Using embryonic-origin, cell-free extracts that promote nucleosomal assembly, the process of establishing activated chromatin could be dissected. Interestingly, now that repression of transcription could be recapitulated in vitro, regulated transcription approaching the levels of gene activation observed in vivo could be achieved. In vitro transcription of chromatin-free templates suffered from the high background signal of basal transcription and addition of most trans-activator proteins had little or no impact on expression. Since basal transcription is essentially nonexistent in vivo, and now was repressed in vitro, regulation of transcription by multiple activators, such as Gal4-VP16 and HIV Tat protein, as examples, was possible in the test tube (Kamakaka et al., 1993; Steger et al., 1998). Biochemical analyses of chromatin structure and function suggested that multiple enzymatic complexes, working at the level of chromatin structure alteration, were required for regulation of gene expression. This work interfaced with the findings of geneticists, who found that suppression of specific mutations, which caused loss of regulated gene expression, was sometimes achieved by genes encoding modifiers or components of chromatin structure. These studies laid the groundwork, linking structural modification of chromatin and regulation of gene expression in vivo.

0 0

Post a comment