Laloraya S, Guacci V, Koshland D
Howard Hughes Medical Institute, Carnegie Institution of Washington, Baltimore, Maryland 21210, USA.
J Cell Biol. 2000 Nov 27;151(5):1047-56. doi: 10.1083/jcb.151.5.1047.
We identified the chromosomal addresses of a cohesin subunit, Mcd1p, in vivo by chromatin immunoprecipitation coupled with high resolution PCR-based chromosomal walking. The mapping of new Mcd1p-binding sites (cohesin-associated regions [CARs]) in single-copy sequences of several chromosomes establish their spacing ( approximately 9 kb), their sequestration to intergenic regions, and their association with AT-rich sequences as general genomic properties of CARs. We show that cohesins are not excluded from telomere proximal regions, and the enrichment of cohesins at the centromere at mitosis reflects de novo loading. The average size of a CAR is 0.8-1.0 kb. They lie at the boundaries of transcriptionally silenced regions, suggesting they play a direct role in defining the silent chromatin domain. Finally, we identify CARs in tandem (rDNA) and interspersed repetitive DNA (Ty2 and subtelomeric repeats). Each 9-kb rDNA repeat has a single CAR proximal to the 5S gene. Thus, the periodicity of CARs in single-copy regions and the rDNA repeats is conserved. The presence and spacing of CARs in repetitive DNA has important implications for genomic stability and chromosome packaging/condensation.
我们通过染色质免疫沉淀结合基于高分辨率PCR的染色体步移技术，在体内确定了黏连蛋白亚基Mcd1p的染色体定位。在几条染色体的单拷贝序列中新的Mcd1p结合位点（黏连蛋白相关区域[CARs]）的定位确定了它们的间距（约9 kb）、它们在基因间区域的隔离以及它们与富含AT序列的关联，这些都是CARs的一般基因组特性。我们发现黏连蛋白并不排除在端粒近端区域，有丝分裂时着丝粒处黏连蛋白的富集反映了从头加载。一个CAR的平均大小为0.8 - 1.0 kb。它们位于转录沉默区域的边界，表明它们在定义沉默染色质结构域中起直接作用。最后，我们在串联（rDNA）和散布的重复DNA（Ty2和亚端粒重复序列）中鉴定出CARs。每个9 kb的rDNA重复序列在5S基因近端有一个单一的CAR。因此，单拷贝区域中CARs的周期性和rDNA重复序列是保守的。重复DNA中CARs的存在和间距对基因组稳定性和染色体包装/凝聚具有重要意义。
