A distal locus control region (LCR) regulates the human growth hormone (hGH) gene cluster. Four DNaseI hypersensitive sites, HSI, II, III, and V located between 14.5 to 32 kb 5'to the pituitary hGH-N gene promoter, mark the determinants of this hGH LCR. These determinants act in concert to establish robust and appropriately regulated expression of hGH-N in pituitary somatotropes. HSI and HSII, at -14.5 to -15.5, are specific to pituitary chromatin and constitute the primary determinants of hGH-N transgene expression in somatotropes. The core elements of HSI, comprising an array of binding sites for the pituitary-enriched transfactor Pit-1, integrate multiple sets of epigenetic regulatory modifications in the process of hGH-N activation. These HSI-dependent activities include: 1) establishing of a 32kb domain of core histone hyperacetylation encompassing the hGH LCR and extending to the hGH-N promoter, 2) generation of a robust PolII 'domain of transcription'within the LCR and adjacent CD79b gene that is necessary for full levels of hGH- N expression, and 3) organization of higher-order chromatin structure that juxtaposes the LCR/CD79b domain of transcription and the hGH-N promoter during the process of hGH-N transcriptional enhancement. Remarkably, the powerful chromatin-based activities of the Pit-1 array at HSI can be functionally distinguished from the activity of the Pit-1 array at the hGH-N promoter. The HSI Pit-1 specificity is mediated, in part, by its unique binding site sequence. HSI activity appears to act in synergy with the adjacent HSII. HSII may be responsible for augmenting HSI activity via extension of epigenetic modifications or by enhancing PolII occupancy and non-coding transcription within the LCR. The role of the LCR in the activation and enhancement of hGH-N expression may be followed by an equally important role in maintaining high levels of hGH-N expression throughout adult life. In this proposal, individual components of LCR action will be characterized using a combination of in vitro, cell-based, and mouse transgenic methodologies. The temporal order of LCR-dependent alterations at the hGH locus will be determined in a novel set of tissue culture lines that are arrested at defined stages of somatotrope differentiation. A naturally occurring mutation of the human Pit-1 protein that selectively blocks hGH-N expression will be used to further extend our understanding of the basis for the specificity of Pit-1 action at the hGH LCR.
The Specific Aims of this proposal focus on these major aspects of hGH LCR function with the ultimate goal of extending our understanding of hGH-N activation and expression and in generalizing our findings to pathways and mechanisms of long-range transcriptional activation in the eukaryotic genome. These findings will eventually be correlated with an array of pathologic defects in hGH expression, both inherited and acquired, and with physiologic alterations in hGH expression that occur in response to environmental stress and ageing.

Public Health Relevance

Knowledge gained from these studies of the human Growth Hormone gene will further define the pathways involved in the formation and function of the human pituitary, extend our understanding of genetic and acquired defects in Growth Hormone gene expression that are observed in human populations, and suggest novel avenues for diagnosis and therapy of these and associated disorders.

Agency
National Institute of Health (NIH)
Institute
Eunice Kennedy Shriver National Institute of Child Health & Human Development (NICHD)
Type
Research Project (R01)
Project #
5R01HD025147-22
Application #
8197328
Study Section
Molecular and Cellular Endocrinology Study Section (MCE)
Program Officer
Ilekis, John V
Project Start
1989-05-01
Project End
2014-11-30
Budget Start
2011-12-01
Budget End
2012-11-30
Support Year
22
Fiscal Year
2012
Total Cost
$476,083
Indirect Cost
$175,288
Name
University of Pennsylvania
Department
Internal Medicine/Medicine
Type
Schools of Medicine
DUNS #
042250712
City
Philadelphia
State
PA
Country
United States
Zip Code
19104
Tsai, Yu-Cheng; Cooke, Nancy E; Liebhaber, Stephen A (2014) Tissue specific CTCF occupancy and boundary function at the human growth hormone locus. Nucleic Acids Res 42:4906-21
Yoo, Eung Jae; Cooke, Nancy E; Liebhaber, Stephen A (2013) Identification of a secondary promoter within the human B cell receptor component gene hCD79b. J Biol Chem 288:18353-65
Ho, Yugong; Shewchuk, Brian M; Liebhaber, Stephen A et al. (2013) Distinct chromatin configurations regulate the initiation and the maintenance of hGH gene expression. Mol Cell Biol 33:1723-34
Nair, Ramya; Lauks, Juliane; Jung, SangYong et al. (2013) Neurobeachin regulates neurotransmitter receptor trafficking to synapses. J Cell Biol 200:61-80
Ho, Yugong; Liebhaber, Stephen A; Cooke, Nancy E (2011) The role of the hGH locus control region in somatotrope restriction of hGH-N gene expression. Mol Endocrinol 25:877-84
Sizova, Daria; Ho, Yugong; Cooke, Nancy E et al. (2010) Research resource: T-antigen transformation of pituitary cells captures three novel cell lines in the Pit-1 lineage. Mol Endocrinol 24:2232-40
Ho, Yugong; Tadevosyan, Aleksey; Liebhaber, Stephen A et al. (2008) The juxtaposition of a promoter with a locus control region transcriptional domain activates gene expression. EMBO Rep 9:891-8
Kimura, Atsushi P; Sizova, Daria; Handwerger, Stuart et al. (2007) Epigenetic activation of the human growth hormone gene cluster during placental cytotrophoblast differentiation. Mol Cell Biol 27:6555-68
Cajiao, Isabela; Sargent, Rachel; Elstrom, Rebecca et al. (2007) Igbeta(CD79b) mRNA expression in chronic lymphocytic leukaemia cells correlates with immunoglobulin heavy chain gene mutational status but does not serve as an independent predictor of clinical severity. Am J Hematol 82:712-20
Yoo, Eung Jae; Cajiao, Isabela; Kim, Jeong-Seon et al. (2006) Tissue-specific chromatin modifications at a multigene locus generate asymmetric transcriptional interactions. Mol Cell Biol 26:5569-79

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