Phosphorylation of the carboxy-terminal repeat domain in RNA polymerase II by cyclin-dependent kinases is sufficient to inhibit transcription.

Cdc2 kinase triggers the entry of mammalian cells into mitosis, the only cell cycle phase in which transcription is globally repressed. We show here that Cdc2 kinase phosphorylates components of the RNA polymerase II transcription machinery including the RNA polymerase II carboxy-terminal repeat domain (CTD). To test specifically the effect of CTD phosphorylation by Cdc2 kinase, we used a yeast in vitro transcription extract that is dependent on exogenous RNA polymerase II that contains a CTD. Phosphorylation was carried out using immobilized Cdc2 so that the kinase could be removed from the phosphorylated polymerase. ATP gamma S and Cdc2 kinase were used to produce an RNA polymerase IIO that was not detectably dephosphorylated in the transcription extract. RNA polymerase IIO produced in this way was defective in promoter-dependent transcription, suggesting that phosphorylation of the CTD by Cdc2 kinase can mediate transcription repression during mitosis. In addition, we show that phosphorylation of pol II with the human TFIIH-associated kinase Cdk7 also decreases transcription activity despite a different pattern of CTD phosphorylation by this kinase. These results extend previous findings that RNA polymerase IIO is defective in preinitiation complex formation. Here we demonstrate that phosphorylation of the CTD by cyclin-dependent kinases with different phosphoryl acceptor specificities can inhibit transcription in a CTD-dependent transcription system.

Protein-DNA interactions associated with the onset of testis-specific expression of the mammalian Pgk-2 gene.

We have identified difference in protein-DNA interactions associated with the promoter of the mammalian spermatogenesis-specific Pgk-2 gene in expressing and nonexpressing cells, using a band shift assay. We compared DNA-binding activities in nuclear protein extracts from expressing adult testis cells versus nonexpressing prepuberal testis cells and nonexpressing somatic cells. One or two DNA-binding activities were found to be uniquely associated with the expressed state of Pgk-2, while a third appears to be associated with the nonexpressed state. All three of these activities map to a region within the first 40 bp upstream from the core promoter of this gene. The Pgk-2 core promoter lacks a TATA box but contain a GC box and a CAAT box. We show that the GC box binds the ubiquitous transcription factor Sp1 and that the CAAT box binds CTF-1, both of which are present in extracts from all three tissue types tested. These results suggest that tissue-specific transcription of the Pgk-2 gene is associated with changes in protein-DNA interactions occurring within a 40-bp enhancer region and that different arrays of protein-DNA interactions in this region are associated with the actively expressed state of the Pgk-2 gene in spermatocytes and spermatids and with the terminally repressed state of Pgk-2 in somatic cells.

Mutagenic treatments result in inactivation of expression of a transfected bacterial gene integrated into a human cell line.

The cell line E2 is a SV40-transformed human fibroblast cell line containing a single integrated copy of the bacterial guanine phosphoribosyl transferase (gpt) gene. Treatment of E2 with ultraviolet light (UV) or ethyl methanesulphonate (EMS) induced the formation of Gpt- derivatives. Several induced derivatives have been isolated, and the structure, expression and revertibility of the gpt gene have been analysed. In the majority of cases the Gpt- phenotype resulted from switching off the gpt gene, in most instances by methylation, but in a few cases by phenotypic switching. Thus mutagenic treatment can result in the inactivation of gene expression in human cells. In a small proportion of Gpt- derivatives the gpt sequences were deleted.

Inactivation of a transfected gene in human fibroblasts can occur by deletion, amplification, phenotypic switching, or methylation.

Plasmids containing the bacterial gpt gene under control of the simian virus 40 promoter were transfected into a simian virus 40-transformed human fibroblast line. Two transfectants, E2 and C10, which contain stably integrated single copies of the gpt gene, were isolated. These two lines produce Gpt- variants spontaneously with a frequency of about 10(-4). We carried out a detailed molecular analysis of the spectrum of alterations which gave rise to the Gpt- phenotype in these variants. DNA from 14 of 19 Gpt- derivatives of one of the cell lines (E2) contains deletions or rearrangements of gpt-containing sequences. In four of the remaining five lines, the Gpt- phenotype was correlated with reduced levels of expression rather than with changes in the gross structure of the gpt gene, and it was possible to reactivate the gpt gene. In one Gpt- line, gpt mRNA was present at normal levels, but no active enzyme was produced. Spontaneous Gpt- derivatives of the other cell line (C10) produced a completely different spectrum of alterations. Very few deletions were found, but several derivatives contained additional extrachromosomal gpt sequences, and, remarkably, in two other Gpt- lines, gpt-containing sequences were amplified more than 100-fold. The phenotypes of the majority of the Gpt- derivatives of C10 could be attributed to alterations in gene expression caused by methylation.