title: POU1F1-mediated activation of hGH-N by deoxyribonuclease I hypersensitive site II of the human growth hormone locus control region.
abstract: The human growth hormone gene (hGH-N) is regulated by a distal locus control region (LCR) composed of five deoxyribonuclease I hypersensitive sites (HSs). The region encompassing HSI and HSII contains the predominant pituitary somatotrope-specific hGH-N activation function of the LCR. This activity was attributed primarily to POU1F1 (Pit-1) elements at HSI, as linkage to HSI was sufficient for properly regulated hGH-N expression in transgenic mice, while HSII alone had no activity. However, the presence of HSII in conjunction with HSI further enhanced hGH-N transgene expression, indicating additional determinants of pituitary hGH-N activation in the HSII region, but limitations of transgenic models and previous ex vivo systems have prevented the characterization of HSII. In the present study, we employ a novel minichromosome model of the hGH-N regulatory domain and show that HSII confers robust POU1F1-dependent activation of hGH-N in this system. This effect was accompanied by POU1F1-dependent histone acetylation and methylation throughout the minichromosome LCR/hGH-N domain. A series of in vitro DNA binding experiments revealed that POU1F1 binds to multiple sites at HSII, consistent with a direct role in HSII function. Remarkably, POU1F1 binding was localized in part to the 3' untranslated region of a primate-specific LINE-1 (long interspersed nuclear element 1) retrotransposon, suggesting that its insertion during primate evolution may have conferred function to the HSII region in the context of pituitary GH gene regulation. These observations clarify the function of HSII, expanding the role of POU1F1 in hGH LCR activity, and provide insight on the molecular evolution of the LCR.
authors.affiliation: Department of Biochemistry and Molecular Biology, Brody School of Medicine at East Carolina University, Greenville, NC 27834, USA.
authors.forename: Tamra L
authors.initials: TL
authors.lastname: Hunsaker
authors.forename: Holly S
authors.initials: HS
authors.lastname: Jefferson
authors.forename: J Kaitlin
authors.initials: JK
authors.lastname: Morrison
authors.forename: Andrew J
authors.initials: AJ
authors.lastname: Franklin
authors.forename: Brian M
authors.initials: BM
authors.lastname: Shewchuk
mesh_terms: D020413:3' Untranslated Regions; D000107:Acetylation; D000818:Animals; D001483:Base Sequence; D001665:Binding Sites / Q000235:genetics; D002460:Cell Line; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism; D003850:Deoxyribonuclease I / Q000378:metabolism*; D004285:Dogs; D005786:Gene Expression Regulation; D005809:Genes, Regulator; D006657:Histones / Q000378:metabolism; D019382:Human Growth Hormone / Q000235:genetics* / Q000378:metabolism*; D006801:Humans; D019901:Locus Control Region; D020084:Long Interspersed Nucleotide Elements; D008745:Methylation; D051379:Mice; D008969:Molecular Sequence Data; D010902:Pituitary Gland / Q000378:metabolism; D011485:Protein Binding / Q000235:genetics; D051381:Rats; D050436:Regulatory Elements, Transcriptional / Q000235:genetics*; D016415:Sequence Alignment; D050817:Transcription Factor Pit-1 / Q000378:metabolism*; D015533:Transcriptional Activation / Q000235:genetics; D019076:Transgenes
chemical_list: D020413:3' Untranslated Regions; D004268:DNA-Binding Proteins; D006657:Histones; C494187:POU1F1 protein, human; D050817:Transcription Factor Pit-1; D019382:Human Growth Hormone; D003850:Deoxyribonuclease I