The molecular role of corepressors is poorly understood. Here, we studied the transcriptional function of the corepressor SMRT during terminal adipogenesis. Genome-wide DNA-binding profiling revealed that this corepressor is predominantly located in active chromatin regions and that most distal SMRT binding events are lost after differentiation induction. Promoter-proximal tethering of SMRT in preadipocytes is primarily mediated by KAISO through the conserved TCTCGCGAGA motif. Further characterization revealed that KAISO, similar to SMRT, accelerates the cell cycle and increases fat accumulation upon knockdown, identifying KAISO as an adipogenic repressor that likely modulates the mitotic clonal expansion phase of this process. SMRT-bound promoter-distal sites tend to overlap with C/EBPβ-bound regions, which become occupied by proadipogenic transcription factors after SMRT clearance. This reveals a role for SMRT in masking enhancers from proadipogenic factors in preadipocytes. Finally, we identified SMRT as an adipogenic gatekeeper as it directly fine-tunes transcription of pro- and antiadipogenic genes.

The cellular abundance of transcription factors (TFs) is an important determinant of their regulatory activities. Deriving TF copy numbers is therefore crucial to understanding how these proteins control gene expression. We describe a sensitive selected reaction monitoring-based mass spectrometry assay that allowed us to determine the copy numbers of up to ten proteins simultaneously. We applied this approach to profile the absolute levels of key TFs, including PPAR and RXR, during terminal differentiation of mouse 3T3-L1 pre-adipocytes. Our analyses revealed that individual TF abundance differs dramatically (from 250 to >300,000 copies per nucleus) and that their dynamic range during differentiation can vary up to fivefold. We also formulated a DNnA binding model for PPAR based on TF copy number, binding energetics and local chromatin state. This model explains the increase in PPAR binding sites during the final differentiation stage that occurs despite a concurrent saturation in PPAR copy number. Originally published at: Simicevic, Jovan; Schmid, Adrien W; Gilardoni, Paola A; Zoller, Benjamin; Raghav, Sunil K; Krier, Irina; Gubelmann, Carinne; Lisacek, Frédérique; Naef, Felix; Moniatte, Marc; Deplancke, Bart (2013). Absolute quantification of transcription factors during cellular differentiation using multiplexed targeted proteomics. Nature Methods, 10(6):570-576.

The genomic loci occupied by RNA polymerase (RNAP) III have been characterized in human culture cells by genomewide chromatin immunoprecipitations, followed by deep sequencing (ChIP-seq). These studies have shown that onlỹ 40% of the annotated 622 human tRNA genes and pseudogenes are occupied by RNAP-III, and that these genes are often in open chromatin regions rich in active RNAP-II transcription units. We have used ChIP-seq to characterize RNAP-III-occupied loci in a differentiated tissue, the mouse liver. Our studies define the mouse liver RNAP-III-occupied loci including a conserved mammalian interspersed repeat (MIR) as a potential regulator of an RNAP-III subunit-encoding gene. They reveal that synteny relationships can be established between a number of human and mouse RNAP-III genes, and that the expression levels of these genes are significantly linked. They establish that variations within the A and B promoter boxes, as well as the strength of the terminator sequence, can strongly affect RNAP-III occupancy of tRNA genes. They reveal correlations with various genomic features that explain the observed variation of 81% of tRNA scores. In mouse liver, loci represented in the NCBI37/mm9 genome assembly that are clearly occupied by RNAP-III comprise 50 Rn5s (5S RNA) genes, 14 known non-tRNA RNAP-III genes, nine Rn4.5s (4.5S RNA) genes, and 29 SINEs. Moreover, out of the 433 annotated tRNA genes, half are occupied by RNAP-III. Transfer RNA gene expression levels reflect both an underlying genomic organization conserved in dividing human culture cells and resting mouse liver cells, and the particular promoter and terminator strengths of individual genes.[Supplemental material is available for this article.]RNA polymerase III (RNAP-III) synthesizes short RNAs involved in essential cellular processes, including protein synthesis, RNA maturation, and transcriptional control, but until recently, the full extent of the genomic loci occupied, and therefore probably transcribed, by RNAP-III in vivo, was unknown. Several groups have now used the ChIP-seq technique, i.e., chromatin immunoprecipitation followed by deep sequencing, to localize genome-wide RNAP-III and some of its transcription factors in several human cultured cell lines. These experiments have revealed a relatively modest number of new RNAP-III transcription units, from a few 10s to about 200, depending on the criteria applied (Barski et al. 2010;Canella et al. 2010;Moqtaderi et al. 2010;Oler et al. 2010). In addition, most previously known RNAP-III genes were occupied by RNAP-III. Thus, in such cells RNAP-III and some of its transcription factors occupied 17 RN5S loci annotated on chromosome 1 in the NCBI/hg18 genome assembly, the VTRNA1-1, VTRNA1-2, VTRNA1-3, and VTRNA2-1 (hsa-mir-886) genes coding for vault RNAs, three SRP genes, 14 SNAR genes, five RNU6 genes, the RNU6ATAC gene, the RN7SK, RMRP, and RPPH1 genes, and the RNY1, RNY3, RNY4, and RNY5 (hsa-mir-1975) genes. Noticeably, however, a large fraction of the annotated tRNA genes was devoid of...