Identification ofXenopusCENP-A and an Associated Centromeric DNA Repeat

Abstract

Kinetochores are the proteinaceous complexes that assemble on centromeric DNA and direct eukaryotic chromosome segregation. The mechanisms by which higher eukaryotic cells define centromeres are poorly understood. Possible molecular contributors to centromere specification include the underlying DNA sequences and epigenetic factors such as binding of the centromeric histone centromere protein A (CENP-A). Frog egg extracts are an attractive system for studying centromere definition and kinetochore assembly. To facilitate such studies, we cloned a Xenopus laevis homologue of CENP-A (XCENP-A). We identified centromere-associated DNA sequences by cloning fragments of DNA that copurified with XCENP-A by chromatin immunoprecipitation. XCENP-A associates with frog centromeric repeat 1 (Fcr1), a 174-base pair repeat containing a possible CENP-B box. Southern blots of partially digested genomic DNA revealed large ordered arrays of Fcr1 in the genome. Fluorescent in situ hybridization with Fcr1 probes stained most centromeres in cultured cells. By staining lampbrush chromosomes, we specifically identified the 11 (of 18) chromosomes that stain consistently with Fcr1 probes.

High-resolution mapping and transcriptional activity analysis of chicken centromere sequences on giant lampbrush chromosomes

“…Although the centromere-specific variant of histone H3 (CENP-A) was not detected in avian and amphibian lampbrush chromosomes (Edwards and Murray 2005;Krasikova and Gaginskaia 2010), recently identified sequences binding to CENP-A in chicken somatic cells were successfully mapped at high resolution on chicken lampbrush chromosomes. At the lampbrush stage, CENP-A interacting tandem repeats and nonrepetitive centromeric sequences in all cases were assigned adjacent to centromere granule enriched with cohesin subunits.…”

“…According to data reported by Edwards and Murray (2005), CENP-A protein is not concentrated at centromeres of Xenopus lampbrush chromosomes, and the position of a centromere can be defined by presence of centromeric repeats that are known to interact with CENP-A at other stages of the cell cycle and in other tissues. Importantly, in both avian and amphibian lampbrush chromosomes, pericentromeric compact chromomeres usually bear pairs of lateral loops, often very long, where RNA-polymerase II transcribes fragments of satellite DNA arrays (Diaz et al 1981;Baldwin and Macgregor 1985;Barsacchi-Pilone et al 1986;Solovei et al 1996;Krasikova et al 2006;Deryusheva et al 2007).…”

De Novo Kinetochore Assembly Requires the Centromeric Histone H3 Variant

“…One hallmark of all kinetochores is the essential CenH3 (Palmer et al, 1987;Stoler et al, 1995;Buchwitz et al, 1999;Henikoff et al, 2000;Takahashi et al, 2000;Sanyal and Carbon, 2002;Talbert et al, 2002;Zhong et al, 2002;Edwards and Murray, 2005). CenH3s contain a unique N terminus and a well-conserved C terminus that is highly homologous to histone H3 (Malik and Henikoff, 2003).…”

“…Outer kinetochore complexes include the conserved NDC80 (Ndc80, Spc24, Spc25, and Nuf2) and DAM1 (Dam1, Ask1, Duo1, Dad1, Dad2, Dad3, Dad4, Spc19, Spc34, and Hsk3) complexes. DAM1 is considered to be the outermost complex because it requires microtubules and all other complexes for kinetochore localization (Enquist-Newman et al, 2001;Janke et al, 2002;Li et al, 2002).One hallmark of all kinetochores is the essential CenH3 (Palmer et al, 1987;Stoler et al, 1995;Buchwitz et al, 1999;Henikoff et al, 2000;Takahashi et al, 2000;Sanyal and Carbon, 2002;Talbert et al, 2002;Zhong et al, 2002;Edwards and Murray, 2005). CenH3s contain a unique N terminus and a well-conserved C terminus that is highly homologous to histone H3 (Malik and Henikoff, 2003).…”

Working map of the lampbrush chromosomes of Xenopus tropicalis: A new tool for cytogenetic analyses

“…One would be the comparison of the position of centromeres on these two types of chromosomes. Position of the centromeres on LBCs of X. tropicalis could now be determined by in situ hybridization of centromeric repeat one (Fcr1) probe as for X. laevis (Edwards and Murray, 2005) or by injecting transcripts of the myc-tagged CENP-C protein in the X. tropicalis oocyte (Joseph Gall, Carnegie Institution, personal communication). The other would be to carry out in situ hybridization of specific probes for single copy genes on both sets of chromosomes, a technique which has been applied successfully to X. tropicalis mitotic chromosomes (Krylov et al, 2007).…”