RESUMO
Sp1 and Sp3 belong to the specificity proteins (Sp)/Krüppel-like transcription factor family. They are closely related, ubiquitously expressed, and recognize G-rich DNA motifs. They are thought to regulate generic processes such as cell-cycle and growth control, metabolic pathways, and apoptosis. Ablation of Sp1 or Sp3 in mice is lethal, and combined haploinsufficiency results in hematopoietic defects during the fetal stages. Here, we show that in adult mice, conditional pan-hematopoietic (Mx1-Cre) ablation of either Sp1 or Sp3 has minimal impact on hematopoiesis, whereas the simultaneous loss of Sp1 and Sp3 results in severe macrothrombocytopenia. This occurs in a cell-autonomous manner as shown by megakaryocyte-specific (Pf4-Cre) double-knockout mice. We employed flow cytometry, cell culture, and electron microscopy and show that although megakaryocyte numbers are normal in bone marrow and spleen, they display a less compact demarcation membrane system and a striking inability to form proplatelets. Through megakaryocyte transcriptomics and platelet proteomics, we identified several cytoskeleton-related proteins and downstream effector kinases, including Mylk, that were downregulated upon Sp1/Sp3 depletion, providing an explanation for the observed defects in megakaryopoiesis. Supporting this notion, selective Mylk inhibition by ML7 affected proplatelet formation and stabilization and resulted in defective ITAM receptor-mediated platelet aggregation.
Assuntos
Plaquetas/citologia , Megacariócitos/citologia , Fator de Transcrição Sp1/genética , Fator de Transcrição Sp3/genética , Animais , Azepinas/química , Plaquetas/metabolismo , Medula Óssea/metabolismo , Citometria de Fluxo , Lectinas Tipo C/metabolismo , Camundongos , Camundongos Knockout , Naftalenos/química , Agregação Plaquetária , Glicoproteínas da Membrana de Plaquetas/metabolismo , Proteoma , Transdução de Sinais , Fator de Transcrição Sp1/metabolismo , Fator de Transcrição Sp3/metabolismo , Baço/metabolismo , Trombocitopenia/metabolismo , Fatores de Transcrição/metabolismoRESUMO
RUNX1 is known to be an essential transcription factor for generating hematopoietic stem cells (HSC), but much less is known about its role in the downstream process of hematopoietic differentiation. RUNX1 has been shown to be part of a large transcription factor complex, together with LDB1, GATA1, TAL1, and ETO2 (N. Meier et al., Development 133:4913-4923, 2006) in erythroid cells. We used a tagging strategy to show that RUNX1 interacts with two novel protein partners, LSD1 and MYEF2, in erythroid cells. MYEF2 is bound in undifferentiated cells and is lost upon differentiation, whereas LSD1 is bound in differentiated cells. Chromatin immunoprecipitation followed by sequencing (ChIP-seq) and microarray expression analysis were used to show that RUNX1 binds approximately 9,000 target sites in erythroid cells and is primarily active in the undifferentiated state. Functional analysis shows that a subset of the target genes is suppressed by RUNX1 via the newly identified partner MYEF2. Knockdown of Myef2 expression in developing zebrafish results in a reduced number of HSC.
Assuntos
Subunidade alfa 2 de Fator de Ligação ao Core/metabolismo , Células Eritroides/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Hematopoese , Complexos Multiproteicos/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Oxirredutases N-Desmetilantes/metabolismo , Proteínas Repressoras/metabolismo , Proteínas de Peixe-Zebra/metabolismo , Animais , Linhagem Celular Tumoral , Subunidade alfa 2 de Fator de Ligação ao Core/genética , DNA/metabolismo , Técnicas de Silenciamento de Genes , Histona Desmetilases , Camundongos , Morfolinos/administração & dosagem , Morfolinos/genética , Proteínas do Tecido Nervoso/genética , Ligação Proteica , Proteínas Repressoras/genética , Peixe-Zebra/embriologia , Proteínas de Peixe-Zebra/genéticaRESUMO
The HMG-box transcription factor Sox2 plays a role throughout neurogenesis and also acts at other stages of development, as illustrated by the multiple organs affected in the anophthalmia syndrome caused by SOX2 mutations. Here we combined proteomic and genomic approaches to characterize gene regulation by Sox2 in neural stem cells. Chd7, a chromatin remodeling ATPase associated with CHARGE syndrome, was identified as a Sox2 transcriptional cofactor. Sox2 and Chd7 physically interact, have overlapping genome-wide binding sites and regulate a set of common target genes including Jag1, Gli3 and Mycn, genes mutated in Alagille, Pallister-Hall and Feingold syndromes, which show malformations also associated with SOX2 anophthalmia syndrome or CHARGE syndrome. Regulation of disease-associated genes by a Sox2-Chd7 complex provides a plausible explanation for several malformations associated with SOX2 anophthalmia syndrome or CHARGE syndrome. Indeed, we found that Chd7-haploinsufficient embryos showed severely reduced expression of Jag1 in the developing inner ear.
Assuntos
Proteínas de Ligação a DNA/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Células-Tronco Neurais/metabolismo , Fatores de Transcrição SOXB1/metabolismo , Animais , Anoftalmia/genética , Síndrome CHARGE/genética , Proteínas de Ligação ao Cálcio/metabolismo , Orelha Interna/metabolismo , Proteínas Hedgehog/metabolismo , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Proteína Jagged-1 , Proteínas de Membrana/metabolismo , Camundongos , Mutação , Receptores Notch/metabolismo , Proteínas Serrate-JaggedRESUMO
Balanced chromosomal rearrangements can cause disease, but techniques for their rapid and accurate identification are missing. Here we demonstrate that chromatin conformation capture on chip (4C) technology can be used to screen large genomic regions for balanced and complex inversions and translocations at high resolution. The 4C technique can be used to detect breakpoints also in repetitive DNA sequences as it uniquely relies on capturing genomic fragments across the breakpoint. Using 4C, we uncovered LMO3 as a potentially leukemogenic translocation partner of TRB@. We developed multiplex 4C to simultaneously screen for translocation partners of multiple selected loci. We identified unsuspected translocations and complex rearrangements. Furthermore, using 4C we detected translocations even in small subpopulations of cells. This strategy opens avenues for the rapid fine-mapping of cytogenetically identified translocations and inversions, and the efficient screening for balanced rearrangements near candidate loci, even when rearrangements exist only in subpopulations of cells.