Transforming Growth Factor ß-Induced Proliferative Arrest Mediated by TRIM26-Dependent TAF7 Degradation and Its Antagonism by MYC.

Recognition of gene promoters by RNA polymerase II is mediated by general transcription factor IID (TFIID), which has been thought to be a static complex and to play a passive role in the regulation of gene expression under the instruction of gene-specific transcription factors. Here we show that transforming growth factor ß (TGF-ß) induced degradation of the TFIID subunit TAF7 in cultured mouse mammary epithelial cells and that this effect was required for proliferative arrest in response to TGF-ß stimulation. TGF-ß stimulated transcription of the gene for the ubiquitin ligase TRIM26, which was shown to ubiquitylate TAF7 and thereby to target it for proteasomal degradation. Sustained exposure of cells to TGF-ß resulted in recovery from proliferative arrest in association with amplification of the Myc proto-oncogene, with MYC inhibiting TRIM26 induction by TGF-ß. Our data thus show that TFIID is not simply a general mediator of transcription but contributes to the regulation of transcription in response to cell stimulation, playing a key role in the cytostatic function of TGF-ß.

Geminin is an indispensable inhibitor of Cdt1 in mouse embryonic stem cells.

Geminin is implicated in regulation of the cell cycle and differentiation. Although loss of Geminin triggers unscheduled DNA rereplication as a result of interruption of its interaction with Cdt1 in some somatic cancer cells, whether such cell cycle regulation also operates in embryonic stem cells (ESCs) has remained unclear. To characterize the Geminin-Cdt1 axis in ESCs and compare it with that in somatic cells, we established conditional knockout (KO) of Geminin in mouse ESCs and mouse embryonic fibroblasts (MEFs). Geminin KO ESCs manifest a large flattened morphology, develop polyploidy accompanied by DNA damage and G2 -M checkpoint activation, and subsequently undergo apoptosis. Rereplication in Geminin KO ESCs was attenuated by inhibition of G2 -M checkpoint signaling or by expression of wild-type Geminin, but not by expression of a Geminin mutant that does not bind to Cdt1, indicating the importance of sequestration of Cdt1 by Geminin in G2 phase. In contrast, Geminin KO MEFs did not manifest disturbance of the cell cycle unless they were treated to force abnormal accumulation of Cdt1. Together, our results indicate that Geminin is a key inhibitor of Cdt1 in mouse ESCs, but that it plays a backup role in MEFs to compensate for accidental up-regulation of Cdt1.

Lack of Transcription Triggers H3K27me3 Accumulation in the Gene Body.

Trimethylated H3K27 (H3K27me3) is associated with transcriptional repression, and its abundance in chromatin is frequently altered in cancer. However, it has remained unclear how genomic regions modified by H3K27me3 are specified and formed. We previously showed that downregulation of transcription by oncogenic Ras signaling precedes upregulation of H3K27me3 level. Here, we show that lack of transcription as a result of deletion of the transcription start site of a gene is sufficient to increase H3K27me3 content in the gene body. We further found that histone deacetylation mediates Ras-induced gene silencing and subsequent H3K27me3 accumulation. The H3K27me3 level increased gradually after Ras activation, requiring at least 35 days to achieve saturation. Such maximal accumulation of H3K27me3 was reversed by forced induction of transcription with the dCas9-activator system. Thus, our results indicate that changes in H3K27me3 level, especially in the body of a subset of genes, are triggered by changes in transcriptional activity itself.

Ras-induced changes in H3K27me3 occur after those in transcriptional activity.

Oncogenic signaling pathways regulate gene expression in part through epigenetic modification of chromatin including DNA methylation and histone modification. Trimethylation of histone H3 at lysine-27 (H3K27), which correlates with transcriptional repression, is regulated by an oncogenic form of the small GTPase Ras. Although accumulation of trimethylated H3K27 (H3K27me3) has been implicated in transcriptional regulation, it remains unclear whether Ras-induced changes in H3K27me3 are a trigger for or a consequence of changes in transcriptional activity. We have now examined the relation between H3K27 trimethylation and transcriptional regulation by Ras. Genome-wide analysis of H3K27me3 distribution and transcription at various times after expression of oncogenic Ras in mouse NIH 3T3 cells identified 115 genes for which H3K27me3 level at the gene body and transcription were both regulated by Ras. Similarly, 196 genes showed Ras-induced changes in transcription and H3K27me3 level in the region around the transcription start site. The Ras-induced changes in transcription occurred before those in H3K27me3 at the genome-wide level, a finding that was validated by analysis of individual genes. Depletion of H3K27me3 either before or after activation of Ras signaling did not affect the transcriptional regulation of these genes. Furthermore, given that H3K27me3 enrichment was dependent on Ras signaling, neither it nor transcriptional repression was maintained after inactivation of such signaling. Unexpectedly, we detected unannotated transcripts derived from intergenic regions at which the H3K27me3 level is regulated by Ras, with the changes in transcript abundance again preceding those in H3K27me3. Our results thus indicate that changes in H3K27me3 level in the gene body or in the region around the transcription start site are not a trigger for, but rather a consequence of, changes in transcriptional activity.