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Please read the abstract in the section 00front of this document / Dissertation (MMed Vet (FER))--University of Pretoria, 1999. / Veterinary Tropical Diseases / unrestricted

A dissertation submitted to the Faculty of Health Sciences, University of the Witwatersrand, Johannesburg, in fulfilment of the requirements for the degree of Master of Sciences in Medicine Johannesburg 2005 / Objectives: To document baseline pituitary-adrenal hormonal and related metabolic variables in 16 female patients with burnout. Then, following stress management intervention, to compare the changes with an equal number of untreated control subjects. / IT2017

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Indiana University-Purdue University Indianapolis (IUPUI) / LHX3 is a transcription factor important in pituitary and nervous system development. Patients with mutations in coding regions of the gene have combined pituitary hormone deficiency (CPHD) that causes growth, fertility, and metabolic problems. Promoter and intronic elements of LHX3 important for basal gene expression in vitro have been identified, but the key regulatory elements necessary for in vivo expression were unknown. With these studies, I sought to elucidate how LHX3 gene expression is regulated in vivo. Based on sequence conservation between species in non-coding regions, I identified a 7.9 kilobase (kb) region 3' of the human LHX3 gene as a potential regulatory element. In a beta galactosidase transgenic mouse model, this region directed spatial and temporal expression to the developing pituitary gland and spinal cord in a pattern consistent with endogenous LHX3 expression. Using a systematic series of deletions, I found that the conserved region contains multiple nervous system enhancers and a minimal 180 base pair (bp) enhancer that direct expression to both the pituitary and spinal cord in transgenic mice. Within this minimal enhancer, TAAT/ATTA sequences that are characteristic of homeodomain protein binding sites are required to direct expression. I performed DNA binding experiments and chromatin immunoprecipitation assays to reveal that the ISL1 and PITX1 proteins specifically recognize these elements in vitro and in vivo. Based on in vivo mutational analyses, two tandem ISL1 binding sites are required for enhancer activity in the pituitary and spine and a PITX1 binding site is required for spatial patterning of gene expression in the pituitary. Additional experiments demonstrated that these three elements cannot alone direct gene expression, suggesting a combination of factors is required for enhancer activity. This study reveals that the key regulatory elements guiding developmental regulation of the human LHX3 gene lie in this conserved downstream region. Further, this work implicates ISL1 as a new transcriptional regulator of LHX3 and describes a possible mechanism for the regulation of LHX3 by a known upstream factor, PITX1. Identification of important regulatory regions will also enable genetic screening in candidate CPHD patients and will thereby facilitate patient treatment and genetic counseling.