The retinoblastoma tumor suppressor protein (pRb) is involved in mitotic exit, promoting the arrest of myoblasts, and myogenic differentiation. However, it is unclear how permanent cell cycle exit is maintained in differentiated muscle. Using RNA interference, expression profiling, and chromatin immunoprecipitations, we show that pRb is essential for cell cycle exit and the differentiation of myoblasts and is also uniquely required to maintain this arrest in myotubes. Remarkably, we also uncover a function for the pRb-related proteins p107 and p130 as enforcers of a G2/M phase checkpoint that prevents progression into mitosis in cells that have lost pRb. We further demonstrate that pRb effects permanent cell cycle exit in part by maintaining trimethylation of histone H3 lysine 27 (H3K27) on cell cycle genes. H3K27 trimethylation silences other genes, including Cyclin D1, in a pRb-independent but polycomb-dependent manner. Thus, our data distinguish two distinct chromatin-based regulatory mechanisms that lead to terminal differentiation.

Recruitment of TATA-binding protein (TBP) is central to activation of transcription by RNA polymerase II (pol II). This depends upon co-activator proteins including TBP-associated factors (TAFs). Yeast Mot1p was identi®ed as a general transcriptional repressor in genetic screens and is also found associated with TBP. To obtain insight into Mot1p function in vivo, we determined the mRNA expression pro®le of the mot1-1 temperature-sensitive (Ts) strain. Unexpectedly, this indicated that Mot1p mostly plays a positive role for transcription. For one potential activation target, HXT2, we analyzed promoter recruitment of Mot1p, TBP, Taf1p (Taf130p) and pol II by chromatin immunoprecipitation assays. Whereas TBP becomes stably associated upon activation of the HXT2 and HXT4 promoters, Mot1p showed only a transient association. TBP recruitment was compromised in two different mot1 mutant strains, but was only moderately affected in a taf1 Ts strain. Together, our data indicate that Mot1p can assist in recruitment of TBP on promoters during gene activation in vivo.

Transcription activation in yeast (Saccharomyces cerevisiae) involves ordered recruitment of transcription factor complexes, such as TFIID, SAGA, and Mot1p. Previously, we showed that both Mot1p and Taf1p are recruited to the HXT2 and HXT4 genes, which encode hexose transporter proteins. Here, we show that SAGA also binds to the HXT2 and HXT4 promoters and plays a pivotal role in the recruitment of Mot1p and Taf1p. The deletion of either SPT3 or SPT8 reduces Mot1p binding to HXT2 and HXT4. Surprisingly, the deletion of GCN5 reduces Taf1p binding to both promoters. When GCN5 is deleted in spt3⌬ or spt8⌬ strains, neither Mot1p nor Taf1p binds, and this results in a diminished recruitment of TATA binding protein and polymerase II to the HXT4 but not the HXT2 promoter. This is reflected by the SAGA-dependent expression of HXT4. In contrast, SAGA-independent induction of HXT2 suggests a functional redundancy with other factors. A functional interplay of different SAGA subunits with Mot1p and Taf1p was supported by phenotypic analysis of MOT1 SAGA or TAF1/SAGA double mutant strains, which revealed novel genetic interactions between MOT1 and SPT8 and between TAF1 and GCN5. In conclusion, our data demonstrate functional links between SAGA, Mot1p, and TFIID in HXT gene regulation.

We previously showed that the Spt-Ada-Gcn5-acetyltransferase (SAGA) complex is recruited to the activated HXT2 and HXT4 genes and plays a role in the association of TBP-associated factors. Using the HXT2 and HXT4 genes, we now present evidence for a functional link between Snf1p-dependent activation, recruitment of the SAGA complex, histone H3 removal, and H3 acetylation. Recruitment of the SAGA complex is dependent on the release of Ssn6p-Tup1p repression by Snf1p. In addition, we found that the Gcn5p subunit of the SAGA complex preferentially acetylates histone H3K18 on the HXT2 and HXT4 promoters and that Gcn5p activity is required for removal of histone H3 from the HXT4 promoter TATA region. In contrast, histone H3 removal from the HXT2 promoter does not require Gcn5p. In conclusion, although similar protein complexes are involved, induction of HXT2 and HXT4 displays important mechanistic differences.The yeast Saccharomyces cerevisiae utilizes glucose fermentation to generate metabolic energy. To optimize this process, glucose concentrations are carefully monitored, and gene expression is tightly linked to glucose availability. When glucose is present, genes involved in the uptake and fermentation of glucose are actively transcribed. However, glucose can also mediate repressive signals for genes involved in processing non-fermentable carbon sources and genes involved in the uptake and processing of alternative carbon sources. This latter process is known as glucose repression (1, 2).A central component in the glucose repression pathway is the Tup1p-Ssn6p complex. The Ssn6p-Tup1p complex has no DNA binding activity and depends on sequence-specific factors such as Mig1p for association to its target promoters (3, 4). The Ssn6p-Tup1p repressor complex genetically interacts with components of the Mediator complex and is associated with histone deacetylation (HDAC) 2 enzymes (5-9). Moreover, deletion of either SSN6 or TUP1 results in an altered chromatin organization of RNR3, FLO1, SUC2, and a-cell-specific genes (10 -14). A direct molecular link between Tup1p and chromatin organization was suggested by the interaction of the Tup1p repression domain with the N-terminal tails of histone H3 and H4 (15). In addition, Tup1p preferentially binds hypoacetylated histones on a subset of genes in vivo (16,17).To ensure efficient influx of glucose, yeast cells can express different hexose transporter (HXT) genes. The HXT2 and HXT4 genes encode high affinity glucose transporters, whose expression is repressed by high levels of glucose (18). Repression of the HXT2 and HXT4 genes is mediated by both subunits of the Ssn6p-Tup1p complex, because deletion of either SSN6 or TUP1 results in promoter activation of these genes under non-inducing conditions (18,19). To release Ssn6p-Tup1p-mediated repression under inducing conditions (i.e. low glucose), the HXT2 and HXT4 genes require Snf1p function (18). Snf1p encodes a serine/threonine kinase and is required for the expression of glucose-repressed genes (20, 21). Snf1p kinase phosp...