RESUMEN
p53 is a central mediator of cellular stress responses, and its precise regulation is essential for the normal progression of hematopoiesis. MYSM1 is an epigenetic regulator essential for the maintenance of hematopoietic stem cell (HSC) function, hematopoietic progenitor survival, and lymphocyte development. We recently demonstrated that all developmental and hematopoietic phenotypes of Mysm1 deficiency are p53-mediated and rescued in the Mysm1(-/-)p53(-/-) mouse model. However, the mechanisms triggering p53 activation in Mysm1(-/-) HSPCs, and the pathways downstream of p53 driving different aspects of the Mysm1(-/-) phenotype remain unknown. Here we show the transcriptional activation of p53 stress responses in Mysm1(-/-) HSPCs. Mechanistically, we find that the MYSM1 protein associates with p53 and colocalizes to promoters of classical p53-target genes Bbc3/PUMA (p53 upregulated modulator of apoptosis) and Cdkn1a/p21. Furthermore, it antagonizes their p53-driven expression by modulating local histone modifications (H3K27ac and H3K4me3) and p53 recruitment. Using double-knockout mouse models, we establish that PUMA, but not p21, is an important mediator of p53-driven Mysm1(-/-) hematopoietic dysfunction. Specifically, Mysm1(-/-)Puma(-/-) mice show full rescue of multipotent progenitor (MPP) viability, partial rescue of HSC quiescence and function, but persistent lymphopenia. Through transcriptome analysis of Mysm1(-/-)Puma(-/-) MPPs, we demonstrate strong upregulation of other p53-induced mediators of apoptosis and cell-cycle arrest. The full viability of Mysm1(-/-)Puma(-/-) MPPs, despite strong upregulation of many other pro-apoptotic mediators, establishes PUMA as the essential non-redundant effector of p53-induced MPP apoptosis. Furthermore, we identify potential mediators of p53-dependent but PUMA-independent Mysm1(-/-)hematopoietic deficiency phenotypes. Overall, our study provides novel insight into the cell-type-specific roles of p53 and its downstream effectors in hematopoiesis using unique models of p53 hyperactivity induced by endogenous stress. We conclude that MYSM1 is a critical negative regulator of p53 transcriptional programs in hematopoiesis, and that its repression of Bbc3/PUMA expression is essential for MPP survival, and partly contributes to maintaining HSC function.
Asunto(s)
Proteínas Reguladoras de la Apoptosis/metabolismo , Endopeptidasas/metabolismo , Células Madre Hematopoyéticas/citología , Células Madre Hematopoyéticas/metabolismo , Células Madre Multipotentes/citología , Células Madre Multipotentes/metabolismo , Proteína p53 Supresora de Tumor/metabolismo , Proteínas Supresoras de Tumor/metabolismo , Animales , Apoptosis , Supervivencia Celular , Endopeptidasas/deficiencia , Endopeptidasas/genética , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Transactivadores , Proteína p53 Supresora de Tumor/deficiencia , Proteína p53 Supresora de Tumor/genética , Proteasas Ubiquitina-EspecíficasAsunto(s)
Ecocardiografía , Fibroma/diagnóstico por imagen , Atrios Cardíacos/diagnóstico por imagen , Neoplasias Cardíacas/diagnóstico por imagen , Válvula Mitral/diagnóstico por imagen , Diagnóstico Diferencial , Fibroma/complicaciones , Neoplasias Cardíacas/complicaciones , Humanos , Embolia Intracraneal/etiología , Masculino , Persona de Mediana Edad , Células Neoplásicas CirculantesRESUMEN
A comparative study of two doubly 13C labeled amphiphilic transmembrane peptides was undertaken to determine the potential of rotational resonance for measuring internuclear distances through the direct dipolar coupling in the presence of motion. The two peptides, having the sequence acetyl-K2-G-L16-K2-A-amide, differed only in the position of 13C labels. The first peptide, [1-13C]leu(11):[alpha-13C]leu(12), had labels on adjacent residues, at the carbonyl of leu(11) and the alpha carbon of leu(12). The second, [1-13C]leu(8):[alpha-(13)/C]leu(11), was labeled on consecutive turns of the alpha-helical peptide. The internuclear distance between labeled positions of the first peptide, which for an ideal alpha helix has a value of 2.48 A, is relatively independent of internal flexibility or peptide conformational change. The dipolar coupling between these two nuclei is sensitive to motional averaging by molecular reorientation, however, making this peptide ideal for investigating these motions. The internuclear distance between labels on the second peptide has an expected static ideal alpha-helix value of 4.6 A, but this is sensitive to internal flexibility. In addition, the dipolar coupling between these two nuclei is much weaker because of their larger separation, making this peptide a much more difficult test of the rotational resonance technique. The dipolar couplings between the labeled nuclei of these two peptides were measured by rotational resonance in the dry peptide powders and in multilamellar dispersions with dimyristoylphosphatidylcholine in the gel phase, at -10 degrees C, and in the fluid phase, at 40 degrees C. The results for the peptide having adjacent labels can be readily interpreted in terms of a simple model for the peptide motion. The results for the second peptide show that, in the fluid phase, the motionally averaged dipolar coupling is too small to be measured by rotational resonance. Rotational resonance, rotational echo double resonance, and related techniques can be used to obtain reliable and valuable dipolar couplings in static solid and membrane systems. The interpretation of these couplings in terms of internuclear distances is straightforward in the absence of molecular motion. These techniques hold considerable promise for membrane protein structural studies under conditions, such as at low temperatures, where molecular motion does not modulate the dipolar couplings. However, a typical membrane at physiological temperatures exhibits complex molecular motions. In the absence of an accurate and detailed description of both internal and whole body molecular motions, it is unlikely that techniques of this type, which are based on extracting distances from direct internuclear dipolar couplings, can be used to study molecular structure under these conditions. Furthermore, the reduction in the strengths of the dipolar couplings by these motions dramatically reduces the useful range of distances which can be measured.
Asunto(s)
Proteínas de la Membrana/química , Péptidos/química , Secuencia de Aminoácidos , Isótopos de Carbono , Fenómenos Químicos , Química Física , Cristalización , Dimiristoilfosfatidilcolina/química , Geles , Membrana Dobles de Lípidos/química , Modelos Químicos , Modelos Moleculares , TermodinámicaRESUMEN
13C-NMR is used to probe the motion and orientation of the carboxyl group in a 70% potassium palmitate/30% water mixture. An increase in delta sigma with increasing temperature corresponds to a change of orientation of the carboxyl group. Comparison with 2H-NMR and lineshape simulations show that near 57 degrees C the molecules exchange between two orientations at a rate between 10-500 Hz.