Chromatin and chromosomes

Recent advances in our understanding of chromosome behavior have been made possible by the development of cutting-edge cytological and molecular techniques. Presentations in this Minisymposium applied these techniques to a diverse set of topics. Sarah Elgin (Washington University) described her lab’s studies on the 1360 transposable element in Drosophila melanogaster. Previously, they showed that transgenes possessing this element promoted heterochromatin formation when inserted at ectopic sites in the genome. Now, using site-specific integration technology, they performed structure/function analyses and mapped the region responsible for silencing to the transposon’s transcription start sites. She also discussed her continued studies on the connection between heterochromatin proteins and RNA interference machinery. Knockdown of Piwi, an Argonaute/Piwi family member that binds Piwi-interacting RNAs and Heterochromatin Protein 1a (HP1a), in the female germline caused loss of HP1a and H3K9me2/3. This resulted in increased expression of the telomeric element HeT-A, demonstrating a role for Piwi in gene silencing in the female germline. Yasushi Hiraoka (Osaka University) identified the Schizosaccharomyces pombe Sme2 locus as a chromosome-pairing site during meiosis. A genomic fragment of Sme2 containing the TATA box and transcription start site is both necessary for pairing at the endogenous location and sufficient to induce pairing at ectopic genomic sites. Sme2 encodes a noncoding RNA that remains associated with the locus, possibly serving as a scaffold for proteins involved in pairing. Kristine Willis (Georgetown University) spoke about the relationship between gene activation and positioning within the Saccharomyces cerevisiae nucleus. By tracking both the position and the protein product of an inducible reporter gene, she finds that the reporter gene requires movement to the nuclear periphery before activation. In a subset of the cells, the activated gene remains at the periphery, while in other cells the gene moves to the interior and remains active. Interestingly, mutations in chromatin-remodeling factors caused defects in retention at the periphery, suggesting a requirement for a remodeled chromatin state at the periphery. Using S. cerevisiae as a model to study chromosome segregation, Min-Hao Kuo (Michigan State University) discovered that histone H3 monitors mitotic tension between sister chromatids at pericentromeres. Mutation of several amino acids within the H3 “tension-sensing motif” caused the loss of Shugoshin (Sgo1) from pericentromeres and chromosome segregation defects. GCN5, a histone acetyltransferase, suppressed the Sgo1 recruitment and segregation defects. A model was proposed whereby GCN5 acts as a negative regulator of Sgo1 spreading, limiting localization to the pericentric region. Carl Schildkraut (Albert Einstein College of Medicine) used single-molecule analysis of replicated DNA to examine replication forks and origins. Studies of replication forks stalled by hydroxyurea revealed that phosphorylation of replication protein A reduced single-stranded DNA and stimulated DNA synthesis to maintain replication fork integrity. When applied to studies of mouse telomeres, this technique revealed that chromosome ends replicate bidirectionally from sites surrounding and within telomeric repeats. Deborah Lannigan (University of Virginia) identified a function for the extracellular signal–regulated kinase 8 (ERK8) in DNA replication. ERK8 shares sequence identity with mitogen-stimulated kinases and was hypothesized to have self-phosphorylating activity, yet its role in cell biology was unknown. Knockdown of ERK8 reduced levels of proliferating cell nuclear antigen (PCNA), resulting in increased DNA breaks. Adding back excess PCNA rescued this defect. Substitutions within the phosphatidylinositol phosphate (PIP) domain of ERK8 recapitulated the ERK8 knockdown phenotype, suggesting this domain as a site of interaction. Knockdown of HDM2, an E3 ligase with a PIP domain, increased the levels of PCNA and rescued ERK8 knockdown defects, suggesting that recruitment of ERK8 to chromatin protects PCNA at the replication fork. Collectively, these presentations introduced American Society for Cell Biology Annual Meeting attendees to the latest technologies used to reveal fundamental principles that govern chromosome biology.