Chromatin structure and function: the heretical path to an RNA transcription factor

This review represents a synthesis of the work of the author and her collaborators through 40 years of research aimed at an understanding of chromatin composition and functional arrangement. It describes the progressive experimental stages, starting with autoradiography and protein analysis and continuing on to a more functional approach testing the template properties of intact nuclei, as well as nuclei depleted of, or reconstituted with, defined fractions extracted from the chromatin of other cell lines or tissues. As new questions were raised at each phase of these studies, the investigation was shifted from chromosomal proteins to the role of a small RNA that coextracted with one protein fraction and whose properties suggested a transcription-activating function. The active RNA was identified as a class III RNA, designated as 7SK. Its properties suggested a role in the activation of two oncogenes, the SV40 T-antigen and the mammalian C-myc gene. A detailed analysis of the c-myc gene expression during transformation induction in temperature-sensitive mammalian cells finally culminated in in vivo evidence for a role of 7SK in c-myc deregulation, using cells transfected with antisense oligonucleotides to block 7SK activity. This was followed by an investigation of promoter targeting by 7SK RNP using electrophoretic mobility shift assays with whole or 7SK-depleted cell extracts. Taken together, these studies indicate that 7SK RNP participates in transformation-dependent deregulation of the c-myc gene by activation of two c-myc minor promoters. The implications of these findings are discussed.