A large fraction of the animal genome is maintained in a transcriptionally repressed state throughout development. By generating viable Dnmt1 ؊/؊ mouse cells we have been able to study the effect of DNA methylation on both gene expression and chromatin structure. Our results confirm that the underlying methylation pattern has a profound effect on histone acetylation and is the major effector of me-H3(K4) in the animal genome. We demonstrate that many methylated genes are subject to additional repression mechanisms that also impact on histone acetylation, and the data suggest that late replication timing may play an important role in this process.

Circadian clocks in mammals are built on a negative feedback loop in which the heterodimeric transcription factor circadian locomotor output cycles kaput (CLOCK)-brain, muscle Arnt-like 1 (BMAL1) drives the expression of its own inhibitors, the PERIOD and CRYPTOCHROME proteins. Reactivation of CLOCK-BMAL1 occurs at a specific time several hours after PERIOD and CRYPTOCHROME protein turnover, but the mechanism underlying this process is unknown. We found that mouse BMAL1 complexes include TRAP150 (thyroid hormone receptor-associated protein-150; also known as THRAP3). TRAP150 is a selective coactivator for CLOCK-BMAL1, which oscillates under CLOCK-BMAL1 transcriptional control. TRAP150 promotes CLOCK-BMAL1 binding to target genes and links CLOCK-BMAL1 to the transcriptional machinery at target-gene promoters. Depletion of TRAP150 caused low-amplitude, long-period rhythms, identifying it as a positive clock element. The activity of TRAP150 defines a positive feedback loop within the clock and provides a potential mechanism for timing the reactivation of circadian transcription. C ircadian clocks are endogenous oscillators that drive daily rhythms of physiology and behavior. The mammalian clock, intrinsic to most cells and tissues (1, 2), is built on a conserved negative feedback loop that generates circadian rhythms at the molecular level (3). The core positive element of the clock is the heterodimeric transcription factor circadian locomotor output cycles kaput (CLOCK)-brain, muscle Arnt-like 1 (BMAL1), which drives transcription of Period (Per) and Cryptochrome (Cry) genes from E-box sites (4). PER and CRY proteins, acting as negative elements of the clock, enter the nucleus, associate with CLOCK-BMAL1 (5) at E-box sites (6), and suppress the transcriptional activity of CLOCK-BMAL1 in part by recruiting the SIN3-HDAC histone deacetylase complex (6) and inhibiting transcriptional termination (7). Turnover of PERs and CRYs ends the negative-feedback phase of the cycle (8-11). An interlocked feedback loop involving REV-ERBα and -β (nuclear receptor subfamily 1, group D, members 1 and 2, respectively) contributes to clock function (12)(13)(14).Reactivation of CLOCK-BMAL1 transcription of circadian target genes occurs several hours after the end of negative feedback (15,16), suggesting that the onset of circadian transcription in each cycle by CLOCK-BMAL1 is not simply a passive consequence of the turnover of negative-feedback proteins, but is positively regulated and timed by unknown clock-controlled factors. Evidence that there is active positive regulation of CLOCK-BMAL1 comes from reports showing enhancement of CLOCK-BMAL1 transcriptional activity by chromatin-modifying proteins by CBP/p300 (17), MLL1 (18), and JARID1a (19). Although not previously described, a clock-controlled, rhythmic positive factor for CLOCK-BMAL1 would provide a potential mechanism for precisely setting the onset of transcription each circadian cycle. ResultsTo identify factors associated with CLOCK-BMAL1, we used FLAG antibodies to affinit...

A large fraction of genes in the mammalian genome is repressed in every cell throughout development. Here, we propose that this long-term silencing is carried out by distinct molecular mechanisms that operate in a global manner and, once established, can be maintained autonomously through DNA replication. Both individually and in combination these mechanisms bring about repression, mainly by lowering gene accessibility through closed chromatin structures.

Summary The entire genome is replicated in a programmed manner with specific regions undergoing DNA synthesis at different times in S phase. Active genes generally replicate in early S phase, while repressed genes replicate late, and for some loci this process is developmentally regulated. Using a nuclear microinjection system, we demonstrate that DNA sequences originally packaged into nucleosomes containing deacetylated histones during late S become reassembled with acetylated histones after undergoing replication in early S. Conversely, a change from early to late replication timing is accompanied by repackaging into nucleosomes containing deacetylated histones. This is carried out by differential cell cycle-controlled acetylation and deacetylation of histones H3 and H4. These studies provide strong evidence that switches in replication timing may play a role in the regulation of nucleosome structure during development.

Studying signaling dynamics in single cells in vivo is critical to understanding how cells act and interact in 3D environments. Experimental and computational tools to quantify a circadian reporter in single cells in intact tissues for >1 wk are used to analyze the period, amplitude, and synchrony of circadian rhythms in vivo.