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. 2014 Mar 5;9(3):e90277. doi: 10.1371/journal.pone.0090277

Figure 6.

Figure 6

A, DZNep treatment inhibited Suz12 occupancy and Mecp2 recruitment at the Avp enhancer during hypothalamic-like differentiation of EB5 cells. Chromatin samples from cells treated with DZNep during differentiation were immunoprecipitated with antibodies against Suz12, Mecp2 or H4Ac and recovered DNA analyzed by qPCR for the presence of the Avp enhancer. Cells treated with DZNep (DZ) in the undifferentiated state for 24 hrs (+Lif) showed strongly reduced Suz12 binding. Following 4 days of differentiation (DZ,-Lif 4 days), Mecp2 binding was decreased while H4Ac was increased. In contrast, following 3 days of differentiation an additional 24 hours application of DZNep (-Lif 4days, DZ) did not affect Mecp2 occupancy. B, DZNep treatment led to reduced methylation at the Avp enhancer. MeDIP evidenced reduced 5hmC at the downstream enhancer in response to increasing doses of DZNep, while methylation levels at the promoter and gene body regions did not change. Bars represent standard deviations from the mean (sem), *, P<0.05; from 5 independent experiments performed.