Molecular effects of novel mutations in Hesx1/HESX1 associated with human pituitary disorders

ArticleinDevelopment 128(24):5189-99 · January 2002with4 Reads
Impact Factor: 6.46 · Source: PubMed

The homeobox gene Hesx1/HESX1 has been implicated in the establishment of anterior pattern in the central nervous system (CNS) in a number of vertebrate species. Its role in pituitary development has been documented through loss-of-function studies in the mouse. A homozygous missense point mutation resulting in a single amino acid substitution, Arg160Cys (R160C), is associated with a heritable form of the human condition of septo-optic dysplasia (SOD). We have examined the phenotype of affected members in this pedigree in more detail and demonstrate for the first time a genetic basis for midline defects associated with an undescended or ectopic posterior pituitary. A similar structural pituitary abnormality was observed in a second patient heterozygous for another mutation in HESX1, Ser170Leu (S170L). Association of S170L with a pituitary phenotype may be a direct consequence of the HESX1 mutation since S170L is also associated with a dominant familial form of pituitary disease. However, a third mutation in HESX1, Asn125Ser (N125S), occurs at a high frequency in the Afro-Caribbean population and may therefore reflect a population-specific polymorphism. To investigate the molecular basis for these clinical phenotypes, we have examined the impact of these mutations on the regulatory functions of HESX1. We show that Hesx1 is a promoter-specific transcriptional repressor with a minimal 36 amino acid repression domain which can mediate promoter-specific repression by suppressing the activity of homeodomain-containing activator proteins. Mutations in HESX1 associated with pituitary disease appear to modulate the DNA-binding affinity of HESX1 rather than its transcriptional activity. Wild-type HESX1 binds a dimeric homeodomain site with high affinity (K(d) 31 nM) whilst HESX1(S170L) binds with a 5-fold lower activity (K(d) 150 nM) and HESX1(R160C) does not bind at all. Although HESX1(R160C) has only been shown to be associated with the SOD phenotype in children homozygous for the mutation, HESX1(R160C) can inhibit DNA binding by wild-type HESX1 both in vitro and in vivo in cell culture. This dominant negative activity of HESX1(R160C) is mediated by the Hesx1 repression domain, supporting the idea that the repression domain is implicated in interactions between homeodomain proteins. Our data suggest a possible molecular paradigm for the dominant inheritance observed in some pituitary disorders.

    • "HESX1 has been shown to function as a repressor of PROP1-mediated gene stimulation [14]. To assess the ability of the mutatnt HESX1 to repress transcription, wild-type or mutant HESX1 pCMVmyc expression vectors were transfected into COS1 cells together with PROP1, in the presence of the 6xP3Luc plasmid that contains six copies of consensus P3 target sites common to PROP1 and HESX1 (5'-AGCTTGAGTCTAATTGAATTACTGTAC-3', underlined) [8, 15], and pRL-CMV vector used as an internal control for the transfection. Forty eight hours after transfection, cells were analyzed using a dualluciferase reporter assay system (Promega). "
    [Show abstract] [Hide abstract] ABSTRACT: Heterozygous and/or homozygous HESX1 mutations have been reported to cause isolated growth hormone deficiency (IGHD) or combined pituitary hormone deficiency (CPHD), in association with septo optic dysplasia (SOD). We report a novel heterozygous HESX1 mutation in a CPHD patient without SOD phenotypes. The propositus was a one-year-old Japanese girl. Shortly after birth, she was found to be hypoglycemic. She was diagnosed with central adrenal insufficiency based on low cortisol and ACTH at a time of severe hypoglycemia. Further endocrine studies indicated that the patient also had central hypothyroidism and growth hormone deficiency. Using a next-generation sequencing strategy, we identified a novel heterozygous HESX1 mutation, c.326G>A (p.Arg109Gln). Western blotting and subcellular localization revealed no significant difference between wild type and mutant HESX1. Electrophoretic mobility shift assays showed that the mutant HESX1 abrogated DNA-binding ability. Mutant HESX1 was unable to repress PROP1-mediated activation. In conclusion, this study identified Arg109 as a critical residue in the HESX1 protein and extends our understanding of the phenotypic features, molecular mechanism, and developmental course associated with mutations in HESX1. When multiple genes need to be analyzed for mutations simultaneously, targeted sequence analysis of interesting genomic regions is an attractive approach.
    Preview · Article · Jan 2016 · Endocrine Journal
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    • "See also Figures S2 and S3. dysplasia (Dattani et al., 1998), whereas the N125S variant is a natural polymorphism in the Afro-Caribbean population (Brickman et al., 2001 ) that is not associated with disease. Interestingly , the R160C mutation completely abolishes all interactions, while the N125S variant has a wild-type target profile (Figures 5D and 5E;Table S4). "
    [Show abstract] [Hide abstract] ABSTRACT: Gene regulatory networks (GRNs) comprising interactions between transcription factors (TFs) and regulatory loci control development and physiology. Numerous disease-associated mutations have been identified, the vast majority residing in non-coding regions of the genome. As current GRN mapping methods test one TF at a time and require the use of cells harboring the mutation(s) of interest, they are not suitable to identify TFs that bind to wild-type and mutant loci. Here, we use gene-centered yeast one-hybrid (eY1H) assays to interrogate binding of 1,086 human TFs to 246 enhancers, as well as to 109 non-coding disease mutations. We detect both loss and gain of TF interactions with mutant loci that are concordant with target gene expression changes. This work establishes eY1H assays as a powerful addition to the toolkit of mapping human GRNs and for the high-throughput characterization of genomic variants that are rapidly being identified by genome-wide association studies. Copyright © 2015 Elsevier Inc. All rights reserved.
    Full-text · Article · Apr 2015 · Cell
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    • "OTX2 is also responsible for Hesx1 expression regulation (Diaczok et al. 2008). Hesx1 is the first pituitary-specific TF to be expressed at or before E6 . 5. (Hermesz et al. 1996, Brickman et al. 2001). Hesx1 expression begins in the rostral region and progresses dorsally; the restricted expression of this TF is responsible for Rathke's pouch formation (Hermesz et al. 1996). "
    [Show abstract] [Hide abstract] ABSTRACT: Pituitary organogenesis is a highly complex and tightly regulated process that depends on several transcription factors (TFs), such as PROP1, PIT1 (POU1F1), HESX1, LHX3 and LHX4. Normal pituitary development requires the temporally and spatially organised expression of TFs and interactions between different TFs, DNA and TF co-activators. Mutations in these genes result in different combinations of hypopituitarism that can be associated with structural alterations of the central nervous system, causing the congenital form of panhypopituitarism. This review aims to elucidate the complex process of pituitary organogenesis, to clarify the role of the major TFs, and to compile the lessons learned from functional studies of TF mutations in panhypopituitarism patients and TF deletions or mutations in transgenic animals.
    Full-text · Article · Aug 2012 · Journal of Endocrinology
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