Chromatin changes at the HBA genes in response to HDAC-inhibition. (A) Restriction enzyme accessibility assays performed on nuclei from K562 cells, mock-treated HeLa cells, or HeLa cells treated with 0.2 μmol/L TSA for 8 hours. Nuclei were exposed to no enzyme (0) or increasing concentrations of HinfI. Shown are Southern blots to detect hypersensitive sites at the α-globin promoters, HS-40 and HS-74, as indicated. The limit digested bands are indicated by black arrows, and bands resulting from digestion by HinfI within HSs are indicated by gray arrows. The limit digested fragments analyzed on each membrane are shown at left (H, HinfI; P, PstI; E, EcoRI; Hd, HindIII). The previously mapped extent of the known hypersensitive sites (open bars) and the probes used (black bars) are indicated. Genes are shown as block arrows. In the bottom panel, the HBA promoter HS was analyzed in nuclei from K562 cells, HeLa cells after treatment with TSA, or HeLa cells which were treated with TSA and then cultured for a further 24 hours in the absence of TSA. (B) Left panel: RT-PCR analysis of HBA2 cDNA (31 cycles) and ACTB cDNA (26 cycles) in an EBV-lymphoblastoid culture which was either mock-treated, or treated with 0.2 μmol/L TSA for 4 or 8 hours. Right panels: ChIP experiments carried out on the EBV-lymphoblastoid cells analyzed by RT-PCR, using antibodies against either acetylated histone H3 or histone H4. Immunoprecipitated material was analyzed by real-time PCR at the amplicons indicated surrounding the α-globin locus (shown in Figure 1) or a control amplicon within the 18S rRNA gene. Data indicate the percentage of input material precipitated at each amplicon.