Epigenetic engineering: histone H3K9 acetylation is compatible with kinetochore structure and function

Abstract

SummaryHuman kinetochores are transcriptionally active, producing very low levels of transcripts of the underlying alpha-satellite DNA. However, it is not known whether kinetochores can tolerate acetylated chromatin and the levels of transcription that are characteristic of housekeeping genes, or whether kinetochore-associated 'centrochromatin', despite being transcribed at a low level, is essentially a form of repressive chromatin. Here, we have engineered two types of acetylated chromatin within the centromere of a synthetic human artificial chromosome. Tethering a minimal NF-kB p65 activation domain within kinetochore-associated chromatin produced chromatin with high levels of histone H3 acetylated on lysine 9 (H3K9ac) and an ,10-fold elevation in transcript levels, but had no substantial effect on kinetochore assembly or function. By contrast, tethering the herpes virus VP16 activation domain produced similar modifications in the chromatin but resulted in an ,150-fold elevation in transcripts, approaching the level of transcription of an endogenous housekeeping gene. This rapidly inactivated kinetochores, causing a loss of assembled CENP-A and blocking further CENP-A assembly. Our data reveal that functional centromeres in vivo show a remarkable plasticity -kinetochores tolerate profound changes to their chromatin environment, but appear to be critically sensitive to the level of centromeric transcription.

Pericentric and centromeric transcription: a perfect balance required

“…Epigenetic modifications of engineered human artificial chromosomes (HACs) confirmed the need for a critical balance in transcription levels for properly functioning centromeres (Bergmann et al 2011(Bergmann et al , 2012 and reviewed in this issue). These studies also showed that active transcription, not histone modifications, is a key element to maintaining centromere function.…”

“…1d). Alteration of the HAC centrochromatin to an open chromatin state likewise resulted in disrupted CENP-A loading; the altered chromatin state did not directly affect the HAC's centromere activity, rather the resulting increase in transcription lead to the observed detrimental effects (Bergmann et al 2012) (Fig. 1d).…”

“…While we are gaining a better understanding of the pathway that results in the assembly of CENP-A nucleosomes at centromeres, the precise mechanism for determining the genomic location of CENP-A deposition is unknown. Recent work implicates centromeric transcripts as active participants in CENP-A deposition and centromere function, adding complexity to a pathway previously thought to be restricted to a large protein network (Topp et al 2004;Chueh et al 2009;Ferri et al 2009;Bergmann et al 2011Bergmann et al , 2012. Historically, centromeres were considered simply heterochromatin-rich and thus transcriptionally silent.…”

“…Densely packed heterochromatin is found in most eukaryotic pericentromeres, most commonly marked by di-and trimethylation of lysine residues 9 and 27 of histone H3 (H3K9me2, H3K9me3, H3K27me2, and H3K27me3), histone modifications typically associated with transcriptional silencing (Gopalakrishnan et al 2009). The chromatin encompassing the centromere core, also referred to as "centrochromatin," is distinct from that of pericentromeres and contains the histone H3 variant CENP-A interspersed with histone H3 methylation and dimethylation of lysine 4 and di-and trimethylation of lysine 36 of histone H3 (H3K4me1, H3K4me2, H3K36me2, and H3K36me3), histone modifications associated with transcriptionally active chromatin (Sullivan and Karpen 2004;Gopalakrishnan et al 2009;Bergmann et al 2011Bergmann et al , 2012. Interestingly, transcripts emanating from both the pericentromere and centromere core have been identified in a multitude of organisms (Eymery et al 2009a;Stimpson and Sullivan 2010).…”

“…Interestingly, transcripts emanating from both the pericentromere and centromere core have been identified in a multitude of organisms (Eymery et al 2009a;Stimpson and Sullivan 2010). Moreover, recent studies have identified proteins that interact with these transcripts (Topp et al 2004;Wong et al 2007;Chueh et al 2009;Ferri et al 2009;Du et al 2010;Hsieh et al 2011), analyzed the effects of increased and decreased transcription on genome stability (Bergmann et al 2011;Ohkuni and Kitagawa 2011;Bergmann et al 2012), or identified changes in transcription in stressed or diseased cells (Eymery et al 2009b;Gopalakrishnan et al 2009;Ting et al 2011;Zhu et al 2011). In this review, we will present pericentric and centromeric noncoding RNA transcription and discuss its diverse roles in modified histone recruitment, centromere function and stability, and insulator activity as well as newly discovered correlations between centromere and pericentromere transcription and human disease.…”