Both casein kinase 1 delta (CK1␦) and epsilon (CK1) phosphorylate core clock proteins of the mammalian circadian oscillator. To assess the roles of CK1␦ and CK1 in the circadian clock mechanism, we generated mice in which the genes encoding these proteins (Csnk1d and Csnk1e, respectively) could be disrupted using the Cre-loxP system. Cre-mediated excision of the floxed exon 2 from Csnk1d led to in-frame splicing and production of a deletion mutant protein (CK1␦ ⌬2 ). This product is nonfunctional. Mice homozygous for the allele lacking exon 2 die in the perinatal period, so we generated mice with liver-specific disruption of CK1␦. In livers from these mice, daytime levels of nuclear PER proteins, and PER-CRY-CLOCK complexes were elevated. In vitro, the half-life of PER2 was increased by ϳ20%, and the period of PER2::luciferase bioluminescence rhythms was 2 h longer than in controls. Fibroblast cultures from CK1␦-deficient embryos also had long-period rhythms. In contrast, disruption of the gene encoding CK1 did not alter these circadian endpoints. These results reveal important functional differences between CK1␦ and CK1: CK1␦ plays an unexpectedly important role in maintaining the 24-h circadian cycle length.Circadian rhythms are rhythms in gene expression, metabolism, physiology, and behavior that persist in constant environmental conditions with a cycle length near 24 h. In mammals, the circadian timing system is hierarchical. The primary pacemaker regulating circadian behavioral rhythms is located in the suprachiasmatic nuclei (SCN) of the hypothalamus. Most cell types express circadian clock genes and will express rhythmicity in vitro. In vivo, the SCN entrains peripheral oscillators through a complex set of physiological and hormonal rhythms (31,32,36).At the molecular level, circadian oscillations are governed by a cell-autonomous negative-feedback loop in which transcription factors drive the expression of their own negative regulators, leading to oscillation between periods of transcriptional activation and repression (reviewed in references 32 and 36). The bHLH-PAS containing transcription factors CLOCK or NPAS2 form heterodimers with BMAL1. These heterodimers binds to E-box elements within regulatory regions of Period (Per1, Per2, and Per3) and Cryptochrome (Cry1 and Cry2) genes to stimulate their transcription. Approximately 12 h after transcriptional activation, PER and CRY proteins reach concentrations sufficient to form repressor complexes that inhibit the activity of the CLOCK/NPAS2:BMAL1 heterodimer, reducing the transcription of Per and Cry genes and subsequently relieving PER/CRY-mediated negative feedback. E-box-mediated expression of other transcription factors, including members of the DBP/HLF/TEF and nuclear orphan receptor families (e.g., Rev-Erb␣ and ROR-A), provides a mechanism for clock control of genes with diverse promoters and with gene expression peaks occurring at a variety of phases.Posttranslational modifications of circadian clock proteins play a well-established role in the re...

How embryonic stem cells (ESC) commit to specific cell lineages and ultimately yield all cell types of a fully formed organism remains a major question. ESC differentiation is accompanied by large-scale histone and DNA modifications, but the relations between these two categories of epigenetic changes are not understood. Here we demonstrate the hierarchical interplay between the histone deacetylase, sirtuin 6 (Sirt6), which targets acetylated histone H3 at lysines 9 and 56 (H3K9ac and H3K56ac), and the Tet (Ten-eleven translocation) enzymes, which convert 5-methylcytosine (5mC) into 5-hydroxymethylcytosine (5hmC). ESCs derived from Sirt6 knockout (S6KO) mice are skewed towards neuroectoderm development. This phenotype is associated with derepression of Oct4, Sox2 and Nanog, which in turn causes an upregulation of Tet enzymes and elevated production of 5hmC. Genome-wide analysis revealed an upregulation of neuroectoderm genes marked with 5hmC in S6KO ESCs, thereby implicating Tet enzymes in the neuroectoderm-skewed differentiation phenotype of S6KO ESCs, which is fully rescued upon knockdown of Tets. We demonstrate a new role for Sirt6 as a chromatin regulator safeguarding the balance between pluripotency and differentiation through Tet-dependent regulation of 5hmC levels.

The cspA is a gene of Escherichia coli, whose expression is specifically induced at low temperatures to a level of 13% of total protein synthesis. The CspA protein consisting of 70 amino acid residues has high sequence similarity with eukaryotic Y-box DNA-binding proteins. We found two independent clones from the Kohara miniset phage collection, which hybridized with a DNA fragment containing cspA. DNA sequencing of these clones confirmed that the two genes are highly homologous to cspA. One designated cspB is mapped at 35 min on the E. coli chromosome and encodes a 71-residue protein with 79% identity to CspA, while the other, cspC, is mapped at 40 min and encodes a 69-residue protein with 70% identity. In addition, a DNA sequence upstream of the clpA gene at 19 min published elsewhere contains an open reading frame for a 74-residue protein with 45% identity to CspA. All csp genes were fused in the coding regions with the lacZ gene, and the expression of beta-galactosidase was examined for these hybrid genes upon cold shock. A similar cold-shock induction to cspA was observed for cspB but not cspC and cspD. These results indicate that E. coli has a family of the cspA gene, some of which are induced by cold shock.