Many physiological processes exhibit circadian rhythms driven by cellular clocks composed of interlinked activating and repressing elements. To investigate temporal regulation in this molecular oscillator, we combined mouse genetic approaches and analyses of interactions of key circadian proteins with each other and with clock gene promoters. We show that transcriptional activators control BRD4-PTEFb recruitment to E-box-containing circadian promoters. During the activating phase of the circadian cycle, the lysine acetyltransferase TIP60 acetylates the transcriptional activator BMAL1 leading to recruitment of BRD4 and the pause release factor P-TEFb, followed by productive elongation of circadian transcripts. We propose that the control of BRD4-P-TEFb recruitment is a novel temporal checkpoint in the circadian clock cycle.
Cohesin is a protein complex whose core subunits, Smc1, Smc3, Scc1, and SA1/SA2 form a ring-like structure encircling the DNA. Cohesins play a key role in the expression, repair, and segregation of eukaryotic genomes. Following a catalytic mechanism that is insufficiently understood, Esco1 and Esco2 acetyltransferases acetylate the cohesin subunit Smc3, thereby inducing stabilization of cohesin on DNA. As a prerequisite for structure-guided investigation of enzymatic activity, we determine here the crystal structure of the mouse Esco2/CoA complex at 1.8 Å resolution. We reconstitute cohesin as tri- or tetrameric assemblies and use those as physiologically-relevant substrates for enzymatic assays in vitro. Furthermore, we employ cell-based complementation studies in mouse embryonic fibroblast deficient for Esco1 and Esco2, as a means to identify catalytically-important residues in vivo. These analyses demonstrate that D567/S566 and E491/S527, located on opposite sides of the murine Esco2 active site cleft, are critical for catalysis. Our experiments support a catalytic mechanism of acetylation where residues D567 and E491 are general bases that deprotonate the ε-amino group of lysine substrate, also involving two nearby serine residues - S566 and S527- that possess a proton relay function.
Cohesin is a protein complex encircles the DNA and regulates the separation of sister chromatids during cell division. Following a catalytic mechanism that is insufficiently understood, Esco1 and Esco2 acetyltransferases acetylate Smc3 subunit of cohesin, thereby inducing a stabilization of cohesin on DNA. As a prerequisite for structureguided investigation of enzymatic activity, we determine here the crystal structure of the mouse Esco2/CoA complex at 1.8 Å resolution. We reconstitute the entire cohesin as a tetrameric assembly and use it as a physiologically-relevant substrate for enzymatic assays in vitro. Furthermore, we employ cell-based complementation studies in mouse embryonic fibroblast deficient for Esco1 and Esco2, as a means to identify catalytically-important residues in vivo. These analyses demonstrate that D567/S566 and E491/S527, located on opposite sides of the MmEsco2 active site cleft, are critical for catalysis. Our experiments supports a catalytic mechanism of acetylation where residues D567 and E491 are general bases that deprotonate the εamino group of lysine substrate, via two nearby serine residues -S566 and S527-that possess a proton relay function.
VItranscript elongation. This competition characterizes the repressive limb of the clock cycle and eventually culminates in a complete cessation of BMAL1 acetylation and, thus, to the termination of clock gene transcription. Taken together, the experiments suggest that control of BRD4-P-TEFb recruitment to E-box-containing circadian promoters is a novel temporal checkpoint in the circadian clock cycle. Abbreviations VII Abbreviations AdCre Adenovirus-expressing Cre recombinase AdGFP Adenovirus-expressing green fluorescence protein ANOVA analysis of variance BAC Bacterial artificial chromosome BMAL1 Brain and muscle ARNT-like protein 1 bp base pair BRD4 Bromodomain-containing protein 4 BSA Bovine Serum Albumin CCGs Clock-controlled output genes CDK9 Cyclin-dependent kinase 9 ChIP Chromatin immunoprecipitation CLOCK Circadian locomotor output cycles kaput CMV Cytomegalovirus
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