Correction for Stevenson and Prendergast, Reversible DNA methylation regulates seasonal photoperiodic time measurement

doi:10.1073/pnas.1321493110

PHYSIOLOGY Correction for “Reversible DNA methylation regulates seasonal photoperiodic time measurement,” by Tyler J. Stevenson and Brian J. Prendergast, which appeared in issue 41, October 8, 2013, of Proc Natl Acad Sci USA (110:16651–16656; first published September 25, 2013; 10.1073/pnas.1310643110).

The authors note the following: “Recent high throughput sequencing has indicated that an upstream region of the dio3 promoter sequence in our paper was the result of a PCR fusion error. The reverse primer located –140bp upstream of the start codon was not specific to Siberian hamsters. As a result, the transcription factor binding site analysis and sodium bisulfite-treated DNA sequence analyses in the original publication were incorrect. To correct this error, we have resequenced the dio3 proximal promoter and conducted replications of the transcription factor binding site analyses and sequencing of sodium bisulfite-treated DNA, using primer sequences with confirmed specificity to Siberian hamster DNA. The corrected dio3 promoter sequence exhibited greater homology with mice and human dio3 promoter (revised Fig. S1), a greater number of CpG sites and a higher CpG frequency (revised Fig. S2) than previously reported. Analysis of sodium bisulfite-treated DNA in the acute LD-SD (Fig. 1F) and photorefractory experiments (Fig. 3E) yielded results consistent with the originally-published report: dio3 promoter DNA methylation was reduced in SD (revised Fig. 1F) and increased in SD-R (revised Fig. 3E). Revised transcription factor binding site analyses have also been performed (revised Table S1). See corrected Table S2 for primers used on sodium bisulfite treated DNA. In addition, the reverse primers for the sequencing and MSRE PCR reactions (Table S2) were originally listed in the incorrect (3′-5′) orientation; the correct orientation (5′-3′) now appears in the revised version of Table S2.

“We thank Drs. Hugues Dardente and David Hazlerigg for their assistance in identifying these errors.”

Fig. 1.

Short photoperiods inhibit reproduction and activate hypothalamic mRNA expression via epigenetic mechanisms. Acute transfer from LD to SD photoperiods caused gonadal regression (A), increased hypothalamic dio3 mRNA expression (B), and decreased hypothalamic dnmt1 and dnmt3b mRNA expression (C) after 8 wk. (D) Immunocytochemical localization of DNMT3b (DNMT3b-ir) in the hamster mediobasal hypothalamus (MBH). DNMT3b-ir was evident throughout the MBH and in the ependymal cell (EC) layer along the third ventricle (III). (E) Transfer from LD to SD reduced DNA methylation in the dio3 proximal promoter, as measured using an MSRE assay. (F) Proportion of LD and SD hamsters in which no unmethylated DNA was detected at each of 17 CpG sites in the dio3 proximal promoter, as assessed by direct sequencing of sodium bisulfite-treated DNA. The abscissa (not to scale) depicts the 17 CpG sites from –249 to the start codon of the dio3 proximal promoter. Averaging across the entire promoter region that was sequenced, evidence of unmethylation was evident on 15% of CpG sites in LD (i.e., on 85% of CpG sites examined in LD hamsters, no detectable C-to-T bisulfite conversion occurred), whereas in SD, evidence of unmethylated DNA was present on 42% of CpG sites (χ² = 18.4, P < 0.002). All data in panels A–E are mean ± SEM. *P < 0.05, ***P < 0.005 vs. LD value.

Fig. 3.

Neuroendocrine refractoriness to SD reverses patterns of DNA methylation induced by acute SD exposure. (A) Acute (10 wk, SD) exposure to SD induced gonadal regression, whereas prolonged exposure (42 wk, SD-R) triggered neuroendocrine refractoriness and gonadal recrudescence. Refractoriness in SD-R hamsters was characterized by a complete reversal of hypothalamic dio3 and dnmt3b mRNA expression (B and C) and by remethylation of DNA in the dio3 proximal promoter (D). (E) Proportion of LD, SD, and SD-R hamsters in which no unmethylated DNA was detected in each of 17 CpG sites in the dio3 proximal promoter, as assessed by direct sequencing of sodium bisulfite DNA from the whole hypothalamus. The abscissa (not to scale) depicts the 17 CpG sites from –249 to the start codon of the dio3 proximal promoter. Averaging across the promoter region that was sequenced, evidence of unmethylation was evident on 25% of CpG sites in LD hamsters, whereas in SD hamsters, evidence of unmethylation was present on 39% of CpG sites (χ² = 5.08, P < 0.03). In SD-R hamsters, methylation patterns returned to LD-like values, and evidence of unmethylation was detected on 29% of CpG sites examined (χ² = 0.46, P > 0.40 vs. LD). Five of 17 sites (sites 16, 13, 12, 2, and 1) exhibited reversals in the pattern of methylation in SD-R hamsters. All data in panels A–D are mean ± SEM. *P < 0.05; ***P < 0.005 vs. LD value.