Fig 2: in vivo recruitment of ERa to CSE gene promoterProtein–DNA complexes were in vivo formaldehyde cross-linked in hOBs from male and female donors and subjected to Chromatin immunoprecipitation (ChIP) analysis. Chromatin fragments were subjected to immunoprecipitation with antibodies against hERa or hSp1. After cross-link reversal, the co-immunoprecipitated DNA was detected by semi-quantitative PCR using the primer pairs spanning the proximal 304 bp region (-348/-44) and 244 bp region (-592/-348) on the CSE core promoter. Aliquots of chromatin taken before immunoprecipitation were used as input positive controls, whereas the negative control was the IgG immunoprecipitation. For each sample, levels of recruited ERa or Sp1 in the immunoprecipitates were determined by densitometry of the PCR fragments relative to Input control. Data are presented as mean + SD.

Fig 3: Schematic representation of the 5’-flanking region of the human CSE geneThe transcription factors up to now identified as regulators of CSE transcription are reported as ovals. Putative Estrogen Receptor binding sites are indicated as ERE half sites. Nrf2 (nuclear factor E2-related factor 2, involved in the antioxidant cell response), ATF4 (activating transcription factor 4, binds the cAMP response element), NFAT (the calcium-regulated nuclear factor of activated T cells) and Sp1 (specificity protein 1, an important component of the eukaryotic cellular transcriptional machinery) binding sites are indicated.

Fig 4: CSE gene promoter activityThe core promoter of CSE gene (-592/+139) was cloned into the upstream of firefly luciferase gene (LUC) in the pGL3-Basic vector (pGL3-731). The transfection was performed in hOB samples from two male and two female donors in absence (-) or in presence (+) of ERa expression vector together 10 nM 17-ß-estradiol (E2) for 48 hours. The transfection efficiency was normalized by measuring the total protein in the extracts. The LUC output was normalized against the promoterless pGL3-Basic vector. Results are expressed as mean + SD.