Howard Hughes Medical Institute, Basic Sciences Division, Fred Hutchinson Cancer Research Center, Seattle, Washington 98109, USA.
Genes Dev. 2018 Jan 1;32(1):20-25. doi: 10.1101/gad.307736.117. Epub 2018 Jan 31.
We combined classical salt fractionation with chromatin immunoprecipitation to recover human centromeric chromatin under native conditions. We found that >85% of the total centromeric chromatin is insoluble under conditions typically used for native chromatin extraction. To map both soluble and insoluble chromatin in situ, we combined CUT&RUN (cleavage under targets and release using nuclease), a targeted nuclease method, with salt fractionation. Using this approach, we observed unexpected structural and conformational variations of centromere protein A (CENP-A)-containing complexes on different α-satellite dimeric units within highly homogenous arrays. Our results suggest that slight α-satellite sequence differences control the structure and occupancy of the associated centromeric chromatin complex.
我们将经典的盐析与染色质免疫沉淀相结合，在天然条件下回收人着丝粒染色质。我们发现，在通常用于天然染色质提取的条件下，超过 85%的总着丝粒染色质是不溶的。为了在原位映射可溶性和不溶性染色质，我们将靶向核酸酶切割和释放（CUT&RUN）与盐析相结合，这是一种靶向核酸酶方法。使用这种方法，我们观察到在高度同源的阵列中，不同的α卫星二聚体单元上含有着丝粒蛋白 A（CENP-A）的复合物的结构和构象存在意想不到的变化。我们的结果表明，轻微的α卫星序列差异控制着相关着丝粒染色质复合物的结构和占有率。
