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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 (Cenpa). Cenpa nucleosomes maintain the centromere by designating the site for new Cenpa 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 Xenopus laevis have not been extensively characterized. In this study, we combine Cenpa ChIP-seq with a k-mer based analysis approach to identify the Xenopus laevis centromere repeat sequences. By in situ hybridization, we show that Xenopus laevis centromeres contain diverse repeat sequences, and we map the centromere position on each Xenopus laevis chromosome using the distribution of centromere-enriched k-mers. Our identification of Xenopus 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.

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.

Non-coding RNAs (ncRNAs) are transcribed throughout the genome and provide regulatory inputs to gene expression through their interaction with chromatin. The genomic targets and regulatory mechanisms of most ncRNAs remain unknown. Using chromatin-associated RNA sequencing (ChAR-seq) we obtained a global map of RNA-chromatin interactions, transcriptome and genome-wide, in human embryonic stem cells and definitive endoderm. We defined the overall architecture of the RNA-chromatin interactome that encompasses all long non-coding RNAs, intronic RNAs, as well as unannotated RNAs derived from regulatory elements, transposable elements, and intergenic regions. We show that the interactome is cell state specific, involves functionally diverse classes of RNAs, and can be accurately predicted by a simple model accounting for RNA expression and distance to their DNA targets. We show that nearly all ncRNAs exclusively interact with genes in close three-dimensional proximity to their transcription locus. We identified a small number of RNAs that deviate from that behavior and interact with many loci across the genome. By relating the changes in the interactome during differentiation to changes in gene expression, we demonstrate that activation or repression of individual genes is unlikely to be controlled by the activity of a single ncRNA.

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