Epigenetic Characterization of the Growth Hormone Gene Identifies SmcHD1 as a Regulator of Autosomal Gene Clusters

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

The H19/Igf2 imprinted locus was dis-regulated following SmcHD1 knock-down.

<p><b>A.</b> A number of imprinted genes associated with BWS and SRS were dysregulated in SmcHD1 SH-SY5Y knock-down cells. A graphical representation of the H19/Igf2 locus on the human chromosome at position 11p15.5. Maternally imprinted genes are highlighted in blue, maternally expressed genes are in red, one of the placental-specific imprinted genes is colored in brown and not imprinted genes are in green. A non-coding RNA, Kcnq1ot1 is colored in blue and is typically expressed from the paternal chromosome presumably acting to silence genes normally expressed from the maternal chromosome (M) including Kcnq1 and Cdkn1c. Differentially DNA methylated regions (ICR1, KvDMR1 (ICR2)) are indicated by the trapezoids (solid indicates hypermethylation and open hypomethylation). <b>B.</b> mRNA quantitation of selected genes in the H19/Igf2 locus using RT-qPCR in SmcHD1 SH-SY5Y knock-down cells. The copy numbers are relative to and corrected using β-actin cDNA levels. * indicates P-values <0.05, ** P-values <0.01 and *** P-values <0.001 using an unpaired Students t-test.</p

Comparative levels of GH promoter methylation in mice.

<p><b>A.</b> Above, schematic of the relative position of CpGs in the mouse GH promoter. Middle, pairwise analysis comparing the levels of DNA methylation in wild type (WT) with dwarf (dw) mice (Snell) using bisulfite DNA sequencing. Bottom, combined bisulfite restriction analysis (CoBRA) of the CpG located at position −4 on the same samples. The proportion of non-methylated C nucleotide is indicated by the cleaved FokI products (blue arrow). <b>B.</b> The GH promoter becomes demethylated during mouse development and is coincident with GH gene expression. Above, schematic illustration of the developmental events relating to Pit-1-mediated induction of the GH gene and concomitant loss of GH promoter methylation. Below, pyrosequencing of bisulfite-treated genomic DNA extracted from mouse pituitary, selected from different days of mouse development (e14.5, P0.5 or P14.5) and displayed as the percent methylation of CpG sites in the mouse promoter.</p

Identification of a GH promoter DMR using mouse BAC transgenes.

<p><b>A.</b> A mouse BAC transgene encompassing GH gene was modified by homologous recombination to replace the mouse GH gene with the rat GH proximal promoter and the coding sequence for RFP. A second recombination event using the same BAC transgene was used to delete a putative distal LCR (ΔLCR- GH:RFP). Imaging of dissected mouse pituitary, illustrates RFP expression from the WT-GH:RFP but not ΔLCR-GH:RFP transgenic mice. <b>B.</b> WT-GH:RFP protein mirrored endogenous GH expression. Pituitaries were fixed and embedded in paraffin, the tissue sectioned and subjected to indirect immunofluorescence with anti-GH, anti-Prl and anti-TSHβ antibodies using Cy2-cojugated secondary antibodies. The images were merged with images of RFP expression. Note: RFP was nuclear while the hormones were localized to the cytoplasm. <b>C.</b> Pairwise analysis of the rat GH promoter methylation levels by bisulfite sequencing of pituitary DNA from WT-GH:RFP and ΔLCR-GH:RFP transgenic mice. The number indicates the relative position of the CpG from the transcriptional start site. The n-values indicate the number of clones sequenced from transgenic mice and used for the pairwise statistical analysis.</p

Inhibition of DNA methylation relieves transcriptional repression of GH and characterization of a methyl-DNA binding activity.

<p><b>A.</b> GH gene repression can be relieved by treatment with 5-azaC. Above, schematic illustration of the CpG sites location in the rat GH promoter (the CpGs are numbered according to their relative position from the transcriptional start site). Middle, bisulfite sequencing of genomic DNA extracted from pituitary derived-GH+ (GC cells) or GH− cells (MMQ cells). The level of DNA methylation is displayed as unmethylated (open circles) or methylated (solid circles) from individual clones. Lower, GH− cells (MMQ) were treated with 5-azaC and the level of gene expression compared using RT-qPCR. The solid bars represent 5-azaC treated cells and the open bars, cells cultured under normal conditions. <b>B.</b> A methyl-specific binding protein binds to a region of DNA derived from the mouse GH promoter. Above, DNA sequence of the double-stranded oligonucleotides used in the EMSA. The position of the CpGs relative to the transcriptional start site are indicated and the position of a previously described binding site for the thyroid hormone receptor (TR) is boxed. Lower left, EMSA with MMQ nuclear extracts comparing the bound proteins from either methylated (lane 2) or unmethylated DNA probes (lane 5). A methylated competitor DNA (M) reveals a specific upper methyl-DNA protein-binding component (Lane 3, arrow). Lower right, an EMSA competition assay with nuclear extracts from pituitary-derived cell lines (MMQ, GC and GHFT) as indicated. Methylated or unmethylated (U) competitor oligonucleotides were used to identify the upper shifted component representing a DNA bound protein. All nuclear extracts contained a methyl-DNA binding protein. <b>C.</b> Methylation of CpGs at position −8 and −7 were required for recruiting a methyl DNA binding protein. Above, schematic of the result from the competition assay illustrated below. The shaded area indicates CpGs essential for the methyl DNA binding protein. Below, an EMSA with MMQ nuclear extracts (NE) and a methylated DNA probe with various competitor oligonucleotides as indicated. <b>D.</b> The methyl-DNA binding activity observed with the GH DMR appears to be unique. The sequence of the oligonucleotides used in the competition assay are listed in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0097535#pone.0097535.s006" target="_blank">Table S1</a>. An EMSA with increasing amounts of MMQ NE (6 and 12 µg) and a methylated GH DMR probe optionally in the presence of competitor oligonucleotides (100X) as indicated.</p

Profiling gene expression from cells with knock-down levels of SmcHD1.

<p><b>A.</b> Retroviral shRNA directed towards SmcHD1 efficiently down-regulated SmcHD1 protein levels in HEK293 cells. shRNAs directed towards SmcHD1, a control shRNA or empty plasmid (pQCXIP) was used for retroviral infection HEK293 cells. NEs were prepared from stably infected cells and analyzed by immunoblot with an anti-SmcHD1 antibody. An anti-LSD1 antibody was used as an internal control for loading. <b>B.</b> A disproportionate number of genes were up-regulated on the X-chromosome in SmcHD1 knock-down cells. A pie chart was used to illustrate the percentage of genes on each chromosome that were up- or down- regulated in SmcHD1 knock-down cells. <b>C.</b> Heat map and hierarchal clustering of selected up- and down- regulated genes in SmcHD1 knock-down cells or cells infected with control non-specific NC5 shRNA. Below, scaling of the fold differences of the genes from cells. Intense red indicates up-regulation and intense blue indicates down-regulation.</p