Genomic Imprinting Research Articles

Expert consensus on the prenatal diagnosis and genetic counseling for uniparental disomy-related imprinting disorders

Uniparental disomy (UPD)-related imprinting disorders are a group of congenital disorders which can lead to severe birth defects. Their molecular etiology is the occurrence of UPD in the genomic imprinting regions, which may cause disturbed expression of parent-of-origin imprinted genes. With the widespread applications of genetic testing techniques, the prenatal diagnosis of UPD-related imprinted diseases has gradually become clinical routines. However, due to the complicated pathogenesis of such disorders, currently there is still a lack of standards and norms for the understanding, diagnosis, management and genetic counseling. By referring to the relevant guidelines and consensus, the latest progress of research, and opinions from experts in the relevant fields, the writing group has formulated a consensus over the prenatal diagnosis and genetic counseling for UPD-related imprinting disorders, with an aim to provide a more accurate and rational evaluation in prenatal clinics.

Preconceptional paternal caloric restriction of high-fat diet-induced obesity in Wistar rats dysregulates the metabolism of their offspring via AMPK/SIRT1 pathway

BackgroundObesity is a metabolic syndrome where allelic and environmental variations together determine the susceptibility of an individual to the disease. Caloric restriction (CR) is a nutritional dietary strategy recognized to be beneficial as a weight loss regime in obese individuals. Preconceptional parental CR is proven to have detrimental effects on the health and development of their offspring. As yet studies on maternal CR effect on their offspring are well established but paternal CR studies are not progressing. In current study, the impact of different paternal CR regimes in diet-induced obese male Wistar rats (WNIN), on their offspring concerning metabolic syndrome are addressed.MethodsHigh-fat diet-induced obese male Wistar rats were subjected to caloric restriction of 50% (HFCR-I) and 40% (HFCR-II) and then they were mated with normal females. The male parent’s reproductive function was assessed by sperm parameters and their DNMT’s mRNA expression levels were also examined. The offspring’s metabolic function was assessed by physiological, biochemical and molecular parameters.ResultsThe HFCR-I male parents have shown reduced body weights, compromised male fertility and reduced DNA methylation activity. Further, the HFCR-I offspring showed attenuation of the AMPK/SIRT1 pathway, which is associated with the progression of proinflammatory status and oxidative stress. In line, the HFCR-I offspring also developed altered glucose and lipid homeostasis by exhibiting impaired glucose tolerance & insulin sensitivity, dyslipidemia and steatosis. However, these effects were largely mitigated in HFCR-II offspring. Regarding the obesogenic effects, female offspring exhibited greater susceptibility than male offspring, suggesting that females are more prone to the influences of the paternal diet.ConclusionThe findings highlight that HFCR-I resulted in paternal undernutrition, impacting the health of offspring, whereas HFCR-II largely restored the effects of a high-fat diet on their offspring. As a result, moderate caloric restriction has emerged as an effective weight loss strategy with minimal implications on future generations. This underscores the shared responsibility of fathers in contributing to sperm-specific epigenetic imprints that influence the health of adult offspring.

Imprinted DNA methylation of the H19 ICR is established and maintained in vivo in the absence of Kaiso

BackgroundPaternal allele-specific DNA methylation of the imprinting control region (H19 ICR) controls genomic imprinting at the Igf2/H19 locus. We previously demonstrated that the mouse H19 ICR transgene acquires imprinted DNA methylation in preimplantation mouse embryos. This activity is also present in the endogenous H19 ICR and protects it from genome-wide reprogramming after fertilization. We also identified a 118-bp sequence within the H19 ICR that is responsible for post-fertilization imprinted methylation. Two mutations, one in the five RCTG motifs and the other a 36-bp deletion both in the 118-bp segment, caused complete and partial loss, respectively, of methylation following paternal transmission in each transgenic mouse. Interestingly, these mutations overlap with the binding site for the transcription factor Kaiso, which is reportedly involved in maintaining paternal methylation at the human H19 ICR (IC1) in cultured cells. In this study, we investigated if Kaiso regulates imprinted DNA methylation of the H19 ICR in vivo.ResultsNeither Kaiso deletion nor mutation of Kaiso binding sites in the 118-bp region affected DNA methylation of the mouse H19 ICR transgene. The endogenous mouse H19 ICR was methylated in a wild-type manner in Kaiso-null mutant mice. Additionally, the human IC1 transgene acquired imprinted DNA methylation after fertilization in the absence of Kaiso.ConclusionsOur results indicate that Kaiso is not essential for either post-fertilization imprinted DNA methylation of the transgenic H19 ICR in mouse or for methylation imprinting of the endogenous mouse H19 ICR.

Pathogenic sequence variant and microdeletion affecting HMGA2 in Silver–Russell syndrome: case reports and literature review

Silver–Russell syndrome (SRS) is a representative imprinting disorder characterized by pre- and postnatal growth failure. We encountered two Japanese SRS cases with a de novo pathogenic frameshift variant of HMGA2 (NM_003483.6:c.138_141delinsCT, p.(Lys46Asnfs*16)) and a de novo ~ 3.4 Mb microdeletion at 12q14.2–q15 involving HMGA2, respectively. Furthermore, we compared clinical features in previously reported patients with various genetic conditions leading to compromised IGF2 expression, i.e., HMGA2 aberrations, PLAG1 aberrations, IGF2 aberrations, and H19/IGF2:IG-DMR epimutations (hypomethylations). The results provide further support for HMGA2 being involved in the development of SRS and imply some characteristic features in patients with HMGA2 aberrations.

Imprinting disorders in children conceived with assisted reproductive technology in Sweden

ObjectiveTo assess whether the use of assisted reproductive technology for conception is associated with imprinting disorders in children and the impact of parental factors related to infertility. DesignA nationwide register-based cohort study. SubjectsAll liveborn singletons in Sweden (N = 2 084 127) between 1997-2017 with follow-up to December 31, 2018. ExposureThe use of specific methods implemented in the assisted reproductive technology Main Outcome MeasuresThe International Classification of Diseases version 10 was used to identify three distinct imprinting disorder groups: Prader-Willi/Silver-Russell syndrome, Beckwith-Wiedemann syndrome, and central precocious puberty. The Cox model combined with inverse probability treatment weights were used to estimate weighted hazard ratio (wHR) with 95% confidence interval (CI), accounting for multiple confounders. ResultsA total of 1044 children were diagnosed with the disorders of interest, and 52 of them were conceived with assisted reproductive technology. The overall risk of being diagnosed with any of the studied imprinting disorders was elevated in children conceived with ART compared to all other children (HR 1.84, 95% CI: 1.38-2.45). After adjusting for parental background factors, the association was partially attenuated (wHR 1.50, 95% CI: 0.97-2.32), but remained also in the weighted comparison restricted to children of couples with known infertility (wHR 1.52, 95% CI: 1.05-2.21). For the specific diagnoses of Prader-Willi/Silver-Russell syndrome and Beckwith-Wiedemann syndrome, compared to children of couples with known infertility, children conceived with assisted reproductive technology showed a small excess risk, which could not be distinguished from the null (wHR 1.56 [95% CI: 0.93-2.62] and 1.80 [95% CI: 0.99-3.28], respectively). Further subgroup analysis showed that the combined use of intra-cytoplasmic sperm injection and cryopreserved embryos was associated with higher risk of both Prader-Willi/Silver-Russell syndrome (wHR 4.60, 95% CI: 1.72-12.28) and Beckwith-Wiedemann syndrome (wHR 6.69, 95% CI: 2.09-21.45). The number of central precocious puberty cases in children conceived with assisted reproductive technology was too small (N=3) to make any meaningful inference. ConclusionThe combined use of intra-cytoplasmic sperm injection and cryopreserved embryos was associated with small elevated risks of Prader-Willi/Silver-Russell syndrome and Beckwith-Wiedemann syndrome in children, independent of parental factors related to infertlity.

Effects of Developmental Lead and Phthalate Exposures on DNA Methylation in Adult Mouse Blood, Brain, and Liver: A Focus on Genomic Imprinting by Tissue and Sex.

Maternal exposure to environmental chemicals can cause adverse health effects in offspring. Mounting evidence supports that these effects are influenced, at least in part, by epigenetic modifications. It is unknown whether epigenetic changes in surrogate tissues such as the blood are reflective of similar changes in target tissues such as cortex or liver. We examined tissue- and sex-specific changes in DNA methylation (DNAm) associated with human-relevant lead (Pb) and di(2-ethylhexyl) phthalate (DEHP) exposure during perinatal development in cerebral cortex, blood, and liver. Female mice were exposed to human relevant doses of either Pb () via drinking water or DEHP () via chow for 2 weeks prior to mating through offspring weaning. Whole genome bisulfite sequencing (WGBS) was utilized to examine DNAm changes in offspring cortex, blood, and liver at 5 months of age. Metilene and methylSig were used to identify differentially methylated regions (DMRs). Annotatr and ChIP-enrich were used for genomic annotations and gene set enrichment tests of DMRs, respectively. The cortex contained the majority of DMRs associated with Pb (66%) and DEHP (57%) exposure. The cortex also contained the greatest degree of overlap in DMR signatures between sexes ( and 8 DMRs with Pb and DEHP exposure, respectively) and exposure types ( and 39 DMRs in males and females, respectively). In all tissues, detected DMRs were preferentially found at genomic regions associated with gene expression regulation (e.g., CpG islands and shores, 5' UTRs, promoters, and exons). An analysis of GO terms associated with DMR-containing genes identified imprinted genes to be impacted by both Pb and DEHP exposure. Of these, Gnas and Grb10 contained DMRs across tissues, sexes, and exposures, with some signatures replicated between target and surrogate tissues. DMRs were enriched in the imprinting control regions (ICRs) of Gnas and Grb10, and we again observed a replication of DMR signatures between blood and target tissues. Specifically, we observed hypermethylation of the Grb10 ICR in both blood and liver of Pb-exposed male animals. These data provide preliminary evidence that imprinted genes may be viable candidates in the search for epigenetic biomarkers of toxicant exposure in target tissues. Additional research is needed on allele- and developmental stage-specific effects, as well as whether other imprinted genes provide additional examples of this relationship. https://doi.org/10.1289/EHP14074.

Maternal perceived stress and green spaces during pregnancy are associated with adult offspring gene (NR3C1 and IGF2/H19) methylation patterns in adulthood: A pilot study

BackgroundChanges in NR3C1 and IGF2/H19 methylation patterns have been associated with behavioural and psychiatric outcomes. Maternal mental state has been associated with offspring NR3C1 promotor and IGF2/H19 imprinting control region (ICR) methylation patterns. However, there is a lack of prospective studies with long-term follow-up. Methods52 mother-offspring pairs were studied from 12 to 22 weeks of pregnancy and offspring was followed-up until 28–29 years-of-age. During pregnancy, mothers filled in a Life Event Scale and a Daily Hassles Scale measuring perceived stress; i.e., appraisal or subjectively experienced severity of impact of important life events and of daily hassles in several life domains during pregnancy, respectively. Green space was quantified around the residence, using high-resolution (1 m2) map data. Saliva and blood samples were obtained from the adult offspring. Absolute DNA methylation levels were determined in blood and saliva on four NR3C1 amplicons, and one IGF2/H19 ICR amplicon using a bisulfite PCR and sequencing method. Linear mixed effect models were used to test the associations between perceived stress and green spaces during pregnancy, and adult offspring methylation patterns. ResultsWe found associations between maternal perceived stress during pregnancy and methylation patterns on two out of the four NR3C1 amplicons, measured in blood, from offspring in adulthood, but not with IGF2/H19 methylation. For an interquartile-range (IQR) increase in maternal perceived life event or daily hassles stress scores, absolute methylation levels on several NR3C1 CpG sites were significantly changed (-1.62 % to +5.89 %, p<0.05). Maternal perceived stress scores were not associated with IGF2/H19 methylation, neither in blood nor in saliva. Maternal exposure to green spaces surrounding the residence during the pregnancy was associated with IGF2/H19 ICR methylation (-0.80 % to −1.04 %, p<0.05) in saliva, but not with NR3C1 promotor methylation. ConclusionWe observed significant long-term effects of maternal perceived stress during pregnancy on the methylation patterns of the NR3C1 promotor in offspring well into adulthood. This may imply that maternal psychological distress during pregnancy may influence the regulation of the HPA-axis well into adulthood. Additionally, maternal proximity to green spaces was associated with IGF2/H19 ICR methylation patterns, which is a novel finding.

Fetal growth delay caused by loss of non-canonical imprinting is resolved late in pregnancy and culminates in offspring overgrowth.

Germline epigenetic programming, including genomic imprinting, substantially influences offspring development. Polycomb Repressive Complex 2 (PRC2) plays an important role in Histone 3 Lysine 27 trimethylation (H3K27me3)-dependent imprinting, loss of which leads to growth and developmental changes in mouse offspring. In this study, we show that offspring from mouse oocytes lacking the PRC2 protein Embryonic Ectoderm Development (EED) were initially developmentally delayed, characterised by low blastocyst cell counts and substantial growth delay in mid-gestation embryos. This initial developmental delay was resolved as offspring underwent accelerated fetal development and growth in late gestation resulting in offspring that were similar stage and weight to controls at birth. The accelerated development and growth in offspring from Eed-null oocytes was associated with remodelling of the placenta, which involved an increase in fetal and maternal tissue size, conspicuous expansion of the glycogen-enriched cell population, and delayed parturition. Despite placental remodelling and accelerated offspring fetal growth and development, placental efficiency, and fetal blood glucose levels were low, and the fetal blood metabolome was unchanged. Moreover, while expression of the H3K27me3-imprinted gene and amino acid transporter Slc38a4 was increased, fetal blood levels of individual amino acids were similar to controls, indicating that placental amino acid transport was not enhanced. Genome-wide analyses identified extensive transcriptional dysregulation and DNA methylation changes in affected placentas, including a range of imprinted and non-imprinted genes. Together, while deletion of Eed in growing oocytes resulted in fetal growth and developmental delay and placental hyperplasia, our data indicate a remarkable capacity for offspring fetal growth to be normalised despite inefficient placental function and the loss of H3K27me3-dependent genomic imprinting.

Antitumor progenitor exhausted CD8+ T cells are sustained by TCR engagement.

The durability of an antitumor immune response is mediated in part by the persistence of progenitor exhausted CD8+ T cells (Tpex). Tpex serve as a resource for replenishing effector T cells and preserve their quantity through self-renewal. However, it is unknown how T cell receptor (TCR) engagement affects the self-renewal capacity of Tpex in settings of continued antigen exposure. Here we use a Lewis lung carcinoma model that elicits either optimal or attenuated TCR signaling in CD8+ T cells to show that formation of Tpex in tumor-draining lymph nodes and their intratumoral persistence is dependent on optimal TCR engagement. Notably, attenuated TCR stimulation accelerates the terminal differentiation of optimally primed Tpex. This TCR-reinforced Tpex development and self-renewal is coupled to proximal positioning to dendritic cells and epigenetic imprinting involving increased chromatin accessibility at Egr2 and Tcf1 target loci. Collectively, this study highlights the critical function of TCR engagement in sustaining Tpex during tumor progression.

Maturing hiPSC-cardiomyocytes in cardiac microtissues for accurate preclinical in vitro modelling of long-QT syndrome type 1

Abstract Funding Acknowledgements Type of funding sources: Foundation. Main funding source(s): reNEW - Novo Nordisk Foundation Center for Stem Cell Medicine Background Long-QT syndrome type 1 (LQT1), a cardiac arrhythmia often leading to sudden cardiac death, is due to mutations in KCNQ1 gene. Human iPSC-derived cardiomyocytes (hiPSC-CMs) have been widely used to model LQT1, promoting the concept of their use in preclinical studies. However, KCNQ1 locus is subjected to developmentally regulated genomic imprinting in the heart: in fetal cardiomyocytes the paternal allele is repressed, while both alleles become expressed after birth. Since hiPSC-CMs are typically immature fetal-like cells, residual imprinting may affect evaluation of KCNQ1 mutations in this system. Purpose This study wants to address the imprinting status of KCNQ1 in hiPSC-CMs and propose maturing hiPSC-CMs as a solution for reliable LQT1 modelling. Methods We derived hiPSC-CMs from a LQT1 patient carrying a heterozygous KCNQ1 mutation on the paternal allele and compared their electrical properties with the isogenic corrected hiPSC-CMs by patch clamp. We analysed KCNQ1 expression by ddPCR and DNA methylation. We used three-dimensional cardiac microtissues (MTs) composed by hiPSC-CMs, hiPSC-derived cardiac fibroblasts and endothelial cells to induce cardiomyocte maturation. Results LQT1 and isogenic corrected hiPSC-CMs showed no difference in action potential duration (APD). We hypothesized that residual KCNQ1 imprinting in hiPSC-CMs due to their immaturity was masking the phenotype. When we analyzed different hiPSC-CM lines, we found in all an unbalanced KCNQ1 allelic expression, with paternal allele accounting for 5-15% of the total. We then investigated whether maturing hiPSC-CMs could remove KCNQ1 imprinting. We included hiPSC-CMs in MTs, as this system previously showed to promote functional maturation and upregulation of KCNQ1. Here, we observed a substantial increased expression of the paternal allele and a reduction in the expression of KCNQ1OT1, the long non-coding RNA inducing paternal allele repression. LQT1 hiPSC-CMs dissociated from MTs showed prolonged APD compared to the corrected line. We also demonstrated that this was due to the reduction of the ionic current mediated by KCNQ1 (IKs) in LQT1 hiPSC-CMs compared to the corrected controls, thus revealing the mutation causative effect. Conclusions Our findings show that residual KCNQ1 imprinting in immature hiPSC-CMs can lead to underestimate (or overestimate) functional effects of pathological variants based on the parental allele that carries the mutation. This is overcome by maturation of hiPSC-CMs in tri-cellular cardiac microtissue which promotes KCNQ1 bi-allelic expression and allow dissecting mutation mechanisms. This study brings attention to the epigenetic regulation of hiPSC models and demonstrates the utility of using matured hiPSC-CMs as in vitro preclinical model for cardiac arrhythmias.

Differential 3D genome architecture and imprinted gene expression: cause or consequence?

Imprinted genes provide an attractive paradigm to unravel links between transcription and genome architecture. The parental allele-specific expression of these essential genes - which are clustered in chromosomal domains - is mediated by parental methylation imprints at key regulatory DNA sequences. Recent chromatin conformation capture (3C)-based studies show differential organization of topologically associating domains between the parental chromosomes at imprinted domains, in embryonic stem and differentiated cells. At several imprinted domains, differentially methylated regions show allelic binding of the insulator protein CTCF, and linked focal retention of cohesin, at the non-methylated allele only. This generates differential patterns of chromatin looping between the parental chromosomes, already in the early embryo, and thereby facilitates the allelic gene expression. Recent research evokes also the opposite scenario, in which allelic transcription contributes to the differential genome organization, similarly as reported for imprinted X chromosome inactivation. This may occur through epigenetic effects on CTCF binding, through structural effects of RNA Polymerase II, or through imprinted long non-coding RNAs that have chromatin repressive functions. The emerging picture is that epigenetically-controlled differential genome architecture precedes and facilitates imprinted gene expression during development, and that at some domains, conversely, the mono-allelic gene expression also influences genome architecture.

Large regions of homozygosity in prenatal diagnosis.

Chromosomal microarrays (CMA) incorporate single nucleotide polymorphisms to enable the detection of regions of homozygosity (ROH). Here, we retrospectively analyzed 6288 prenatal cases who performed CMA to explored the clinical implications of large ROH in prenatal diagnosis. We analyzed cases with ROH larger than 10 megabases and reviewed the ultrasound findings; karyotype results and pregnancy follow-up data. Cases with possible imprinting disorders were assessed by methylation-specific multiplex ligation-dependent probe amplification. In total, we identified 50 cases with large ROH and chromosomes 1 and 2 were the most affected. About 59.18% of the ROH cases had ultrasound abnormalities, with the most common findings being ultrasound soft-marker abnormalities. There were seven fetuses had ROH which covered almost the entire chromosome and four had terminal ROH that involved almost the entire long arm of the chromosomes, which indicated uniparental disomy (UPD), of which 70% showed abnormal ultrasound findings. Ten cases with multiple ROH on different chromosomes indicated the third to fifth degree of consanguinity. In this study, we highlighted the clinical relevance of large ROH related to UPD. The analysis of ROH allowed us to gain further understanding of complex cytogenetic and disease mechanisms in prenatal diagnosis.

The role of imprinting genes' loss of imprints in cancers and their clinical implications.

Genomic imprinting plays an important role in the growth and development of mammals. When the original imprint status of these genes is lost, known as loss of imprinting (LOI), it may affect growth, neurocognitive development, metabolism, and even tumor susceptibility. The LOI of imprint genes has gradually been found not only as an early event in tumorigenesis, but also to be involved in progression. More than 120 imprinted genes had been identified in humans. In this review, we summarized the most studied LOI of two gene clusters and 13 single genes in cancers. We focused on the roles they played, that is, as growth suppressors and anti-apoptosis agents, sustaining proliferative signaling or inducing angiogenesis; the molecular pathways they regulated; and especially their clinical significance. It is notable that 12 combined forms of multi-genes' LOI, 3 of which have already been used as diagnostic models, achieved good sensitivity, specificity, and accuracy. In addition, the methods used for LOI detection in existing research are classified into detection of biallelic expression (BAE), differentially methylated regions (DMRs), methylation, and single-nucleotide polymorphisms (SNPs). These all indicated that the detection of imprinting genes' LOI has potential clinical significance in cancer diagnosis, treatment, and prognosis.

The phenomenon of genomic imprinting was discovered 40 years ago.

The phenomenon of genomic imprinting was discovered 40 years ago.

CNS involvement in myotonic dystrophy type 1: does sex play a role?

Myotonic dystrophy type 1 (DM1) is a hereditary neuromuscular disorder affecting the central nervous system (CNS). Although sex differences have been explored in other neuromuscular disorders, research on this topic in DM1 remains limited. The present study aims to analyze sex differences (both the patient's and disease-transmitting parent's sex) with a focus on CNS outcomes. Retrospective data from 146 non-congenital DM1 patients were analyzed, including clinical, molecular, neuropsychological, and neuroradiological data. Sex and inheritance pattern differences were analyzed using t-tests, and ANOVA analyses were conducted to address the interactions. Overall, no significant sex differences were observed except in certain cognitive domains. However, individuals with maternal inheritance showed larger CTG expansion size, lower estimated IQs, and poorer performance on visual memory, executive functions, and language domains than those with paternal inheritance. Notably, IQ performance was independently influenced by inheritance pattern and CTG expansion. This study is the first to delve into sex differences in DM1 with a focus on CNS outcomes. While the results revealed the absence of a sex-specific clinic-molecular profile, more substantial CNS differences were observed between patients with maternal and paternal inheritance patterns. The hypothetical existence of genomic imprinting and its potential mechanism are discussed. These findings hold potential implications for aiding clinical management by improving genetic counseling and predicting disease severity and prognosis.

Novel 14q32.2 paternal deletion encompassing the whole DLK1 gene associated with Temple syndrome

BackgroundTemple syndrome (TS14) is a rare imprinting disorder caused by maternal UPD14, imprinting defects or paternal microdeletions which lead to an increase in the maternal expressed genes and a silencing the paternally expressed genes in the 14q32 imprinted domain. Classical TS14 phenotypic features include pre- and postnatal short stature, small hands and feet, muscular hypotonia, motor delay, feeding difficulties, weight gain, premature puberty along and precocious puberty.MethodsAn exon array comparative genomic hybridization was performed on a patient affected by psychomotor and language delay, muscular hypotonia, relative macrocephaly, and small hand and feet at two years old. At 6 years of age, the proband presented with precocious thelarche. Genes dosage and methylation within the 14q32 region were analyzed by MS-MLPA. Bisulfite PCR and pyrosequencing were employed to quantification methylation at the four known imprinted differentially methylated regions (DMR) within the 14q32 domain: DLK1 DMR, IG-DMR, MEG3 DMR and MEG8 DMR.ResultsThe patient had inherited a 69 Kb deletion, encompassing the entire DLK1 gene, on the paternal allele. Relative hypermethylation of the two maternally methylated intervals, DLK1 and MEG8 DMRs, was observed along with normal methylation level at IG-DMR and MEG3 DMR, resulting in a phenotype consistent with TS14. Additional family members with the deletion showed modest methylation changes at both the DLK1 and MEG8 DMRs consistent with parental transmission.ConclusionWe describe a girl with clinical presentation suggestive of Temple syndrome resulting from a small paternal 14q32 deletion that led to DLK1 whole-gene deletion, as well as hypermethylation of the maternally methylated DLK1-DMR.

Cobalamins Function as Allosteric Activators of an Angelman Syndrome-Associated UBE3A/E6AP Variant.

Genetic aberrations of the maternal UBE3A allele, which encodes the E3 ubiquitin ligase E6AP, are the cause of Angelman syndrome (AS), an imprinting disorder. In most cases, the maternal UBE3A allele is not expressed. Yet, approximately 10 percent of AS individuals harbor distinct point mutations in the maternal allele resulting in the expression of full-length E6AP variants that frequently display compromised ligase activity. In a high-throughput screen, we identified cyanocobalamin, a vitamin B12-derivative, and several alloxazine derivatives as activators of the AS-linked E6AP-F583S variant. Furthermore, we show by cross-linking coupled to mass spectrometry that cobalamins affect the structural dynamics of E6AP-F583S and apply limited proteolysis coupled to mass spectrometry to obtain information about the regions of E6AP that are involved in, or are affected by binding cobalamins and alloxazine derivatives. Our data suggest that dietary supplementation with vitamin B12 can be beneficial for AS individuals.

HDAC4 Genomic Methylation and Subsequent DNA Binding Following Joint Injury Confers a Sustained Epigenetic Memory of Disrupted Muscle Plasticity

Joint injury is associated with protracted atrophy and weakness that poorly recovers; our objective was to define molecular effectors of lost muscle size, strength, and plasticity following joint injury. We hypothesized that epigenetic imprinting of joint injury in muscle impairs muscle plasticity and contributes to poor functional recovery following surgical repair. We recruited a total of sixteen ACL-injured participants from whom we obtained vastus lateralis muscle biopsies Pre-, 7-days post-, and 4-months post- reconstruction surgery (Sx) and strength measures up to 6-months post-Sx. We performed immunohistochemistry, RNA-seq, reduced representation bisulfite sequencing (RRBS), and HDAC4 chromatin immunoprecipitation sequencing (HDAC4 ChIP-seq) to determine the impact of epigenetics and gene expression on histological profiles, muscle size and muscle strength following injury. Binding and Expression Target Analysis (BETA) of DNA methylation revealed a conserved 985bp regulatory genomic region that was differentially methylated upstream of the HDAC4 promoter. We observed 108 (Pre-Sx), 111 (7d post-Sx), and 110 (4-months post-Sx) differentially methylated promoter sites of which 95 sites were common across all timepoints in the injured limb. Additionally, we observed 1293 differentially methylated intragenic sites within the HDAC4 locus that were also common across all timepoints following injury. Altered HDAC4 methylation was associated with a 2.2 and 4.3 fold change in HDAC4 transcript abundance Pre-Sx and 7d post-Sx, respectively (FDR &lt; 0.05). Notably, HDAC4 binding to critical contractile genes coincided with their downregulation 7d post-Sx (Log2 fold change: ACTA1: -2.02; CKM: -0.97; ATP2A1: -2.34; HOMER2: -1.09; all FDR&lt;0.05). Altered transcript abundance of these genes coincided with lower mean muscle fiber cross-sectional area in the injured limb compared to Healthy (Healthy: 4845 ± 213.4μm2; pre-Sx: 4313 ± 190.6μm2, p&lt;0.05; 7d post-Sx: 3685 ± 228.1μm2, p&lt;0.05; 4-months post-Sx: 3422 ± 142.8μm2, p&lt;0.05) and lower peak torque compared to Healthy (Healthy: 174.8 ± 10.72nm; pre-Sx: 135.2 ± 10.19nm, p&lt;0.05; 4-months post-Sx: 82.64 ± 6.80nm, p&lt;0.05; 6-months post-Sx: 105.9 ± 8.26nm, p&lt;0.05) that was sustained throughout follow-up. Our results suggest that joint injury confers HDAC4 promoter-proximal and intragenic epigenetic imprinting by DNA methylation that increases HDAC4 transcript abundance and HDAC4-DNA binding to represses expression of genes regulating muscle contractile function. This HDAC4 gene regulatory program likely contributes to prolonged deficits in muscle size and strength following joint injury. This work was supported by NIH-NIAMS R01 AR072061. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Modulating the epigenetic interplay in exhausted CD8 T cells for effective immunotherapy

Abstract Epigenetic imprinting of terminal exhaustion in chronically stimulated CD8 T cells is a major impediment to T cell immunotherapies during chronic viral infections or cancer. Foundational work establishes that exhausted CD8 T cells (TEX) acquire heritable DNA methylation programs that silence genes critical for T cell stemness/function, restraining responses to immune checkpoint blockade (ICB). However, the molecular interplay between histone post-translational modifications and de novo DNA methylation during the progression to terminal exhaustion remains poorly understood. To address this, we employ our novel in vitro model of human T cell dysfunction and the chronic LCMV infection model to reveal a temporal linkage in which progressive histone changes prime the acquisition of de novo DNA methylation in TEX. Via a selective inhibitor or CRISPR editing in human CD8 T cells, we identify specific histone-modifying enzymes as key epigenetic drivers that orchestrate DNA methylation programming during terminal exhaustion. Notably, targeting the histone-modifying enzymes reinvigorated effector function and cytotoxicity in dysfunctional human T cells. Moreover, combined epigenetic therapy and anti-PD-L1 enhanced the ICB responses of both progenitor and cytolytic TEX during chronic viral infection. Collectively, our findings reveal novel epigenetic drivers of CD8 T cell dysfunction, supporting therapeutic potential of targeting the epigenetic crosstalk underpinning T cell exhaustion.

Selection signatures and landscape genomics analysis to reveal climate adaptation of goat breeds

Goats have achieved global prominence as essential livestock since their initial domestication, primarily owing to their remarkable adaptability to diverse environmental and production systems. Differential selection pressures influenced by climate have led to variations in their physical attributes, leaving genetic imprints within the genomes of goat breeds raised in diverse agroecological settings. In light of this, our study pursued a comprehensive analysis, merging environmental data with single nucleotide polymorphism (SNP) variations, to unearth indications of selection shaped by climate-mediated forces in goats. Through the examination of 43,300 SNPs from 51 indigenous goat breeds adapting to different climatic conditions using four analytical methods: latent factor mixed models (LFMM), F-statistics (Fst), Extended haplotype homozygosity across populations (XPEHH), and spatial analysis method (SAM), A total of 74 genes were revealed to display clear signs of selection, which are believed to be influenced by climatic conditions. Among these genes, 32 were consistently identified by at least two of the applied methods, and three genes (DENND1A, PLCB1, and ITPR2) were confirmed by all four approaches. Moreover, our investigation yielded 148 Gene Ontology (GO) terms based on these 74 genes, underlining pivotal biological pathways crucial for environmental adaptation. These pathways encompass functions like vascular smooth muscle contraction, cellular response to heat, GTPase regulator activity, rhythmic processes, and responses to temperature stimuli. Of significance, GO terms about endocrine regulation and energy metabolic responses, key for local adaptation were also uncovered, including biological processes, such as cell differentiation, regulation of peptide hormone secretion, and lipid metabolism. These findings contribute to our knowledge of the genetic structure of climate-triggered adaptation across the goat genome and have practical implications for marker-assisted breeding in goats.