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Abstract
Testis-specific H2B (TH2B) histone gene of rat is expressed during meiotic event of spermatogenic differentiation. The gene is unusual in that it has conserved the regulatory elements involved in the S phase-specific transcription of somatic H2B genes as well as the S phase-specific stabilization of histone mRNA. Genomic sequencing revealed that all analyzed CpG sites in the promoter region of TH2B gene are methylated in somatic tissues but not in testis. During spermatogenesis, these CpG sites are unmethylated as early as spermatogonia type A and up to sperm. Thus, there is a good correlation between DNA hypomethylation and germ cell-specific expression of TH2B gene. Results obtained from in vivo DNase footprinting and DNA mobility shift experiments are consistent with the hypothesis that DNA methylation inhibits gene activity by preventing the binding of transcription factors to their recognition sequences. The results show that (i) the binding of ubiquitous transcription factors to the promoter region of TH2B gene may be blocked in nuclei of liver, and (ii) DNA methylation can directly interfere with the binding of transcription factors recognizing a hexamer (ACGTCA) motif. In vitro DNA methylation and transfection experiments demonstrated that expression of TH2B gene is inhibited by DNA methylation in vivo. These findings indicate that DNA methylation may play a key role in the transcriptional repression of germ cell-specific TH2B gene.
Highlights
Testis-specific H2B (THPB) histone gene of rat is rat (Kim et al, 1987).Our previous studies showed that TH2B expressed during meiotic event of spermatogenic dif- gene is expressed only in testis, in contrast to its counterpart, ferentiation
Genomic sequencing revealed that all analyzed CpG sites in the promoter region of TH2B gene are methylated in somatic tissues but not in testis.During spermatogenesis, these CpG sites areunmethylatedas early as spermatogoniatype A and up to sperm
Our results presented here indicate that DNA methylation may play a causal role in the repression of TH2B gene in somatic cells
Summary
For the synthesis of methylated oligonucleotides, 5-methylcytosine phosphoamidite replaced cytosine at four CpG sites indicated in bold. (>60 days), and spermatogonia type A and Sertoli cells from testes Nuclear proteins were extracted from isolated nuclei as described of Day 8 prepubertal rats. The plasmid was isolated from sperm nuclei as described previously Barberius et al (1987), and nuclei from fractionated spermatogenic mixture contained 10 pg of DNA and 20 units of SssI methylase in cells were isolated as described previously TCC-ACT-CCA-ACG-TCT-3', Fig. 1).As an unmethylated control, isothiocyanate-cesium chloride method (Chirgwin et al, 1979), and the pTB plasmid containing TH2B gene (insert; 3.8 kb) was treated DNA waspurified from isolated nuclei as describedpreviously (Weinas described (Saluz and Jost, 1989). The probe was a 310bp HinfI-MspI DNA fragment recognizing the intergenic sequences between THZA and THZB genes
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