Genes

A population-specific missense variant, rs373863828 (G > A, p.Arg457Gln), has been identified in Pacific Islanders and is associated with increased body mass index and lower odds of type 2 diabetes. However, the mechanism underlying these associations is unknown. The variant is located in exon 5 of CREBRF, a gene which is rapidly upregulated in response to nutrient deprivation. This study aimed to assess the effect of rs373863828 on gene expression in adipose tissue from Samoan adults from the Soifua Manuia (''Good Health'') study. Needle subcutaneous adipose (upper buttock) biopsies were obtained in Apia, Samoa, from fasting Samoans without diabetes. RNA isolated from adipose tissue (n=91, 65.9% female) was subjected to bulk paired-end mRNA-sequencing (2x150 base pair). Differential gene expression among the three rs373863828 genotypes (GG, AG, AA) was assessed using a likelihood-ratio test implemented in DESeq2, and overrepresentation analysis for significant differentially expressed genes (DEGs) (padj < 0.1) was performed with clusterProfiler and oRG.DB. Bulk expression was deconvoluted to determine the cell type composition of the adipose tissue. Twenty-two DEGs were associated with rs373863828 genotype including CBX7 (padj=0.026 ) and SELENOM (padj= 0.004), both of which have roles in adipogenesis and metabolism. A single expression profile comprising 13 of the 22 DEGs corresponding to CREBRF genotype was identified, and overrepresentation analysis supported this finding, with terms such as ''keratinocyte differentiation'' and ''skin development'' enriched among significant DEGs. However, PERMANOVA of adipose cell type proportions showed no difference (p > 0.05) among the three genotypes. rs373863828 genotype was associated with expression of 22 genes in whole subcutaneous adipose tissue of this sample of fasting Samoan adults without diabetes. Future work should assess cell- and tissue-specific rs373863828 genotype effects using a combination of single-cell approaches and human cell models with perturbations associated with nutritional stress.

Background: Short-read whole-genome sequencing (sWGS) has limitations in characterizing tandem-repeat expansions and variants in highly homologous or pseudogene-associated regions. Long-read sequencing can address some of these challenges but often benefits from high-molecular-weight DNA. TruPath Genome incorporates spatial proximity information into a short-read sequencing framework. This study evaluated its performance in characterizing selected complex genomic variants using archived clinical DNA specimens. Methods: Ten archived peripheral blood DNA specimens were analyzed, comprising seven cases with disease-associated short tandem repeat (STR) expansions and three cases involving homologous or pseudogene-associated loci (STRC, SMN1/SMN2, and CYP21A2). TruPath results were compared with previously available orthogonal assay results, including triplet repeat-primed PCR (TP-PCR), multiplex ligation-dependent probe amplification (MLPA), quantitative PCR (qPCR), and sWGS-based ExpansionHunter analysis. Results: TruPath generated repeat-length estimates across the evaluated STR loci. For two FMR1 full-mutation alleles, TruPath estimated 284 and 295 CGG repeats, respectively, while TP-PCR reported expansions exceeding 200 repeats. In a DMPK case, TruPath estimated an expanded allele of 1,203 CTG repeats, compared with a TP-PCR result exceeding 100 repeats. At the STRC and SMN1/SMN2 loci, TruPath findings were consistent with available orthogonal results for deletion and copy-number status. At the CYP21A2 locus, TruPath reconstructed the RCCX gene arrangement and assigned the I2G variant and a chimeric allele to different haplotypes. Conclusions: In this small series of archived clinical DNA specimens, TruPath Genome enabled characterization of selected repeat expansions and homologous or pseudogene-associated variants. The findings support further evaluation of proximity-mapped sequencing as a complementary approach for complex genomic regions, particularly when conventional short-read sequencing is limited.

Individuals carrying a G4C2 repeat expansion in C9orf72 can develop amyotrophic lateral sclerosis (ALS) and/or frontotemporal dementia (FTD), at various and unpredictable ages. The contribution of the expansion to this extreme clinical variability remained a matter of debate. In this work, we validated the use of optical genome mapping to efficiently size this expansion from blood and applied this method to a cohort of 54 repeat expansion carriers including patients with FTD (n = 23), ALS (n = 22) and presymptomatic individuals (n = 9). Despite the high variability in expansion profiles, distinct groups can be inferred. Although consensus repeat profiles were similar between FTD and ALS patients, relationships between repeat number, indexes of repeat instability, and the age at sampling differed between clinical conditions. An age at sampling-dependent increase of repeat instability in blood was observed in presymptomatic carriers and FTD patients (p = 0.01 and p = 0.04 respectively), in contrast to patients with ALS (p = 0.9). The proportion of shorter repeats ([500-1500 repeats]) increased with the age at sampling in ALS patients but decreased in presymptomatic and FTD cohorts, while the proportion of larger repeats (>2000 G4C2), in particular those exceeding 4000 repeats, increased along with the age at sampling in presymptomatic carriers and FTD cases (p <1.10-e4 and p = 0.006 respectively) conversely to patients with ALS (p = 0.98). In conclusion, this work strongly supports different dynamics of repeat instability of the C9orf72 pathogenic expansion in blood linked to the clinical evolution. These findings provide new insights into the relationship between expansion profiles and clinical variability.

CBX1-related syndrome is characterized by developmental delay, hypotonia, autistic features, and mild dysmorphic features. This syndrome is caused by heterozygous missense variants in CBX1, which encodes heterochromatin protein 1 beta (HP1{beta}). These variants are situated within the chromodomain of HP1{beta}, a critical region that mediates the interaction between HP1{beta} and trimethylated H3K9. While a prior study identified three individuals with pathogenic CBX1 variants, the variant and clinical spectrum of CBX1-related syndrome remains largely unknown. Here we report three patients with novel de novo heterozygous variants in CBX1. Patients with CBX1 p.(Leu40Pro) and p.(Cys60Arg) manifested with global developmental delay, while the patient with CBX1 p.(Cys60Gly) primarily exhibited speech delay without concurrent motor developmental delay. All variants reside within chromodomain of the HP1{beta} protein. Interestingly, in vitro cellular assays revealed that CBX1 p.(Cys60Gly) variant showed an intermediate cellular phenotype between the wild type and the CBX1 p.(Leu40Pro) and p.(Cys60Arg) variants, suggesting a possible genotype-phenotype correlation. This report provides further evidence that missense variants within the chromodomain of HP1{beta} cause a syndromic neurodevelopmental disorder, CBX1-related syndrome.

ABSTRACT Objective: Skeletal muscle growth rate differs substantially among diverse pig breeds. In the present study, RNA-seq was performed to identify differentially expressed genes (DEGs) across pig breeds with distinct growth rates. CCND2 showed obvious differential expression among pig breeds. Although CCND2 is essential for cell cycle control, its biological function in skeletal muscle differentiation has rarely been reported. This work aims to identify DEGs in pig breeds with different growth rates and explore CCND2 role in porcine skeletal muscle development. Methods: RNA-seq was performed to identify differentially expressed genes (DEGs) across pig breeds with distinct growth rates. Weighted gene co-expression network analysis (WGCNA) was used to identify gene modules potentially associated with skeletal muscle growth and development. Quantitative reverse transcription polymerase chain reaction (qRT-PCR) analysis was performed to validate the role of CCND2 in skeletal muscle differentiation. Results We identified 5,416, 4,846, and 2,777 DEGs in the Duroc pig (DU) vs. Jiangquan black pig (JQ), DU vs. Laiwu pigs (LW), and JQ vs. LW comparison groups, respectively. WGCNA identified two gene modules (lightgreen and darkmagenta) potentially associated with skeletal muscle growth and development. CCND2 exhibited differential expression in comparisons between DU vs JQ and DU vs LW, with higher expression observed in Duroc pigs. qRT-PCR analysis revealed that overexpression of CCND2 at day 6 (D6) of cell differentiation significantly increased the expression levels of CCND2 gene and markers of myogenic differentiation (p < 0.05). Conversely, knockdown of CCND2 significantly decreased the expression levels of CCND2 gene and myogenic differentiation markers (p < 0.05). Conclusions This study identified breed-specific DEGs and key gene modules in porcine skeletal muscle and verified that CCND2 promotes myogenic differentiation. These findings provide a genetic theoretical basis for further investigations into skeletal muscle growth and development. Keywords pig, skeletal muscle, transcriptome, WGCNA, CCND2

Background: Short-rib polydactyly syndrome type IV (Beemer-Langer syndrome) is a lethal skeletal ciliopathy that can result from biallelic pathogenic variants in KIAA0586 (TALPID3). Earlier clinical observations suggested an unusually high occurrence of this phenotype in Romani families from the Carpathian Basin. Methods: Whole-exome sequencing was performed in affected individuals from five independent families, and the frequency of KIAA0586 c.1815G>A was assessed in a Hungarian Romani genetic database of 500 individuals. Results: The affected individuals were homozygous for c.1815G>A, a splice-disrupting variant previously shown to cause exon 14 skipping. Four families self-identified as Roma from Hungary and one affected individual was from Turkey. Six heterozygous carriers were identified among 500 individuals in the Hungarian Romani cohort, corresponding to an allele frequency of 0.6%. This was markedly higher than the frequencies cited from ALFA (0.01%) and gnomAD (0.0014%). The Hungarian-Romani and Turkish affected individuals also shared genotypes at informative markers flanking the variant, supporting a shared ancestral haplotype. Conclusions: These observations strengthen evidence that KIAA0586 c.1815G>A is a founder allele enriched in at least some Romani populations. The data support consideration of ancestry-informed carrier testing in appropriately counseled families. Historical admixture may explain the occurrence of the same haplotype in a Turkish case, but this remains a hypothesis requiring broader population-genomic confirmation.

Background: More than five years after the COVID-19 outbreak, the infection by the SARS-CoV-2 virus should still be considered as a global threat with highly heterogeneous clinical outcomes from asymptomatic to fatal depending on existing genetic and phenotypic inter-individual and inter-population variability. Our objective is to identify risk loci associated with COVID-19 in an admixed Latin American population. Methods: We performed a genome-wide association study in the framework of the Chilean C19-GenoNet team, analyzing a cohort of 3,417 individuals recruited from the northernmost to the southernmost regions of the country, who presented variations in the severity of their COVID-19 clinical manifestations. Results: Our investigation replicated the extensively reported association of several variants at the 3p21.31 locus with COVID-19 severity and found a suggestive association for a variant within the FOXP4 gene at 6p21.1 with COVID-19 hospitalization. Both associations appear to be shared with other populations. Importantly, our study identified three previously unreported loci associated with COVID-19 severity or hospitalization at 7q21.11, 16p13.12 and 6p25.3, and one additional locus at 4p13 associated with SARS-CoV-2 positivity, a proxy for infection susceptibility. Conclusions: these new risk loci may represent population-specific effects in Chilean and, potentially, other Latin American admixed groups. Our study highlights the importance of expanding efforts to study underrepresented and genetically different populations, which may provide new clues on the genetic basis of human diseases and may help to anticipate global responses to emerging viral infections and future pandemics.

Non-coding variants altering mRNA splicing are increasingly recognized as important cause of Mendelian disorders. Deep intronic variants (DIV) that activate cryptic exons (CEs) cause ~20% inherited retinal diseases (IRD) cases, yet it remains challenging to predict intronic variant pathogenicity accurately, thus we used a high throughput splicing assay (HTSA) to measure the effects of rare deep intronic variants in recessive IRDs with one confirmed pathogenic variant. 640 very rare deep intronic variants were chosen in trans-position with known mutation from 76 patients. We tested them with HTSA, which consists of a split-GFP minigene, separated by an SMN1 gene intron into which the 270bp sequences flanking the DIVs were cloned. The plasmid library was transfected into 293HEK cells, and 48 hours later total RNA was extracted from the transfected cells. RNA transcripts produced by HTSA minigene were amplified by RT-PCR and sequenced. The intron sequences spliced between the two GFP exons were identified and quantified. 98 variants activated CE >100 times more in altered oligo compared to reference oligo and were classified as pathogenic in this experimental setting, validated in longer context, 26 variants were classified as VUS. Interestingly, only 6 from 90 variants (6,6%) were predicted to be pathogenic by in silico algorithms (Splice AI) which leaves room for significant improvement of prediction algorithms. The results of this experiment confirmed diagnosis for 50 of 78 patients (64%). So, 19,4% of deep intronic variants were shown to cause CE activation. This can lead to conclusion that significant portion of undiagnosed patients with recessive IRDs carry pathogenic intronic mutation which causes the disease.

Purpose A significant proportion of cases with rare inherited retinal disease (IRD) remain genetically unresolved following short-read whole genome sequencing (WGS). This study aimed to increase the diagnostic yield of two cohorts in a research setting. One cohort was previously screened by short-read WGS (n=120 cases), while the second cohort (n=28 cases) was not included in any previous short-read WGS studies. In contrast to the larger cohort, these cases only underwent either exome sequencing (ES) or single-molecule molecular inversion probe (smMIPs) sequencing as pre-screening in earlier studies. For all cases, we performed a stepwise, case by case reanalysis in which short-read WGS was generated (n=28 cases) and (re)analyzed short-read WGS data using an optimized approach. For a subset of cases (n=20) that remained unresolved, this was followed by long read WGS. Together, these steps aimed to improve the diagnostic yield in these cohorts. Methods Short-read WGS data of 148 IRD-cases were (re)analyzed using updated allele frequency databases, new variant caller and variant predictor tools, as well as updated and extended gene-panels to identify causal single nucleotide variants (SNVs) and structural variants (SVs). For 20 genetically unresolved cases with sufficient high-quality DNA available, long-read WGS was performed. Results The (re)analysis of short-read WGS data in the 120 previously WGS-screened unresolved cases resulted in a diagnostic yield of 15% (18/120). For the 28 individuals without prior WGS, performing WGS analysis within this study provided an additional yield of 32% (9/28). Furthermore, long-read WGS contributed two genetic diagnoses among the 20 long-read WGS-sequenced probands. Amongst other reasons, new genetic diagnoses were attributed to pathogenic variants in genes newly associated with IRDs and in genes that were not included in the gene panels applied in previous WGS studies. The Mobile Element Locator Tool facilitated the identification of two pathogenic Alu insertions in short-read WGS. Long-read WGS identified two additional pathogenic variants. Conclusion These results support periodic reanalysis of existing short-read WGS data as a key component of IRD genetic diagnostics, with selective long-read WGS representing a valuable additional approach for unresolved cases. Together, these complementary strategies offer a practical framework for improving molecular diagnosis and narrowing the remaining diagnostic gap in IRDs.

Fibroblasts are readily obtained and expanded and may provide an alternative cell source for cultivated seafood if their latent myogenic potential can be accessed. We examined whether transient exposure to 5-azacytidine (AZA), a cytidine analogue known to convert mouse embryonic fibroblasts into myoblasts, elicits a persistent muscle gene program in embryo-derived zebrafish (Danio rerio) Z3 fibroblasts. Cells were exposed to 0, 2.5, 5 or 10 M AZA for 48 h and maintained in drug-free medium for 3 weeks. RNA-seq identified 744 genes that increased and 670 that decreased consistently across the concentration series. Increased genes encompassed myogenic regulators, fusion factors, sarcomeric components and excitation-contraction machinery, whereas decreased genes were enriched for extracellular-matrix and adhesion functions. In a separate six-dose RT-qPCR experiment (0-20 M AZA) with three biological replicates per concentration, all 19 selected muscle genes were significantly increased at 1.25 M, the lowest concentration tested, whereas 9 of 12 extracellular-matrix/fibroblast genes were significantly decreased. Several muscle genes plateaued or declined at higher concentrations, and multinucleated myotubes were not observed. A separate 5-aza-2'-deoxycytidine (decitabine) series showed a comparable muscle-associated response at 0.25 M. These findings indicate that zebrafish fibroblasts retain access to a myogenic-like transcriptional program, and they provide a model for identifying food-compatible approaches to control fish-cell lineage plasticity for cultivated seafood.

BackgroundN6-Methyladenosine (m6A) regulates RNA fate in flowering plants, but the repertoire and expression patterns of m6A-related genes remain incompletely characterized in liverworts. Here, we characterized these gene families in the liverwort Marchantia polymorpha within a comparative framework spanning diverse plant lineages. ResultsA comparative survey of 119 plant genomes using sequence similarity and conserved-domain criteria identified 18 candidate m6A-related loci in M. polymorpha, comprising seven writer-related genes, five AlkB homolog genes, and six YTH- or CPSF30-related genes. Across the surveyed genomes, canonical writer components generally had low copy numbers, whereas AlkB homolog and reader-related families showed greater copy-number variation. Phylogenetic and domain analyses supported the family assignments of the M. polymorpha candidates. Eight of the 18 candidate loci showed detectable collinearity with Marchantia quadrata. Putative promoter analysis identified motifs annotated as hormone-, light-, and stress-responsive, with hormone-responsive motifs accounting for the largest share of predicted sites. Public transcriptome analysis revealed variation in candidate gene expression across developmental stages and tissues. Quantitative reverse-transcription PCR showed higher transcript abundance for all six YTH- or CPSF30-related genes in thalli than in gemmae or gemma cups. Cold, heat, osmotic, salt, and dark treatments elicited gene-specific expression responses. Darkness increased transcript abundance of several candidates, including MpMTA, MpALKBH1A, and MpECT2. A predicted protein-association network suggested links between subsets of the candidates and RNA-processing proteins. ConclusionsThese findings define a candidate m6A-related gene repertoire in M. polymorpha and identify tissue- and stress-responsive genes for targeted functional studies of RNA methylation in this liverwort.

In eukaryotic cells, the positive transcription elongation factor b (P-TEFb) plays a critical role in the transition of RNAPII from the paused state to actively transcribing mode. Although three different cyclin (Cyc) T (CycT1, T2a, and T2b) interact with CDK9 to form functional P-TEFb, a majority of P-TEFb complexes are comprised of the CycT1 and CDK9. To analyze the role of the CycT1 subunit of P-TEFb on cellular transcription, we established HEK293T cells lacking CycT1 proteins (CycT1-KO cells). While no apparent growth defects were observed with CYcT1-KO cells, the level of CDK9 was decreased and no compensatory over-expression of CycT2 was observed, suggesting that the reduced level of functional P-TEFb complexes is sufficient for the normal cell growth. Reporter gene assays indicate that NF-kappaB-dependent transcription induced by PMA was impaired in CycT1-KO cells although the function of NF-kappaB (P65) per se was not affected. On the other hand, AP1-dependent transactivation induced by PMA was unaffected in CycT1-KO cells. Moreover, transcription stimulated by JQ1, a strong P-TEFb inducer, was reduced in CycT1-KO cells. Data of biochemical analysis indicate that P-TEFb with CycT1 (P-TEFb (CDK9:CycT1)) was efficiently released from 7SKsnRNP by JQ1 while P-TEFb (CDK9:CycT2) was not affected by JQ1. Transcriptome analysis indicated that the lack of CycT1 had a minor effect on the steady-state transcription. However, expression of JQ1-dependent genes was severely reduced in CycT1-KO cells. Interestingly, referring to gene expression profiles of 33 different types of cancer revealed that 11 out of 13 top JQ1-dependent genes were also highly upregulated in Pancreatic Adenocarcinoma. From these results, we conclude that although many genes can be regulated by both P-TEFb (CDK9:CycT1) and P-TEFb (CDK9:CycT2), CycT1 plays a critical role in regulating highly inducible genes, which are also aberrantly regulated in a particular type of cancer.

Chromatin profiling methods, such as ChIP-seq (chromatin immunoprecipitation followed by sequencing), are used to characterize the genomic localization of DNA-associated proteins. While ChIP and ChIP-seq have been used for decades, an orthogonal approach, CUT&Tag (Cleavage Under Targets and Tagmentation), is gaining popularity as an efficient and cost-effective alternative. Although previous comparative studies note discrepancies in signal-to-noise ratios and detection bias at certain genomic regions, many differences between ChIP-seq and CUT&Tag results remain largely unreconciled. Here, we systematically investigate the disagreeing signals captured by these two methods across well annotated genomic regions. We assess multiple histone mark profiles generated by different groups in two cell lines (K562 and MCF-7). Overall, our analysis indicates that compared to ChIP-seq, CUT&Tag may have limited sensitivity in low-GC environments and, as previously observed, increased signal in hyper-accessible chromatin. Notably, within GC-poor regions of active gene bodies and Polycomb-repressed domains, CUT&Tag exhibits a loss of H3K36me3 and H3K27me3 signal, respectively, where occupancy of these histone marks is otherwise expected. Further, active promoters with discrepant H3K4me3 and H3K27ac signal between the two assays differ systematically in GC content and chromatin accessibility. Promoters differentially enriched for CUT&Tag signal relative to ChIP-seq generally show higher, broader GC-content profiles and higher DNase-seq and ATAC-seq signal, while promoters enriched for ChIP-seq signal harbor the opposite characteristics. A local bias for high GC content and/or chromatin accessibility in CUT&Tag may also explain its differing signal patterns at nucleosome-depleted regions (NDRs) compared to ChIP-seq and MNase-seq promoter profiles. Altogether, our results highlight that studies focused on profiling the intensity and structure of histone modification occupancy can be sensitive to potential biases of CUT&Tag to GC content and chromatin accessibility. These characteristics should be accounted for when selecting a chromatin profiling approach, analyzing and interpreting data as well as drawing biological conclusions.

Background: Shotgun metagenomic pipelines routinely discard host-derived reads, which can exceed 90% of reads in low-biomass clinical samples. Whether these reads support mitochondrial heteroplasmy detection, and at what allele fraction, has not been established. Methods: Shotgun metagenomic data from 47 upper respiratory tract swabs from healthy adults in Nagpur, India, were analysed. Human Reads were recruited against the rCRS and realigned competitively to GRCh38 to remove nuclear mitochondrial segments (NUMTs). Kraken2 was used as a microbial control. Heteroplasmy was called against a pooled, substitution-specific beta-binomial error model and a leave-one-out panel of normals. The detection limit was evaluated from the substitution spectrum against gnomAD v3.1. Results: Complete mitochondrial genomes were recovered from all samples (mean depth 433X, range 9-3,029X). Competitive realignment relocated 3.22% of recruited reads to nuclear loci; microbial cross-mapping removed 0.045%. Error rates varied 12.8-fold across substitution classes, with T>A being the highest. The 134 calls had a Ti/Tv of 0.43, compared with 3.51 in gnomAD, and T>A accounted for 42.5% of calls. T>A allele fractions never exceeded 4.72%, whereas transitions reached 90.81%. At a 5% threshold, T>A calls disappeared, and Ti/Tv rose to 2.20 before plateauing; 16 calls exceeded this limit. Macrohaplogroups were assigned in 46 of 47 samples (M 22, R 23, N 1). Conclusions: Metagenomic host reads support complete mitochondrial recovery, haplogroup typing, and heteroplasmy detection for allele fractions > 5%. Below this, a T>A-dominated error process persists after NUMT removal that background-fitted models do not capture.

Background: Dysfunction of the KCND3-encoded Kv4.3 channel, which carries the transient outward potassium current (Ito), has long been suspected to contribute to the pathophysiology of Brugada Syndrome (BrS). Although recent genome-wide association studies (GWAS) have implicated KCND3, the role of rare variation in this gene remains uncertain and clinically unactionable. Methods: Genome sequencing was performed in 983 BrS probands from Europe and Thailand. Putative gain-of-function (GoF) rare variants in KCND3 identified in patients were functionally characterized by patch-clamp analysis of Kv4.3 current properties using a HEK293 cell heterozygous model. Biobank datasets were utilized to evaluate rare-common variant interaction at the KCND3 locus. Results: Eight BrS cases carried six different rare KCND3 variants which clustered at the Kv4.3 C-terminus, with terminal exon rare variants significantly enriched in cases versus study (p=0.004) and population (p=2.0e-6) controls. Four of the five KCND3 case variants studied demonstrated significant GoF effects versus wild-type and putative benign variants, including increased Ito amplitude (p.Leu586Val, p.Ala634Thr) and slower inactivation (p.Gly600Arg, p.Ala654Thr, p.Ala634Thr). Analysis of p.Gly600Arg carriers in the UK Biobank and All of Us revealed a significant enrichment of the protective KCND3 expression-reducing allele of the GWAS lead single-nucleotide polymorphism rs12090194, suggesting a possible epistatic common-rare variant interaction modifies penetrance in carriers. Conclusions: GoF rare variants at the C-terminus of KCND3 are associated with BrS in a small proportion of cases. Determination of the rare and common variant status in BrS patients and their relatives may provide information about disease risk.

Heavy metal contamination of aquatic environments poses persistent risks to ecosystems and human health. Aquatic macrophytes such as Pistia stratiotes are increasingly used for phytoremediation due to their capacity to accumulate contaminants without displaying symptoms of abiotic stress. However, the molecular mechanisms underlying their metal tolerance remain poorly understood. Metallothioneins (MTs) are cysteine-rich proteins involved in metal binding and detoxification in plants. In this study, genome-wide analysis was performed to identify and characterise candidate MT genes in P. stratiotes and to assess their transcriptional response to copper exposure. Six novel MT genes (PsMT2f, PsMT3a-d, and PsMT4) were identified and classified into MT2, MT3, and MT4 subfamilies based on conserved cysteine motifs and domain architecture. Quantitative real-time PCR showed differential and tissue-specific expression of MT genes in roots and leaves following exposure to copper for up to 4 h. These findings provide the first comprehensive characterisation of the MT gene family in P. stratiotes and demonstrate their potential role in copper tolerance, supporting the application of this species in the phytoremediation of metal-contaminated aquatic systems.

Background: X-chromosome inactivation (XCI) evolved independently in eutherian and marsupial mammals, where it is orchestrated by the unrelated long non-coding RNAs Xist and RSX, respectively. Xist organizes a repressive nuclear compartment through multivalent RNA-protein interactions, but whether RSX exploits similar biophysical principles remains unknown. A recent paper has identified bona fide RSX interacting proteins. Results: We integrated proteome-scale RNA-protein interaction prediction, experimental validation, phase-separation propensity analysis, functional annotation and comparative RNA-structure modelling to characterize the RSX interaction landscape. Using catRAPID, we ranked 1,168 RNA-binding proteins from the native Monodelphis domestica proteome. Predictions were significantly enriched for experimentally identified RSX interactors, with 4.85-fold enrichment among the top 50 candidates (P approximately 1.6 x 10-5), increasing to approximately eightfold for proteins shared by the experimental RSX and Xist interactomes (P approximately 2 x 10-6). Among 30 high-confidence RSX interactors, 13 were experimentally supported, 17 were previously unrecognized candidates and 17 exhibited high phase-separation propensity. The network was enriched in ribonucleoprotein granules and nuclear bodies and converged on m6A regulators and SR-family splicing factors. Comparative modelling detected no conserved secondary or tertiary architecture between RSX and Xist. Conclusions: RSX and Xist appear to have converged not through RNA sequence or global structure, but through recruitment of related, condensation-prone protein networks. These findings identify interaction-network and biophysical convergence as a potential principle of lncRNA-mediated chromosome regulation and provide testable candidates for determining whether RSX establishes a condensate-like compartment on the marsupial inactive X.

Two rounds of whole-genome duplication (2R-WGD) in the vertebrate lineage are thought to have driven gene diversification. Mammals have four genes encoding five nebulin superfamily proteins (LASP1, LASP2/NEBL, NEB, and NRAP) hypothesised to have arisen from invertebrate LASP via WGD. Here, we examined the evolutionary fate of the nebulin superfamily after an additional WGD, teleost-specific genome duplication (TGD). Genome and transcriptome analysis of zebrafish (Danio rerio) and medaka (Oryzias latipes), of which the complete genomes were recently determined, showed only lasp family genes retained duplicate copies. Zebrafish retained two LASP2/NEBL-derived ohnologs; one (LASP2L) had been misidentified as a nebulette gene in the Zebrafish Information Network (ZFIN) despite lacking nebulette-specific exons. Another (LASP2/NEBL) is newly identified in this study because it lies in a newly determined region of the latest genome assembly. Contrastingly, medaka retained two LASP1 ohnologs and one LASP2/NEBL. The duplicated lasp family proteins showed overlapping and specific tissue expression patterns. We confirmed all three zebrafish lasp proteins bound actin and co-localized with actin-rich structures when ectopically expressed in cultured fibroblasts. This supports that TGD-derived lasp duplicates underwent lineage-specific retention and expression divergence in teleosts whilst conserving core actin-binding properties.

Environmental exposures can induce epigenetic modifications that persist across generations, potentially contributing to the transmission of environmentally-induced phenotypes. However, the extent to which such persistence of molecular changes are maintained independently of genetic mechanisms remains unclear. In this study, we investigated the evolution and genetic determinism of DNA methylation (DNAm) variation across three successive generations of Japanese quails (G0 to G2) belonging to two epilines, defined according to whether their female ancestor had received genistein supplementation (epi+) or not (epi-). Using reduced representation bisulfite sequencing (RRBS), we characterised CpG methylation patterns and identified differentially methylated cytosines (DMCs) and regions (DMRs) between control and genistein-supplemented epilines. Out of 112,745 CpG sites analysed, no DMCs or DMRs were detected between epilines in the first generation following exposure (G0), whereas 621 DMCs (36 DMRs) were identified in G1, and 1,381 DMCs (101 DMRs) in G2. Despite this progressive increase of differential methylation sites, mean differences in methylation rate between epilines were globally small. Similarly, genome-wide genetic differentiation between epilines was very limited. Overall, SNP-based heritability of DNA methylation was 0.19 indicating that genetic variation accounted for only a small part of the global variation of DNAm levels. By contrast, DNAm at differentially methylated CpG sites was extensively under genetic control, with a mean SNP-based heritability > 0.5, and meQTL analysis further identified significant associations between SNPs and methylation levels. Together, these results indicate that local DNAm differentiation in quail is under substantial genetic regulation, while the limited genetic differentiation between groups suggests that genetic variation alone can not fully explain the progressive accumulation of methylation differences following an ancestral exposure to genistein. These results support DNAm as a plausible molecular candidate mechanism in the multigenerational response to environmental exposure and highlight the complex interplay between genetic and epigenetic regulations in the inheritance of phenotypes.

The teleost whole-genome duplication (TGD) contributed to functional diversification of opsins. Some TGD paralogs, including those of parapinopsin (PP), Vertebrate Ancient (VA) opsin, and long wavelength-sensitive (LWS) opsin, show different absorption spectra and/or expression patterns. However, our knowledge of detailed evolutionary processes and mechanisms by which TGD contributed to opsin diversification is still limited. Here, we report that TGD paralogs of parietopsin (PT1 and PT2) are retained in extant species. While most species, including the zebrafish, have only PT1, several characins, including the red piranha (Pygocentrus nattereri), retain both PT1 and PT2. The Mexican tetra (Astyanax mexicanus) and catfishes have only PT2. To assess the degree of functional diversification between PT1 and PT2, we characterized spectral properties and expression patterns. Maximum absorption spectra differ slightly among PTs. Those of red piranha PT1, PT2, Mexican tetra PT2, and Japanese catfish (Silurus asotus) PT2 were located at 517 nm, 528 nm, 517 nm, and ~535 nm, respectively. Fluorescence in situ hybridization showed that (1) piranha PT1 and PT2 are expressed in the same pineal cells, and (2) they are also co-expressed with PP1. Based on these findings, functional diversification of opsins after gene duplication is discussed.