Fig 7.

CTCF promotes sense transcription at the SAMD4A promoter and interacts with TFIIB. HUVECs were treated with TNF-α for 0 to 60 min. (A) Levels of sense and antisense transcripts at the SAMD4A promoter determined by qRT-PCR. HUVECs were treated with siRNAs that knock down CTCF (KD) or mock treated (mock) prior to stimulation with TNF-α for 0 or 30 min and isolation of total RNA. Next, the levels of sense and antisense transcripts from regions S0, S1, and Sb were quantified after first-strand cDNA synthesis (using sense or antisense primers indicated in the diagram). Knocking down CTCF reduces overall transcription levels at all three sites at both times; it also reduces the sense/antisense ratio in favor of antisense transcripts copied from S0 and S1 (yellow highlights). *, difference significant (P < 0.01, two-tailed unpaired Student t test; n = 4). (B) CTCF and TFIIB bind to the same promoter and 3′ end fragments of SAMD4A, assessed by sequential ChIP (ReChIP-qPCR), where an anti-TFIIB was used first and then an anti-CTCF (and vice versa). Then, the percent enrichments (relative to the input ± the SD; n = 6) of three SAMD4A regions were determined. The results indicate that bound CTCF and TFIIB on S1, S3, and S4 at 0 min but not at 60 min. The 3′UTR (Z2) of ZFPM2 does not bind CTCF or TFIIB and serves as a negative control. *, value significantly greater than the corresponding obtained with ZFPM2 (P < 0.01, two-tailed unpaired Student t test). (C) CTCF and TFIIB coimmunoprecipitate. Immunoprecipitations (IPs) were carried out (in 125 or 250 mM NaCl) using anti-TFIIB (top) or anti-CTCF (bottom), before CTCF (top) or TFIIB (bottom) were detected by immunoblotting (using loadings of 1× and 2×); a nonspecific immunoglobulin (IgG) and inputs (using loadings of 0.1× and 0.2×) are included as controls. CTCF is detected after pulling down TFIIB (top) and TFIIB after pulling down CTCF (bottom). This indicates the two interact in vivo.