Fig. 4.

The upstream CpGI likely comprises a transcriptional enhancer. (A and B) ChIP analysis of CpGI 1 for (A) H3K4me1, H3K4me3, and H3 or (B) H3K27ac, H3, and IgG in αT3-1 cells, as in Fig. 2 (n = 2–4; Fig. S3). (C) Total αT3-1 RNA was reverse transcribed (RT) for qPCR of the regions marked. Controls lacked reverse transcription (normalized to gDNA standards; n = 3; Fig. S4). (D–G) Levels of eRNA were measured similarly in (D) αT3-1 and LβT2 cells; (E) WT and shTet2 αT3-1 cells; (F) LβT2 cells with or without GnRH treatment; and (G) primary gonadotrope cells (n = 3–6). (H and I) A 3C assay was carried out in Dpn2-digested DNA from αT3-1 cells, and chimeric fragments were detected by using nested forward primers (nos. 842 and 843 or nos. 844 and 845) targeting the functional TSS or upstream of the canonical exon 1, with various primers targeting the upstream region, as detailed in Fig. S4. (H) Amplicons were resolved by electrophoresis and identity confirmed by sequencing or (I) measured by qPCR using standard curves of the cloned chimeric fragments (n = 4–10); a t test was used to compare interaction of the pairs of regions.