Abstract
We have previously described a 3.5-Mb domain of enhance scaffold/matrix attachment region (S/MAR) at a human neocentromere, and normal expression of underlying genes within this region. We also reported that partial inhibition of histone deacetylation using 33 nmtrichostatin A (TSA) resulted in a shift in the position of the CENP-A-binding domain within the neocentromere, with no noticeable effects on mitotic segregation function. In this study, 33 nM TSA caused a reduction in the size of the enhanced S/MAR domain of one-half to 1.7 Mb. Treatment with a DNA-intercalating drug distamycin A (DST) at 75 microg/ml resulted in a size reduction of the enhanced S/MAR domain at the neocentromere of two-thirds to 1.2 Mb, and that of the CENP-A-binding domain of 40%, from 330 to 196 kb, with no significant shift in the position of the latter domain. Other DST effects include mitotic chromosomal missegregation, reduction in the levels of Topo IIalpha, CENP-A, CENP-C, and HP1alpha, and an increase in mitotic checkpoint protein BubR1. TSA or DST treatment similarly resulted in a significant reduction, by approximately 20 and 50%, respectively, in the size of the enhanced S/MAR domain at the alpha-satellite DNA of a native chromosome 10 centromere. Transcriptional competence within the neocentromere is overall not noticeably altered by either TSA or DST treatment, as is evident from the absence of any significant increase or decrease in the expression levels of 47 underlying genes tested. These results suggest that a substantial contraction of the S/MAR domain may not be deleterious to centromere function, that disruption of the S/MAR domain directly affects the binding properties of a host of scaffold/matrix and centromeric/pericentric proteins, and that the overall competence and regulation of transcription at the neocentromeric chromatin is similar to those found at the corresponding normal genomic sites.
Highlights
The centromere of the eukaryotes is essential for the proper segregation of replicated sister chromatids during cell division
We have investigated the effects of Trichostatin A (TSA)-induced histone hyperacetylation on scaffold/matrix attachment and gene expression at the mardel[10] neocentromere using a moderate dose of 33 nM TSA
scaffold/matrix attachment region (S/MAR) analysis involved the differential isolation of scaffold/matrix attached and non-attached DNA from cell lines containing the neocentromeric mardel[10] chromosome (M10) or normal chromosome 10 (N10), and comparing the hybridisation profiles of these DNA fractions across a genomic array
Summary
The centromere of the eukaryotes is essential for the proper segregation of replicated sister chromatids during cell division. It appears as the primary constriction on condensed chromosomes and is the point of microtubule spindle attachment. The lack of any repetitive satellite DNA at neocentromeres supported the proposal of an epigenetic model for centromere chromatin formation and maintenance [8,9]. The induction of histone hyperacetylation using the deacetylase inhibitor Trichostatin A (TSA) causes chromosome loss and the disruption of the fission yeast heterochromatin protein Swi association from pericentric regions, interfering with the transcriptional repression in centromeric heterochromatin resulting in the expression of reporter genes [14]. In human cells, prolonged treatment with TSA disrupts Swi homologue HP1 localisation and causes centromeric defects resulting in chromosomal missegregation [11]
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