The role of DNA methylation in regulation of the murine Lhx3 gene

Abstract

LHX3 is a LIM-homeodomain transcription factor with critical roles in pituitary and nervous system development. Mutations in the LHX3 gene are associated with pediatric diseases featuring severe hormone deficiencies, hearing loss, developmental delay, and other symptoms. The mechanisms that govern LHX3/Lhx3 transcription are poorly understood. In this study, we examined the role of DNA methylation in the expression status of the mouse Lhx3 gene. Pituitary cells that do not normally express Lhx3 (Pit-1/0 cells) were treated with 5-aza-2'-deoxycytidine, a demethylating reagent. This treatment leads to activation of Lhx3 gene expression suggesting that methylation contributes to Lhx3 regulation. Treatment of Pit-1/0 pituitary cells with a combination of a demethylating reagent and a histone deacetylase inhibitor led to rapid activation of Lhx3 expression, suggesting possible crosstalk between DNA methylation and histone modification processes. To assess DNA methylation levels, treated and untreated Pit-1/0 genomic DNAs were subjected to bisulfite conversion and sequencing. Treated Pit-1/0 cells had decreased methylation at specific sites in the Lhx3 locus compared to untreated cells. Chromatin immunoprecipitation assays demonstrated interactions between the MeCp2 methyl binding protein and Lhx3 promoter regions in the Pit-1/0 cell line. Overall, this study demonstrates that DNA methylation patterns of the Lhx3 gene are associated with its expression status.

Keywords: 5-aza-dc; 5′-aza-2′-deoxycytidine; CPHD; Chromatin; CpG; DNA methyltransferase; DNMT; FSHβ; GnRH-R; HDAC; LHX3; LIM-homeodomain protein 3; MBP; NF1; PRL; Promoter; SP1; TSA; TSS; Transcription; alpha glycoprotein subunit; combined pituitary hormone deficiency; cytosine-phosphate bond-guanine; follicle-stimulating hormone; gonadotropin-releasing hormone receptor; histone deacetylase; methyl-CpG binding protein; nuclear factor 1; prolactin; specificity protein 1; transcription start site; trichostatin A; αGSU.

Figure 1. Lhx3 mRNA and protein are expressed in mouse pituitary Pit-1/Triple but not Pit-1/0 cells

(A) Lhx3a or Lhx3b mRNA was amplified by RT-PCR in Pit-1/0 and Pit-0/Triple cells. Beta actin primers were used in a positive control to test cDNA integrity. (B) Protein levels of LHX3 were determined by western blotting of whole cell protein extracts of Pit-1/0 and Pit-1/Triple cells. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) antibodies were used as positive controls. Each experiment was performed three times with similar results.

Figure 2. Lhx3a promoter and its methylation pattern

(A) The upper diagram represents the mouse Lhx3 gene, with black rectangles signifying protein-coding exons and white areas denoting untranslated exonic regions. Two upstream promoters (aP and bP) produce the Lhx3a and Lhx3b mRNAs. The lower diagram below shows potential sites of methylation in the Lhx3a promoter, spanning from position −166 to +166 with the major transcription start site (TSS) set to position 0. Each vertical black bar indicates a CG base pair. The positions of SP1 binding sites (Yaden et al., 2006) are shown. (B) The methylation pattern of the Lhx3a promoter in mouse pituitary Pit-1/0 cells was determined by bisulfite sequencing (see materials and methods). Black boxes indicate methylated CpGs and open boxes indicate non methylated CpGs. Horizontal rows in each diagram represent individual sequenced clones from experiments. The overall percentage of observed methylation at each position is noted.

Figure 3. Lhx3b promoter and its methylation pattern

(A) The upper diagram represents the mouse Lhx3 gene, with black rectangles signifying protein-coding exons and white areas denoting untranslated exonic regions. Two upstream promoters (aP and bP) produce the Lhx3a and Lhx3b mRNAs. The diagram below shows sites of potential methylation in the Lhx3b promoter, spanning from position −235 to +85 with the transcription start site (TSS) set at position 0. The positions of SP1 binding sites are shown. (B) The methylation pattern of the Lhx3b promoter in mouse pituitary Pit-1/0 cells was determined by bisulfite sequencing (materials and methods). Black boxes indicate methylated CpGs and open boxes indicate non methylated CpGs.

Figure 4. Lhx3 promoter methylation patterns in Pit-1/Triple cells

(A) The methylation pattern of the Lhx3a promoter in mouse pituitary Pit-1/Triple cells was determined by bisulfite sequencing. Black boxes indicate methylated CpGs and open boxes indicate non methylated CpGs. Horizontal rows in each diagram represent individual sequenced clones from experiments. The overall percentage of observed methylation at each position is noted. (B) The methylation pattern of the Lhx3b promoter in mouse pituitary Pit-1/Triple cells.

Figure 5. Lhx3 expression is activated in mouse pituitary Pit-1/0 cells after treatment with 5’-aza-dc

Pit-1/0 cells were treated with 2.5 µM 5-aza-dc and RNA was harvested 24 hours (A) or 48 hours (B) after treatment and RT-PCR was performed. (C) Pit-1/0 cells were treated with 2.5 µM 5-aza-dc and genomic DNA was harvested for bisulfite conversion 48 hours post treatment. The methylation patterns of the Lhx3a (top) and Lhx3b (bottom) promoters were determined by bisulfite sequencing as described in the materials and methods. Black boxes indicate methylated CpGs and open boxes indicate non methylated CpGs.

Figure 6. Lhx3 expression after treatment with 5’-aza-dc plus TSA in Pit-1/0 cells

(A) Mouse pituitary Pit-1/0 cells were treated with 2.0 µM 5-aza-dc plus 100 nM TSA and RNA was harvested 24 hours after treatment and RT-PCR was performed. (B), (C) Pit-1/0 cells were treated with 2.0 µM 5-aza-dc plus 100 nM TSA and genomic DNA was harvested for bisulfite conversion analysis at 24 hours post treatment. The methylation pattern of the Lhx3a (B) and Lhx3b (C) promoters were determined by bisulfite sequencing as described in the materials and methods. Black boxes indicate methylated CpGs and open boxes indicate non methylated CpGs.

Figure 7. MeCP2 is associated with the Lhx3 gene in Pit-1/0 cells

(A) MeCP2 and MBD2 were detected in mouse pituitary Pit-1/0 cells by western blotting. GAPDH was used as a positive control. Two independent cell extracts are shown. (B, C) ChIP assays were carried out with chromatin from Pit-1/0 cells treated with 5-aza-dc, 5-aza-dc plus TSA or DMSO (solvent control), using anti-MeCP2, anti-MBD2 (negative control), anti-acetylated histone H3 (acH3) and IgG (negative control) antibodies and primers to the Lhx3a and Lhx3b promoters. A representative experiment of three independent, biological replicates is shown. Numbers indicate fold changes for untreated samples compared to IgG negative controls. Asterisks (*) indicate significant differences to IgG negative controls at P<0.05.