Human centromere repositioning activates transcription and opens chromatin fibre structure [TTseq]

Human centromeres appear as constrictions on mitotic chromosomes and form a platform for kinetochore assembly in mitosis. Biophysical experiments led to a suggestion that repetitive DNA at centromeric regions form a compact scaffold necessary for function, but this was revised when neocentromeres were discovered on non-repetitive DNA. To test whether centromeres have a special chromatin structure we have analysed the architecture of a neocentromere. Centromere formation is accompanied by RNA pol II recruitment and active transcription to form a decompacted, negatively supercoiled domain enriched in ‘open’ chromatin fibres. In contrast, centromerisation causes a spreading of repressive epigenetic marks to surrounding regions, delimited by H3K27me3 polycomb boundaries and divergent genes. This flanking domain is transcriptionally silent and partially remodelled to form ‘compact’ chromatin, similar to satellite-containing DNA sequences, and exhibits genomic instability. We suggest transcription disrupts chromatin to provide a foundation for kinetochore formation whilst compact pericentromeric heterochromatin generates mechanical rigidity. TT-Seq was used to examine nascent transcription accross the neocentromere region in HybNeo3 and GM10253A cells. TTseq is an adaptation of 4sU-Seq whereby the labeled RNA is fragmented prior to 4sU-purification and results in an enrichment of transient transcripts. TTseq was performed on 8 samples, 2 control untreated samples for each cell line GM10253A and HybNeo3 and also 2 exosome RNAi knockdown treated samples for each cell line.