Chromatin states have to be faithfully duplicated during DNA replication to maintain cell identity. It is unclear whether or how ATP-dependent chromatin-remodelling factors are involved in this process. Here we provide evidence that the Williams syndrome transcription factor (WSTF) is targeted to replication foci through direct interaction with the DNA clamp PCNA, an important coordinator of DNA and chromatin replication. WSTF, in turn, recruits imitation switch (ISWI)-type nucleosome-remodelling factor SNF2H to replication sites. These findings reveal a novel recruitment mechanism for ATP-dependent chromatin-remodelling factors that is fundamentally different from the previously documented targeting by sequence-specific transcriptional regulators. RNA-interference-mediated depletion of WSTF or SNF2H causes a compaction of newly replicated chromatin and increases the amount of heterochromatin markers, including HP1beta. This increase in the amount of HP1beta protein is mediated by progression through S phase and is not the result of an increase in HP1beta mRNA levels. We propose that the WSTF-ISWI complex has a role in the maintenance of chromatin structures during DNA replication.

Chromatin remodelling complexes containing the nucleosome‐dependent ATPase ISWI were first isolated from Drosophila embryos (NURF, CHRAC and ACF). ISWI was the only common component reported in these complexes. Our purification of human CHRAC (HuCHRAC) shows that ISWI chromatin remodelling complexes can have a conserved subunit composition in completely different cell types, suggesting a conserved function of ISWI. We show that the human homologues of two novel putative histone‐fold proteins in Drosophila CHRAC are present in HuCHRAC. The two human histone‐fold proteins form a stable complex that binds naked DNA but not nucleosomes. HuCHRAC also contains human ACF1 (hACF1), the homologue of Acf1, a subunit of Drosophila ACF. The N‐terminus of mouse ACF1 was reported as a heterochromatin‐targeting domain. hACF1 is a member of a family of proteins with a related domain structure that all may target heterochromatin. We discuss a possible function for HuCHRAC in heterochromatin dynamics. HuCHRAC does not contain topoisomerase II, which was reported earlier as a subunit of Drosophila CHRAC.

Error-free genome duplication and segregation are ensured through the timely activation of ubiquitylation enzymes. The anaphase-promoting complex or cyclosome (APC/C), a multisubunit E3 ubiquitin ligase, is regulated by phosphorylation. However, the mechanism remains elusive. Using systematic reconstitution and analysis of vertebrate APC/Cs under physiological conditions, we show how cyclin-dependent kinase 1 (CDK1) activates the APC/C through coordinated phosphorylation between Apc3 and Apc1. Phosphorylation of the loop domains by CDK1 in complex with p9/Cks2 (a CDK regulatory subunit) controlled loading of coactivator Cdc20 onto APC/C. A phosphomimetic mutation introduced into Apc1 allowed Cdc20 to increase APC/C activity in interphase. These results define a previously unrecognized subunit-subunit communication over a distance and the functional consequences of CDK phosphorylation. Cdc20 is a potential therapeutic target, and our findings may facilitate the development of specific inhibitors.

The histone fold is a structural motif with which two related proteins interact and is found in complexes involved in wrapping DNA, the nucleosome, and transcriptional regulation, as in NC2. We reveal a novel function for histone-fold proteins: facilitation of nucleosome remodeling. ACF1-ISWI complex (ATP-dependent chromatin assembly and remodeling factor [ACF]) associates with histone-fold proteins (CHRAC-15 and CHRAC-17 in the human chromatin accessibility complex [CHRAC]) whose functional relevance has been unclear. We show that these histone-fold proteins facilitate ATP-dependent nucleosome sliding by ACF. Direct interaction of the CHRAC-15/17 complex with the ACF1 subunit is essential for this process. CHRAC-17 interacts with another histone-fold protein, p12, in DNA polymerase epsilon, but CHRAC-15 is essential for interaction with ACF and enhancement of nucleosome sliding. Surprisingly, CHRAC-15/17, p12/CHRAC-17, and NC2 complexes facilitate ACF-mediated chromatin assembly by a mechanism different from nucleosome sliding enhancement, suggesting a general activity of H2A/H2B type histone-fold complexes in chromatin assembly.

Fission yeast Atf1 is a member of the ATF/CREB basic leucine zipper (bZIP) family of transcription factors with strong homology to mammalian ATF2. Atf1 regulates transcription in response to stress stimuli and also plays a role in controlling heterochromatin formation and recombination. However, its DNA binding independent role is poorly studied. Here, we report that Atf1 has a distinct role in regulating the anaphasepromoting complex/cyclosome (APC/C) ubiquitin ligase. We have identified atf1؉ as a dose-dependent suppressor of apc5-1, a mutation causing mitotic arrest. Remarkably, the suppression is not dependent upon the bZIP domain and is therefore independent of the ability of Atf1 to bind DNA. Interestingly, Atf1 physically binds the APC/C in vivo. Furthermore, we show that addition of purified Atf1 proteins into a cell-free system stimulates ubiquitylation of cyclin B and securin by the APC/C. These results reveal a novel role for Atf1 in cell cycle control through protein-protein interaction.Ubiquitylation is a post-translational modification that occurs through the action of an enzymatic cascade consisting of three enzymes E1, E2, and E3, and typically controls the proteolysis, localization, or activity of a protein (1). It is a tightly regulated, highly specific and temporally controlled process, and thus plays an important role in many processes such as the cell cycle, signal transduction, transcription, DNA repair, development, and regulation of the immune response.The anaphase-promoting complex/cyclosome (APC/C) 2 is a large (1.5 MDa) multisubunit E3 ubiquitin ligase, which has essential functions in key events during the cell cycle, such as chromosome segregation and mitotic exit by degrading securin/Cut2/Pds1 and cyclin B/Cdc13/Clb2, respectively (2-4). The APC/C belongs to the RING finger family of E3 ubiquitin ligases, which include Ubr1, c-Cbl, and Skp1-Cullin 1-F-box (SCF) complex (5), but unlike other members of this family, it is exceptionally large and complex, consisting of at least 11 conserved subunits. The APC/C appears to consist of two separable subcomplexes which associate independently with the scaffold subunit, Apc1/Cut4 (6). The first subcomplex includes a RING finger subunit Apc11 and a cullin domain subunit Apc2. This subcomplex is capable of ubiquitylating proteins to some extent, but lacks substrate specificity (7-9). The specificity seems to rely on the second subcomplex, consisting of three subunits Apc3/Nuc2/Cdc27, Apc6/Cut9/Cdc16, and Apc8/Cut23/Cdc23, which contain a tetratricopeptide repeat (TPR) domain. A member of the Fizzy family of APC/C activators, Cdh1, has been shown to bind Apc3/Cdc27 via its C-terminal IR motifs and recruit a substrate to the APC/C (10 -12). However, the exact roles of the remaining subunits and other TPR subunits, such as Apc5 and Apc7, are unknown.The activity of the APC/C is regulated in part by the association of the Fizzy family of WD40-containing activator proteins (2-4). There are two major activators during the cell cycle, Fizzy/Slp1/Cdc2...