Identification and characterization of centromeric sequences inXenopus laevis

Abstract

Centromeres play an essential function in cell division by specifying the site of kinetochore formation on each chromosome for mitotic spindle attachment. Centromeres are defined epigenetically by the histone H3 variant CEntromere Protein A (CENP-A). CENP-A nucleosomes maintain the centromere by designating the site for new CENP-A assembly after dilution by replication. Vertebrate centromeres assemble on tandem arrays of repetitive sequences but the function of repeat DNA in centromere formation has been challenging to dissect due to the difficulty in manipulating centromeres in cells.Xenopus laevis egg extracts assemble centromeres in vitro , providing a system for studying centromeric DNA functions. However, centromeric sequences in X. laevis have not been extensively characterized. In this study we combine CENP-A ChIP-seq with a k-mer based analysis approach to identify the X. laevis centromere repeat sequences. By in situ hybridization we show that X. laevis centromeres contain diverse repeat sequences and we map the centromere position on each X. laevis chromosome using the distribution of centromere enriched k-mers. Our identification of X. laevis centromere sequences enables previously unapproachable centromere genomic studies. Our approach should be broadly applicable for the analysis of centromere and other repetitive sequences in any organism.

The molecular architecture of CenH3-deficient holocentromeres in Lepidoptera is dependent on transcriptional and chromatin dynamics

“…This approach revealed that the proportion of transposon sequences underlying CENP-T domains is not significantly different to that in the rest of the genome ( Figure 1F). Second, as a complementary approach, we searched de novo for any centromere-enriched repeats as described (Smith et al, 2020). Consistent with the first approach, these analyses also revealed that CENP-T-permissive sites in the B. mori genome are not enriched for repetitive DNA sequences ( Figure S1F).…”

Conserved chromatin and repetitive patterns reveal slow genome evolution in frogs

“…10). The X. tropicalis centromere sequence is different from centromericassociated repeats found in X. laevis 68,69 , suggesting the sequences evolve rapidly after speciation but are maintained within the species.…”

Molecular conflicts disrupting centromere maintenance contribute to Xenopus hybrid inviability

“…Recent characterization of X. laevis centromere sequences by chromatin immunoprecipitation with CENP-A antibodies and sequencing analysis (ChIP-seq) revealed a family of related sequences found in distinct combinations and abundances on different X. laevis chromosomes. 26 However, X. laevis centromeres 3L and 4L did not possess any distinguishing features in terms of size or composition. Thus, differences in core centromere DNA sequences do not appear to drive the specific chromosome mis-segregation events and genome loss observed in the inviable X. tropicalis/X.…”

“…CENP-A ChIP-seq and data analysis CENP-A MNase ChIP-seq was performed as previously described. 26 Briefly, livers were extracted from adult X. borealis animals and flash frozen. Upon thawing, livers were diced on ice, rinsed in PBS, and buffer 1 (2.5 mM EDTA, 0.5 M EGTA, 15 mM Tris-HCl pH 7.4, 15 mM NaCl, 60 mM KCl, 15 mM sodium citrate 0.5 mM spermidine, 0.15 mM spermine, 340 mM sucrose, supplemented with 0.1 mM PMSF) was added and the tissue dounced using pestle A 12 times.…”

“…The recent detailed characterization of X. laevis centromere sequences allowed us to test whether the centromere assembly defects observed in egg extract occurred on the same chromosomes disrupted in hybrid embryos. 25,26 Fluorescence in situ hybridization (FISH) probes in combination with CENP-A immunofluorescence identified X. laevis chromosomes 3L and 4L, the two chromosomes that lose large genomic regions in hybrid embryos, as those that also lose centromeric CENP-A staining when replicated in X. tropicalis egg extract (Figures S2C-S2E). Thus, the in vitro system reproduces incompatibilities likely to underlie chromosome mis-segregation and ultimately genome loss observed in vivo.…”