RESUMO
Transposable elements (TEs) are abundant in the human genome, and they provide the sources for genetic and functional diversity. The regulation of TEs expression and their functional consequences in physiological conditions and cancer development remain to be fully elucidated. Previous studies suggested TEs are repressed by DNA methylation and chromatin modifications. The effect of 3D chromatin topology on TE regulation remains elusive. Here, by integrating transcriptome and 3D genome architecture studies, we showed that haploinsufficient loss of NIPBL selectively activates alternative promoters at the long terminal repeats (LTRs) of the TE subclasses. This activation occurs through the reorganization of topologically associating domain (TAD) hierarchical structures and recruitment of proximal enhancers. These observations indicate that TAD hierarchy restricts transcriptional activation of LTRs that already possess open chromatin features. In cancer, perturbation of the hierarchical chromatin topology can lead to co-option of LTRs as functional alternative promoters in a context-dependent manner and drive aberrant transcriptional activation of novel oncogenes and other divergent transcripts. These data uncovered a new layer of regulatory mechanism of TE expression beyond DNA and chromatin modification in human genome. They also posit the TAD hierarchy dysregulation as a novel mechanism for alternative promoter-mediated oncogene activation and transcriptional diversity in cancer, which may be exploited therapeutically.
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INTRODUCTION: The pathogenesis of thymic epithelial tumors remains largely unknown. We previously identified GTF2I L424H as the most frequently recurrent mutation in thymic epithelial tumors. Nevertheless, the precise role of this mutation in tumorigenesis of thymic epithelial cells is unclear. METHODS: To investigate the role of GTF2I L424H mutation in thymic epithelial cells in vivo, we generated and characterized a mouse model in which the Gtf2i L424H mutation was conditionally knocked-in in the Foxn1+ thymic epithelial cells. Digital spatial profiling was performed on thymomas and normal thymic tissues with GeoMx-mouse whole transcriptome atlas. Immunohistochemistry staining was performed using both mouse tissues and human thymic epithelial tumors. RESULTS: We observed that the Gtf2i mutation impairs development of the thymic medulla and maturation of medullary thymic epithelial cells in young mice and causes tumor formation in the thymus of aged mice. Cell cycle-related pathways, such as E2F targets and MYC targets, are enriched in the tumor epithelial cells. Results of gene set variation assay analysis revealed that gene signatures of cortical thymic epithelial cells and thymic epithelial progenitor cells are also enriched in the thymomas of the knock-in mice, which mirrors the human counterparts in The Cancer Genome Atlas database. Immunohistochemistry results revealed similar expression pattern of epithelial cell markers between mouse and human thymomas. CONCLUSIONS: We have developed and characterized a novel thymoma mouse model. This study improves knowledge of the molecular drivers in thymic epithelial cells and provides a tool for further study of the biology of thymic epithelial tumors and for development of novel therapies.
Assuntos
Neoplasias Epiteliais e Glandulares , Timoma , Neoplasias do Timo , Fatores de Transcrição TFIII , Fatores de Transcrição TFII , Animais , Humanos , Camundongos , Mutação , Neoplasias Epiteliais e Glandulares/genética , Neoplasias Epiteliais e Glandulares/patologia , Timoma/genética , Timoma/patologia , Neoplasias do Timo/genética , Neoplasias do Timo/patologia , Fatores de Transcrição TFII/genética , Fatores de Transcrição TFIII/genéticaRESUMO
Proper lymphopoiesis and immune responses depend on the spatiotemporal control of multiple processes, including gene expression, DNA recombination and cell fate decisions. High-order 3D chromatin organization is increasingly appreciated as an important regulator of these processes and dysregulation of genomic architecture has been linked to various immune disorders, including lymphoid malignancies. In this review, we present the general principles of the 3D chromatin topology and its dynamic reorganization during various steps of B and T lymphocyte development and activation. We also discuss functional interconnections between architectural, epigenetic and transcriptional changes and introduce major key players of genomic organization in B/T lymphocytes. Finally, we present how alterations in architectural factors and/or 3D genome organization are linked to dysregulation of the lymphopoietic transcriptional program and ultimately to hematological malignancies.
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Linfócitos B/imunologia , Transformação Celular Neoplásica/imunologia , Montagem e Desmontagem da Cromatina , Leucemia/imunologia , Ativação Linfocitária , Linfoma/imunologia , Linfopoese , Linfócitos T/imunologia , Animais , Linfócitos B/metabolismo , Linfócitos B/patologia , Diferenciação Celular , Linhagem da Célula , Proliferação de Células , Transformação Celular Neoplásica/genética , Transformação Celular Neoplásica/metabolismo , Transformação Celular Neoplásica/patologia , Epigênese Genética , Regulação Leucêmica da Expressão Gênica , Humanos , Leucemia/genética , Leucemia/metabolismo , Leucemia/patologia , Linfoma/genética , Linfoma/metabolismo , Linfoma/patologia , Fenótipo , Linfócitos T/metabolismo , Linfócitos T/patologiaRESUMO
Linker histone H1 proteins bind to nucleosomes and facilitate chromatin compaction1, although their biological functions are poorly understood. Mutations in the genes that encode H1 isoforms B-E (H1B, H1C, H1D and H1E; also known as H1-5, H1-2, H1-3 and H1-4, respectively) are highly recurrent in B cell lymphomas, but the pathogenic relevance of these mutations to cancer and the mechanisms that are involved are unknown. Here we show that lymphoma-associated H1 alleles are genetic driver mutations in lymphomas. Disruption of H1 function results in a profound architectural remodelling of the genome, which is characterized by large-scale yet focal shifts of chromatin from a compacted to a relaxed state. This decompaction drives distinct changes in epigenetic states, primarily owing to a gain of histone H3 dimethylation at lysine 36 (H3K36me2) and/or loss of repressive H3 trimethylation at lysine 27 (H3K27me3). These changes unlock the expression of stem cell genes that are normally silenced during early development. In mice, loss of H1c and H1e (also known as H1f2 and H1f4, respectively) conferred germinal centre B cells with enhanced fitness and self-renewal properties, ultimately leading to aggressive lymphomas with an increased repopulating potential. Collectively, our data indicate that H1 proteins are normally required to sequester early developmental genes into architecturally inaccessible genomic compartments. We also establish H1 as a bona fide tumour suppressor and show that mutations in H1 drive malignant transformation primarily through three-dimensional genome reorganization, which leads to epigenetic reprogramming and derepression of developmentally silenced genes.
Assuntos
Transformação Celular Neoplásica/genética , Cromatina/química , Cromatina/genética , Histonas/deficiência , Histonas/genética , Linfoma/genética , Linfoma/patologia , Alelos , Animais , Linfócitos B/metabolismo , Linfócitos B/patologia , Autorrenovação Celular , Cromatina/metabolismo , Montagem e Desmontagem da Cromatina/genética , Epigênese Genética , Regulação Neoplásica da Expressão Gênica , Inativação Gênica , Genes Supressores de Tumor , Centro Germinativo/patologia , Histonas/metabolismo , Humanos , Linfoma/metabolismo , Camundongos , Mutação , Células-Tronco/metabolismo , Células-Tronco/patologiaRESUMO
The hierarchical three-dimensional folding of the mammalian genome constitutes an important regulatory layer of gene expression and cell fate control during processes such as development and tumorigenesis. Accumulating evidence supports the existence of complex topological assemblies in which multiple genes and regulatory elements are frequently interacting with each other in the 3D nucleus. Here, we will discuss the nature, organizational principles, and potential function of such assemblies, including the recently reported enhancer "hubs," "cliques," and FIREs (frequently interacting regions) as well as multi-contact hubs. We will also review recent studies that investigate the role of transcription factors (TFs) in driving the topological genome reorganization and hub formation in the context of cell fate transitions and cancer. Finally, we will highlight technological advances that enabled these studies, current limitations, and future directions necessary to advance our understating in the field.
Assuntos
Núcleo Celular/metabolismo , Montagem e Desmontagem da Cromatina , Cromatina/metabolismo , Fatores de Transcrição/metabolismo , Animais , Sítios de Ligação , Núcleo Celular/genética , Cromatina/genética , Epigênese Genética , Humanos , Conformação de Ácido Nucleico , Regiões Promotoras Genéticas , Conformação Proteica , Fatores de Transcrição/genética , Transcrição GênicaRESUMO
Recent studies have shown that mutations at non-coding elements, such as promoters and enhancers, can act as cancer drivers. However, an important class of non-coding elements, namely CTCF insulators, has been overlooked in the previous driver analyses. We used insulator annotations from CTCF and cohesin ChIA-PET and analyzed somatic mutations in 1,962 whole genomes from 21 cancer types. Using the heterogeneous patterns of transcription-factor-motif disruption, functional impact, and recurrence of mutations, we developed a computational method that revealed 21 insulators showing signals of positive selection. In particular, mutations in an insulator in multiple cancer types, including 16% of melanoma samples, are associated with TGFB1 up-regulation. Using CRISPR-Cas9, we find that alterations at two of the most frequently mutated regions in this insulator increase cell growth by 40%-50%, supporting the role of this boundary element as a cancer driver. Thus, our study reveals several CTCF insulators as putative cancer drivers.
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Fator de Ligação a CCCTC/genética , Fator de Ligação a CCCTC/metabolismo , Animais , Proteínas de Ciclo Celular/genética , Proteínas Cromossômicas não Histona/genética , Proteínas de Ligação a DNA/genética , Regulação da Expressão Gênica/genética , Regulação Neoplásica da Expressão Gênica/genética , Genoma Humano , Humanos , Mutação , Neoplasias/genética , Neoplasias/metabolismo , Regiões Promotoras Genéticas/genética , Proteínas Repressoras/genética , CoesinasRESUMO
Self-renewal and pluripotency of the embryonic stem cell (ESC) state are established and maintained by multiple regulatory networks that comprise transcription factors and epigenetic regulators. While much has been learned regarding transcription factors, the function of epigenetic regulators in these networks is less well defined. We conducted a CRISPR-Cas9-mediated loss-of-function genetic screen that identified two epigenetic regulators, TAF5L and TAF6L, components or co-activators of the GNAT-HAT complexes for the mouse ESC (mESC) state. Detailed molecular studies demonstrate that TAF5L/TAF6L transcriptionally activate c-Myc and Oct4 and their corresponding MYC and CORE regulatory networks. Besides, TAF5L/TAF6L predominantly regulate their target genes through H3K9ac deposition and c-MYC recruitment that eventually activate the MYC regulatory network for self-renewal of mESCs. Thus, our findings uncover a role of TAF5L/TAF6L in directing the MYC regulatory network that orchestrates gene expression programs to control self-renewal for the maintenance of mESC state.
Assuntos
Células-Tronco Embrionárias/metabolismo , Redes Reguladoras de Genes , Células-Tronco Pluripotentes Induzidas/metabolismo , Proteínas Proto-Oncogênicas c-myc/genética , Fatores Associados à Proteína de Ligação a TATA/genética , Animais , Sistemas CRISPR-Cas , Ciclo Celular/genética , Proliferação de Células , Reprogramação Celular , Embrião de Mamíferos , Células-Tronco Embrionárias/citologia , Epigênese Genética , Fibroblastos/citologia , Fibroblastos/metabolismo , Edição de Genes , Regulação da Expressão Gênica , Células HEK293 , Histonas/genética , Histonas/metabolismo , Humanos , Células-Tronco Pluripotentes Induzidas/citologia , Camundongos , Cultura Primária de Células , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Proteínas Proto-Oncogênicas c-myc/metabolismo , Transdução de Sinais , Fatores Associados à Proteína de Ligação a TATA/metabolismoRESUMO
The generation of induced pluripotent stem cells (iPSCs) from differentiated cells following forced expression of OCT4, KLF4, SOX2, and C-MYC (OKSM) is slow and inefficient, suggesting that transcription factors have to overcome somatic barriers that resist cell fate change. Here, we performed an unbiased serial shRNA enrichment screen to identify potent repressors of somatic cell reprogramming into iPSCs. This effort uncovered the protein modifier SUMO2 as one of the strongest roadblocks to iPSC formation. Depletion of SUMO2 both enhances and accelerates reprogramming, yielding transgene-independent, chimera-competent iPSCs after as little as 38 hr of OKSM expression. We further show that the SUMO2 pathway acts independently of exogenous C-MYC expression and in parallel with small-molecule enhancers of reprogramming. Importantly, suppression of SUMO2 also promotes the generation of human iPSCs. Together, our results reveal sumoylation as a crucial post-transcriptional mechanism that resists the acquisition of pluripotency from fibroblasts using defined factors.
Assuntos
Diferenciação Celular/genética , Células-Tronco Pluripotentes Induzidas/metabolismo , Proteínas Proto-Oncogênicas c-myc/genética , Proteínas Modificadoras Pequenas Relacionadas à Ubiquitina/genética , Reprogramação Celular/genética , Regulação da Expressão Gênica no Desenvolvimento/genética , Humanos , Células-Tronco Pluripotentes Induzidas/citologia , Fator 4 Semelhante a Kruppel , RNA Interferente Pequeno/genéticaRESUMO
The reprogramming of somatic cells into induced pluripotent stem cells (iPSCs) upon overexpression of OCT4, KLF4, SOX2 and c-MYC (OKSM) provides a powerful system to interrogate basic mechanisms of cell fate change. However, iPSC formation with standard methods is typically protracted and inefficient, resulting in heterogeneous cell populations. We show that exposure of OKSM-expressing cells to both ascorbic acid and a GSK3-ß inhibitor (AGi) facilitates more synchronous and rapid iPSC formation from several mouse cell types. AGi treatment restored the ability of refractory cell populations to yield iPSC colonies, and it attenuated the activation of developmental regulators commonly observed during the reprogramming process. Moreover, AGi supplementation gave rise to chimera-competent iPSCs after as little as 48 h of OKSM expression. Our results offer a simple modification to the reprogramming protocol, facilitating iPSC induction at unparalleled efficiencies and enabling dissection of the underlying mechanisms in more homogeneous cell populations.
Assuntos
Ácido Ascórbico/farmacologia , Reprogramação Celular/genética , Células-Tronco Embrionárias/citologia , Células-Tronco Pluripotentes Induzidas/citologia , Piridinas/farmacologia , Pirimidinas/farmacologia , Animais , Antioxidantes/farmacologia , Apoptose , Pontos de Checagem do Ciclo Celular , Diferenciação Celular/genética , Proliferação de Células , Células Cultivadas , Quinase 3 da Glicogênio Sintase/antagonistas & inibidores , Glicogênio Sintase Quinase 3 beta , Proteínas de Fluorescência Verde/genética , Fator 4 Semelhante a Kruppel , Fatores de Transcrição Kruppel-Like/biossíntese , Fatores de Transcrição Kruppel-Like/genética , Camundongos , Fator 3 de Transcrição de Octâmero/biossíntese , Fator 3 de Transcrição de Octâmero/genética , Proteínas Proto-Oncogênicas c-myc/biossíntese , Proteínas Proto-Oncogênicas c-myc/genética , Fatores de Transcrição SOXB1/biossíntese , Fatores de Transcrição SOXB1/genéticaRESUMO
Induced pluripotency is a powerful tool to derive patient-specific stem cells. In addition, it provides a unique assay to study the interplay between transcription factors and chromatin structure. Here, we review the latest insights into chromatin dynamics that are inherent to induced pluripotency. Moreover, we compare and contrast these events with other physiological and pathological processes that involve changes in chromatin and cell state, including germ cell maturation and tumorigenesis. We propose that an integrated view of these seemingly diverse processes could provide mechanistic insights into cell fate transitions in general and might lead to new approaches in regenerative medicine and cancer treatment.
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Reprogramação Celular , Cromatina/genética , Cromatina/metabolismo , Animais , Carcinogênese/genética , Diferenciação Celular/genética , Fusão Celular , Metilação de DNA , Epigênese Genética , Células Germinativas/citologia , Células Germinativas/metabolismo , Histonas/metabolismo , Humanos , Células-Tronco Pluripotentes Induzidas/citologia , Células-Tronco Pluripotentes Induzidas/metabolismo , Técnicas de Transferência Nuclear , Transdução de Sinais , Fatores de Transcrição/metabolismoRESUMO
Although transcriptional regulation of stem cell pluripotency and differentiation has been extensively studied, only a small number of studies have addressed the roles for posttranslational modifications in these processes. A key mechanism of posttranslational modification is ubiquitination by the ubiquitin-proteasome system (UPS). Here, using shotgun proteomics, we map the ubiquitinated protein landscape during embryonic stem cell (ESC) differentiation and induced pluripotency. Moreover, using UPS-targeted RNAi screens, we identify additional regulators of pluripotency and differentiation. We focus on two of these proteins, the deubiquitinating enzyme Psmd14 and the E3 ligase Fbxw7, and characterize their importance in ESC pluripotency and cellular reprogramming. This global characterization of the UPS as a key regulator of stem cell pluripotency opens the way for future studies that focus on specific UPS enzymes or ubiquitinated substrates.
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Reprogramação Celular/genética , Células-Tronco Pluripotentes/citologia , Células-Tronco Pluripotentes/metabolismo , Complexo de Endopeptidases do Proteassoma/metabolismo , Ubiquitina/metabolismo , Animais , Proteínas de Ciclo Celular/metabolismo , Diferenciação Celular , Proliferação de Células , Células-Tronco Embrionárias/citologia , Células-Tronco Embrionárias/metabolismo , Proteínas F-Box/metabolismo , Proteína 7 com Repetições F-Box-WD , Regulação da Expressão Gênica no Desenvolvimento , Proteínas de Fluorescência Verde/metabolismo , Meia-Vida , Camundongos , Estabilidade Proteica , Proteólise , Proteoma/metabolismo , Proteínas Proto-Oncogênicas c-myc/metabolismo , RNA Interferente Pequeno/metabolismo , Transativadores/metabolismo , Ubiquitina-Proteína Ligases/metabolismoRESUMO
L3mbtl2 has been implicated in transcriptional repression and chromatin compaction but its biological function has not been defined. Here we show that disruption of L3mbtl2 results in embryonic lethality with failure of gastrulation. This correlates with compromised proliferation and abnormal differentiation of L3mbtl2(-/-) embryonic stem (ES) cells. L3mbtl2 regulates genes by recruiting a Polycomb Repressive Complex1 (PRC1)-related complex, resembling the previously described E2F6-complex, and including G9A, Hdac1, and Ring1b. The presence of L3mbtl2 at target genes is associated with H3K9 dimethylation, low histone acetylation, and H2AK119 ubiquitination, but the latter is neither dependent on L3mbtl2 nor sufficient for repression. Genome-wide studies revealed that the L3mbtl2-dependent complex predominantly regulates genes not bound by canonical PRC1 and PRC2. However, some developmental regulators are repressed by the combined activity of all three complexes. Together, we have uncovered a highly selective, essential role for an atypical PRC1-family complex in ES cells and early development.
Assuntos
Desenvolvimento Embrionário , Proteínas Nucleares/metabolismo , Células-Tronco Pluripotentes/metabolismo , Complexo Repressor Polycomb 1/metabolismo , Fatores de Transcrição/metabolismo , Proteínas Supressoras de Tumor/metabolismo , Animais , Diferenciação Celular , Proliferação de Células , Montagem e Desmontagem da Cromatina/genética , Corpos Embrioides/citologia , Corpos Embrioides/metabolismo , Genoma/genética , Camundongos , Complexos Multiproteicos/metabolismo , Proteínas Nucleares/química , Células-Tronco Pluripotentes/citologia , Complexo Repressor Polycomb 2 , Ligação Proteica/genética , Estrutura Terciária de Proteína , Proteínas Repressoras/metabolismo , Fatores de Transcrição/química , Transcrição Gênica , Proteínas Supressoras de Tumor/química , Dedos de ZincoRESUMO
BACKGROUND: Upregulation of nuclear factor kappa B (NFκB) activity and neuroendocrine differentiation are two mechanisms known to be involved in prostate cancer (PC) progression to castration resistance. We have observed that major components of these pathways, including NFκB, proteasome, neutral endopeptidase (NEP) and endothelin 1 (ET-1), exhibit an inverse and mirror image pattern in androgen-dependent (AD) and -independent (AI) states in vitro. METHODS: We have now investigated for evidence of a direct mechanistic connection between these pathways with the use of immunocytochemistry (ICC), western blot analysis, electrophoretic mobility shift assay (EMSA) and proteasome activity assessment. RESULTS: Neuropeptide (NP) stimulation induced nuclear translocation of NFκB in a dose-dependent manner in AI cells, also evident as reduced total inhibitor κB (IκB) levels and increased DNA binding in EMSA. These effects were preceded by increased 20 S proteasome activity at lower doses and at earlier times and were at least partially reversed under conditions of NP deprivation induced by specific NP receptor inhibitors, as well as NFκB, IκB kinase (IKK) and proteasome inhibitors. AD cells showed no appreciable nuclear translocation upon NP stimulation, with less intense DNA binding signal on EMSA. CONCLUSIONS: Our results support evidence for a direct mechanistic connection between the NPs and NFκB/proteasome signaling pathways, with a distinct NP-induced profile in the more aggressive AI cancer state.
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Rejeição de Enxerto/genética , Rejeição de Enxerto/imunologia , Células-Tronco Pluripotentes Induzidas/imunologia , Células-Tronco Pluripotentes Induzidas/transplante , Animais , Reprogramação Celular/genética , Reprogramação Celular/imunologia , Células-Tronco Pluripotentes Induzidas/citologia , Células-Tronco Pluripotentes Induzidas/metabolismo , Camundongos , Teratoma/genética , Teratoma/imunologia , Transplante Homólogo/imunologia , Transplante Isogênico/imunologia , Regulação para Cima/genética , Regulação para Cima/imunologiaRESUMO
BACKGROUND: Castration-resistance in prostate cancer (PC) is a critical event hallmarking a switch to a more aggressive phenotype. Neuroendocrine differentiation and upregulation of NFκB transcriptional activity are two mechanisms that have been independently linked to this process. METHODS: We investigated these two pathways together using in vitro models of androgen-dependent (AD) and androgen-independent (AI) PC. We measured cellular levels, activity and surface expression of Neutral Endopeptidase (NEP), levels of secreted Endothelin-1 (ET-1), levels, sub-cellular localisation and DNA binding ability of NFκB, and proteasomal activity in human native PC cell lines (LnCaP and PC-3) modelling AD and AI states. RESULTS: At baseline, AD cells were found to have high NEP expression and activity and low secreted ET-1. In contrast, they exhibited a low-level activation of the NFκB pathway associated with comparatively low 20S proteasome activity. The AI cells showed the exact mirror image, namely increased proteasomal activity resulting in a canonical pathway-mediated NFκB activation, and minimal NEP activity with increased levels of secreted ET-1. CONCLUSIONS: Our results seem to support evidence for divergent patterns of expression of the NFκB/proteasome pathway with relation to components of the NEP/neuropeptide axis in PC cells of different level of androgen dependence. NEP and ET-1 are inversely and directly related to an activated state of the NFκB/proteasome pathway, respectively. A combination therapy targeting both pathways may ultimately prove to be of benefit in clinical practice.
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Induced pluripotent stem cells (iPSCs) have been derived from various somatic cell populations through ectopic expression of defined factors. It remains unclear whether iPSCs generated from different cell types are molecularly and functionally similar. Here we show that iPSCs obtained from mouse fibroblasts, hematopoietic and myogenic cells exhibit distinct transcriptional and epigenetic patterns. Moreover, we demonstrate that cellular origin influences the in vitro differentiation potentials of iPSCs into embryoid bodies and different hematopoietic cell types. Notably, continuous passaging of iPSCs largely attenuates these differences. Our results suggest that early-passage iPSCs retain a transient epigenetic memory of their somatic cells of origin, which manifests as differential gene expression and altered differentiation capacity. These observations may influence ongoing attempts to use iPSCs for disease modeling and could also be exploited in potential therapeutic applications to enhance differentiation into desired cell lineages.
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Diferenciação Celular , Linhagem da Célula , Células-Tronco Pluripotentes Induzidas/citologia , Células-Tronco Pluripotentes Induzidas/metabolismo , Animais , Linfócitos B/citologia , Células Cultivadas , Corpos Embrioides/citologia , Corpos Embrioides/metabolismo , Epigenômica , Fibroblastos/citologia , Fibroblastos/metabolismo , Perfilação da Expressão Gênica , Células-Tronco Hematopoéticas/citologia , Células-Tronco Hematopoéticas/metabolismo , Camundongos , Músculo Esquelético/citologia , Células-Tronco/citologia , Transcrição GênicaRESUMO
GATA-1 and GATA-2 control proliferation and differentiation of hematopoietic progenitor cells via transcriptional regulation. In this issue of Molecular Cell, Jing et al. (2008) demonstrate that GATA factor exchange on the Kit locus directs a transcriptional switch by reconfiguring chromatin loops.