RESUMO
Multipotent progenitor cells confirm their T cell-lineage identity in the CD4-CD8- double-negative (DN) pro-T cell DN2 stages, when expression of the essential transcription factor Bcl11b begins. In vivo and in vitro stage-specific deletions globally identified Bcl11b-controlled target genes in pro-T cells. Proteomics analysis revealed that Bcl11b associated with multiple cofactors and that its direct action was needed to recruit those cofactors to selective target sites. Regions near functionally regulated target genes showed enrichment for those sites of Bcl11b-dependent recruitment of cofactors, and deletion of individual cofactors relieved the repression of many genes normally repressed by Bcl11b. Runx1 collaborated with Bcl11b most frequently for both activation and repression. In parallel, Bcl11b indirectly regulated a subset of target genes by a gene network circuit via the transcription inhibitor Id2 (encoded by Id2) and transcription factor PLZF (encoded by Zbtb16); Id2 and Zbtb16 were directly repressed by Bcl11b, and Id2 and PLZF controlled distinct alternative programs. Thus, our study defines the molecular basis of direct and indirect Bcl11b actions that promote T cell identity and block alternative potentials.
Assuntos
Linfopoese/imunologia , Células Precursoras de Linfócitos T/imunologia , Proteína com Dedos de Zinco da Leucemia Promielocítica/biossíntese , Proteínas Repressoras/imunologia , Proteínas Supressoras de Tumor/imunologia , Animais , Diferenciação Celular/imunologia , Regulação da Expressão Gênica/imunologia , Proteína 2 Inibidora de Diferenciação/biossíntese , Proteína 2 Inibidora de Diferenciação/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Células Precursoras de Linfócitos T/citologia , Proteína com Dedos de Zinco da Leucemia Promielocítica/imunologiaRESUMO
Runt domain-related (Runx) transcription factors are essential for early T cell development in mice from uncommitted to committed stages. Single and double Runx knockouts via Cas9 show that target genes responding to Runx activity are not solely controlled by the dominant factor, Runx1. Instead, Runx1 and Runx3 are coexpressed in single cells; bind to highly overlapping genomic sites; and have redundant, collaborative functions regulating genes pivotal for T cell development. Despite stable combined expression levels across pro-T cell development, Runx1 and Runx3 preferentially activate and repress genes that change expression dynamically during lineage commitment, mostly activating T-lineage genes and repressing multipotent progenitor genes. Furthermore, most Runx target genes are sensitive to Runx perturbation only at one stage and often respond to Runx more for expression transitions than for maintenance. Contributing to this highly stage-dependent gene regulation function, Runx1 and Runx3 extensively shift their binding sites during commitment. Functionally distinct Runx occupancy sites associated with stage-specific activation or repression are also distinguished by different patterns of partner factor cobinding. Finally, Runx occupancies change coordinately at numerous clustered sites around positively or negatively regulated targets during commitment. This multisite binding behavior may contribute to a developmental "ratchet" mechanism making commitment irreversible.
Assuntos
Linhagem da Célula/imunologia , Subunidade alfa 2 de Fator de Ligação ao Core/genética , Subunidade alfa 3 de Fator de Ligação ao Core/genética , Células Precursoras de Linfócitos T/imunologia , Linfócitos T/imunologia , Transcriptoma , Animais , Diferenciação Celular , Linhagem da Célula/genética , Subunidade alfa 2 de Fator de Ligação ao Core/imunologia , Subunidade alfa 3 de Fator de Ligação ao Core/imunologia , Feminino , Perfilação da Expressão Gênica , Regulação da Expressão Gênica , Subunidade alfa de Receptor de Interleucina-2/genética , Subunidade alfa de Receptor de Interleucina-2/imunologia , Masculino , Camundongos , Células Precursoras de Linfócitos T/citologia , Cultura Primária de Células , Proteínas Repressoras/genética , Proteínas Repressoras/imunologia , Linfócitos T/classificação , Linfócitos T/citologia , Proteínas Supressoras de Tumor/genética , Proteínas Supressoras de Tumor/imunologiaRESUMO
Genetic mutations or regulatory failures underlie cellular malfunction in many diseases, including colorectal cancer and inflammatory bowel diseases. However, mutational defects alone fail to explain the complexity of such disorders. Epigenetic regulation-control of gene action through chemical and structural changes of chromatin-provides a platform to integrate multiple extracellular inputs and prepares the cellular genome for appropriate gene expression responses. Coregulation by polycomb repressive complex 2-mediated trimethylation of lysine 27 on histone 3 and DNA methylation has emerged as one of the most influential epigenetic controls in colorectal cancer and many other diseases, but molecular details remain inadequate. Here we review the molecular interplay of these epigenetic features in relation to gastrointestinal development, homeostasis, and disease biology. We discuss other epigenetic mechanisms pertinent to the balance of trimethylation of lysine 27 on histone 3 and DNA methylation and their actions in gastrointestinal cancers. We also review the current molecular understanding of chromatin control in the pathogenesis of inflammatory bowel diseases.
Assuntos
Metilação de DNA/genética , Epigênese Genética/genética , Neoplasias Gastrointestinais/genética , Histonas/genética , Células-Tronco/fisiologia , Cromatina/genética , Humanos , Doenças Inflamatórias Intestinais/genética , Lisina/genética , Complexo Repressor Polycomb 2/genética , Processamento de Proteína Pós-TraducionalRESUMO
T-cell development from hematopoietic progenitors depends on multiple transcription factors, mobilized and modulated by intrathymic Notch signaling. Key aspects of T-cell specification network architecture have been illuminated through recent reports defining roles of transcription factors PU.1, GATA-3, and E2A, their interactions with Notch signaling, and roles of Runx1, TCF-1, and Hes1, providing bases for a comprehensively updated model of the T-cell specification gene regulatory network presented herein. However, the role of lineage commitment factor Bcl11b has been unclear. We use self-organizing maps on 63 RNA-seq datasets from normal and perturbed T-cell development to identify functional targets of Bcl11b during commitment and relate them to other regulomes. We show that both activation and repression target genes can be bound by Bcl11b in vivo, and that Bcl11b effects overlap with E2A-dependent effects. The newly clarified role of Bcl11b distinguishes discrete components of commitment, resolving how innate lymphoid, myeloid, and dendritic, and B-cell fate alternatives are excluded by different mechanisms.
Assuntos
Diferenciação Celular/genética , Redes Reguladoras de Genes , Proteínas Repressoras/fisiologia , Linfócitos T/citologia , Proteínas Supressoras de Tumor/fisiologia , Animais , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Receptores Notch , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismo , Transdução de Sinais , Proteínas Supressoras de Tumor/genética , Proteínas Supressoras de Tumor/metabolismoRESUMO
As early T cell precursors transition from multipotentiality to T lineage commitment, they change expression of multiple transcription factors. It is unclear whether individual transcription factors directly control choices between T cell identity and some alternative fate or whether these factors mostly affect proliferation or survival during the normal commitment process. Here, we unraveled the impacts of deleting individual transcription factors at two stages in early T cell development, using synchronized in vitro differentiation systems, single-cell RNA-seq with batch indexing, and controlled gene-disruption strategies. First, using a customized method for single-cell CRISPR disruption, we defined how the early-acting transcription factors Bcl11a, Erg, Spi1 (PU.1), Gata3, and Tcf7 (TCF1) function before commitment. The results revealed a kinetic tug of war within individual cells between T cell factors Tcf7 and Gata3 and progenitor factors Spi1 and Bcl11a, with an unexpected guidance role for Erg. Second, we tested how activation of transcription factor Bcl11b during commitment altered ongoing cellular programs. In knockout cells where Bcl11b expression was prevented, the cells did not undergo developmental arrest, instead following an alternative path as T lineage commitment was blocked. A stepwise, time-dependent regulatory cascade began with immediate-early transcription factor activation and E protein inhibition, finally leading Bcl11b knockout cells toward exit from the T cell pathway. Last, gene regulatory networks of transcription factor cross-regulation were extracted from the single-cell transcriptome results, characterizing the specification network operating before T lineage commitment and revealing its links to both the Bcl11b knockout alternative network and the network consolidating T cell identity during commitment.
Assuntos
Redes Reguladoras de Genes , Linfócitos T , Diferenciação Celular/genética , Linhagem da Célula/genética , Linfócitos T/metabolismo , Proteínas Supressoras de Tumor/genéticaRESUMO
PU.1 (encoded by Spi1), an ETS-family transcription factor with many hematopoietic roles, is highly expressed in the earliest intrathymic T cell progenitors but must be down-regulated during T lineage commitment. The transcription factors Runx1 and GATA3 have been implicated in this Spi1 repression, but the basis of the timing was unknown. We show that increasing Runx1 and/or GATA3 down-regulates Spi1 expression in pro-T cells, while deletion of these factors after Spi1 down-regulation reactivates its expression. Leveraging the stage specificities of repression and transcription factor binding revealed an unconventional but functional site in Spi1 intron 2. Acute Cas9-mediated deletion or disruption of the Runx and GATA motifs in this element reactivates silenced Spi1 expression in a pro-T cell line, substantially more than disruption of other candidate elements, and counteracts the repression of Spi1 in primary pro-T cells during commitment. Thus, Runx1 and GATA3 work stage specifically through an intronic silencing element in mouse Spi1 to control strength and maintenance of Spi1 repression during T lineage commitment.
Assuntos
Subunidade alfa 2 de Fator de Ligação ao Core/metabolismo , Fator de Transcrição GATA3/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Linfócitos T/metabolismo , Transativadores/metabolismo , Animais , Sequência de Bases , Sítios de Ligação , Linhagem da Célula , Subunidade alfa 2 de Fator de Ligação ao Core/química , Fator de Transcrição GATA3/química , Deleção de Genes , Perfilação da Expressão Gênica , Inativação Gênica , Loci Gênicos , Íntrons/genética , Camundongos Endogâmicos C57BL , Proteínas Repressoras/metabolismo , Proteínas Supressoras de Tumor/metabolismoRESUMO
Notch signaling is the dominant intercellular signaling input during the earliest stages of T cell development in the thymus. Although Notch1 is known to be indispensable, we show that it does not mediate all Notch signaling in precommitment stages: Notch2 initially works in parallel to promote early murine T cell development and antagonize other fates. Notch-regulated target genes before and after T lineage commitment change dynamically, and we show that this partially reflects shifts in genome-wide DNA binding by RBPJ, the transcription factor activated by complex formation with the Notch intracellular domain. Although Notch signaling and transcription factor PU.1 can activate some common targets in precommitment T progenitors, Notch signaling and PU.1 activity have functionally antagonistic effects on multiple targets, delineating separation of pro-T cells from alternative PU.1-dependent fates. These results define a distinct mechanism of Notch signal response that distinguishes the initial stages of murine T cell development.
Assuntos
Proteínas Proto-Oncogênicas/genética , Receptor Notch1/genética , Receptor Notch2/genética , Linfócitos T/metabolismo , Transativadores/genética , Animais , Diferenciação Celular , Regulação da Expressão Gênica no Desenvolvimento/imunologia , Humanos , Camundongos , Transdução de Sinais/genética , Linfócitos T/imunologiaRESUMO
The zinc finger transcription factor, Bcl11b, is expressed in T cells and group 2 innate lymphoid cells (ILC2s) among hematopoietic cells. In early T-lineage cells, Bcl11b directly binds and represses the gene encoding the E protein antagonist, Id2, preventing pro-T cells from adopting innate-like fates. In contrast, ILC2s co-express both Bcl11b and Id2. To address this contradiction, we have directly compared Bcl11b action mechanisms in pro-T cells and ILC2s. We found that Bcl11b binding to regions across the genome shows distinct cell type-specific motif preferences. Bcl11b occupies functionally different sites in lineage-specific patterns and controls totally different sets of target genes in these cell types. In addition, Bcl11b bears cell type-specific post-translational modifications and organizes different cell type-specific protein complexes. However, both cell types use the same distal enhancer region to control timing of Bcl11b activation. Therefore, although pro-T cells and ILC2s both need Bcl11b for optimal development and function, Bcl11b works substantially differently in these two cell types.