RESUMEN
Adenovirus transformed cells have a dedifferentiated phenotype. Eliminating E1A in transformed human embryonic kidney cells derepressed â¼2600 genes, generating a gene expression profile closely resembling mesenchymal stem cells (MSCs). This was associated with a dramatic change in cell morphology from one with scant cytoplasm and a globular nucleus to one with increased cytoplasm, extensive actin stress fibers, and actomyosin-dependent flattening against the substratum. E1A-induced hypoacetylation at histone H3 Lys27 and Lys18 (H3K27/18) was reversed. Most of the increase in H3K27/18ac was in enhancers near TEAD transcription factors bound by Hippo signaling-regulated coactivators YAP and TAZ. E1A causes YAP/TAZ cytoplasmic sequestration. After eliminating E1A, YAP/TAZ were transported into nuclei, where they associated with poised enhancers with DNA-bound TEAD4 and H3K4me1. This activation of YAP/TAZ required RHO family GTPase signaling and caused histone acetylation by p300/CBP, chromatin remodeling, and cohesin loading to establish MSC-associated enhancers and then superenhancers. Consistent results were also observed in primary rat embryo kidney cells, human fibroblasts, and human respiratory tract epithelial cells. These results together with earlier studies suggest that YAP/TAZ function in a developmental checkpoint controlled by signaling from the actin cytoskeleton that prevents differentiation of a progenitor cell until it is in the correct cellular and tissue environment.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/genética , Proteínas E1A de Adenovirus/metabolismo , Diferenciación Celular/genética , Silenciador del Gen , Péptidos y Proteínas de Señalización Intracelular/genética , Fosfoproteínas/genética , Citoesqueleto de Actina/metabolismo , Adenoviridae , Animales , Células Cultivadas , Células HEK293 , Humanos , Ratas , Transducción de Señal , Transactivadores , Factores de Transcripción , Proteínas Coactivadoras Transcripcionales con Motivo de Unión a PDZ , Proteínas Señalizadoras YAPRESUMEN
Although aberrant protein aggregation has been conclusively linked to dozens of devastating amyloid diseases, scientists remain puzzled about the molecular features that render amyloid fibrils or small oligomers toxic. Here, we report a previously unobserved type of amyloid fibril that tests as cytotoxic: one in which the strands of the contributing ß-sheets are out of register. In all amyloid fibrils previously characterized at the molecular level, only in-register ß-sheets have been observed, in which each strand makes its full complement of hydrogen bonds with the strands above and below it in the fibril. In out-of-register sheets, strands are sheared relative to one another, leaving dangling hydrogen bonds. Based on this finding, we designed out-of-register ß-sheet amyloid mimics, which form both cylindrin-like oligomers and fibrils, and these mimics are cytotoxic. Structural and energetic considerations suggest that out-of-register fibrils can readily convert to toxic cylindrins. We propose that out-of-register ß-sheets and their related cylindrins are part of a toxic amyloid pathway, which is distinct from the more energetically favored in-register amyloid pathway.
Asunto(s)
Amiloide/química , Rojo Congo/farmacología , Cristalografía por Rayos X/métodos , Colorantes Fluorescentes/farmacología , Humanos , Enlace de Hidrógeno , Microscopía Electrónica de Transmisión/métodos , Modelos Moleculares , Conformación Molecular , Péptidos/química , Conformación Proteica , Estructura Secundaria de Proteína , Proteínas/química , Termodinámica , Difracción de Rayos X , Microglobulina beta-2/químicaRESUMEN
OBJECTIVE: To isolate and identify the differentially expressed genes in spermatogenesis for the understanding molecular mechanism of spermatogenesis. METHODS: Screening of the cDNA library, Northern blot, expression and purification in E. coli with GST expression system, immunocytochemical staining of testis sections were used. RESULTS: (1) A cDNA fragment designated as RSD-7 was isolated from rat testis cDNA library. It was 1,238 bp in length, coding a protein of 232 amino acids with the GenBank accession number AF315467. The encoding protein of RSD-7 cDNA had a Ubiquitin-like domain. (2) Northern blot indicated that RSD-7 was uniquely expressed in rat testis, and in the testis RSD-7 emerged on the 30th postnatal day and expressed until 120th postnatal day. (3) Expression and purification of RSD-7 protein in E. coli with GST expression system and were used to obtain anti-RSD-7 antibody. (4) Immunolocalization of RSD-7 in rat testis revealed that it is expressed only in Sertoli cells. CONCLUSIONS: Transcription pattern of RSD-7 and localization of RSD-7 protein in testis have been made, which established the base for the functional study of RSD-7.
Asunto(s)
Proteínas de Escherichia coli/biosíntesis , Proteínas Represoras/biosíntesis , Espermatogénesis , Testículo/metabolismo , Ubiquitinas/biosíntesis , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Clonación Molecular , ADN Complementario/biosíntesis , ADN Complementario/genética , Escherichia coli/genética , Proteínas de Escherichia coli/genética , Masculino , Datos de Secuencia Molecular , Conejos , Ratas , Ratas Wistar , Proteínas Represoras/genética , Células de Sertoli/metabolismo , Ubiquitinas/genéticaRESUMEN
Oncogenic transformation by adenovirus small e1a depends on simultaneous interactions with the host lysine acetylases p300/CBP and the tumor suppressor RB. How these interactions influence cellular gene expression remains unclear. We find that e1a displaces RBs from E2F transcription factors and promotes p300 acetylation of RB1 K873/K874 to lock it into a repressing conformation that interacts with repressive chromatin-modifying enzymes. These repressing p300-e1a-RB1 complexes specifically interact with host genes that have unusually high p300 association within the gene body. The TGF-β, TNF-, and interleukin-signaling pathway components are enriched among such p300-targeted genes. The p300-e1a-RB1 complex condenses chromatin in a manner dependent on HDAC activity, p300 lysine acetylase activity, the p300 bromodomain, and RB K873/K874 and e1a K239 acetylation to repress host genes that would otherwise inhibit productive virus infection. Thus, adenovirus employs e1a to repress host genes that interfere with viral replication.
Asunto(s)
Adenoviridae/genética , Proteínas E1A de Adenovirus/metabolismo , Proteína de Retinoblastoma/metabolismo , Factores de Transcripción p300-CBP/metabolismo , Adenoviridae/fisiología , Proteínas E1A de Adenovirus/genética , Transformación Celular Viral , Células Cultivadas , Quimiocina CXCL1/metabolismo , Cromatina/metabolismo , Regulación de la Expresión Génica , Interacciones Huésped-Patógeno , Humanos , Regiones Promotoras Genéticas , ARN Mensajero/genética , ARN Mensajero/metabolismo , Análisis de Secuencia de ARN , Transducción de Señal , Replicación ViralRESUMEN
DNA methylation is fundamental for the stability and activity of genomes. Drosophila melanogaster and vertebrates establish a global DNA methylation pattern of their genome during early embryogenesis. Large-scale analyses of DNA methylation patterns have uncovered revealed that DNA methylation patterns are dynamic rather than static and change in a gene-specific fashion during development and in diseased cells. However, the factors and mechanisms involved in dynamic, postembryonic DNA methylation remain unclear. Methylation of lysine 9 in histone H3 (H3-K9) by members of the Su(var)3-9 family of histone methyltransferases (HMTs) triggers embryonic DNA methylation in Arthropods and Chordates. Here, we demonstrate that Drosophila SETDB1 (dSETDB1) can mediate DNA methylation and silencing of genes and retrotransposons. We found that dSETDB1 tri-methylates H3-K9 and binds methylated CpA motifs. Tri-methylation of H3-K9 by dSETDB1 mediates recruitment of DNA methyltransferase 2 (Dnmt2) and Su(var)205, the Drosophila ortholog of mammalian "Heterochromatin Protein 1", to target genes for dSETDB1. By enlisting Dnmt2 and Su(var)205, dSETDB1 triggers DNA methylation and silencing of genes and retrotransposons in Drosophila cells. DSETDB1 is involved in postembryonic DNA methylation and silencing of Rt1b{} retrotransposons and the tumor suppressor gene retinoblastoma family protein 1 (Rb) in imaginal discs. Collectively, our findings implicate dSETDB1 in postembryonic DNA methylation, provide a model for silencing of the tumor suppressor Rb, and uncover a role for cell type-specific DNA methylation in Drosophila development.
Asunto(s)
Metilación de ADN/genética , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/embriología , Drosophila melanogaster/genética , Embrión no Mamífero/metabolismo , Silenciador del Gen , Animales , Homólogo de la Proteína Chromobox 5 , Proteínas Cromosómicas no Histona/metabolismo , ADN (Citosina-5-)-Metiltransferasas/metabolismo , Proteínas de Drosophila/química , Ojo/embriología , Ojo/metabolismo , Heterocromatina/metabolismo , N-Metiltransferasa de Histona-Lisina , Histonas/metabolismo , Lisina/metabolismo , Unión Proteica , Estructura Terciaria de Proteína , Secuencias Reguladoras de Ácidos Nucleicos/genética , Proteína de Retinoblastoma/metabolismo , Retroelementos/genéticaRESUMEN
Homeotic genes contain cis-regulatory trithorax response elements (TREs) that are targeted by epigenetic activators and transcribed in a tissue-specific manner. We show that the transcripts of three TREs located in the Drosophila homeotic gene Ultrabithorax (Ubx) mediate transcription activation by recruiting the epigenetic regulator Ash1 to the template TREs. TRE transcription coincides with Ubx transcription and recruitment of Ash1 to TREs in Drosophila. The SET domain of Ash1 binds all three TRE transcripts, with each TRE transcript hybridizing with and recruiting Ash1 only to the corresponding TRE in chromatin. Transgenic transcription of TRE transcripts restores recruitment of Ash1 to Ubx TREs and restores Ubx expression in Drosophila cells and tissues that lack endogenous TRE transcripts. Small interfering RNA-induced degradation of TRE transcripts attenuates Ash1 recruitment to TREs and Ubx expression, which suggests that noncoding TRE transcripts play an important role in epigenetic activation of gene expression.