The role of the pituitary-specific element DNAse I hypersensitive site II in the human growth hormone locus control region
Multicellular organisms face a challenge with regard to gene expression: as each individual nucleus contains the same genome, how can genes be differentially regulated across development and between different tissues? A variety of regulatory regions beyond proximal promoters have been identified which contribute to this intricate process. Locus control regions (LCRs) constitute one category of long-range regulators. These sequences are usually comprised of multiple determinants and have the unique ability to confer tissue-specific, copy-number-dependent expression to a gene, regardless of its location in the genome. An LCR has been found to regulate the pituitary-secreted human growth hormone ( hGH) gene and four neighboring placenta-specific genes. The five genes in the cluster share over 90% sequence homology, which makes the strict temporal and spatial regulation accomplished by the hGH LCR a particularly interesting model system for the study of complex gene regulation. Research on this region has focused on elucidating the roles of individual LCR elements — as defined by short sequences hypersensitive to Deoxyribonuclease I (DNase I HS) — in promoting highly-regulated gene expression. This thesis explores the specific roles of pituitary-specific DNase I HS in regulating growth hormone in vivo, particularly that of Hypersensitive Site II (HSII), which is located ∼15 kb upstream from the gene target. The deletion of HSII results in a 75% decrease in hGH-Ntranscripts, demonstrating that it is necessary for full expression of the gene. Results also indicate that HSII is required for the recruitment of a pituitary transcription factor, Pit-1, to the hGH-N gene promoter and for occupancy of the elongating form of RNA Polymerase II at the gene. Through a study in a ‘neutral’ milieu, the HSII region is also implicated in the establishment of a promoter-independent H4 histone acetylated domain, which could represent an initiating event in the expression of hGH. The study also highlights the codependency of the individual LCR elements and the gene promoter in regulating target genes. These investigations contribute to the growing field of study of eukaryotic gene expression, and increase our knowledge of one of the many incredibly complex regulatory networks in these organisms.