K562 cells were nucleofected with 0

K562 cells were nucleofected with 0.5 g of siGENOME SMARTpool targeting either TFII-I, HDAC3, USF2, or 0.5 g of siCONTROL nontargeting pool (Neg.) or mock transfected. also been shown to interact with a conserved E-box located in LCR element HS2 (4, 14). A mutation of this E-box impairs the enhancer function of HS2. We have previously shown that TFII-I and USF interact with the core promoter of the -globin gene in vitro and in vivo (22). The interaction of TFII-I was more pronounced in erythroid cells in which the -globin gene is repressed. Here we demonstrate that reducing TFII-I activity by RNA interference (RNAi) or the expression of a dominant-negative mutant leads to the derepression of the -globin gene in erythroid cells. CM-272 Moreover, we show that TFII-I is complexed with histone deacetylase 3 (HDAC3) and that both proteins together interact with the -globin core promoter. A reduction of USF activity by expression of a dominant-negative protein causes a decrease of RNA polymerase (Pol) II recruitment to the globin gene locus and the repression of -globin gene transcription. The results support the hypothesis that antagonistic activities exerted by TFII-I and USF during embryonic and adult erythroid development contribute to the stage-specific expression of the -globin gene. MATERIALS AND METHODS Construction of protein expression vectors. The plasmid pCMV/A-USF was obtained from Charles Vinson (NIH) and has been described previously (35). The A-USF open reading frame, including an N-terminal hemagglutinin (HA) tag, was amplified by PCR. The reaction was carried out using Elongase (Invitrogen), 0.4 mM forward primer (5-GCGCCTTAAGCTCCACCATGGCGTATCC-3) containing an AflII restriction site (underlined), 0.4 mM reverse primer (5-GCGCTCTAGAAGAAGCTTTTAGTTGCTGTCATTC-3) containing a XbaI restriction site (underlined), 1.5 CM-272 mM Mg2+, and 5 ng pCMV/A-USF as a template in a final volume of 50 l. PCR conditions were as follows: 94C for 30 s, 35 cycles of 94C for 30 s, 61C for 30 s, and 68C for 30 s, with a final extension at 68C for 2 min. The amplified product and pcDNA4/TO (Invitrogen) were digested with AflII and XbaI and subsequently ligated to create pTO/A-USF. pET11d/USF1 was digested with BamHI and XbaI to S1PR4 isolate the USF1 open reading frame containing an N-terminal Flag tag. The fragment was subcloned into pcDNA4/TO and then properly oriented by digestion with PmeI and ligation back into pcDNA4/TO to create pTO/USF1. pET11d/p70 and pET11d/TFII-I were CM-272 digested with NcoI and SpeI to isolate the open reading frames. The fragments were subcloned into the pLitmus 29 vector (New England Biolabs). A subsequent digest with BamHI and SpeI isolated the open reading frames, which were subcloned into the pcDNA 3.1(+) vector (Invitrogen). The open reading frames were properly oriented by digestion with PmeI and ligation back into pcDNA 3.1(+). CM-272 The open reading frames CM-272 were isolated from pcDNA 3.1(+) by digestion with AflII and XbaI and subcloned into pcDNA4/TO, thus creating pTO/p70 and pTO/TFII-I. pTO/HA-p70, expressing an HA-tagged p70 mutant and containing a neomycin resistance gene, was created by amplifying the p70 open reading frame by PCR using pTO/p70 as a template and the forward primer containing a BamHI site (5-GCGCGGATCCCATGGCCCAAGTTGCAATGTCC-3) and the reverse primer containing an XbaI site (5-GCGCTCTAGAGTTCAGGTTTTTTAACAACGAAC-3). The PCR product and pTO/A-USF were digested with BamHI and XbaI and subsequently ligated, creating pcDNA4/TO/HA-p70. This construct was digested with XbaI and BstZ171 to remove the Zeocin resistance gene. pCMV-566 (obtained from Charles Vinson) was digested with PciI, filled in using a Klenow reaction, and then digested with XbaI to isolate a fragment containing the neomycin resistance gene. These two fragments were then ligated to create pTO/HA-p70. pTO, the empty control vector, was created by digesting pcDNA4/TO with XbaI and BstZ171. pCMV-566 was digested with PciI, filled in using a Klenow reaction, and digested with XbaI. The.