Figure 4. ChIP and gene expression analysis of Cenp-A spreading into euchromatin on del(17).

(A) ChIP-qPCR showed that Cenp-A is enriched at D17Z1 on the normal HSA17, and to lesser extent on del(17). Five genomic sites distal to the breakpoint and a control genomic site present only on the normal HSA17 were interrogated by ChIP to measure the genomic extent of Cenp-A spreading. All five euchromatic HSA17 short arm sites were enriched from Cenp-A on the del(17) compared to the normal HSA17. Enrichment was determined based on % input compared to D17Z1 (the typical site of Cenp-A enrichment on HSA17). Error bars represent SEM. Significant differences between Cenp-A enrichment at each genomic site on the del(17) and normal 17 were determined using a Student’s t-test. (B) Summary schematic of chromatin fiber and ChIP results showing the spreading of Cenp-A chromatin into ~300kb of HSA17 short arm euchromatin. Additional Genome Browser tracks are included below Cenp-A fiber and ChIP data to illustrate that Cenp-A chromatin spreads over a genomic region that includes genes/RNA transcripts and transposable elements and is largely characterized by open chromatin (ie. DNase hypersensitive sites, DNase HSS). (C) RT-qPCR analysis of the ELAC2 gene that is present with the repositioned centrmeric chromatin domain (genomic site 1, Figure 4A) revealed that ELAC2 transcription is reduced on the del(17) compared to the normal 17. RNA was extracted in duplicate from each cell line, and RT-qPCR was done in triplicate for each sample. Relative expression was calculated as a ratio of ELAC2 expression over beta actin expression, and the normal HSA17 was adjusted to 1. Error bars = SEM. A Student’s t-test was used to determine that gene expression was significantly different between the normal 17 and del(17) (p < 0.0001).