Figure 3.

Reversal of amplification primers in the 3C assay confirms the specific association between hGH-N and the HSI,II region. (A) Map of primer positions for 3C using the HSII region primer as the anchor. The symbols are as in Fig 2A. (B) HSI-dependent association between HSI,II and the hGH-N promoter, and evidence for a pituitary (Pit)-specific interaction between HSII and HSIII-HSV regions. The semiquantitative PCR analyses of 3C ligation products were normalized to parallel analyses of the endogenous ERCC3 locus (C). Each bar represents the mean±s.d. of four independent assays of the indicated chromatin samples: hGH/P1 pituitary chromatin (black bars), hGH/P1(ΔHSI) pituitary chromatin (grey bars) and control liver chromatin (white bars). Lane C contains a PCR analysis of ligation products of BglII-digested hGH/P1 plasmid DNA and represents random ligation of equimolar quantities of each of the BglII fragments in the locus under the conditions of the assay. The ligation efficiency shown on the Y axis was calculated using the equation (signal_tissue/signal_{random control})/(signal_{ERCC3 ligation}/signal_{ERCC3 loading}). (C) 3C analysis of the control ERCC3 locus. The indicated head-to-head ligation frequency of two adjacent BglII fragments released from the ubiquitously expressed ERCC3 locus during the 3C analysis was determined and used as an internal control for each 3C assay. PCR within a unique BglII fragment was used as the loading control for each analysis. (D) A looping model of pituitary-specific hGH-N activation. The 3C studies of the hGH/P1 and hGH/P1(ΔHSI) transgene loci in the pituitary and the hGH/P1 transgene locus in the liver are summarized in a diagrammatic format. 3C, chromosome conformation capture; hGH, human growth hormone.