The mechanisms ensuring specific incorporation of CENP-A at centromeres are poorly understood. Mis16 and Mis18 are required for CENP-A localization at centromeres and form a complex that is conserved from fission yeast to human. Fission yeast sim1 mutants that alleviate kinetochore domain silencing are defective in Scm3(Sp), the ortholog of budding yeast Scm3(Sc). Scm3(Sp) depends on Mis16/18 for its centromere localization and like them is recruited to centromeres in late anaphase. Importantly, Scm3(Sp) coaffinity purifies with CENP-A(Cnp1) and associates with CENP-A(Cnp1) in vitro, yet localizes independently of intact CENP-A(Cnp1) chromatin and is differentially released from chromatin. While Scm3(Sc) has been proposed to form a unique hexameric nucleosome with CENP-A(Cse4) and histone H4 at budding yeast point centromeres, we favor a model in which Scm3(Sp) acts as a CENP-A(Cnp1) receptor/assembly factor, cooperating with Mis16 and Mis18 to receive CENP-A(Cnp1) from the Sim3 escort and mediate assembly of CENP-A(Cnp1) into subkinetochore chromatin.

Summary: Cytosine methylation is an epigenetic mark that promotes gene silencing and plays an important role in genome defence against transposons and invading DNA viruses. Previous data showed that the largest family of single‐stranded DNA viruses, Geminiviridae, prevents methylation‐mediated transcriptional gene silencing (TGS) by interfering with the proper functioning of the plant methylation cycle. Here, we describe a novel counter‐defence strategy used by geminiviruses, which reduces the expression of the plant maintenance DNA methyltransferases, METHYLTRANSFERASE 1 (MET1) and CHROMOMETHYLASE 3 (CMT3), in both locally and systemically infected tissues. We demonstrated that the virus‐mediated repression of these two maintenance DNA methyltransferases is widespread among geminivirus species. Additionally, we identified Rep (Replication associated protein) as the geminiviral protein responsible for the repression of MET1 and CMT3, and another viral protein, C4, as an ancillary player in MET1 down‐regulation. The presence of Rep suppressed TGS of an Arabidopsis thaliana transgene and of host loci whose expression was strongly controlled by CG methylation. Bisulfite sequencing analyses showed that the expression of Rep caused a substantial reduction in the levels of DNA methylation at CG sites. Our findings suggest that Rep, the only viral protein essential for replication, displays TGS suppressor activity through a mechanism distinct from that thus far described for geminiviruses.

The histone H3 variant CENP-A is the most favored candidate for an epigenetic mark that specifies the centromere. In fission yeast, adjacent heterochromatin can direct CENP-A^Cnp1 chromatin establishment, but the underlying features governing where CENP-A^Cnp1 chromatin assembles are unknown. We show that, in addition to centromeric regions, a low level of CENP-A^Cnp1 associates with gene promoters where histone H3 is depleted by the activity of the Hrp1^Chd1 chromatin-remodeling factor. Moreover, we demonstrate that noncoding RNAs are transcribed by RNA polymerase II (RNAPII) from CENP-A^Cnp1 chromatin at centromeres. These analyses reveal a similarity between centromeres and a subset of RNAPII genes and suggest a role for remodeling at RNAPII promoters within centromeres that influences the replacement of histone H3 with CENP-A^Cnp1.

Geminiviruses are small DNA viruses that replicate in nuclei of infected plant cells after accumulation of host replication machinery. Tomato golden mosaic virus (TGMV) and Tomato yellow leaf curl Sardinia virus (TYLCSV) encode a protein, RepAC1 (or Rep), that is essential for viral replication. Rep/RepAC1 is an oligomeric protein that binds to double-stranded DNA, catalyzes cleavage and ligation of single-stranded DNA, and is sufficient for host induction. It also interacts with several host proteins, including the cell cycle regulator, retinoblastoma, and essential components of the cell DNA replication machinery, like proliferating nuclear cell antigen (PCNA) and RFC-1. To identify other cellular proteins that interact with Rep/RepAC1 protein, a Nicotiana benthamiana cDNA library was screened with a yeast two-hybrid assay. The host cell sumoylation enzyme, NbSCE1 ( N. benthamiana SUMO-conjugating enzyme, homolog to Saccharomyces cerevisiae UBC9), was found to interact specifically with RepAC1. Mapping studies localized the interaction to the N-terminal half of RepAC1. Effects on geminivirus replication were observed in transgenic plants with altered levels of SUMO, the substrate for UBC9.

Specialized chromatin containing CENP-A nucleosomes instead of H3 nucleosomes is found at all centromeres. However, the mechanisms that specify the locations at which CENP-A chromatin is assembled remain elusive in organisms with regional, epigenetically regulated centromeres. It is known that normal centromeric DNA is transcribed in several systems including the fission yeast, Schizosaccharomyces pombe. Here, we show that factors which preserve stable histone H3 chromatin during transcription also play a role in preventing promiscuous CENP-A^Cnp1 deposition in fission yeast. Mutations in the histone chaperone FACT impair the maintenance of H3 chromatin on transcribed regions and promote widespread CENP-A^Cnp1 incorporation at non-centromeric sites. FACT has little or no effect on CENP-A^Cnp1 assembly at endogenous centromeres where CENP-A^Cnp1 is normally assembled. In contrast, Clr6 complex II (Clr6-CII; equivalent to Rpd3S) histone deacetylase function has a more subtle impact on the stability of transcribed H3 chromatin and acts to prevent the ectopic accumulation of CENP-A^Cnp1 at specific loci, including subtelomeric regions, where CENP-A^Cnp1 is preferentially assembled. Moreover, defective Clr6-CII function allows the de novo assembly of CENP-A^Cnp1 chromatin on centromeric DNA, bypassing the normal requirement for heterochromatin. Thus, our analyses show that alterations in the process of chromatin assembly during transcription can destabilize H3 nucleosomes and thereby allow CENP-A^Cnp1 to assemble in its place. We propose that normal centromeres provide a specific chromatin context that limits reassembly of H3 chromatin during transcription and thereby promotes the establishment of CENP-A^Cnp1 chromatin and associated kinetochores. These findings have important implications for genetic and epigenetic processes involved in centromere specification. Centromeres are the chromosomal locations at which kinetochores, the machinery that directs chromosome segregation, are assembled. In most eukaryotes, centromere location is epigenetically determined, meaning that the underlying DNA sequence does not dictate where they are formed. The genome is packaged in particles called nucleosomes, composed of histone proteins. Centromere DNA is wrapped around unusual nucleosomes that differ from those elsewhere in the genome because the histone H3-like CENP-A replaces the normal histone H3 component. We used fission yeast to investigate where CENP-A^Cnp1 nucleosomes are formed in cells containing excess CENP-A^Cnp1 and how the formation of these non-centromeric CENP-A nucleosomes is controlled. H3 nucleosomes are disassembled and reassembled during transcription by RNA polymerase II (RNAPII). We show that the normal process of reassembling robust H3 chromatin on RNAPII genes is required to prevent CENP-A^Cnp1 assembly in its place. Centromeres allow a low level of RNAPII transcription, and our analyses suggest that DNA sequences and chromatin contexts at centromeres may limit the activities required to stabilize and reassemble H3 chromatin during transcription in order to promote the establishment of CENP-A^Cnp1 chromatin and associated kinetochores.