title: Enhancer-blocking activity is associated with hypersensitive site V sequences in the human growth hormone locus control region.
abstract: Activation of the human growth hormone gene (hGH-N) is linked to a locus control region (LCR) containing four (I-III, V) hypersensitive sites (HS). Pit-1 binding to HS I/II is required for efficient pituitary expression. However, inclusion of HS III and V, located about 28 and 32 kb upstream of the hGH-N gene, respectively, is also required for consistent hGH-N expression levels in vivo. HS V is referred to as a boundary for the hGH LCR, but no specific enhancer blocking or barrier function is reported. We examined a 547 bp fragment containing HS V sequences (nucleotides -32,718/-32,172 relative to hGH-N) for enhancer-blocking activity using a well-established transient gene transfer system and assessed these sequences for CCCTC binding factor (CTCF), which is linked to enhancer-blocking activity. The 547 bp HS V fragment decreased enhancer activity with a reverse-orientation preference when inserted between HS III enhancer sequences and a minimal thymidine kinase promoter (TKp). These sequences are associated with CTCF in human pituitary and nonpituitary chromatin. Enhancer-blocking activity with an orientation preference was further localized to a 45 bp sub-fragment, with evidence of CTCF and upstream binding factor 1 (USF1) binding; USF1 is linked more closely with barrier function. The presence of yin and yang 1 (Yy1) that cooperates with CTCF in the regulation of X-chromosome inactivation was also seen. A decrease in CTCF and Yy1 RNA levels was associated with a significant reduction in enhancer-blocking activity. Assessment of CpG-dinucleotides in the TKp indicates that the presence of HS V sequences are associated with an increased incidence of CpG-dinucleotide methylation of the GC box region. These data support association of CTCF and enhancer-blocking activity with HS V that is consistent with a role as a (LCR) boundary element and also implicates Yy1 in this process.
authors.affiliation: Department of Physiology, University of Manitoba, Winnipeg, Manitoba, Canada.
authors.forename: Yan
authors.initials: Y
authors.lastname: Jin
authors.forename: Kevin
authors.initials: K
authors.lastname: Oomah
authors.forename: Peter A
authors.initials: PA
authors.lastname: Cattini
mesh_terms: D001483:Base Sequence; D000076246:CCCTC-Binding Factor; D047369:Chromatin Immunoprecipitation; D018899:CpG Islands; D019175:DNA Methylation / Q000235:genetics; D004274:DNA, Recombinant / Q000235:genetics; D004742:Enhancer Elements, Genetic / Q000235:genetics*; D005260:Female; D005786:Gene Expression Regulation; D057809:HEK293 Cells; D006367:HeLa Cells; D019382:Human Growth Hormone / Q000235:genetics*; D006801:Humans; D038101:Insulator Elements; D019901:Locus Control Region / Q000235:genetics*; D008969:Molecular Sequence Data; D010902:Pituitary Gland / Q000378:metabolism; D010920:Placenta / Q000378:metabolism; D011247:Pregnancy; D011485:Protein Binding; D025941:Protein Interaction Mapping; D034622:RNA Interference; D034741:RNA, Small Interfering / Q000494:pharmacology; D012097:Repressor Proteins / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism; D014162:Transfection; D050986:YY1 Transcription Factor / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism
chemical_list: D000076246:CCCTC-Binding Factor; C000619697:CTCF protein, human; D004274:DNA, Recombinant; D034741:RNA, Small Interfering; D012097:Repressor Proteins; D050986:YY1 Transcription Factor; C494698:YY1 protein, human; D019382:Human Growth Hormone