High throughput mapping of origins of replicationand histone H3 actylation in human cells

Mapping origins of replication has been challenging in higher eukaryotes. We have developed a rapid, genome-wide method to map origins in asynchronous human cells by combining the nascent strand abundance assay with a highly-tiled microarray platform, and we validated the technique by two independent assays. We applied this method to analyze the enrichment of nascent DNA in three 50 kb regions containing known origins of replication in the MYC, LMNB2, and HBB genes, a 200 kb region containing the rare fragile site, FRAXA, and a 1075 kb region on chromosome 22, and detected most of the known origins, as well as 28 new origins. Surprisingly, the 28 new origins were small in size, and predominantly located within genes. Our study also revealed a strong correlation between origin replication timing and chromatin acetylation. Keywords: origin mapping, nascent strand DNA, histone acetylaction mapping, ChIP-chip Goal #1: Map the origins of DNA replication in human cells. This experiment is based on the fact that during S phase, short newly replicated DNA fragments (300bp-1kb) are generated only at the origins of replication. By combining the nascent strand assay with a tiled microarray platform, we have developed a rapid, non-PCR based, high-throughput approach to map active origins in asynchronous human cells. Two independent biological samples were analyzed. On the array design, four control regions, MYC (chr8), LMNB2 (chr19), HBB (chr11), and FRAXA (chrX), containing known origins were used as positive controls. Goal #2: Map the hyperacetylated histone H3 (K9/K14) regions in human cells for comparison with the origin mapping data. For this purpose, we combined the ChIP assay with the microarray technology. Two independent biological samples were analyzed. On the array design, two control regions, the hyperacetylated region, TUBA1 (chr2), and the hypoacetylated region, HET405 (chr9), were used as positive and negative controls, respectively, for our ChIP-on chip experiments. Note: For statistical purposes, each probe was duplicated on the array, creating four data sets, FWD1, FWD2, REV1, and REV2. To summarize our array design, 93% of the five regions on chromosomes 8, 11, 19, 22 and X were covered at the highest density with a 20 nt overlap between probes; for the remaining 7%, the gaps were 507±864 nt in length (range 31-9817 nt).