LINE Retrotransposon RNA Is an Essential Structural and Functional Epigenetic Component of a Core Neocentromeric Chromatin

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Abstract
We have previously identified and characterized the phenomenon of ectopic human centromeres, known as neocentromeres. Human neocentromeres form epigenetically at euchromatic chromosomal sites and are structurally and functionally similar to normal human centromeres. Recent studies have indicated that neocentromere formation provides a major mechanism for centromere repositioning, karyotype evolution, and speciation. Using a marker chromosome mardel(10) containing a neocentromere formed at the normal chromosomal 10q25 region, we have previously mapped a 330-kb CENP-A–binding domain and described an increased prevalence of L1 retrotransposons in the underlying DNA sequences of the CENP-A–binding clusters. Here, we investigated the potential role of the L1 retrotransposons in the regulation of neocentromere activity. Determination of the transcriptional activity of a panel of full-length L1s (FL-L1s) across a 6-Mb region spanning the 10q25 neocentromere chromatin identified one of the FL-L1 retrotransposons, designated FL-L1b and residing centrally within the CENP-A–binding clusters, to be transcriptionally active. We demonstrated the direct incorporation of the FL-L1b RNA transcripts into the CENP-A–associated chromatin. RNAi-mediated knockdown of the FL-L1b RNA transcripts led to a reduction in CENP-A binding and an impaired mitotic function of the 10q25 neocentromere. These results indicate that LINE retrotransposon RNA is a previously undescribed essential structural and functional component of the neocentromeric chromatin and that retrotransposable elements may serve as a critical epigenetic determinant in the chromatin remodelling events leading to neocentromere formation. The centromere is an essential chromosomal structure for the correct segregation of chromosomes during cell division. Normal human centromeres comprise a 171-bp α-satellite DNA arranged into tandem and higher-order arrays. Neocentromeres are fully functional centromeres that form epigenetically on noncentromeric regions of the chromosomes, with recent evidence indicating an important role they play in centromere repositioning, karyotype evolution, and speciation. Neocentromeres contain fully definable DNA sequences and provide a tractable system for the molecular analysis of the centromere chromatin. Here, the authors investigate the role of epigenetic determinants in the regulation of neocentromere structure and function. They identify that a retrotransposable DNA element found within the neocentromere domain is actively transcribed and that the transcribed RNA is essential for the structural and functional integrity of the neocentromere. This study defines a previously undescribed epigenetic determinant that regulates the neocentromeric chromatin and provides insight into the mechanism of neocentromere formation and centromere repositioning.