24-hour rhythms of DNA methylation and their relation with rhythms of RNA expression in the human dorsolateral prefrontal cortex
Authors: Lei Yu
Amanda J Myers
Gyan P Srivastava
Jishu Xu
Lori B Chibnik
David A Bennett
Philip L De Jager
Julie A Schneider
Andrew S P Lim
Aron S Buchman
Journal: PLoS Genetics (2014)
Discipline: MOLECULAR BIOLOGY & GENETICS
Keywords: Circadian rhythms; DNA methylation; RNA expression; Human brain tissues; Transcription start sites
Affiliations: Division of Neurology, Department of Medicine, Sunnybrook Health Sciences Centre, University of Toronto, Toronto, Ontario, Canada., Program in Translational Neuropsychiatric Genomics, Department of Neurology, Brigham and Women's Hospital, Harvard Medical School, Boston, Massachusetts, United States of America; Program in Medical and Population Genetics, Broad Institute, Cambridge, Massachusetts, United States of America., Rush Alzheimer Disease Center and Department of Neurological Sciences, Rush University, Chicago, Illinois, United States of America., Department of Psychiatry and Behavioral Sciences, Miller School of Medicine, University of Miami, Miami, Florida, United States of America.

Abstract:  Circadian rhythms modulate the biology of many human tissues, including brain tissues, and are driven by a near 24-hour transcriptional feedback loop. These rhythms are paralleled by 24-hour rhythms of large portions of the transcriptome. The role of dynamic DNA methylation in influencing these rhythms is uncertain. While recent work in Neurospora suggests that dynamic site-specific circadian rhythms of DNA methylation may play a role in modulating the fungal molecular clock, such rhythms and their relationship to RNA expression have not, to our knowledge, been elucidated in mammalian tissues, including human brain tissues. We hypothesized that 24-hour rhythms of DNA methylation exist in the human brain, and play a role in driving 24-hour rhythms of RNA expression. We analyzed DNA methylation levels in post-mortem human dorsolateral prefrontal cortex samples from 738 subjects. We assessed for 24-hour rhythmicity of 420,132 DNA methylation sites throughout the genome by considering methylation levels as a function of clock time of death and parameterizing these data using cosine functions. We determined global statistical significance by permutation. We then related rhythms of DNA methylation with rhythms of RNA expression determined by RNA sequencing. We found evidence of significant 24-hour rhythmicity of DNA methylation. Regions near transcription start sites were enriched for high-amplitude rhythmic DNA methylation sites, which were in turn time locked to 24-hour rhythms of RNA expression of nearby genes, with the nadir of methylation preceding peak transcript expression by 1-3 hours. Weak ante-mortem rest-activity rhythms were associated with lower amplitude DNA methylation rhythms as were older age and the presence of Alzheimer's disease. These findings support the hypothesis that 24-hour rhythms of DNA methylation, particularly near transcription start sites, may play a role in driving 24-hour rhythms of gene expression in the human dorsolateral prefrontal cortex, and may be affected by age and Alzheimer's disease. 

Retracted: No

PubPeer comments:
 2021 Expression of Concern. https: journals.plos.org plosgenetics article?id=10.1371 journal.pgen.1009470 Following publication of this article [1], concerns were raised regarding the methodology used for the reported analyses and whether the conclusions are fully supported. The article included a reporting error pertaining to methods used for the analyses reported in Fig 2A,B of [1]. The Results and Materials and Methods sections incorrectly state, u201cwe performed a series of analyses comparing the observed data to 10,000 permuted null datasets generated by randomly shuffling the times of death in our data while preserving the correlation between DNA methylation sites.u201d The authors have clarified that the analyses permuted time of death within each block of 500 contiguous CpG; the permutation is independent across the blocks, preserving local within block correlations. Thus, the Results and Materials and Methods statements should have read,  we performed a series of analyses comparing the observed data to 10,000 permuted null datasets generated by randomly shuffling the times of death in blocks of 500 contiguous CpGs in our data while preserving the within block correlation between DNA methylation sites.u201d The authors initially presumed that the block-wise permutation would yield identical results to an unblocked permutation. However, subsequent computations by the authors confirm that p-values obtained using this block-wise permutation approach are sensitive to block size. Methodological concerns were raised pertaining to the use of a block-wise permutation approach, the use of averaged distributions for permuted null datasets, and use of shuffled times of death to compare distribution of nadir times. The authors acknowledge that when these aspects of the methodology are adjusted, some of the analyses reported in Figs 2 and 6 no longer yield statistically significant results. The authors are cooperating with PLOS Genetics Editors to address these issues. In the meantime, the PLOS Genetics Editors issue this Expression of Concern. Reference 1.Lim ASP, Srivastava GP, Yu L, Chibnik LB, Xu J, Buchman AS, et al. (2014) 24-Hour Rhythms of DNA Methylation and Their Relation with Rhythms of RNA Expression in the Human Dorsolateral Prefrontal Cortex. PLoS Genet 10(11): e1004792. pmid:25375876 , 
 Retraction: 24-Hour Rhythms of DNA Methylation and Their Relation with Rhythms of RNA Expression in the Human Dorsolateral Prefrontal Cortex https: journals.plos.org plosgenetics article?id=10.1371 journal.pgen.1009895 After publication of this article [1], concerns were raised regarding the validity of the conclusions. Specifically, the p-values used to support Fig 2 [1] are based on a permutation approach that was incorrectly reported, as outlined in [2], and, when repeated using a more robust approach, does not support the central conclusion regarding rhythmicity of methylation. In addition, comparison of nadir time distributions between groups as carried out for Fig 6 [1] is recognized to be insufficient to support conclusions about relationships between sex, age, Alzheimeru2019s disease, or 24-hour activity rhythms and the timing of rhythms of methylation. The authors recognize the weaknesses present in the analytical approach used in this article that were uncovered after the paper was published; an inadvertent error led to results which were significant and specifically affects the results shown in Figs 2 and 6, and from which some of the conclusions of the article were derived. In light of the above issues with the analytic approach and the concern that a number of key conclusions are not fully supported by the published analyses in [1], the authors and the PLOS Genetics Editors retract this article. All authors agreed with the retraction. ,