Dynamic Reprogramming of DNA Methylation at an Epigenetically Sensitive Allele in Mice

There is increasing evidence in both plants and animals that epigenetic marks are not always cleared between generations. Incomplete erasure at genes associated with a measurable phenotype results in unusual patterns of inheritance from one generation to the next, termed transgenerational epigenetic inheritance. The Agouti viable yellow (A(vy)) allele is the best-studied example of this phenomenon in mice. The A(vy) allele is the result of a retrotransposon insertion upstream of the Agouti gene. Expression at this locus is controlled by the long terminal repeat (LTR) of the retrotransposon, and expression results in a yellow coat and correlates with hypomethylation of the LTR. Isogenic mice display variable expressivity, resulting in mice with a range of coat colours, from yellow through to agouti. Agouti mice have a methylated LTR. The locus displays epigenetic inheritance following maternal but not paternal transmission; yellow mothers produce more yellow offspring than agouti mothers. We have analysed the DNA methylation in mature gametes, zygotes, and blastocysts and found that the paternally and maternally inherited alleles are treated differently. The paternally inherited allele is demethylated rapidly, and the maternal allele is demethylated more slowly, in a manner similar to that of nonimprinted single-copy genes. Interestingly, following maternal transmission of the allele, there is no DNA methylation in the blastocyst, suggesting that DNA methylation is not the inherited mark. We have independent support for this conclusion from studies that do not involve direct analysis of DNA methylation. Haplo-insufficiency for Mel18, a polycomb group protein, introduces epigenetic inheritance at a paternally derived A(vy) allele, and the pedigrees reveal that this occurs after zygotic genome activation and, therefore, despite the rapid demethylation of the locus

A genome-wide screen for modifiers of transgene variegation identifies genes with critical roles in development

BACKGROUND: Some years ago we established an N-ethyl-N-nitrosourea screen for modifiers of transgene variegation in the mouse and a preliminary description of the first six mutant lines, named MommeD1-D6, has been published. We have reported the underlying genes in three cases: MommeD1 is a mutation in SMC hinge domain containing 1 (Smchd1), a novel modifier of epigenetic gene silencing; MommeD2 is a mutation in DNA methyltransferase 1 (Dnmt1); and MommeD4 is a mutation in Smarca 5 (Snf2h), a known chromatin remodeler. The identification of Dnmt1 and Smarca5 attest to the effectiveness of the screen design. RESULTS: We have now extended the screen and have identified four new modifiers, MommeD7-D10. Here we show that all ten MommeDs link to unique sites in the genome, that homozygosity for the mutations is associated with severe developmental abnormalities and that heterozygosity results in phenotypic abnormalities and reduced reproductive fitness in some cases. In addition, we have now identified the underlying genes for MommeD5 and MommeD10. MommeD5 is a mutation in Hdac1, which encodes histone deacetylase 1, and MommeD10 is a mutation in Baz1b (also known as Williams syndrome transcription factor), which encodes a transcription factor containing a PHD-type zinc finger and a bromodomain. We show that reduction in the level of Baz1b in the mouse results in craniofacial features reminiscent of Williams syndrome. CONCLUSIONS: These results demonstrate the importance of dosage-dependent epigenetic reprogramming in the development of the embryo and the power of the screen to provide mouse models to study this process

COVID‐19 Vaccine Response in People with Multiple Sclerosis

ObjectiveThe purpose of this study was to investigate the effect of disease modifying therapies on immune response to severe acute respiratory syndrome-coronavirus 2 (SARS-CoV-2) vaccines in people with multiple sclerosis (MS).MethodsFour hundred seventy-three people with MS provided one or more dried blood spot samples. Information about coronavirus disease 2019 (COVID-19) and vaccine history, medical, and drug history were extracted from questionnaires and medical records. Dried blood spots were eluted and tested for antibodies to SARS-CoV-2. Antibody titers were partitioned into tertiles with people on no disease modifying therapy as a reference. We calculated the odds ratio of seroconversion (univariate logistic regression) and compared quantitative vaccine response (Kruskal Wallis) following the SARS-CoV-2 vaccine according to disease modifying therapy. We used regression modeling to explore the effect of vaccine timing, treatment duration, age, vaccine type, and lymphocyte count on vaccine response.ResultsCompared to no disease modifying therapy, the use of anti-CD20 monoclonal antibodies (odds ratio = 0.03, 95% confidence interval [CI] = 0.01–0.06, p [less than] 0.001) and fingolimod (odds ratio = 0.04; 95% CI = 0.01–0.12) were associated with lower seroconversion following the SARS-CoV-2 vaccine. All other drugs did not differ significantly from the untreated cohort. Both time since last anti-CD20 treatment and total time on treatment were significantly associated with the response to the vaccination. The vaccine type significantly predicted seroconversion, but not in those on anti-CD20 medications. Preliminary data on cellular T-cell immunity showed 40% of seronegative subjects had measurable anti-SARS-CoV-2 T cell responses.InterpretationSome disease modifying therapies convey risk of attenuated serological response to SARS-CoV-2 vaccination in people with MS. We provide recommendations for the practical management of this patient group. ANN NEUROL 202

An ENU mutagenesis screen to identify modifiers of gene silencing in the mouse

There is increasing evidence that the epigenotype of an organism contributes to phenotype. With the aim of understanding more about the factors involved in the initiation, maintenance and reprogramming of epigenetic state, our laboratory has designed an ENU mutagenesis screen to recover modifiers of gene silencing in the mouse. In this thesis I describe the dominant and recessive screens 1 performed which recovered a total of nine mutant lines which showed alterations in gene silencing, some of these lines had increased transgene expression (suppressors of variegation), and others had decreased transgene expression (enhancers of variegation). These mutant lines were able to modify gene silencing at a variegating transgene and were therefore named Modifiers of murine metastable epialleles (Mommes). I worked extensively with three dominant lines recovered from this and a previously performed, identical screen; Momme D4, Momme D5 and Momme D7. The mutations underlying all three of these lines were mapped to discrete genomic regions and in the case of Momme D4, a mutation was found in the Snf2h gene, which encodes a compenent of chromatin remodelling complexes. The three dominant lines examined all displayed dose—dependent effects, in all cases homozgyosity for the mutations was lethal. Stochastic effects on viability were also found in association with heterozygosity for the mutations. Two of the mutant lines, Momme D4 and Momme D7 affected expression at a single copy endogenous allele known to be sensitive to epigenetic state, agouti viable yellow, A”. Breeding studies have revealed that these mutations display unusual maternal and paternal effects, whereby wildtype offspring of heterozygous parents are affected. One of the recessive mutant lines, Momme R1, was studied at some depth. Age-associated loss of fertility and susceptibility to development of ovarian teratomas was seen in homozygous females. A mutation was identified in the Foxo3a gene of the mutant mice, this gene encodes a transcription factor with roles in cell cycle control, apoptosis and DNA repair. The mechanism by which loss of Foxo3a affects transgene silencing is not clear and these mice provide a platform to investigate this further. In summary, several mouse models have been produced which represent mutations that alter gene silencing in the mouse. By studying the behaviour of these mutant colonies l have found evidence that the untransmitted genotype of the parent can influence the phenotype of the offspring

Methylation of the <i>A^vy</i> Allele in 12.5-dpc Embryos following Paternal Transmission

<p>The 12.5-dpc embryos produced from an <i>A^vy/a</i> sire mated with an <i>a/a</i> dam. Samples were digested and subjected to Southern transfer as described in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.0020049#pgen-0020049-g001" target="_blank">Figure 1</a>. A range of methylation states were observed, evidenced by the varying amounts of the 9-kb BamHI band remaining after HpaII digestion. This indicates that the methylation is likely to be reset by this stage of development.</p

Methylation of the <i>A^vy</i> Allele in Mature Sperm

<p>(A) Expression of the <i>A^vy</i> allele is controlled by an IAP, inserted into pseudoexon 1a (grey box). A cryptic promoter within the 3′ LTR of the IAP (black arrows) directs transcription of the <i>agouti</i> coding exons. The BamHI (B) and MspI (M) sites are shown in the region of the unique 400-bp probe B. Tail and mature sperm from a yellow and a pseudoagouti male were collected. DNA was prepared and samples digested with BamHI followed by MspI or its isoschizomer HpaII, transferred and hybridised with the <i>agouti</i> probe [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.0020049#pgen-0020049-b003" target="_blank">3</a>]. The <i>A^vy</i> allele produces a 9-kb BamHI band, while the <i>a</i> allele produces a 3.3-kb band. Membranes were stripped and rehybridised with a murine <i>α-globin</i> probe to check for equal digestion within the tissue samples (shown in [B]). These results represent experiments performed on sperm and tail DNA from seven yellow and five pseudoagouti males, a further one of each are shown in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.0020049#pgen-0020049-sg001" target="_blank">Figure S1</a>. Mature sperm were isolated from both epididymes of the male (each sample contained in the order of 10⁶ to 10⁷ spermatocytes). Sperm samples were checked by light microscopy and found to be greater than 95% spermatocytes. The methylation state of the tissues is indicated by the ratio of the 9-kb BamHI band to the 7-kb band remaining after HpaII digestion. The 9-kb band is marked by an asterisk. The methylation state of the sperm reflects the phenotype of the father rather than the range of phenotypes seen in the offspring.</p

Methylation of the <i>A^vy</i> Allele following Paternal Transmission

<div><p>The methylation status of each CpG dinucleotide was determined by sequencing PCR clones of bisulfite-converted DNA [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.0020049#pgen-0020049-b004" target="_blank">4</a>]. Each line represents an individual clone, theoretically from one cell, and each circle an individual CpG. Open circles indicate an unmethylated CpG, and closed circles a methylated CpG. Each block of lines represents the clones derived from the sperm of one adult male mouse, one set of ten zygotes, ten two-cell embryos or one blastocyst. The percentage of methylation in each dataset is shown (calculated from the number of methylated CpGs divided by the total CpGs sequenced, multiplied by 100). The position of each circle is representative of the relative location along the length of the PCR product. Any clones with greater than 5% non-CpG methylation were excluded from the dataset, and these clones made up less than 5% of all clones sequenced. These clones tended to have very high levels of non-CpG methylation, an indication of incomplete bisulfite conversion. Zygotes, two-cell embryos, or blastocysts were collected from yellow or pseudoagouti <i>A^vy/a</i> sires mated to <i>a/a</i> dams, as indicated. Clones were only included in the zygote or blastocyst samples if they could be distinguished from others in the sample by CpG or low-level non-CpG methylation.</p><p>(A) The IAP LTR pseudoexon 1a junction shown in detail. The bisulfite sequencing primers are shown [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.0020049#pgen-0020049-b004" target="_blank">4</a>]. The PCR product contains 11 CpG dinucleotides, depicted as circles, all of which are in the LTR.</p><p>(B) Data obtained from the sperm of four pseudoagouti males, and 10 × 10 zygotes, 10 × 10 two-cell embryos or 10 × 1 blastocysts collected from pseudoagouti <i>A^vy/a</i> sires mated to <i>a/a</i> dams.</p><p>(C) Data obtained from the sperm of four yellow male and 9 × 1 blastocysts collected from yellow <i>A^vy/a</i> sires mated to <i>a/a</i> dams. These data indicate that the <i>A^vy</i> allele is subject to rapid demethylation immediately postfertilisation following paternal transmission.</p></div

Methylation of the <i>A^vy</i> Allele following Maternal Transmission

<div><p>The methylation status of each CpG dinucleotide was determined by sequencing PCR clones of bisulfite-converted DNA, as described in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.0020049#pgen-0020049-g002" target="_blank">Figure 2</a> [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.0020049#pgen-0020049-b004" target="_blank">4</a>]. Each block of lines represents the clones derived from one bisulfite conversion of ten cells (oocytes or zygotes). The percentage of methylation in each dataset is shown (calculated from the number of methylated CpGs divided by the total CpGs sequenced, multiplied by 100). Clones were only included in the samples if they could be distinguished from others in the sample by CpG or non-CpG methylation. Any clones with higher than 5% non-CpG methylation (an indication of incomplete bisulfite conversion) were excluded from the dataset, and these clones made up less than 5% of all clones sequenced.</p><p>(A) and (D) Unfertilised oocytes from pseudoagouti or yellow <i>A^vy/a</i> females, respectively. DNA methylation at the <i>A^vy</i> allele does not appear to have been reset during oogenesis.</p><p>(B) Zygotes from pseudoagouti <i>A^vy/a</i> dams mated to <i>a/a</i> sires.</p><p>(C) Blastocysts from pseudoagouti <i>A^vy/a</i> dams mated to <i>a/a</i> sires. The <i>A^vy</i> allele is not subject to rapid demethylation immediately postfertilisation following maternal inheritance, but is completely cleared of DNA methylation before blastocyst formation.</p></div

Pedigrees of Crosses between <i>Mel18^+/−</i> and Mice Carrying the <i>A^vy</i> Allele

<div><p><i>A^vy/a</i> C57BL/6J sires of the indicated phenotype were mated with <i>Mel18^+/−</i> C57BL/6J dams. Data were produced from at least five different mating pairs in each case. Offspring not carrying the <i>A^vy</i> allele have been omitted. All coat colour phenotypes were scored by one observer, before the analysis of <i>Mel18</i> genotype.</p><p>(A) There was no significant shift in the proportion of phenotypes observed between <i>Mel18^+/−</i> and <i>Mel18^+/+</i> littermates from yellow sires.</p><p>(B) There was a significant shift towards the pseudoagouti phenotype in <i>Mel18^+/−</i> compared with <i>Mel18^+/+</i> littermates from pseudoagouti sires (<i>p</i> = 0.006). This shift produced a statistically significant difference in the range of <i>Mel18^+/−</i> offspring observed from yellow and pseudoagouti sires (<i>p</i> = 0.0002); the <i>Mel18^+/−</i> offspring of pseudoagouti sires are more likely to be pseudoagouti than those of yellow sires, i.e., transgenerational epigenetic inheritance is observed. Epigenetic inheritance was not observed in the <i>Mel18^+/+</i> littermates.</p></div