DNA Methylation Supports Intrinsic Epigenetic Memory in Mammalian Cells

We have investigated the role of DNA methylation in the initiation and maintenance of silenced chromatin in somatic mammalian cells. We found that a mutated transgene, in which all the CpG dinucleotides have been eliminated, underwent transcriptional silencing to the same extent as the unmodified transgene. These observations demonstrate that DNA methylation is not required for silencing. The silenced CpG-free transgene exhibited all the features of heterochromatin, including silencing of transcriptional activity, delayed DNA replication, lack of histone H3 and H4 acetylation, lack of H3-K4 methylation, and enrichment in tri-methyl-H3-K9. In contrast, when we tested for transgene reactivation using a Cre recombinase-mediated inversion assay, we observed a marked difference between a CpG-free and an unmodified transgene: the CpG-free transgene resumed transcription and did not exhibit markers of heterochromatin whereas the unmodified transgene remained silenced. These data indicate that methylation of CpG residues conferred epigenetic memory in this system. These results also suggest that replication delay, lack of histone H3 and H4 acetylation, H3-K4 methylation, and enrichment in tri-methyl-H3-K9 are not sufficient to confer epigenetic memory. We propose that DNA methylation within transgenes serves as an intrinsic epigenetic memory to permanently silence transgenes and prevent their reactivation

Deficiencies tested in the chromosome <i>2R</i> candidate region.

<p>The figure depicts genes implicated in this study and a previous genome-wide association study of sleep, plotted along the chromosome, along with tested deficiency lines. Not all genes in the region are plotted, and the plot is not to scale. Polymorphisms are indicated at the bottom as triangles. Lavender triangles, night sleep <i>CV</i>_E; Purple triangles, night sleep from the GWAS; black triangles, both night sleep <i>CV</i>_E and night sleep; blue triangles, night sleep from the artificial selection study. Deficiency mapping implicated at least two quantitative trait loci (QTL) in the region. Blue bars denote genes within the first QTL; orange bars denote the second QTL.</p

Selection for long and short sleep duration in <i>Drosophila melanogaster</i> reveals the complex genetic network underlying natural variation in sleep

<div><p>Why do some individuals need more sleep than others? Forward mutagenesis screens in flies using engineered mutations have established a clear genetic component to sleep duration, revealing mutants that convey very long or short sleep. Whether such extreme long or short sleep could exist in natural populations was unknown. We applied artificial selection for high and low night sleep duration to an outbred population of <i>Drosophila melanogaster</i> for 13 generations. At the end of the selection procedure, night sleep duration diverged by 9.97 hours in the long and short sleeper populations, and 24-hour sleep was reduced to 3.3 hours in the short sleepers. Neither long nor short sleeper lifespan differed appreciably from controls, suggesting little physiological consequences to being an extreme long or short sleeper. Whole genome sequence data from seven generations of selection revealed several hundred thousand changes in allele frequencies at polymorphic loci across the genome. Combining the data from long and short sleeper populations across generations in a logistic regression implicated 126 polymorphisms in 80 candidate genes, and we confirmed three of these genes and a larger genomic region with mutant and chromosomal deficiency tests, respectively. Many of these genes could be connected in a single network based on previously known physical and genetic interactions. Candidate genes have known roles in several classic, highly conserved developmental and signaling pathways—EGFR, Wnt, Hippo, and MAPK. The involvement of highly pleiotropic pathway genes suggests that sleep duration in natural populations can be influenced by a wide variety of biological processes, which may be why the purpose of sleep has been so elusive.</p></div

Examples of allele frequency trajectories observed across generations.

<p>Each plot shows the minor allele frequency for the long- and short-sleep selection schemes plotted against generation. Minor allele frequency was defined using the combined allele frequencies for all populations prior to selection (i.e., generation 0). (A), divergent trajectory at position 14,976,564 on chromosome <i>3L</i>; (B) similar trajectory at position 6,300,126 on chromosome <i>X</i>. Dark blue triangles indicate the average minor allele frequencies for long sleep Replicate 1 and Replicate 2 populations; Dark red squares indicate average minor allele frequencies for short sleep Replicate 1 and Replicate 2 populations. (C) box plots for all of the polymorphisms significant for the logistic regression versus generation. Blue, long sleep; red, short sleep.</p

Deficiencies tested in the chromosome <i>2R</i> candidate region.

<p>The figure depicts genes implicated in this study and a previous genome-wide association study of sleep, plotted along the chromosome, along with tested deficiency lines. Not all genes in the region are plotted, and the plot is not to scale. Polymorphisms are indicated at the bottom as triangles. Lavender triangles, night sleep <i>CV</i>_E; Purple triangles, night sleep from the GWAS; black triangles, both night sleep <i>CV</i>_E and night sleep; blue triangles, night sleep from the artificial selection study. Deficiency mapping implicated at least two quantitative trait loci (QTL) in the region. Blue bars denote genes within the first QTL; orange bars denote the second QTL.</p

Effects of mutations on night sleep and correlated sleep traits.

<p>The figure shows the mean and standard error of sleep phenotypes. (A) Night sleep. (B) Day sleep. (C) Night average bout length. (D) Sleep latency. (E) Day bout number. (F) Night sleep trans-heterozygotes. Yellow bars indicate crosses with significant dominance and/or epistasis effects and maternal effects; green bars indicate crosses with significant maternal effects. M, male; F, female. *, <i>P</i> <0.05; **, <i>P</i> <0.01; ****, <i>P</i> <0.0001.</p

Sleep patterns of <i>Minos</i> mutants after 12 hours of sleep deprivation.

<p>Average minutes of sleep per 30 minutes are plotted for control (dotted lines) and deprived (solid lines) flies. Purple shading indicates the application of the shaking stimulus. Shaded bars along the x-axis indicate day (white) and night (black). (A) <i>sgg</i>. (B) <i>fz</i>. (C) <i>CG33156</i>. (D) <i>w</i>¹¹¹⁸{5905}.</p

Polymorphisms with significant allele frequency changes across generations.

<p>Polymorphisms with significant allele frequency changes across generations.</p

Phenotypic response to artificial selection for night sleep duration.

<p>(A), combined-sex average night sleep duration ± SE is plotted for each generation of selection; (B), combined-sex night sleep coefficient of environmental variation (<i>CV</i>_E) is plotted for each generation of selection; (C) and (D), combined-sex cumulative selection differential (ΣS) versus combined-sex cumulative response (ΣR) for (C) long sleep and (D) short sleep populations; (E), combined-sex cumulative differential (ΣD) versus combined-sex cumulative response (ΣR) for the control populations. Light blue and dark blue triangles indicate Replicate 1 and Replicate 2 populations selected for long sleep; Light red and dark red squares indicate Replicate 1 and Replicate 2 populations selected for short sleep; and light gray and black circles indicate Replicate 1 and Replicate 2 control populations.</p