Epigenetics & Chromatin

BackgroundLamins are major regulators of the spatial and functional organization of chromatin. Lamins at the nuclear periphery form the lamina that anchors heterochromatin to the nuclear envelope. A subpool of A-type lamins localizes in the nuclear interior, where they also bind to euchromatic genomic regions. A-type lamin properties and chromatin association are regulated by lamin-associated polypeptide 2alpha (LAP2). Here we systematically analyze, how LAP2 depletion affects chromatin organization, accessibility and gene expression on a genome-wide level. ResultsLAP2 depletion in mouse dermal fibroblasts positively and negatively affects chromatin accessibility and gene expression throughout the genome, which correlates with changes in chromatin association of A-type lamins and the nucleosomal remodeler proteins BRG1 and CHD4. In particular, A-type lamins bind to open chromatin regions close to BRG1 and CHD4 binding sites and deregulated genes, but do not directly accumulate on genes and BRG1 and CHD4-enriched sites. Unsupervised clustering of the datasets on LAP2-bound genomic regions confirms spreading of A-type lamins to active chromatin regions containing deregulated genes and an enrichment of chromatin remodelers on a subset of these genomic regions. ConclusionsLAP2 depletion in fibroblasts leads to a gross rearrangement of chromatin. Genome-wide chromatin reorganization is linked to spreading of A-type lamins to active chromatin regions and accompanied by a restriction of chromatin remodelers to a subset of active genomic regions. These changes correlate with changes in chromatin accessibility and gene expression throughout the genome, particularly in regions where lamin binding is gained in LAP2 knockout versus wildtype cells.

DNMT3A is a de novo DNA methyltransferase whose recruitment to chromatin regulates its function. Missense mutations within the chromatin-binding PWWP domain are associated with diverse human disorders, yet how mutations in the same domain produce distinct phenotypes remains unclear. Here we systematically characterise 19 clinically reported mutations in the PWWP domain of DNMT3A that are associated with Heyn-Sproul-Jackson syndrome (HESJAS), paraganglioma (PG) and clonal haematopoiesis (CH). We show that all PWWP-domain mutations associated with HESJAS abolished interaction with H3K36me2 modified nucleosomes, defining this as a consistent biochemical feature of HESJAS. In contrast, mutations from all disease classes differentially altered DNA binding of the PWWP domain, driven by alterations in the net charge of the domain. However, these effects are largely overcome by inclusion of the DNNMT3A1 N-terminal region, which is absent from its embryonic isoform, suggesting that PWWP mutations may differentially affect DNMT3A function through development. Changes in the thermal stability of the isolated PWWP domain mutants did not directly translate into altered stability of full-length DNMT3A1 in cells. We show that HESJAS mutations can affect the intramolecular interaction between the PWWP and adjacent ADD domain, an interaction proposed to contribute to the autoinhibitory function of the ADD domain. However, not all mutations behaved in the same way, suggesting that multiple factors govern the intramolecular autoinhibition of DNMT3A. Together, this study advances our understanding of the molecular mechanisms by which DNMT3A PWWP-domain mutations are mechanistically heterogeneous, providing a biochemical framework that contributes to distinct disease phenotypes.

The first step in initiating DNA replication is binding of the origin recognition complex (ORC) to chromosomes. Metazoan ORC is recruited to chromatin via the Orc1 intrinsically disordered region (IDR) whose DNA and chromatin binding activity are regulated by Cyclin Dependent Kinase (CDK) phosphorylation. ORC is also enriched in heterochromatin where it is required for the formation and maintenance of a silenced chromatin state. ORCs recruitment to heterochromatin is developmentally and cell cycle regulated but the underlying regulatory mechanism remains unknown. We hypothesized that CDK-dependent phosphorylation of the Orc1 IDR underpins regulated recruitment to heterochromatin. Using bioinformatic analyses, we find that the Drosophila Orc1 IDR (Orc1IDR) contains an exceptionally high density of CDK phospho-sites and, despite considerable sequence variation, the density of sites, but not their position, is conserved. In vitro DNA binding and phase separation experiments reveal that phosphorylation tunes Orc1IDR function in a rheostat-like fashion. Using phospho-mimetic variants, we find that constitutive phosphorylation not only weakens interphase chromatin binding but fully inhibits partitioning of Orc1IDR into heterochromatin. Finally, we use phospho-mimetic variants to probe the importance of site-specific phosphorylation and find that the precise position of sites can be changed provided the new sites are equitably distributed across the sequence. These studies demonstrate that phosphorylation tunes the biochemical properties of the Orc1 IDR to control DNA binding, phase separation, and, consequentially, heterochromatin recruitment. This work suggests that localized dephosphorylation of the DNA binding Orc1 IDR may underlie recruitment of ORC to specific genomic loci.

Developmental genes are frequently regulated by multiple enhancers distributed across large cis-regulatory regions. How these enhancers communicate with their target promoter and how their interactions are shaped by distinct developmental transcriptional environments remain incompletely understood. Here, we investigate the chromatin organization of the Drosophila shavenbaby locus, a developmental gene controlled by seven distal enhancers. Tissue-specific UMI-4C revealed extensive enhancer-promoter and enhancer-enhancer interactions, including in cell populations where individual enhancers are inactive. Quantitative three-dimensional DNA-FISH revealed compact enhancer-promoter hubs enriched in shavenbaby-expressing cells, yet also present in non-expressing cells and prior to expression. Perturbation of shavenbaby regulators, transcription factors, and architectural proteins revealed that multiple factors contribute to hub organization. Their relative contributions differed between epidermal populations, indicating that similar hubs can be supported by different combinations of regulators. Perturbations that reduced hub organization were frequently associated with reduced shavenbaby-dependent trichome formation. Together, our results identify a robust, multi-factorial enhancer-promoter hub that is shaped by distinct regulatory inputs across developmental contexts.

Cohesin is a large multisubunit protein complex that plays essential roles in cell proliferation, genome organisation, and gene regulation in metazoans. Germline mutations in cohesin subunits or regulators cause a group of human developmental disorders collectively known as cohesinopathies. Increasing evidence indicates that individual cohesin subunits can confer distinct molecular functions to the complex; for example, STAG1 and STAG2 have both overlapping and non-overlapping roles in genome organisation. The zebrafish tailbud provides an excellent developmental model for investigating the coordination of cell proliferation and differentiation, processes in which cohesin has crucial functions. We previously demonstrated that loss of Stag2 disrupts Wnt signalling and mesoderm patterning in the zebrafish tailbud. Here, we show that, unlike mammals, zebrafish can tolerate complete loss of Stag1 from embryogenesis through to adulthood. In contrast to Stag2 deficiency, loss of Stag1 impairs cell cycle progression, activates p53 signalling, and induces a metabolic shift towards catabolism. BMP signalling is reduced in Stag1-deficient embryos and is accompanied by expansion of BMP antagonist chordin expression. Stag1 loss also alters chromatin accessibility at the chordin locus and affects accessibility at chromatin domain boundaries. We propose that modulation of growth and signalling pathways compensates for the absence of Stag1, allowing embryonic development to proceed correctly. Together, these findings reveal distinct contributions of Stag1 and Stag2 to cell-cycle regulation, chromatin architecture, and developmental signalling during vertebrate embryogenesis.

In eukaryotes, DNA replication requires the origin recognition complex (ORC), a six-subunit assembly that promotes replisome formation on chromosomal origins. Orc6 is the smallest and least evolutionarily conserved among all ORC subunits. In Drosophila, Orc6 binds tightly with the core ORC(1-5) and is required for DNA binding and replication initiation, whereas in Xenopus and human systems Orc6 loosely associates with the rest of the complex resulting in some differences for replication-associated activities. Despite these variations, Orc6 remains essential for viability in all species. In current study we analyzed specific residues within the C-terminal 11 helix that is critical for stable association of Orc6 with the ORC complex in Drosophila. Human Orc6 lacks these residues, however it possesses a strong nuclear localization signal (NLS) that is absent in Drosophilidae. We propose that this NLS drives human protein to the nucleus and compensates for weaker Orc6-ORC(1-5) interactions by increasing the nuclear concentration of Orc6 and shifting the equilibrium toward formation of the fully assembled ORC complex at the DNA.

Mammalian preimplantation development requires precisely coordinated lineage decisions to establish the trophectoderm (TE), inner cell mass (ICM), epiblast (EPI), and primitive endoderm (PrE). Glucose metabolism and epigenetic regulation are increasingly recognised as key determinants of lineage specification during preimplantation development. However, how glucose-dependent metabolic cues interface with epigenetic mechanisms to regulate embryonic cell fate remains poorly understood. Here, we investigated the role of glucose in regulating protein methylation by protein arginine methyltransferases (PRMT) in bovine preimplantation development. PRMT1 and its associated histone mark H4R3me2a were detected throughout bovine oocyte maturation and embryo development. Pharmacological inhibition of Type I PRMTs using two structurally distinct inhibitors, GSK3368715 and MS023, markedly reduced global protein asymmetric dimethylarginine (ADMA) and H4R3me2a levels. PRMT inhibition impaired blastocyst cell proliferation, reduced total cell number, and disrupted both first and second lineage decisions, as demonstrated by decreased CDX2- and SOX2-positive TE and ICM cells and reduced NANOG- and GATA6-positive EPI and PrE cell allocation. Mechanistically, Type I PRMT inhibition downregulated key components of the Hippo-associated TE programme, including YAP, TEAD4, and TFAP2C. Consistent effects were observed in mouse embryos, where MS023 treatment reduced ADMA, CDX2, YAP, and TFAP2C expression and impaired TE and ICM allocation. Collectively, our findings identify Type I PRMT-mediated ADMA as an essential epigenetic regulator of early mammalian lineage specification and support a conserved ADMA-Hippo regulatory axis linking arginine methylation to embryonic cell fate decisions. In briefType I protein arginine methyltransferase (PRMT)-mediated asymmetric dimethylarginine (ADMA) is required for proper lineage specification during mammalian preimplantation development. ADMA depletion disrupts Hippo signalling, cell proliferation, and trophectoderm and inner cell mass allocation in bovine and mouse embryos.

Chromatin accessibility has long been used as a marker for regions of DNA with regulatory potential. Fiber-seq detects chromatin accessibility on individual DNA fibers, enabling analyses beyond the identification of the accessible chromatin regions (ACRs). By providing single molecule level high resolution, Fiber-seq provides unprecedented qualitative descriptions, including potential categorizations of ACRs, identification of internal transcription factor footprints and nucleosome positioning within individual DNA fibers. As with all tools, the power of this technique depends on careful experimental design and data analysis -- incorrect usage will result in incorrect conclusions. Here we offer guidelines and flag potential pitfalls when generating and analyzing Fiber-seq data, such as (1) the optimum levels of adenosine methylation per-fiber, (2) the power of per-fiber state inference, (3) the importance of controlling for read depth and methylation rates when comparing across samples, (4) the limitations of long-read sequence mapping, and (5) suggestions for identification of differentially accessible peaks across samples.

IntroductionFetal development represents a critical window during which genetic and environmental influences shape lifelong health. DNA methylation (DNAm) is a candidate underlying mechanism. While individual prenatal exposures have been related to DNAm, no studies have investigated the broader prenatal exposome, nor incorporated genetics with the exposome. Here, we integrated the prenatal exposome and genetics as predictors of DNAm at birth. MethodsWe used data from the Dutch Generation R (n=2282) and English Avon Longitudinal Study of Parents and Children (ALSPAC; n=809) cohorts. We performed epigenome-wide elastic net regression, using Generation R for model development/internal validation and ALSPAC for external validation, to predict DNAm at each CpG site. We used three models: Model 1 included 42 prenatal exposures, Model 2 additionally included child sex, gestational age and birth weight, and Model 3 further included meQTLs. ResultsIn Model 1, the prenatal exposome explained on average 0.7% of DNAm variation across 347 validated CpGs (0.1% of tested CpGs). This increased to 40,044 CpGs (10.2%) with 1.3% of variation explained in Model 2, and 91,305 CpGs (23.2%) with 3.0% of variation explained in Model 3. In Model 1, prenatal smoking was the largest predictor, followed by delivery characteristics, among which meconium-stained amniotic fluid was a novel finding. In Model 3, typically both SNPs and multiple prenatal exposures were selected. DiscussionWe find that genomic associations with cord blood DNAm are stronger and more widespread than prenatal exposures, although typically, the prenatal exposome explains additional variation in DNAm beyond genetic influences.

Adversity in early life has lasting effects on the physiology and behavior of exposed individuals and their descendants. In mice, early-life stress alters the RNA content of adult sperm, and this RNA is sufficient to transmit some of the effects to the offspring who were never exposed. However, sperm cells are not yet formed during the early postnatal window in which the exposure occurs. Spermatogonial cells (SPGs), which give rise to them, are present at that time, but whether they respond to the exposure and maintain a molecular signature of it into adulthood is unknown. Here we show that early-life stress alters both the transcriptome and the chromatin accessibility of mouse SPGs, and that a molecular signature of the exposure remains detectable in adulthood. One day after exposure ended, the transcriptional response was extensive, with proliferation and nucleosome-organization programs coordinately up-regulated. In adulthood, the transcriptional response was modest and dominated by coordinately down-regulated gene programs. Single-cell profiling of the whole testis localized the adult response to spermatogonial stem cells (SSCs) and to genes involved in spermatogenesis. At the chromatin level, accessibility shifted one day after exposure at binding motifs for signal-responsive transcription factor families, and in adulthood at a different set of families, in both cases at primed enhancers. These data demonstrate that SPGs respond to an early postnatal environmental exposure and identify them as a candidate origin of the molecular changes later found in adult sperm.

Nuclear pore complexes (NPCs) are nuclear envelope (NE)-embedded protein assemblies that mediate nucleocytoplasmic exchange and interact with the genome, including binding of an NPC component Nup93 to Polycomb chromatin domains. Here, we investigated the in vivo relevance of this relationship in Drosophila, which unusually contains two distinct paralogs of Nup93. Interestingly, we identified a Nup93-2-specific tumorigenic phenotype in larval wings, where depletion of Nup93-2, but not Nup93-1, led to tumor-like overgrowth, reminiscent of Polycomb mutations. Consistently, our transcriptomic analysis revealed a wide-spread loss of gene silencing in Nup93-2-depleted wings, particularly in a Nup93-bound Polycomb domain spanning genes for activators of JAK/STAT signaling. Nup93 paralogs were not found to differ in their effect on NPC biogenesis but strikingly, showed differences in subnuclear localization patterns. While Nup93-1 co-localized exclusively with fully assembled NPCs, Nup93-2 exhibited only partial co-localization and was found at additional NE locations in a tissue-specific manner. Together, our results identify an in vivo silencing role of a Nup93 paralog and suggest that Nup93-2 may form a unique NE-associated complex that targets a subset of Polycomb domains containing growth-promoting genes.

Both global and local chromatin structure influence the heterogeneous distribution of somatic mutations across the cancer genome. For instance, at the local scale, DNA regions bound by transcription factors (TF) and other chromatin-associated proteins display elevated somatic mutation rates, due to variable DNA damage and repair at these protein-bound sites. However, the contribution of TF co-binding towards the variations in somatic mutation rates remains largely unexplored. Here, we combine somatic mutations from whole-genome sequencing of liver cancers with ChIP-seq profiles for over 150 TFs and chromatin-associated proteins in the human liver cancer cell line HepG2, to systematically examine how TF co-occupancy shapes local mutational landscapes. We show that somatic mutation rates at binding sites vary substantially across distinct TFs and co-binding combinations. Furthermore, the magnitude and spatial distribution of somatic mutation rates at TF binding sites differ across promoters and enhancers, likely influenced by the local chromatin accessibility and architecture. Finally, we identify NFIA (Nuclear Factor IA) as a distinct exception, maintaining elevated mutation rates across its binding sites independent of local co-binding context. Together, these findings reveal that combinatorial TF co-occupancy and local chromatin architecture are associated with differences in somatic mutation rates across regulatory regions in liver cancer.

DNA methylation undergoes predictable changes with age, and preimplantation embryos are known to undergo global epigenetic reprogramming. However, the specific fate of age associated methylation signatures during early development has not been systematically quantified. Using published human sperm age associated differentially methylated regions (DMRs) as a feature space, we integrated single cell methylome and transcriptome data to develop the Transgenerational Reset Operator (TRO), a computational framework for profiling preimplantation stages. We found that the morula stage represents the nadir of age associated methylation entropy while retaining high developmental potency, distinguishing it from a simple demethylation endpoint. Dynamical modelling revealed that independent DMR drift fails to recapitulate the morula state, requiring a coordinated, structured correction concentrated in specific DMR subsets and modules with marked directional sensitivity. Independent chromatin accessibility data supported a stage specific methylation accessibility coupling at morula, albeit with modest effect sizes. Cross species mouse and orthogonal multiomic evidence suggested partial conservation but with weight dependence and heterogeneity. Collectively, our study defines morula as a computational "ground zero" candidate for age associated methylation features and proposes a testable hypothesis of developmental regulation, while emphasizing that matched parental offspring perturbation experiments are needed to establish causal mechanisms.

During development, pluripotent stem cells generate diverse cell types through gene regulatory networks orchestrated by combinations of transcription factors (TFs). Following terminal differentiation, however, cellular identities become remarkably stable and resistant to TF-mediated perturbation, yet the mechanisms underlying this stability remain poorly understood. Here, we identify Polycomb repressive complexes (PRCs) as key regulators of neuronal identity maintenance. Although PRCs are well known for repressing the promoters of developmental genes during early cell fate specification, we unexpectedly find that PRC-mediated H3K27me3 expands into megabase-scale domains during neuronal maturation that align with topologically associating domains (TADs). These H3K27me3 "mega-domains" selectively encompass genes associated with alternative neural and non-neural lineages. While depletion of H3K27me3 in mature neurons has only modest effects on basal gene expression, it significantly increases neuronal activity-dependent c-FOS binding and induction of lineage-inappropriate genes within these mega-domains. Our findings reveal a previously unrecognized role for PRCs in establishing TAD-scale repressive chromatin domains during neuronal maturation, thereby safeguarding neuronal identity from external stimuli through broad silencing of alternative cell fate programs. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=124 SRC="FIGDIR/small/743812v1_ufig1.gif" ALT="Figure 1"> View larger version (25K): org.highwire.dtl.DTLVardef@1e32ee3org.highwire.dtl.DTLVardef@1b3e27org.highwire.dtl.DTLVardef@8d83eforg.highwire.dtl.DTLVardef@d0d5bc_HPS_FORMAT_FIGEXP M_FIG Graphical Abstract C_FIG

Cell confluency is a major determinant of cell-cell communication, protein interactions, access to nutrients, and signalling dynamics, thereby significantly impacting biological outcomes. Lung cancer cells like A549 are widely used as screening models for scientific studies wherein their growth in vitro progress from non-confluent to confluent growth. In this study, we investigated the transcriptomic adaptations associated with the transition of A549 cells from baseline non-confluent to confluent growth. Comparative transcriptomic analysis between confluent and cells at baseline identified 815 upregulated and 671 downregulated transcripts. Pathway enrichment analysis of deregulated transcripts in confluent cells revealed enhanced cholesterol and sterol biosynthetic pathways, along with suppression of chromosomal segregation and mitotic pathways. At confluency, an increased expression of glucose transporters (SLC2, SLC60, and SL37 families) and glycolytic pathways, and a decrease in amino acid transporters (SLC1, SLC7, SLC38, and SLC36) and amino acid metabolic pathways is observed. A reduced one-carbon metabolic signature (SHMT2, DHFR, and MTHFD2) and enhanced fatty acid precursor synthesis (HMGCLL1, ALDH6A1, and AASS) were also observed at confluency. 1H NMR profiling of culture media revealed higher glucose and glutamine utilisation with lactate accumulation during culture maturation. Collectively, the data suggest transcriptome-level rewiring in A549 cells with preferential biosynthesis of lipids and sterols at confluency and underscore the importance of considering culture maturity in cancer biology, metabolism, and therapeutic studies.

Abstract. Introduction: Preterm birth drives adverse perinatal maternal and infant health outcomes through heterogeneous symptoms, severity, and etiologies. Delivery prior to reaching 37 weeks of gestation may result from medically indicated intervention for pregnancy complications or spontaneously in the absence of prior symptoms. Placental tissue collected following preterm birth exhibits differential DNA methylation compared to full-term placentas and may indicate pregnancy health during gestation. Placental DNA currently has limited utility for assessing health of ongoing pregnancy, as sampling placental tissue during gestation increases the risk of infection and miscarriage. Risks associated with placental sampling during pregnancy limit the use of DNA methylation in clinical preterm birth prediction. Assessing preterm birth risk during gestation requires non-invasive methods for characterizing placental DNA methylation. Results: We quantified genome-wide DNA methylation patterns of hypermethylated cell-free DNA in pregnant (n = 99) and non-pregnant (n = 93) plasma using cell-free methylated DNA immunoprecipitation sequencing (cfMeDIP-seq). In each sample, we assessed DNA methylation status in 300-bp genomic windows, examining both sequencing read counts and calculated absolute molar DNA amount. Known hypermethylated placental regions, including RASSF1, STAT5A, and ERG promoters showed significantly increased odds of detection in pregnant samples, suggesting enrichment of cell-free placental DNA. Of the 536,444 300-bp windows examined, 173,071 (32%) showed significant enrichment in pregnant plasma. Linear modeling identified 107,505 differentially methylated regions (DMRs) associated with pregnancies later diagnosed with intrauterine growth restriction (IUGR) (n = 22). Alu elements showed increased representation in these DMRs than expected, while other repetitive elements exhibited underrepresentation. Discussion: These results demonstrate cfMeDIP-seq's ability to enrich for cell-free placental DNA and characterize cell-free DNA methylation signatures of pregnancies complicated by IUGR. Enrichment of cell-free placental DNA enables non-invasive profiling of placental DNA methylation from maternal plasma. Detectable epigenetic signatures in maternal plasma may identify pregnancies at elevated risk for preterm birth before clinical symptoms appear. Our findings further highlight the potential of cell-free placental DNA for monitoring pregnancy health.

Butterfly wings present a tremendous gallery of colorful patterns, offering a unique opportunity to study how developmental pattern formation processes evolve. We still do not understand the genetic basis of several key aspects of wing pattern development, however. Three paralogous POU domain transcription factors nubbin, ventral veinless (vvl), and pdm3 are all known wing development genes in Drosophila melanogaster. Here we combine gene expression and knockout approaches to show that each of these genes plays multiple novel wing patterning roles in the common buckeye butterfly, Junonia coenia. We found that nubbin controls eyespot pattern determination via a non-cell autonomous repressor-like effect originating at the wing veins, such that nubbin knockouts have larger eyespots. nubbin also regulates pigment identity and scale morphology across the wings. We also found that vvl regulates pigment identity of the discal bands and ventral hindwing. Last, we found that pdm3 is required for determining the outer rings of eyespot patterns, where it is co-expressed with spalt and the lncRNA ivory. pdm3 is also necessary for determining wing margin stripes, where it is again co-expressed with spalt, leading us to propose that the eyespot and wing margin gene regulatory networks could be homologous. Finally, pdm3 affects pigmentation of the ventral hindwing, phenocopying the seasonally-plastic color switch in J. coenia. Together, our work shows that POU domain transcription factors play diverse roles in butterfly wing pattern development and highlights nubbin as one of the first genes implicated in the repressive function of wing veins in color pattern determination. Highlights- Gene expression and knockouts reveal three POU factors regulate butterfly wing color pattern - nubbin regulates eyespot development, likely via a repressor from the wing veins - nubbin controls scale color and morphology across the wing - pdm3 coordinates eyespot development and is co-expressed with spalt and ivory - Expression of genes in the eyespot and wing margin suggests network homology

Background. DNA methylation patterns in cord blood are robustly associated with birthweight in the general population. However, it remains unknown whether these associations extend to clinically relevant populations, such as preterm neonates or those born small for gestational age, and whether they directly reflect birthweight or are driven indirectly by genetic, familial, maternal, and obstetric factors. Methods. We calculated a birthweight methylation profile score (MPSBW) using weights of 835 CpGs previously associated with birthweight in the general population and evaluated its association with birthweight in 67 monochorionic (MC) twin pairs including 134 neonates (97% born preterm) from the Twinlife study. MC twin pairs are identical twins sharing a single placenta, often unequally, which can result in unequal resource distribution and differential fetal growth. Results. We examined the association between within-pair differences in birthweight and MPSBW, thereby estimating the association independent of factors shared equally by co-twins. A 500-gram increase in birthweight was associated with a 0.256 SD increase in MPSBW (p<0.005) in this population of preterm neonates. Adjustment for polygenic score for birthweight (PGSBW) confirmed that the observed epigenetic associations were not driven by common genetic variation underlying birthweight. Interestingly, a similar effect size (0.226 SD per 500 g birthweight increase; p<0.05) was observed in the within-pair analysis, which controls for all shared influences within a twin pair. Conclusion DNA methylation is associated with individual differences in birthweight in a high-risk clinical population of MC twins, independent of shared genetic, familial or maternal influences.

THAP9 is a transposable element-derived gene which encodes a protein that is homologous to the active Drosophila P-element transposase (DmTNP). Both THAP9 and DmTNP possess a C-terminal domain (CTD) which is functionally uncharacterized. Sequence and structural analysis suggest that the THAP9-CTD has a novel fold which is only found in THAP9 homologs. To explore the evolutionary history and characteristics of this novel domain, exhaustive phylogenetic analysis (using MSA, structure prediction, MSTA-based clustering) was performed. THAP9-CTD homologs were more widely distributed throughout the animal kingdom in comparison to DmTNP-CTD homologs which were restricted to arthropods. Moreover, the THAP9-CTD homologs were more conserved, especially among mammals and birds and their average length increased in a class-specific manner. Comparison with the DmTNP-CTD homologs demonstrates that although their respective CTDs may have evolved independently, they both surprisingly share similar secondary structure elements consisting of three conserved helical regions made of hydrophobic residues that are predicted to make up a conserved core. The role of the respective CTDs were further investigated by creating truncation mutants lacking the CTD. Interestingly both THAP9 and DmTNP truncation mutants are still capable of DNA excision and integration suggesting that their respective CTDs are not essential for DNA transposition. Moreover, CTD truncation favours DNA integration in THAP9: this suggests that CTD acquisition during evolution may have led to THAP9 domestication as observed in other transposable element-derived genes like Rag1 and piggybac, which have similar terminal regulatory domains.

Epigenetic modifications, including histone modifications and DNA methylation, direct gene expression programs during the growth and development of eukaryotic organisms. In Arabidopsis thaliana, the chromatin remodeler PICKLE (PKL), a homolog of animal CHD3, plays a critical role during these processes. Previous studies of PKL have painted a complex picture of its function, including both activating and repressive roles, however, how PKL can control both transcriptional silencing and activation is unknown. We have identified a group of J-domain-containing proteins (DNAJs), usually known for their roles in protein folding, that guide PKL recruitment to various gene promoters by bridging PKL to transcription factors. Mutation of PKL's DNAJ-interacting domain disrupts PKL association with chromatin. Once recruited to transcription factor-bound sites, PKL coordinates with the histone chaperone ATRX to deposit HISTONE3.3 (H3.3) at targeted loci, which also negatively affects the accumulation of H3 lysine 27 trimethylation (H3K27me3) at promoters. We found that transcriptional outcome of PKL binding depends not only on the changes in H3.3 occupancy but also on the pre-existing chromatin context at PKL-targeted sites. Our findings outline a new mechanism for CHD3 chromatin remodeler recruitment and function.