The validity of such an in vivo chromatin approach derives from the fact that in several known imprinting control-elements, a specialized organization of chromatin characterized by nuclease hypersensitivity is present on only one of the two parental chromosome (3). A systematic search for allele-specific chromatin features constitutes an alternative strategy to identify elements that regulate imprinting. So far, sequence elements that regulate parental allele-specific gene expression have been identified in a number of imprinted loci, either because of their differential DNA methylation or through functional studies in transgenic mice (1,2). To further our understanding of genomic imprinting it will be essential to identify key control elements, and to investigate their regulation by both epigenetic modifications (such as DNA methylation) and trans-acting factors. Probing chromatin structure with nuclease sensitivity assays. A combination of DNase I and micrococcal nuclease sensitivity assays indicates that the characteristic erythroid-specific hypersensitivity of HS4 to DNase I is the result of tissue-specific alterations in both nucleosome positioning and tertiary DNA structure. Similarly arranged NF-E2 and GATA binding sites are present in the other HSs of the human LCR, as well as in the homologous mouse and goat sequences and the chicken beta-globin enhancer. This analysis indicates that binding sites for the hematopoietic transcription factors NF-E2 and GATA-1 are required for the formation of the characteristic chromatin structure of the HS following stable transfection into murine erythroleukemia cells. We now report a mutational analysis of the HS4 HS-forming element (HSFE). This element binds six proteins in vitro. Previously, we identified a 101 bp element that is required for the formation of this HS. We have used 5' HS4 as a model to study the formation of these HSs. The active elements of the LCR coincide with strong erythroid-specific DNase I- hypersensitive sites (HSs). The beta-like globin genes require the upstream locus control region (LCR) for proper expression. Stamatoyannopoulos, J A Goodwin, A Joyce, T Lowrey, C H NF-E2 and GATA binding motifs are required for the formation of DNase I hypersensitive site 4 of the human beta-globin locus control region.
HS1, HS2 and HS3 were also sensitive for micrococcal nuclease. The increase in sensitivity at HS1 and HS2 after T3treatment in vivo was successfully reproduced in a cell-free system by in vitro treatment with T3. We conclude that transcriptional activation of the gene by T3and Dex have very similar mechanisms, but that at the inflammation stage they become slightly different. Three new sites appeared after turpentine oil treatment, while the sensitivities of HS3 and HS4 increased. After T3treatment the sensitivity of HS1 and HS2 increased and after dexamethasone (Dex) treatment that of all four sites did so. They were designated HS1, HS2, HS3 and HS4 (3'->5'). Four DNase I hypersensitive sites were observed in the 5'-upstream region of the rat AGP gene of liver cells. Following these treatments, the chromatin structure of this gene was analyzed by means of digestion with DNase I or micrococcal nuclease. Transcription of the ratalpha1-acid glycoprotein (AGP) gene is activated by glucocorticoid, thyroid hormone (T3) and cytokines. Matsukawa, T Kawasaki, H Tanaka, M Ohba, Y
Analysis of chromatin structure of rat alpha1-acid glycoprotein gene changes in DNase I hypersensitive sites after thyroid hormone, glucocorticoid hormone and turpentine oil treatment.
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