Single molecules were localized and tracked using SLIMfast, a custom-written MATLAB implementation of the MTT algorithm (Serg et al., 2008), using the following algorithm settings: Localization Rabbit Polyclonal to NDUFA4 error: 10?6.25; deflation loops: 0; Blinking (frames); 1; maximum number of competitors: 3; maximal expected diffusion constant (m2/s): 20. NCBI Gene Expression Omnibus (accession no: “type”:”entrez-geo”,”attrs”:”text”:”GSE109964″,”term_id”:”109964″GSE109964) The following previously published dataset was used: Tveves SSTjian R2016Global accessibility of mitotic chromosomeshttp://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=”type”:”entrez-geo”,”attrs”:”text”:”GSE85184″,”term_id”:”85184″GSE85184Publicly available at the NCBI Gene Expression Omnibus (accession no: “type”:”entrez-geo”,”attrs”:”text”:”GSE85184″,”term_id”:”85184″GSE85184) Abstract Maintenance of transcription programs is challenged during mitosis when chromatin becomes condensed and transcription is silenced. How do the daughter cells Varespladib methyl re-establish the original transcription program? Here, we report that the TATA-binding protein (TBP), a key component of the core transcriptional machinery, Varespladib methyl remains bound globally to active promoters in mouse embryonic stem cells during mitosis. Using live-cell single-molecule imaging, we observed that TBP mitotic binding Varespladib methyl is highly stable, with an average residence time of minutes, in stark contrast to typical TFs with residence times of seconds. To test the functional effect of mitotic TBP binding, we used a drug-inducible degron system and found that TBP promotes the association of RNA Polymerase II with mitotic chromosomes, and Varespladib methyl facilitates transcriptional reactivation following mitosis. These results suggest that the core transcriptional machinery promotes efficient transcription maintenance globally. and is also shown on Figure 6D. We still observe high levels of intronic reads in asynchronous (A) samples, despite near complete degradation of TBP. Our results are reminiscent of a previous study that has observed Pol II transcription in the absence of TBP in mouse blastocyst cells (Martianov et al., 2002). However, we observed a marked decrease in transcription reactivation in M60 samples following TBP degradation in mitosis (Figure 6D). This decrease occurs globally as most genes show decreased nascent chr-RNA levels (Figure 6F), suggesting a specific role for TBP in transcriptional reactivation following mitosis. Intriguingly, we see that TBP degradation has an effect on tRNA (Pol III genes) but not on rRNA (Pol I genes) expression (Figure 6figure supplement 3). Although this result may suggest differential roles for TBP among the three different polymerases, more stringent nascent RNA analysis will be needed to further inform this line of research. To examine the kinetics of transcriptional reactivation following mitosis, we calculated the log2 ratio of reads in the 30- and 60 min conditions relative to mitotically arrested samoles (M30/M and M60/M, respectively), for both untreated and TBP-degraded samples. In this way, we can observe the change in RNA levels relative to mitotic cells as a function of time after release from mitosis. Globally, the untreated M30/M sample shows no overall change in RNA levels whereas M60/M samples show massive increase in both upstream and downstream transcription at the TSS (Figure 7figure supplement 1). In contrast, TBP-degraded samples show delayed transcription levels evident in the M60/M sample (Figure 7figure supplement 1). To determine if these changes are driven by differences in TBP levels, we measured the average change in RNA levels when genes are clustered by the three groups as determined by TBP ChIP-seq k-means clustering shown in Figure 3D. We observed no effect between the three groups (Figure 7figure supplement 2), further suggesting that the minor changes we observed by TBP ChIP-seq are likely due to changes in cyclical gene expression. We next performed unbiased k-means clustering on the untreated M30/M data with k?=?3. Cluster 1 includes 5504 genes that show an increase in transcription whereas cluster 3 includes 6693 genes that show a decrease in transcription relative to mitotic cells. The remaining genes (cluster 2) show no change in transcription (Figure 7A,B). Ordering the M60/M data using the same three clusters shows that cluster one increases in transcription the earliest and the fastest whereas clusters 2 and 3 lag behind. We then ordered the M30/M and M60/M data from TBP-degraded samples (Figure 7A,B). This analysis shows that the early changes in transcription seen in untreated samples are dampened in TBP-degraded samples with the biggest effect on cluster 1. Using GO term analysis, cluster one is enriched for genes involved in metabolism of RNA, cell cycle, and transcription factor activity (Figure 7C). These genes are also generally highly expressed in the asynchronous population (Figure 7figure supplement 2), suggesting that these genes represent the global transcription program of the cells. In contrast, cluster three is enriched for genes involved in basic cellular processes such as microtubule cytoskeleton organization and regulation of GTPase activity, but also for genes.