Figure 4.

H3.3 is enriched at amplification origins before the initiation of DNA replication. (A) Experimental scheme for H3.3A-GFP ChIP-qPCR with or without UAS:dacapo (UAS:dap) expression. (B) H3.3 is still enriched at the amplicons when replication initiation is blocked by dap. Anti-GFP ChIP-qPCR analysis of stage 10 follicle cell nuclei after heat induction of H3.3A-GFP from flies with (gray bars) or without (green bars) coexpression of the CDK2 inhibitor dap. Ratio of DNA in the pellet vs. input was normalized to pellet/input ratio at negative control locus 93E/F. The hsp70 locus is a positive control. Note that, because the H3.3A-GFP transgene was hemizygous in these genetic crosses, H3.3A-GFP enrichment at the amplicons is lower than in previous experiments. The DAFC-66D amplicon and position of primers (black bars) are shown below. The other amplicons assayed were DAFC—7F, 22B, 30B, 34B and 62D. The occupancy of H3.3A-GFP was not significantly different from UAS:dap expression, except for DAFC-62D (* P-value of 0.04, as calculated by ratio paired t-test, see Table S2). Values represent the average of three biological replicates, and error bars represent SD.