Abstract
Regulatory elements for the mouse growth hormone (GH) gene are located distally in a putative locus control region (LCR) in addition to key elements in the promoter proximal region. The role of promoter DNA methylation for GH gene regulation is not well understood. Pit-1 is a POU transcription factor required for normal pituitary development and obligatory for GH gene expression. In mammals, Pit-1 mutations eliminate GH production resulting in a dwarf phenotype. In this study, dwarf mice illustrated that Pit-1 function was obligatory for GH promoter hypomethylation. By monitoring promoter methylation levels during developmental GH expression we found that the GH promoter became hypomethylated coincident with gene expression. We identified a promoter differentially methylated region (DMR) that was used to characterize a methylation-dependent DNA binding activity. Upon DNA affinity purification using the DMR and nuclear extracts, we identified structural maintenance of chromosomes hinge domain containing -1 (SmcHD1). To better understand the role of SmcHD1 in genome-wide gene expression, we performed microarray analysis and compared changes in gene expression upon reduced levels of SmcHD1 in human cells. Knock-down of SmcHD1 in human embryonic kidney (HEK293) cells revealed a disproportionate number of up-regulated genes were located on the X-chromosome, but also suggested regulation of genes on non-sex chromosomes. Among those, we identified several genes located in the protocadherin β cluster. In addition, we found that imprinted genes in the H19/Igf2 cluster associated with Beckwith-Wiedemann and Silver-Russell syndromes (BWS & SRS) were dysregulated. For the first time using human cells, we showed that SmcHD1 is an important regulator of imprinted and clustered genes.
Highlights
Plasma growth hormone (GH) levels decline with age and contribute to decreased somatotropic axis signaling (GH releasing hormone [GHRH], GH and insulin-like growth factor -1 [IGF-1]) [1]
In order to study GH gene expression in real-time using mouse pituitary, we generated a series of bacterial artificial chromosome (BAC) transgenic mice where the mouse GH gene was substituted with a homologous rat GH promoter and the coding sequence for red fluorescent reporter gene and another that contained an additional deletion in a putative upstream regulatory element or locus control region (LCR, DLCR-GH:RFP)
To show that the RFP protein expression closely paralleled GH expression using the wild type littermates (WT) BAC, we labeled pituitary tissue sections from mice with antiGH, anti-Prl and anti- thyroid stimulating hormone b (TSHb) antibodies and visualized the presence of each cytoplasmic hormone with a fluorescein isothiocyanate (FITC)-conjugated secondary antibody. These results demonstrated that pituitary of transgenic mice containing WT BAC transgenes is a suitable model system to study promoter DNA methylation levels compared to the DLCR version of the BAC transgene, which lacked RFP expression
Summary
Plasma growth hormone (GH) levels decline with age and contribute to decreased somatotropic axis signaling (GH releasing hormone [GHRH], GH and insulin-like growth factor -1 [IGF-1]) [1]. The mammalian GH gene is expressed only in the pituitary and is dependent on the expression of a functional homeodomain containing transcription factor, the pituitary-specific Pit-1 protein (POU1-F1) [3]. A distal locus control region (LCR) located ,14.5 kb upstream of the human GH-N gene is required for gene expression [4]. It is characterized by a series of pituitary-specific DNase I hypersensitive sites (HS) when expressed. The region representing the homologous LCR in rodent models is relatively uncharacterized, while the human LCR encompassing HSI and HSII represents an intergenic sequence that is homologous to mouse and rat genomic sequence
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