H2A.Z is an essential histone variant implicated in the regulation of key nuclear events. However, the metazoan chaperones responsible for H2A.Z deposition and its removal from chromatin remain unknown. Here we report the identification and characterization of the human protein ANP32E as a specific H2A.Z chaperone. We show that ANP32E is a member of the presumed H2A.Z histone-exchange complex p400/TIP60. ANP32E interacts with a short region of the docking domain of H2A.Z through a new motif termed H2A.Z interacting domain (ZID). The 1.48 Å resolution crystal structure of the complex formed between the ANP32E-ZID and the H2A.Z/H2B dimer and biochemical data support an underlying molecular mechanism for H2A.Z/H2B eviction from the nucleosome and its stabilization by ANP32E through a specific extension of the H2A.Z carboxy-terminal α-helix. Finally, analysis of H2A.Z localization in ANP32E(-/-) cells by chromatin immunoprecipitation followed by sequencing shows genome-wide enrichment, redistribution and accumulation of H2A.Z at specific chromatin control regions, in particular at enhancers and insulators.

CPEB is a sequence-specific RNA-binding protein that controls the polyadenylation-induced translation of mos and cyclin B1 mRNAs in maturing Xenopus oocytes. CPEB activity requires not only the phosphorylation of S174, but also the synthesis of a heretofore-unknown upstream effector molecule. We show that the synthesis of RINGO/Spy, an atypical activator of cyclin-dependent kinases (cdks), is necessary for CPEB-directed polyadenylation. Deletion analysis and mRNA reporter assays show that a cis element in the RINGO/Spy 3 UTR is necessary for translational repression in immature (G2-arrested) oocytes. The repression is mediated by 3 UTR Pumilio-Binding Elements (PBEs), and by its binding protein Pumilio 2 (Pum2). Pum2 also interacts with the Xenopus homolog of human Deleted for Azoospermia-like (DAZL) and the embryonic poly(A)-binding protein (ePAB). Following the induction of maturation, Pum2 dissociates not only from RINGO/Spy mRNA, but from XDAZL and ePAB as well; as a consequence, RINGO/Spy mRNA is translated. These results demonstrate that a reversible Pum2 interaction controls RINGO/Spy mRNA translation and, as a result, CPEB-mediated cytoplasmic polyadenylation.

Eukaryotic circadian clocks are built on transcriptional feedback loops. In mammals, the PERIOD (PER) and CRYPTOCHROME (CRY) proteins accumulate, form a large nuclear complex (PER complex), and repress their own transcription. We found that mouse PER complexes included RNA helicases DDX5 and DHX9, active RNA polymerase II large subunit, Per and Cry pre-mRNAs, and SETX, a helicase that promotes transcriptional termination. During circadian negative feedback, RNA polymerase II accumulated near termination sites on Per and Cry genes but not on control genes. Recruitment of PER complexes to the elongating polymerase at Per and Cry termination sites inhibited SETX action, impeding RNA polymerase II release and thereby repressing transcriptional reinitiation. Circadian clock negative feedback thus includes direct control of transcriptional termination.

At the core of the mammalian circadian clock is a negative feedback loop in which the dimeric transcription factor CLOCK-BMAL1 drives processes that in turn suppress its transcriptional activity. To gain insight into the mechanisms of circadian feedback, we analyzed mouse protein complexes containing BMAL1. Receptor for activated C kinase-1 (RACK1) and protein kinase C-alpha (PKCalpha) were recruited in a circadian manner into a nuclear BMAL1 complex during the negative feedback phase of the cycle. Overexpression of RACK1 and PKCalpha suppressed CLOCK-BMAL1 transcriptional activity, and RACK1 stimulated phosphorylation of BMAL1 by PKCalpha in vitro. Depletion of endogenous RACK1 or PKCalpha from fibroblasts shortened the circadian period, demonstrating that both molecules function in the clock oscillatory mechanism. Thus, the classical PKC signaling pathway is not limited to relaying external stimuli but is rhythmically activated by internal processes, forming an integral part of the circadian feedback loop.

CENP-A is a histone variant, which replaces histone H3 at centromeres and confers unique properties to centromeric chromatin. The crystal structure of CENP-A nucleosome suggests flexible nucleosomal DNA ends, but their dynamics in solution remains elusive and their implication in centromere function is unknown. Using electron cryo-microscopy, we determined the dynamic solution properties of the CENP-A nucleosome. Our biochemical, proteomic, and genetic data reveal that higher flexibility of DNA ends impairs histone H1 binding to the CENP-A nucleosome. Substituting the 2-turn αN-helix of CENP-A with the 3-turn αN-helix of H3 results in compact particles with rigidified DNA ends, able to bind histone H1. In vivo replacement of CENP-A with H3-CENP-A hybrid nucleosomes leads to H1 recruitment, delocalization of kinetochore proteins, and significant mitotic and cytokinesis defects. Our data reveal that the evolutionarily conserved flexible ends of the CENP-A nucleosomes are essential to ensure the fidelity of the mitotic pathway.