Interplay between chromatin marks in development and disease.

DNA methylation (DNAme) and histone post-translational modifications (PTMs) have important roles in transcriptional regulation. Although many reports have characterized the functions of such chromatin marks in isolation, recent genome-wide studies reveal surprisingly complex interactions between them. Here, we focus on the interplay between DNAme and methylation of specific lysine residues on the histone H3 tail. We describe the impact of genetic perturbation of the relevant methyltransferases in the mouse on the landscape of chromatin marks as well as the transcriptome. In addition, we discuss the specific neurodevelopmental growth syndromes and cancers resulting from pathogenic mutations in the human orthologues of these genes. Integrating these observations underscores the fundamental importance of crosstalk between DNA and histone H3 methylation in development and disease.

Paternal MTHFR deficiency leads to hypomethylation of young retrotransposons and reproductive decline across two successive generations.

5,10-Methylenetetrahydrofolate reductase (MTHFR) is a crucial enzyme in the folate metabolic pathway with a key role in generating methyl groups. As MTHFR deficiency impacts male fertility and sperm DNA methylation, there is the potential for epimutations to be passed to the next generation. Here, we assessed whether the impact of MTHFR deficiency on testis morphology and sperm DNA methylation is exacerbated across generations in mouse. Although MTHFR deficiency in F1 fathers has only minor effects on sperm counts and testis weights and histology, F2 generation sons show further deterioration in reproductive parameters. Extensive loss of DNA methylation is observed in both F1 and F2 sperm, with >80% of sites shared between generations, suggestive of regions consistently susceptible to MTHFR deficiency. These regions are generally methylated during late embryonic germ cell development and are enriched in young retrotransposons. As retrotransposons are resistant to reprogramming of DNA methylation in embryonic germ cells, their hypomethylated state in the sperm of F1 males could contribute to the worsening reproductive phenotype observed in F2 MTHFR-deficient males, compatible with the intergenerational passage of epimutations.

Reduced Euchromatin histone methyltransferase 1 causes developmental delay, hypotonia, and cranial abnormalities associated with increased bone gene expression in Kleefstra syndrome mice.

Haploinsufficiency of Euchromatin histone methyltransferase 1 (EHMT1), a chromatin modifying enzyme, is the cause of Kleefstra syndrome (KS). KS is an intellectual disability (ID) syndrome, with general developmental delay, hypotonia, and craniofacial dysmorphisms as additional core features. Recent studies have been focused on the role of EHMT1 in learning and memory, linked to the ID phenotype of KS patients. In this study we used the Ehmt1(+/-) mouse model, and investigated whether the core features of KS were mimicked in these mice. When comparing Ehmt1(+/-) mice to wildtype littermates we observed delayed postnatal growth, eye opening, ear opening, and upper incisor eruption, indicating a delayed postnatal development. Furthermore, tests for muscular strength and motor coordination showed features of hypotonia in young Ehmt1(+/-) mice. Lastly, we found that Ehmt1(+/-) mice showed brachycephalic crania, a shorter or bent nose, and hypertelorism, reminiscent of the craniofacial dysmorphisms seen in KS. In addition, gene expression analysis revealed a significant upregulation of the mRNA levels of Runx2 and several other bone tissue related genes in P28 Ehmt1(+/-) mice. Runx2 immunostaining also appeared to be increased. The mRNA upregulation was associated with decreased histone H3 lysine 9 dimethylation (H3K9me2) levels, the epigenetic mark deposited by Ehmt1, in the promoter region of these genes. Together, Ehmt1(+/-) mice indeed recapitulate KS core features and can be used as an animal model for Kleefstra syndrome. The increased expression of bone developmental genes in the Ehmt1(+/-) mice likely contributes to their cranial dysmorphisms and might be explained by diminished Ehmt1-induced H3K9 dimethylation.

Hippocampal dysfunction in the Euchromatin histone methyltransferase 1 heterozygous knockout mouse model for Kleefstra syndrome.

Euchromatin histone methyltransferase 1 (EHMT1) is a highly conserved protein that catalyzes mono- and dimethylation of histone H3 lysine 9, thereby epigenetically regulating transcription. Kleefstra syndrome (KS), is caused by haploinsufficiency of the EHMT1 gene, and is an example of an emerging group of intellectual disability (ID) disorders caused by genes encoding epigenetic regulators of neuronal gene activity. Little is known about the mechanisms underlying this disorder, prompting us to study the Euchromatin histone methyltransferase 1 heterozygous knockout (Ehmt1(+/-)) mice as a model for KS. In agreement with the cognitive disturbances observed in patients with KS, we detected deficits in fear extinction learning and both novel and spatial object recognition in Ehmt1(+/-) mice. These learning and memory deficits were associated with a significant reduction in dendritic arborization and the number of mature spines in hippocampal CA1 pyramidal neurons of Ehmt1(+/-) mice. In-depth analysis of the electrophysiological properties of CA3-CA1 synapses revealed no differences in basal synaptic transmission or theta-burst induced long-term potentiation (LTP). However, paired-pulse facilitation (PPF) was significantly increased in Ehmt1(+/-) neurons, pointing to a potential deficiency in presynaptic neurotransmitter release. Accordingly, a reduction in the frequency of miniature excitatory post-synaptic currents (mEPSCs) was observed in Ehmt1(+/-) neurons. These data demonstrate that Ehmt1 haploinsufficiency in mice leads to learning deficits and synaptic dysfunction, providing a possible mechanism for the ID phenotype in patients with KS.

BORIS/CTCFL-mediated chromatin accessibility alterations promote a pro-invasive transcriptional signature in melanoma cells.

Melanoma is the deadliest form of skin cancer, due to its tendency to metastasize early. Brother of regulator of imprinted sites (BORIS), also known as CCCTC binding factor-like (CTCFL), is a transcription regulator that becomes ectopically expressed in melanoma. We recently showed that BORIS contributes to melanoma phenotype switching by altering the gene expression program of melanoma cells from an intermediate melanocytic state toward a more mesenchymal-like state. However, the mechanism underlying this transcriptional switch remains unclear. Here, ATAC-seq was used to study BORIS-mediated chromatin accessibility alterations in melanoma cells harboring an intermediate melanocytic state. The gene set that gained promoter accessibility, following ectopic BORIS expression, showed enrichment for biological processes associated with melanoma invasion, while promoters of genes associated with proliferation showed reduced accessibility. Integration of ATAC-seq and RNA-seq data demonstrated that increased chromatin accessibility was associated with transcriptional upregulation of genes involved in tumor progression processes, and the aberrant activation of oncogenic transcription factors, while reduced chromatin accessibility and downregulated genes were associated with repressed activity of tumor suppressors and proliferation factors. Together, these findings indicate that BORIS mediates transcriptional reprogramming in melanoma cells by altering chromatin accessibility and gene expression, shifting the cellular transcription landscape of melanoma cells toward a mesenchymal-like genetic signature.

Sample size justifications in Gait & Posture.

BACKGROUND: Context regarding how researchers determine the sample size of their experiments is important for interpreting the results and determining their value and meaning. Between 2018 and 2019, the journal Gait & Posture introduced a requirement for sample size justification in their author guidelines. RESEARCH QUESTION: How frequently and in what ways are sample sizes justified in Gait & Posture research articles and was the inclusion of a guideline requiring sample size justification associated with a change in practice? METHODS: The guideline was not in place prior to May 2018 and was in place from 25th July 2019. All articles in the three most recent volumes of the journal (84-86) and the three most recent, pre-guideline volumes (60-62) at time of preregistration were included in this analysis. This provided an initial sample of 324 articles (176 pre-guideline and 148 post-guideline). Articles were screened by two authors to extract author data, article metadata and sample size justification data. Specifically, screeners identified if (yes or no) and how sample sizes were justified. Six potential justification types (Measure Entire Population, Resource Constraints, Accuracy, A priori Power Analysis, Heuristics, No Justification) and an additional option of Other/Unsure/Unclear were used. RESULTS: In most cases, authors of Gait & Posture articles did not provide a justification for their study's sample size. The inclusion of the guideline was associated with a modest increase in the percentage of articles providing a justification (16.6-28.1%). A priori power calculations were the dominant type of justification, but many were not reported in enough detail to allow replication. SIGNIFICANCE: Gait & Posture researchers should be more transparent in how they determine their sample sizes and carefully consider if they are suitable. Editors and journals may consider adding a similar guideline as a low-resource way to improve sample size justification reporting.

Prevalence of group B streptococcal colonization in the healthy non-pregnant population: a systematic review and meta-analysis.

BACKGROUND: Colonization and transmission precede invasive group B streptococcal (GBS) disease. Data on GBS colonization prevalence, detection methods and risk factors for carriage are relevant for vaccine development and to understand GBS pathogenesis. OBJECTIVES: To evaluate GBS colonization prevalence after the first week of life in the healthy non-pregnant population. DATA SOURCES: Pubmed/Medline, Embase, Latin American and Caribbean Health Sciences Literature, World Health Organization Library Information System, and Scopus. Search performed 12 January 2021 with search terms related to 'GBS' and 'colonization, epidemiology, prevalence or screening' without restrictions. STUDY ELIGIBILITY CRITERIA: All studies that reported prevalence of GBS colonization (any site) in the healthy population. PARTICIPANTS: All individuals (>6 days of age), with no indication of pregnancy, invasive disease or severe underlying immunological co-morbidities. METHODS: Logit transformation and a random effects model (DerSimonian and Laird) were used to pool colonization estimates. Subgroup analysis and meta-regression on a priori determined subgroups were performed. RESULTS: We included 98 studies with 43 112 participants. Our search identified 9309 studies of which 8831 were excluded based on title and abstract and 380 after reading the full text. Colonization rates varied considerably between studies (I2 = 97%), which could be partly explained by differences in culture methods (R2 = 27%), culture sites (R2 = 24%), continent (R2 = 10%) and participant's age (R2 = 6%). Higher prevalence was found with selective culture methods (19%, 95% CI 16%-23% versus non-selective methods 8%, 95% CI 6%-9%; p < 0.0001). Colonization rates were highest in rectum (19%, 95% CI 15%-24%), vagina (14%, 95% CI 12%-17%) and urethra (9%, 95% CI 5%-18%). In participants with negative rectal cultures, 7% (95% CI 5%-9%) had GBS cultured from another niche. Colonization prevalence was lower in children (6 months to 16 years; 3%, 95% CI 2%-5%) compared with adults (16%, 95% CI 14%-20%; p < 0.0001). Using selective culture methods in adults resulted in a prevalence of 26% (95% CI 19%-33%) rectal, 21% (95% CI 17%-25%) vaginal and 9% (95% CI 6%-14%) urethral colonization. CONCLUSION: The rectum is the most common body site colonized by GBS. The best approach to screen for any GBS colonization is to screen multiple body sites using selective culture methods.

Repression of germline genes by PRC1.6 and SETDB1 in the early embryo precedes DNA methylation-mediated silencing.

Silencing of a subset of germline genes is dependent upon DNA methylation (DNAme) post-implantation. However, these genes are generally hypomethylated in the blastocyst, implicating alternative repressive pathways before implantation. Indeed, in embryonic stem cells (ESCs), an overlapping set of genes, including germline "genome-defence" (GGD) genes, are upregulated following deletion of the H3K9 methyltransferase SETDB1 or subunits of the non-canonical PRC1 complex PRC1.6. Here, we show that in pre-implantation embryos and naïve ESCs (nESCs), hypomethylated promoters of germline genes bound by the PRC1.6 DNA-binding subunits MGA/MAX/E2F6 are enriched for RING1B-dependent H2AK119ub1 and H3K9me3. Accordingly, repression of these genes in nESCs shows a greater dependence on PRC1.6 than DNAme. In contrast, GGD genes are hypermethylated in epiblast-like cells (EpiLCs) and their silencing is dependent upon SETDB1, PRC1.6/RING1B and DNAme, with H3K9me3 and DNAme establishment dependent upon MGA binding. Thus, GGD genes are initially repressed by PRC1.6, with DNAme subsequently engaged in post-implantation embryos.

BORIS/CTCFL promotes a switch from a proliferative towards an invasive phenotype in melanoma cells.

Melanoma is among the most aggressive cancers due to its tendency to metastasize early. Phenotype switching between a proliferative and an invasive state has been suggested as a critical process for metastasis, though the mechanisms that regulate state transitions are complex and remain poorly understood. Brother of Regulator of Imprinted Sites (BORIS), also known as CCCTC binding factor-Like (CTCFL), is a transcriptional modulator that becomes aberrantly expressed in melanoma. Yet, the role of BORIS in melanoma remains elusive. Here, we show that BORIS is involved in melanoma phenotype switching. Genetic modification of BORIS expression in melanoma cells combined with whole-transcriptome analysis indicated that BORIS expression contributes to an invasion-associated transcriptome. In line with these findings, inducible BORIS overexpression in melanoma cells reduced proliferation and increased migration and invasion, demonstrating that the transcriptional switch is accompanied by a phenotypic switch. Mechanistically, we reveal that BORIS binds near the promoter of transforming growth factor-beta 1 (TFGB1), a well-recognized factor involved in the transition towards an invasive state, which coincided with increased expression of TGFB1. Overall, our study indicates a pro-invasive role for BORIS in melanoma via transcriptional reprogramming.