An H3K36 Methylation-Engaging Tudor Motif of Polycomb-like Proteins Mediates PRC2 Complex Targeting

Polycomb repressive complex 2 (PRC2) regulates pluripotency, differentiation and tumorigenesis through catalysis of histone H3 lysine 27 trimethylation (H3K27me3) on chromatin. However, the mechanisms that underlie PRC2 recruitment and spreading on chromatin remain unclear. Here we report that histone H3 lysine 36 trimethylation (H3K36me3)-binding activity is harbored in the Tudor motifs of PRC2-associated polycomblike (PCL) proteins PHF1/PCL1 and PHF19/PCL3. Ectopically expressed PHF1 induced Tudor-dependent stabilization of PRC2 complexes on bulk chromatin and mediated spreading of PRC2 and H3K27me3 into H3K36me3-containing chromatin regions. In murine pluripotent stem cells, we identified coexistence of H3K36me3, H3K27me3, and PHF19/PCL3 at a subset of ‘poised’ developmental genes, and demonstrated that PHF19/PCL3 Tudor function is required for optimal H3K27me3 and repression of these loci. Collectively, our data suggest that PCL recognition of H3K36me3 promotes intrusion of PRC2 complexes into active chromatin regions to promote gene silencing and modulate the chromatin landscape during development

Evaluation of the feasibility, diagnostic yield, and clinical utility of rapid genome sequencing in infantile epilepsy (Gene-STEPS): an international, multicentre, pilot cohort study

BACKGROUND: Most neonatal and infantile-onset epilepsies have presumed genetic aetiologies, and early genetic diagnoses have the potential to inform clinical management and improve outcomes. We therefore aimed to determine the feasibility, diagnostic yield, and clinical utility of rapid genome sequencing in this population. METHODS: We conducted an international, multicentre, cohort study (Gene-STEPS), which is a pilot study of the International Precision Child Health Partnership (IPCHiP). IPCHiP is a consortium of four paediatric centres with tertiary-level subspecialty services in Australia, Canada, the UK, and the USA. We recruited infants with new-onset epilepsy or complex febrile seizures from IPCHiP centres, who were younger than 12 months at seizure onset. We excluded infants with simple febrile seizures, acute provoked seizures, known acquired cause, or known genetic cause. Blood samples were collected from probands and available biological parents. Clinical data were collected from medical records, treating clinicians, and parents. Trio genome sequencing was done when both parents were available, and duo or singleton genome sequencing was done when one or neither parent was available. Site-specific protocols were used for DNA extraction and library preparation. Rapid genome sequencing and analysis was done at clinically accredited laboratories, and results were returned to families. We analysed summary statistics for cohort demographic and clinical characteristics and the timing, diagnostic yield, and clinical impact of rapid genome sequencing. FINDINGS: Between Sept 1, 2021, and Aug 31, 2022, we enrolled 100 infants with new-onset epilepsy, of whom 41 (41%) were girls and 59 (59%) were boys. Median age of seizure onset was 128 days (IQR 46-192). For 43 (43% [binomial distribution 95% CI 33-53]) of 100 infants, we identified genetic diagnoses, with a median time from seizure onset to rapid genome sequencing result of 37 days (IQR 25-59). Genetic diagnosis was associated with neonatal seizure onset versus infantile seizure onset (14 [74%] of 19 vs 29 [36%] of 81; p=0·0027), referral setting (12 [71%] of 17 for intensive care, 19 [44%] of 43 non-intensive care inpatient, and 12 [28%] of 40 outpatient; p=0·0178), and epilepsy syndrome (13 [87%] of 15 for self-limited epilepsies, 18 [35%] of 51 for developmental and epileptic encephalopathies, 12 [35%] of 34 for other syndromes; p=0·001). Rapid genome sequencing revealed genetic heterogeneity, with 34 unique genes or genomic regions implicated. Genetic diagnoses had immediate clinical utility, informing treatment (24 [56%] of 43), additional evaluation (28 [65%]), prognosis (37 [86%]), and recurrence risk counselling (all cases). INTERPRETATION: Our findings support the feasibility of implementation of rapid genome sequencing in the clinical care of infants with new-onset epilepsy. Longitudinal follow-up is needed to further assess the role of rapid genetic diagnosis in improving clinical, quality-of-life, and economic outcomes. FUNDING: American Academy of Pediatrics, Boston Children's Hospital Children's Rare Disease Cohorts Initiative, Canadian Institutes of Health Research, Epilepsy Canada, Feiga Bresver Academic Foundation, Great Ormond Street Hospital Charity, Medical Research Council, Murdoch Children's Research Institute, National Institute of Child Health and Human Development, National Institute for Health and Care Research Great Ormond Street Hospital Biomedical Research Centre, One8 Foundation, Ontario Brain Institute, Robinson Family Initiative for Transformational Research, The Royal Children's Hospital Foundation, University of Toronto McLaughlin Centre

Long-Term Characteristics of Human-Derived Biliary Organoids under a Single Continuous Culture Condition

Organoids have been used to investigate the three-dimensional (3D) organization and function of their respective organs. These self-organizing 3D structures offer a distinct advantage over traditional two-dimensional (2D) culture techniques by creating a more physiologically relevant milieu to study complex biological systems. The goal of this study was to determine the feasibility of establishing organoids from various pediatric liver diseases and characterize the long-term evolution of cholangiocyte organoids (chol-orgs) under a single continuous culture condition. We established chol-orgs from 10 different liver conditions and characterized their multicellular organization into complex epithelial structures through budding, merging, and lumen formation. Immunofluorescent staining, electron microscopy, and single-nucleus RNA (snRNA-seq) sequencing confirmed the cholangiocytic nature of the chol-orgs. There were significant cell population differences in the transcript profiles of two-dimensional and organoid cultures based on snRNA-seq. Our study provides an approach for the generation and long-term maintenance of chol-orgs from various pediatric liver diseases under a single continuous culture condition

Selection constraints on transcribed pseudogenes.

<p>Comparison of nucleotide diversities in human population (A) and cross-species conservations (B) between non-transcribed (‘n’) and transcribed pseudogenes (‘y’). AluY, a young repeats that emerged recently in primates, was used as control. For duplicated pseudogenes, the median diversities for transcribed and non-transcribed are 0. 00051 and 0.00054 (p<0.02, Wilcoxon test), the values for processed pseudogenes are 0.00055 and 0.00064 (p<3e-06, Wilcoxon test).</p

Transcriptional correlations (ρ_pg:g) between pseudogenes and their parents.

<p>A) A heatmap for distribution of ρ_pg:g, including data from separation of processed and duplicated pseudogenes into two groups based on the presence of a coding gene within 20 kb. The coefficients between transcribed pseudogenes and randomly chosen coding genes (top) were used as a control for p-value estimation. Colors represent relative numbers of pseudogenes in each ρ_pg:g range (in Z-score transformation). B) Pseudogenes transcribed in the sense direction (S) exhibited higher ρ_pg:g than those in the antisense (A). C) The transcriptional correlation between pseudogenes and their parents (ρ_pg:g) is inversely correlated to the transcriptional correlation between miRNAs and their putative targets (ρ_miRNA:g). Genes were binned on their ρ_miRNA:g values (x-axis) and then the mean and standard deviation of ρ_pg:g (y-axis) for each group of genes was plotted. D) Expression of parental genes targeted by miRNAs was less affected by miRNA KD than the targeting genes without pseudogenes. Only genes in response to KD (up >1.3 fold) were analyzed here. Y-axis shows the fold change of KD over control. The miRNA targets were experimentally determined by the CLASH analysis <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0093972#pone.0093972-Helwak1" target="_blank">[49]</a>. The middle line in the boxplots mark median and the box lines mark the first and third quartile values (same for boxplots below).</p

Top pseudogene candidates of three different types of predicted functional potentials (ND, not determined). The full lists can be found in Table S1.

<p>Top pseudogene candidates of three different types of predicted functional potentials (ND, not determined). The full lists can be found in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0093972#pone.0093972.s008" target="_blank">Table S1</a>.</p