RESUMO
Myostatin is a negative regulator of the growth and development of skeletal muscle mass. In fish, myostatin is expressed in several organs in addition to skeletal muscle. To understand the mechanisms regulating myostatin gene expression in the sea perch, Lateolabrax japonicus, we examined the methylation status of the myostatin gene promoter region in several tissues (liver, eye, kidney, brain, and heart) isolated from adult specimens. The frequency of methylated cytosines was very low in all tissues, regardless of the level of myostatin expression, suggesting that DNA methylation is not involved in the tissue-specific regulation of myostatin expression. Southern blot analysis of genomic DNA obtained from micrococcal nuclease-treated nuclei showed that chromatin digestion occurs in tissues where the myostatin gene is actively transcribed and that the myostatin gene is protected from micrococcal nuclease in tissues where myostatin is not expressed. The chromatin structure in the myostatin gene region appears to regulate its expression without DNA methylation.
Assuntos
Cromatina/genética , Metilação de DNA/genética , Miostatina/genética , Percas/genética , Regiões Promotoras Genéticas/genética , Região 5'-Flanqueadora/genética , Animais , Sequência de Bases , Southern Blotting , Sequência Conservada/genética , Ilhas de CpG/genética , DNA/isolamento & purificação , Metilação de DNA/efeitos dos fármacos , Genoma/genética , Nuclease do Micrococo/farmacologia , Dados de Sequência Molecular , Oceanos e Mares , Análise de Sequência de DNA , SulfitosRESUMO
To determine the changes in chromatin organization during male pronucleus remodeling, we have compared the composition of nucleoprotein particles (NP-ps) resulting from digestion with endogenous nuclease (ENase) and with micrococcal nuclease (MNase). Whole nuclei were isolated from sea urchin gametes and zygotes containing partially decondensed (15 min postinsemination, p.i.) or a fully decondensed (40 min p.i.) male pronucleus and digested with nucleases. The NP-ps generated were analyzed in agarose gels, and their histone composition was determined. Sperm core histones (SpH) and cleavage stage (CS) variants were identified by Western immunoblots revealed with specific antibodies. A single NP-ps was generated after digestion of sperm nucleus with MNase, which migrated in agarose gels between DNA fragments of 1.78-1.26 Kb. Sperm chromatin remained undigested after incubation in ENases activating buffer, indicating that these nuclei do not contain ENases. One type of NP-ps was obtained by digestion of unfertilized egg nuclei, either with ENase or MNase; the NP-ps was located in the region of the agarose gel corresponding to DNA fragments of 3.4-1.95 Kb [Imschenetzky et al. (1989): Exp Cell Res 182:436-444]. When whole nuclei from zygotes containing the female pronucleus and a partially remodeled male pronucleus were digested with ENase, a single NP-ps was generated, which migrated between DNA fragments of 2.5-1.9 Kb. This particle contained only CS histone variants. Alternatively, when these nuclei were digested with MNase, two NP-ps were generated; the slower migrating NP-ps (s) was located in the same position of the agarose gel as those resulting from ENase digestion and the faster migrating NP-ps (f) migrated between DNA fragments of 1.95-1.26 Kb. It was found that NP-ps (s) contained only CS histone variants, whereas NP-ps (f) were formed by a subset of SpH and by CS histone variants. When nuclei from zygotes containing a fully decondensed male pronucleus were digested either with ENase or MNase, a single type of NP-ps was observed, which migrated in the same position as NP-ps (s) in agarose gels. This particle contained only CS histone variants. On the basis of the histone compositions and on electrophoretic similarities, it was concluded that NP-ps (s) originated from the female pronucleus and that NP-ps (f) were generated from the partially remodeled male pronucleus. Consequently, our results indicate that at an intermediate stage of male pronucleus remodeling the chromatin is formed by NP-ps containing a subset of both SpH and of CS histone variants, whereas at final stages of male pronucleus decondensation chromatin organization is similar to that of the female pronucleus.
Assuntos
Núcleo Celular/química , Histonas/análise , Nucleossomos/química , Espermatozoides/química , Zigoto/química , Animais , Western Blotting , Cromatina/metabolismo , DNA/isolamento & purificação , Eletroforese em Gel de Ágar , Endonucleases/farmacologia , Feminino , Variação Genética , Histonas/genética , Histonas/isolamento & purificação , Masculino , Nuclease do Micrococo/farmacologia , Nucleossomos/metabolismo , Oócitos/química , Ouriços-do-MarRESUMO
The chromatin structure of Entamoeba histolytica was investigated. It was found that this protozoan organizes its chromatin in nucleosome-like particles 10 nm in diameter, but digestion of the chromatin with micrococcal nuclease did not render a regularly spaced DNA ladder in agarose gels. Southern blot analysis of the products of Entamoeba chromatin digestion using total amebic DNA and a non-transcribed repetitive sequence produced a banding pattern characteristic of eukaryotic chromatin with a repetitive size of approximately 130 bp. Conversely, hybridization with two active gene probes, actin and ribosomal RNA, showed that these sequences are not part of the chromatin organized in nucleosomes. It was also found that the basic nuclear proteins differ from histones of higher eukaryotes in electrophoretic mobility. Screening of an E. histolytica HM1-IMSS genomic library with Saccharomyces cerevisiae H3 and H4 genes and attempts to amplify E. histolytica sequences, homologous to these yeast histone genes, gave negative results suggesting that the Entamoeba proteins involved in chromatin organization are not typical histones.