Abstract
Locus control regions (LCRs) comprise sets of DNA elements capable of establishing autonomous chromatin domains that support robust and physiologically appropriate expression of target genes, often working over extensive distances. Human growth hormone (hGH-N) expression in the pituitary is under the regulation of a well characterized LCR containing four DNase I hypersensitive sites (HSs). The two pituitary-specific HS, HSI and HSII, are located 14.5 and 15.5 kb 5' to the hGH-N promoter. HSI is essential for activation of hGH-N during pituitary development and for sustaining robust activity in the adult. To determine whether the closely linked HSII has a role in hGH-N expression, it was deleted from a previously validated hGH/P1 transgene. Analysis of three independent hGH/P1(ΔHSII) transgenic mouse lines revealed that this deletion had no adverse effect on the formation of HSI, yet resulted in a substantial loss (70%) in hGH-N mRNA expression. This loss of expression was accompanied by a corresponding reduction in recruitment of the pituitary-specific transcription factor Pit-1 to the hGH-N promoter and a selective decrease in promoter occupancy of the elongation-linked isoform of RNA polymerase II. Sufficiency of HSI and HSII in LCR activity was explored by establishing two additional sets of mouse transgenic lines in which DNA segments containing these HS were positioned within the λ phage genome. In this "neutral" DNA context, HSII was required for the recruitment of HAT activity. These data establish HSII as a nonredundant component of the hGH LCR essential for establishment of robust levels of hGH-N gene expression.
Highlights
A series of distal DNA elements regulates the human growth hormone gene
HSI and HSII are encompassed within a 1.6-kb BglII fragment4 located 14.5–16.1 kb 5Ј to the Human growth hormone (hGH-N) promoter [17]
HSI is located within a 404-bp subsegment at the 3Ј end of this fragment and HSII has been mapped to the more 5Ј 1.2-kb segment [35]
Summary
A series of distal DNA elements regulates the human growth hormone gene. Results: Deletion of one of these elements, HSII, results in reduced growth hormone expression and changes in several activating phenomena at the locus. The human growth hormone gene (hGH) cluster contains the pituitary somatotrope-specific hGH-N gene and four highly homologous placenta-specific genes (Fig. 1A) This locus has been established as a model for studying the dynamics of LCR activities. A region extending from 14.5 to 32 kb upstream of the hGH-N promoter encompasses five DNase I hypersensitive sites (HS) comprising the hGH LCR (Fig. 1A) [17] This region is required for robust and spatially regulated expression of both the pituitary [17] and placental [3] genes within the cluster. In the case of hGH LCR actions in the pituitary, it has been demonstrated that transcription of the region between the LCR and hGH-N, containing the CD79b gene (Fig. 1A), constitutes an essential step in the pathway of hGH-N gene activation [26]. An analysis of a second set of transgenic lines in which we studied the actions of HSI/II in isolation from the rest of the locus suggests that HSII may be involved in the initial stages of histone modification and gene activation
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