Maternal Spindle Transfer (MST) and Pronuclear Transfer (PNT) are micromanipulation techniques that allow the transfer of the nuclear material from one oocyte or zygote to the cytoplasm of another. The application of these techniques has provided novel insights into nuclear-cytoplasmic interactions necessary for development, as well as fundamental discoveries such as genomic imprinting. Now, after extensive pre-clinical research, PNT and MST are starting to be used clinically for Mitochondrial Donation (also known as mitochondrial replacement therapy) and for patients with a history of repeated IVF failure. Here, we describe the methods for PNT and MST currently used in the mouse model. The techniques are readily adaptable for application to other mammalian species, including humans.

Pancreatic ductal progenitor cells have been proposed to contribute to adult tissue maintenance and regeneration after injury, but the identity of such ductal cells remains elusive. Here, from adult mice, we identify a near homogenous population of ductal progenitor-like clusters, with an average of 8 cells per cluster. They are a rare subpopulation, about 0.1% of the total pancreatic cells, and can be sorted using a fluorescence-activated cell sorter with the CD133highCD71lowFSCmid-high phenotype. They exhibit properties in self-renewal and tri-lineage differentiation (including endocrine-like cells) in a unique 3-dimensional colony assay system. An in vitro lineage tracing experiment, using a novel HprtDsRed/+ mouse model, demonstrates that a single cell from a cluster clonally gives rise to a colony. Droplet RNAseq analysis demonstrates that these ductal clusters express embryonic multipotent progenitor cell markers Sox9, Pdx1, and Nkx6-1, and genes involved in actin cytoskeleton regulation, inflammation responses, organ development, and cancer. Surprisingly, these ductal clusters resist prolonged trypsin digestion in vitro, preferentially survive in vivo after a severe acinar cell injury and become proliferative within 14 days post-injury. Thus, the ductal clusters are the fundamental units of progenitor-like cells in the adult murine pancreas with implications in diabetes treatment and tumorigenicity.

Failure of palatogenesis results in cleft palate, one of the most common congenital disabilities in humans. During the final phases of palatogenesis, the protective function of the peridermal cell layer must be eliminated for the medial edge epithelia to adhere properly, which is a prerequisite for the successful fusion of the secondary palate. However, a deeper understanding of the role and fate of the periderm in palatal adherence and fusion has been hampered due to a lack of appropriate periderm-specific genetic tools to examine this cell type in vivo. Here we used the cytokeratin-6A (Krt-6a) locus to develop both constitutive ( Krt6ai-Cre) and inducible ( Krt6ai-Cre ERT2 ) periderm-specific Cre driver mouse lines. These novel lines allowed us to achieve both the spatial and temporal control needed to dissect the periderm fate on a cellular resolution during palatogenesis. Our studies suggest that, already before the opposing palatal shelves contact each other, at least some palatal periderm cells start to gradually lose their squamous periderm-like phenotype and dedifferentiate into cuboidal cells, reminiscent of the basal epithelial cells seen in the palatal midline seam. Moreover, we show that transforming growth factor–β (TGF-β) signaling plays a critical periderm-specific role in palatogenesis. Thirty-three percent of embryos lacking a gene encoding the TGF-β type I receptor ( Tgfbr1) in the periderm display a complete cleft of the secondary palate. Our subsequent experiments demonstrated that Tgfbr1-deficient periderm fails to undergo appropriate dedifferentiation. These studies define the periderm cell fate during palatogenesis and reveal a novel, critical role for TGF-β signaling in periderm dedifferentiation, which is a prerequisite for appropriate palatal epithelial adhesion and fusion.

Abstract Positive impacts on human health can occasionally arise from adopting techniques and approaches developed for entirely different purposes. Mitochondrial replacement therapy (MRT), also referred to as mitochondrial donation, is an example. Its origins date back to the development of embryo micromanipulation techniques in the 1980s. These techniques, originally designed to investigate how the nucleus and cytoplasm interact to instruct the process of embryonic development, are now on the cusp of being introduced into the clinic as the only realistic hope for preventing the propagation of a debilitating set of genetic diseases. While there is little doubt that the risk associated with carrying out these long-established techniques is far outweighed by the clinical impact of preventing genetic disease, their adoption represents an unprecedented level of intervention in early human development. The authors reassess development of the technologies using a lens which may inform and improve their adoption into clinical practice.

Background Point mutations in histone variant H3.3 (H3.3K27M, H3.3G34R) and the H3.3-specific ATRX/DAXX chaperone complex are frequent events in pediatric gliomas. These H3.3 point mutations affect many chromatin modifications but the exact oncogenic mechanisms are currently unclear. Histone H3.3 is known to localize to nuclear compartments known as promyelocytic leukemia (PML) nuclear bodies, which are frequently mutated and confirmed as oncogenic drivers in acute promyelocytic leukemia. Results We find that the pediatric glioma-associated H3.3 point mutations disrupt the formation of PML nuclear bodies and this prevents differentiation down glial lineages. Similar to leukemias driven by PML mutations, H3.3-mutated glioma cells are sensitive to drugs that target PML bodies. We also find that point mutations in IDH1/2—which are common events in adult gliomas and myeloid leukemias—also disrupt the formation of PML bodies. Conclusions We identify PML as a contributor to oncogenesis in a subset of gliomas and show that targeting PML bodies is effective in treating these H3.3-mutated pediatric gliomas.

Eggs contain about 200,000 mitochondria that generate adenosine triphosphate and metabolites essential for oocyte development. Mitochondria also integrate metabolism and transcription via metabolites that regulate epigenetic modifiers, but there is no direct evidence linking oocyte mitochondrial function to the maternal epigenome and subsequent embryo development. Here, we have disrupted oocyte mitochondrial function via deletion of the mitochondrial fission factor Drp1. Fission-deficient oocytes exhibit a high frequency of failure in peri- and postimplantation development. This is associated with altered mitochondrial function, changes in the oocyte transcriptome and proteome, altered subcortical maternal complex, and a decrease in oocyte DNA methylation and H3K27me3. Transplanting pronuclei of fertilized Drp1 knockout oocytes to normal ooplasm fails to rescue embryonic lethality. We conclude that mitochondrial function plays a role in establishing the maternal epigenome, with serious consequences for embryo development.

Pancreatic ducts function to deliver digestive enzymes into the intestines. Upon injury, ducts can become proliferative and contribute to tissue regeneration; however, the identity of the ductal cells that contribute to these processes is unknown. We combined fluorescence-activated cell sorting, a methylcellulose-containing 3-dimensional culture, droplet RNA-sequencing, and a clonal lineage tracing tool to identify and isolate a distinct subpopulation of pancreatic ductal cells that exhibit progenitor cell properties. These ductal cells are unique in that they form tightly-bound clusters (termed FSCmid-high), with an average of 8 cells per cluster. FSCmid-high clusters comprise only about 0.1% of the total pancreas, are tri-potent for duct, acinar and endocrine lineages, and self-renew robustly in vitro. Transcriptomic analysis of FSCmid-high clusters reveals enrichment for genes involved in cell-cell interactions, organ development, and cancer pathways. FSCmid-high clusters express embryonic pancreatic progenitor markers Sox9, Pdx1, and Nkx6-1 at both transcription and protein levels. FSCmid-high clusters are resistant to enzymatic dissociation and survive severe in vivo acinar injury, which induces formation of ductal rosettes that become proliferative within 14 days. Thus, FSCmid-high clusters represent a small subset of ductal cells with progenitor cell properties. These rare progenitor-like duct cell clusters have implications in pancreas regeneration and tumor initiation/progression.

ABSTRACT Most paediatric diffuse intrinsic pontine gliomas carry a lysine- to methionine-27 (K27M) mutation in the histone variant H3.3 encoded by the ‘H3.3 histone A’ ( H3F3A ) gene. To establish a pre-clinical model of the disease we made a Cre/ loxP conditional H3.3K27M mutation at the orthologous H3f3a locus in mice. Importantly, expression of the mutant transcript is under endogenous H3f3a regulatory control. This system is distinct from others in which H3.3M27 is ectopically expressed, thereby providing a resource for the development of pDIPG models with orthologous regulation of mutant H3.3. Mice in which expression of the mutant transcript was induced with a nestin- cre transgene developed as dwarfs in the presence of intact growth hormone signaling.

Histone H3.3 is an H3 variant which differs from the canonical H3.1/2 at four residues, including a serine residue at position 31 which is evolutionarily conserved. The H3.3 S31 residue is phosphorylated (H3.3 S31Ph) at heterochromatin regions including telomeres and pericentric repeats. However, the role of H3.3 S31Ph in these regions remains unknown. In this study, we find that H3.3 S31Ph regulates heterochromatin accessibility at telomeres during replication through regulation of H3K9/K36 histone demethylase KDM4B. In mouse embryonic stem (ES) cells, substitution of S31 with an alanine residue (H3.3 A31 -phosphorylation null mutant) results in increased KDM4B activity that removes H3K9me3 from telomeres. In contrast, substitution with a glutamic acid (H3.3 E31, mimics S31 phosphorylation) inhibits KDM4B, leading to increased H3K9me3 and DNA damage at telomeres. H3.3 E31 expression also increases damage at other heterochromatin regions including the pericentric heterochromatin and Y chromosome-specific satellite DNA repeats. We propose that H3.3 S31Ph regulation of KDM4B is required to control heterochromatin accessibility of repetitive DNA and preserve chromatin integrity.

Abstract Alternative Lengthening of Telomeres (ALT) is a telomere maintenance pathway utilized in 15% of cancers. ALT cancers are strongly associated with inactivating mutations in ATRX; yet ATRX knockout (KO) alone is insufficient to trigger ALT, suggesting that additional cooperating factors are involved. We identify H3.3G34R and IDH1/2 mutations as two such factors in ATRX-mutated glioblastomas. Both mutations are capable of inactivating histone demethylases, and we identify KDM4B as the key demethylase which is inactivated in ALT. Mouse ES cells inactivated for ATRX, TP53, TERT and KDM4B (KDM4B KO or H3.3G34R) show characteristic features of ALT. Conversely, KDM4B over-expression in ALT cancer cells abrogates ALT-associated features. Our data demonstrate that inactivation of KDM4B, either through H3.3G34R or IDH1/2 mutations, acts in tandem with ATRX mutations to promote ALT in cancers. Citation Format: Lee H. Wong, Maheshi Udugama, Linda Hii, Andrew Garvie, Matthew Garvie, Benjamin Vinod, Lyn Chan, Jeffrey Mann, Philippe Collas, Joanna Voon. Mutations inhibiting KDM4B drive ALT telomere maintenance pathway in ATRX-mutated cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2065.

Beckwith-Wiedemann syndrome (BWS) and Silver-Russell syndrome (SRS) are imprinting disorders manifesting as aberrant fetal growth and severe postnatal-growth-related complications. Based on the insulator model, one-third of BWS cases and two-thirds of SRS cases are consistent with misexpression of insulin-like growth factor 2 (IGF2), an important facilitator of fetal growth. We propose that the IGF2-dependent BWS and SRS cases can be identified by prenatal diagnosis and can be prevented by prenatal intervention targeting IGF2. We test this hypothesis using our mouse models of IGF2-dependent BWS and SRS. We find that genetically normalizing IGF2 levels in a double rescue experiment corrects the fetal overgrowth phenotype in the BWS model and the growth retardation in the SRS model. In addition, we pharmacologically rescue the BWS growth phenotype by reducing IGF2 signaling during late gestation. This animal study encourages clinical investigations to target IGF2 for prenatal diagnosis and prenatal prevention in human BWS and SRS.

PIWI-interacting small RNAs (piRNAs) maintain genome stability in animal germ cells, with a predominant role in silencing transposable elements. Mutations in the piRNA pathway in the mouse uniformly lead to failed spermatogenesis and male sterility. By contrast, mutant females are fertile. In keeping with this paradigm, we previously reported male sterility and female fertility associated with loss of the enzyme HENMT1, which is responsible for stabilising piRNAs through the catalysation of 3 '-terminal 2 '-O-methylation. However, the Henmt1 mutant females were poor breeders, suggesting they could be subfertile. Therefore, we investigated oogenesis and female fertility in these mice in greater detail. Here, we show that mutant females indeed have a 3- to 4-fold reduction in follicle number and reduced litter sizes. In addition, meiosis-II mutant oocytes display various spindle abnormalities and have a dramatically altered transcriptome which includes a down-regulation of transcripts required for microtubule function. This down-regulation could explain the spindle defects observed with consequent reductions in litter size. We suggest these various effects on oogenesis could be exacerbated by asynapsis, an apparently universal feature of piRNA mutants of both sexes. Our findings reveal that loss of the piRNA pathway in females has significant functional consequences.

Alternative Lengthening of Telomeres (ALT) is a telomere maintenance pathway utilised in 15% of cancers. ALT cancers are strongly associated with inactivating mutations in ATRX; yet loss of ATRX alone is insufficient to trigger ALT, suggesting that additional cooperating factors are involved. We identify H3.3G34R and IDH1/2 mutations as two such factors in ATRX-mutated glioblastomas. Both mutations are capable of inactivating histone demethylases, and we identify KDM4B as the key demethylase inactivated in ALT. Mouse embryonic stem cells inactivated for ATRX, TP53, TERT and KDM4B (KDM4B knockout or H3.3G34R) show characteristic features of ALT. Conversely, KDM4B over-expression in ALT cancer cells abrogates ALT-associated features. In this work, we demonstrate that inactivation of KDM4B, through H3.3G34R or IDH1/2 mutations, acts in tandem with ATRX mutations to promote ALT in glioblastomas.

Additional file 2:List of unique tags mapped to repetitive elements and ncRNAs based on the 3' end sequences. (TXT 846 KB)

Background Beckwith-Wiedemann syndrome (BWS) is an imprinting disorder with a population frequency of approximately 1 in 10,000. The most common epigenetic defect in BWS is a loss of methylation (LOM) at the 11p15.5 imprinting centre, KCNQ1OT1 TSS-DMR, and affects 50% of cases. We hypothesised that genetic factors linked to folate metabolism may play a role in BWS predisposition via effects on methylation maintenance at KCNQ1OT1 TSS-DMR. Results Single nucleotide variants (SNVs) in the folate pathway affecting methylenetetrahydrofolate reductase ( MTHFR ), methionine synthase reductase ( MTRR ), 5-methyltetrahydrofolate-homocysteine S -methyltransferase (MTR), cystathionine beta-synthase ( CBS ) and methionine adenosyltransferase ( MAT1A ) were examined in 55 BWS patients with KCNQ1OT1 TSS-DMR LOM and in 100 unaffected cases. MTHFR rs1801133: C>T was more prevalent in BWS with KCNQ1OT1 TSS-DMR LOM ( p < 0.017); however, the relationship was not significant when the Bonferroni correction for multiple testing was applied (significance, p = 0.0036). None of the remaining 13 SNVs were significantly different in the two populations tested. The DNMT1 locus was screened in 53 BWS cases, and three rare missense variants were identified in each of three patients: rs138841970: C>T, rs150331990: A>G and rs757460628: G>A encoding NP_001124295 p.Arg136Cys, p.His1118Arg and p.Arg1223His, respectively. These variants have population frequencies of less than 1 in 1000 and were absent from 100 control cases. Functional characterization using a hemimethylated DNA trapping assay revealed a reduced methyltransferase activity relative to wild-type DNMT1 for each variant ranging from 40 to 70% reduction in activity. Conclusions This study is the first to examine folate pathway genetics in BWS and to identify rare DNMT1 missense variants in affected individuals. Our data suggests that reduced DNMT1 activity could affect maintenance of methylation at KCNQ1OT1 TSS-DMR in some cases of BWS, possibly via a maternal effect in the early embryo. Larger cohort studies are warranted to further interrogate the relationship between impaired MTHFR enzymatic activity attributable to MTHFR rs1801133: C>T, dietary folate intake and BWS.

An array of oncogenic histone point mutations have been identified across a number of different cancer studies. It has been suggested that some of these mutant histones can exert their effects by inhibiting epigenetic writers. Here, we report that the H3.3 G34R (glycine to arginine) substitution mutation, found in paediatric gliomas, causes widespread changes in H3K9me3 and H3K36me3 by interfering with the KDM4 family of K9/K36 demethylases. Expression of a targeted single-copy of H3.3 G34R at endogenous levels induced chromatin alterations that were comparable to a KDM4 A/B/C triple-knockout. We find that H3.3 G34R preferentially binds KDM4 while simultaneously inhibiting its enzymatic activity, demonstrating that histone mutations can act through inhibition of epigenetic erasers. These results suggest that histone point mutations can exert their effects through interactions with a range of epigenetic readers, writers and erasers.

Mutations in PARK2 ( parkin ) can result in Parkinson’s disease (PD). Parkin shares a bidirectional promoter with parkin coregulated gene ( PACRG ) and the transcriptional start sites are separated by only ~200 bp. Bidirectionally regulated genes have been shown to function in common biological pathways. Mice lacking parkin have largely failed to recapitulate the dopaminergic neuronal loss and movement impairments seen in individuals with parkin-mediated PD. We aimed to investigate the function of PACRG and test the hypothesis that parkin and PACRG function in a common pathway by generating and characterizing two novel knockout mouse lines harbouring loss of both parkin and Pacrg or Pacrg alone. Successful modification of the targeted allele was confirmed at the genomic, transcriptional and steady state protein levels for both genes. At 18–20 months of age, there were no significant differences in the behaviour of parental and mutant lines when assessed by openfield, rotarod and balance beam. Subsequent neuropathological examination suggested there was no gross abnormality of the dopaminergic system in the substantia nigra and no significant difference in the number of dopaminergic neurons in either knockout model compared to wildtype mice.

ATRX (alpha thalassemia/mental retardation X-linked) complexes with DAXX to deposit histone variant H3.3 into repetitive heterochromatin. Recent genome sequencing studies in cancers have revealed mutations in ATRX and their association with ALT (alternative lengthening of telomeres) activation. Here we report depletion of ATRX in mouse ES cells leads to selective loss in ribosomal RNA gene (rDNA) copy number. Supporting this, ATRX-mutated human ALT-positive tumors also show a substantially lower rDNA copy than ALT-negative tumors. Further investigation shows that the rDNA copy loss and repeat instability are caused by a disruption in H3.3 deposition and thus a failure in heterochromatin formation at rDNA repeats in the absence of ATRX. We also find that ATRX-depleted cells are reduced in ribosomal RNA transcription output and show increased sensitivity to RNA polymerase I (Pol I) transcription inhibitor CX5461. In addition, human ALT-positive cancer cell lines are also more sensitive to CX5461 treatment. Our study provides insights into the contribution of ATRX loss of function to tumorigenesis through the loss of rDNA stability and suggests the therapeutic potential of targeting Pol I transcription in ALT cancers.

Background: Health coaching is potentially a practical method to assist patients in achieving and maintaining healthy lifestyles. In health coaching, the coach partners with the patient, helping patients discover their own strengths, challenges, and solutions. Methods: Two medical assistants were provided with brief training. The 12-week program consisted of telephone coaching with in-person visits at the beginning and end of the program. Coaching targeted improvements in diet, physical activity, and/or sleep habits using a self-care planning form. Results: A total of 82 subjects enrolled in the program, 72% completed 8 weeks and 49% completed 12 weeks. Subjects who completed assessments at 12 weeks had significant weight loss despite the fact that weight loss was not a study goal. There also were improvements in diet and physical activity. Subject who completed the study were highly satisfied with the program and felt that health coaching should be available in all family medicine clinics. The main barrier providers voiced was remembering to refer patients. The medical providers indicated high satisfaction with the study and valued having coaching available for their patients. Conclusions: Medical assistants can be trained to assist patients with lifestyle changes that are associated with improved health and weight control.