Changes in methylation patterns of kiss1 and kiss1r gene promoters across puberty - PubMed (original) (raw)

Changes in methylation patterns of kiss1 and kiss1r gene promoters across puberty

Amanda K Wyatt et al. Genet Epigenet. 2013.

Abstract

The initiation of mammalian puberty is underpinned by an increase in Kisspeptin (Kiss1) signaling via its receptor (Kiss1r/GPR54) on gonadotropin-releasing hormone (GnRH) neurons. Animals and humans with loss-of-function mutations in Kiss1 or Kiss1r fail to go through puberty. The timing of puberty is dependent on environmental factors, and malleability in puberty timing suggests a mechanism that can translate environmental signals into patterns of Kiss1/Kiss1r gene expression. Epigenetics is a powerful mechanism that can control gene expression in an environment-dependent manner. We investigated whether epigenetic DNA methylation is associated with gene expression changes at puberty. We used bisulfite-PCR-pyrosequencing to define the methylation in the promoters of Kiss1 and Kiss1r before and after puberty in female rats. Both Kiss1 and Kiss1r showed highly significant puberty-specific differential promoter methylation patterns. By identifying key differentially methylated residues associated with puberty, these findings will be important for further studies investigating the control of gene expression across the pubertal transition.

Keywords: Kiss1r; kisspeptin; methylation; neuroendocrine; puberty; reproduction.

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Figures

Figure 1

Figure 1

Details of hypothalamic microdissection and distribution of promoter CpG dinucleotides. A–D. show schematic diagrams of representative brain levels, modified from Allen Mouse Brain Atlas, Allen Institute for Brain Science (

http://mouse.brain-map.org/experiment/thumbnails/100048576?image\_type=atlas

), in accordance with their usage policy (

http://www.alleninstitute.org/Media/policies/citation\_policy\_content.html

) from which tissue was taken. Dotted line demarcates region dissected and processed for DNA extraction and bisulfite sequencing. Rostral (A) and caudal (B) POA, containing GnRH neurons were pooled for Kiss1r analysis. Similarly, rostral (C) and caudal (D) regions of the RP3V were pooled for Kiss1 analysis. POA, preoptic area; RP3V, rostral periventricular region of the third ventricle. E. Schematic drawing of the Kiss1 promoter showing positions of CpG dinucleotides (small vertical lines) and amplification primers (⇀ Forward, ↽ Reverse). +1 is the transcription start site and → indicates the direction of transcription. Note that in the ~2.8 kb 5′ to +1 that only 1 CpG is not covered by the primers. F. Schematic drawing of the Kiss1r promoter showing positions of CpG dinucleotides and amplification primers. Decorations are as in E. Note that in the ~2 kb 5′ to +1 that only 5 CpGs are not covered by the primers.

Figure 2

Figure 2

Details of methylation across entire promoter regions. A. Bar graph showing the percentage of CpG residues within each of the 6 regions across the Kiss1 promoter. Note that the promoter as a whole is mostly methylated irrespective of physiological state. B. Bar graphs showing the percentage of CpG residues within each of the 5 regions spanning the Kiss1r promoter. Note that the promoter as a whole is mostly unmethylated irrespective of physiological state. ND, no data, indicating that insufficient sequencing coverage was obtained for these three regions. This mainly because of the high poly-T content that arises as a consequence of bisulfite conversion. Error bars indicate standard error of the mean.

Figure 3

Figure 3

Kiss1 and Kiss1r mRNA expression before and after puberty. Bar graphs showing the fold-change, relative to pre-puberty levels, in gene expression for Kiss1 (A) and Kiss1r (B) before (white bars) and after (black bars) puberty. Numbers at base of bars indicate number of brains assayed. *, P < 0.05, Students t-test.

Figure 4

Figure 4

Differential methylation in the Kiss1 and Kiss1r promoters across puberty. (A) Bar graph showing the percentage of CpGs that are methylated (y-axis) within region 4 of the Kiss1 promoter before (white) and after (black) puberty. *P < 0.05, Student’s t-test. (**B–D**) Bar graphs showing the percentage of CpGs that are unmethylated (y-axis) within regions 2 (**B**), 4 (**C**), and 5 (**D**) of the _Kiss1r_ promoter before (white) and after (black) puberty. Note that regions 2 and 4 showed a decrease in methylation after (black bars) puberty compared with before (white bars; *, _P_ < 0.05, Student’s t-test), whereas region 5 showed and increase in methylation after puberty (*, _P_ < 0.05, Student’s t-test). Error bars indicate standard error of the mean. Due to the large number of reads sequenced per PCR reaction per animal, power analysis indicates >90% power in this experiment.

Figure 5

Figure 5

Predicted binding sites for transcription factors relative to sites of differential methylation for Kiss1 (A) and Kiss1r (B) promoters. Nucleotide numbers relative to the start of transcription are shown for each region examined. Each transcription factor predicted from the TESS analysis to bind to sites of differential methylation is shown. Sequences beneath transcription factor names denote core nucleotide binding sequence. Methylated residues within the core motif are shown with *. For NF-E, −4 indicates that the methylated C lies 4 nucleotides upstream of the 5′-most core sequence nucleotide. Factors are shown bound (present) when the promoter region is unmethylated and absent when the promoter regions is methylated. For each gene a the pre-puberty state (i) and the post-puberty state (ii) is shown. Dark horizontal bar represents promoter region sequenced, 2.8 kb for Kiss1 and 2.2 kb for Kiss1r.

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