|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, but limitations of transgenic models and previous ex vivo systems have prevented the characterization of HSII and many open questions remain for how POU1F1 elements 15 kb upstream regulate hGH-N expression. In the present study, a novel minichromosome model of the hGH-N regulatory domain was employed, showing 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. 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. Also within the present study, several POU1F1 element-specific protein-DNA interaction studies were employed to identify potential cofactors that may be involved in POU1F1-dependent chromatin modification in the hGH locus. These studies showed that DNA-bound POU1F1 interacts with the nucleosome remodeling complex subunit Brg1 and with the histone acetyltransferase GCN5. These observations provide the first direct evidence for HSI POU1F1-mediated cofactor recruitment in the distal activation of hGH-N by a chromatin-modification mechanism. Finally, an inducible POU1F1-expression system was developed in conjunction with the hGH-N minichromosome model, to allow the temporal resolution of POU1F1-mediated histone modifications leading to POU1F1-dependent hGH-N transcription. These observations provide insight on the molecular mechanism underlying the long-range activation of hGH-N by the distal LCR.