genes in immature cells but not in fully mature non-neuronal cells. To understand the molecular basis of this progressive restriction of cellular plasticity, we screened for C. elegans mutants in which ectopically expressed CHE-1 is able to induce neuronal effector genes in epidermal cells. We identified a ubiquitin hydrolase, usp-48, that restricts cellular plasticity with a notable cellular specificity. Even though we find usp-48 to be very broadly expressed in all tissue types, usp-48 null mutants specifically make epidermal cells susceptible to CHE-1-mediated activation of neuronal target genes. We screened for additional genes that allow epidermal cells to be at least partially reprogrammed by ectopic che-1 expression and identified many additional proteins that restrict cellular plasticity of epidermal cells, including a chromatin-related factor (H3K79 methyltransferase, DOT-1.1), a transcription factor (nuclear hormone receptor NHR-48), two MAPK-type protein kinases (SEK-1 and PMK-1), a nuclear localized O-GlcNAc transferase (OGT-1) and a member of large family of nuclear proteins related to the Rb-associated LIN-8 chromatin factor. These findings provide novel insights into the control of cellular plasticity.
Assuntos
Plasticidade Celular/genética , Cromatina/genética , Proteínas Quinases/genética , Fatores de Transcrição/genética , Sequência de Aminoácidos , Animais , Caenorhabditis elegans/genética , Diferenciação Celular , Reprogramação Celular/genética , Cromatina/metabolismo , Biologia Computacional/métodos , Epiderme/metabolismo , Regulação da Expressão Gênica , Histonas/metabolismo , Humanos , Hibridização in Situ Fluorescente , Família Multigênica , Mutagênese , Mutação , Transporte Proteico , Interferência de RNA , Transgenes , Proteases Específicas de Ubiquitina/química , Proteases Específicas de Ubiquitina/genética , Proteases Específicas de Ubiquitina/metabolismo , Sequenciamento Completo do GenomaRESUMO
How specific cell types can be directly converted into other distinct cell types is a matter of intense investigation with wide-ranging basic and biomedical implications. Here, we show that removal of the histone 3 lysine 27 (H3K27) methyltransferase Polycomb repressor complex 2 (PRC2) permits ectopically expressed, neuron-type-specific transcription factors ("terminal selectors") to convert Caenorhabditis elegans germ cells directly into specific neuron types. Terminal-selector-induced germ-cell-to-neuron conversion can be observed not only upon genome-wide loss of H3K27 methylation in PRC2(-) animals but also upon genome-wide redistribution of H3K27 methylation patterns in animals that lack the H3K36 methyltransferase MES-4. Manipulation of the H3K27 methylation status not only permits conversion of germ cells into neurons but also permits hlh-1/MyoD-dependent conversion of germ cells into muscle cells, indicating that PRC2 protects the germline from the aberrant execution of multiple distinct somatic differentiation programs. Taken together, our findings demonstrate that the normally multistep process of development from a germ cell via a zygote to a terminally differentiated somatic cell type can be short-cut by providing an appropriate terminal selector transcription factor and manipulating histone methylation patterns.