BMC Genomic Data

MYC oncoprotein binding at promoters and enhancers influences RNA polymerase II (RNAPII)-driven gene expression. Numerous genes also bind MYC near their transcriptional end sites (TESs). This often allows direct promoter-TES contact via looping and further regulates total and 'read-through' transcription that extends beyond standard termination sites. We aimed here to better clarify the rules governing TES associated MYC and/or RNAPII binding cross-talk in human and murine cells. Using ChIPseq and RNAseq datasets from the ENCODE portal and elsewhere, MYC and RNAPII binding profiles were found to differ around TESs and transcriptional start sites (TSSs). Variations in E box flanking sequences likely accounted for the somewhat lower affinities of MYC for TES-associated sites. Motifs for numerous other transcription factors were also observed to cluster non-randomly and in close proximity to MYC and RNAPII binding site peak summits. On average, genes with TES-proximal MYC or RNAPII sites were more highly expressed than those without, although co-binding tended to be suppressive. Both normal and neoplastic proliferative stimuli altered the MYC and RNAPII binding patterns of many genes, indicating that 'category switching' was common, subject to disparate external signals and often reversible. Functionally related gene sets with high levels of read-through transcription were uniformly marked by significant amounts of TES-associated MYC and/or RNAPII binding. These findings indicate that, both independently and together, MYC and RNAPII binding near TESs dynamically impact total and read-through transcription while also coordinating the expression of many common purpose gene sets.

Interest in microbiota-host co-metabolism and the effects of its derived co-metabolites on biological processes is increasing rapidly. In addition to their demonstrated associations with mammalian metabolic health and cognition, microbiota-host co-metabolites (MHCMs) represent lifelong contributors to the endogenous exposome. We have previously shown the MHCM trimethylamine N-oxide (TMAO) to exert beneficial effects on murine blood-brain barrier integrity and cognition. Here we investigated whether these positive neural effects of TMAO extended to humans, analysing how TMAO exposure associates with neurodevelopmental outcomes in children and whether an in vitro human neuronal-astrocyte co-culture could contribute to further investigation of the underlying mechanism(s) and neuronal processes related to these associations. In a cohort study of childhood mental health (N=1,203), TMAO was associated with fewer internalising problems, while its precursor microbial metabolite trimethylamine was associated with more behavioural problems in both the cross-sectional and an independent longitudinal study from 1 to 15 years of age (N=630-820). Given prior associations between TMAO exposure and exposure to the environmental pollutants mercury and arsenic, we investigated how the effects of TMAO interacted with these known neurotoxicants. TMAO had a protective effect, modifying the relationship between arsenic exposure and poorer neurodevelopmental outcomes. Furthermore, TMAO activated synaptogenesis-related gene expression and was functionally protective against the negative effects of mercury in our in vitro model. Together, our findings emphasise the importance of interdisciplinary approaches to evaluate associations and potential pathways of MHCMs (endogenous) and environmental (exogenous) metabolites on neurodevelopment in exposome studies.

Developmental processes depend on carefully coordinated gene expression. Expression is modulated by the binding of transcription factors (TFs) to cis-regulatory elements (CREs), like enhancers and promoters. Many computational and experimental approaches have been developed to find CREs, particularly enhancers, in the genome, each with strengths and caveats. Given the increasing availability of ATAC-seq data and methods to find TF binding therein, we hypothesized that we could use TF footprinting tools to find clusters of TF binding events within accessible chromatin that may act as CREs. Using Drosophila anterior-posterior patterning network as a test bed, we used a digital genomic footprinting tool (DGT), TOBIAS, on previously published early embryo ATAC-seq data to characterize the TF footprint landscape of 16 TFs essential for embryonic patterning. Even in this system, with its extensive enhancer annotation, most footprinted TF binding sites lie outside of known enhancers, with intergenic and intronic regions hosting the highest TF footprint count, albeit at low density. To find potential novel enhancers, we identified high-density TF footprint clusters that are highly conserved and overlap with active enhancer histone mark signals. Five high confidence candidates were selected for reporter assay validation and all five were found to drive spatially patterned expression in the embryo. This study shows that even in a highly characterized system, the analysis of footprinted TF binding sites in ATAC-seq data can uncover new regulatory regions and suggests this approach may be helpful in using existing ATAC-seq data to find novel CREs. ARTICLE SUMMARYGiven the increasing availability of ATAC-seq datasets, workflows to exploit the data to uncover new cis-regulatory elements (CREs), including enhancers, are valuable. Using early anterior-posterior patterning in the Drosophila embryo as a test case, we find that previously published transcription factor footprinting tools and ATAC-seq data can be analyzed to yield new candidate CREs. Experimental validation confirms the activity of selected candidate CREs, suggesting that existing data can be analyzed to find novel regulatory elements.

Background Complex traits are influenced by numerous variants, most of which have regulatory effects on gene expression that can be mediated by DNA methylation. Molecular QTL mapping is an approach that aims to dissect these effects. However, obtaining molecular phenotypes on a large scale is challenging, particularly in livestock species. In cattle, an epigenotyping array called EpiChip has recently been developed in the European RUMIGEN project. The EpiChip, which contains 43,317 CpG sites distributed all over the bovine genome, enables large-scale measurement of DNA methylation. This study aims to characterize the genetic determinism of blood DNA methylation in cows by estimating heritability and mapping cis- and trans-methylation QTLs (meQTLs). Results Whole blood samples from 4,457 genotyped Holstein cows were epigenotyped. Across all CpG sites, the heritability estimates averaged 24.6%. The local meQTL mapping at sequence-level for variable CpG sites (SD > 2.5%; n = 28,806) detected cis-meQTLs for 80.1% of the CpG sites, with sentinel SNPs located close to their associated CpGs. A two-step analysis was also conducted to identify long-range associations, with a particular focus on trans-meQTL hotspots. First, we identified CpG-SNP trans-associations using medium-density genotypes (50k SNPs) that revealed 31,846 SNPs with significant effects on 1 to 530 trans-CpG sites. Then, regions associated with at least 34 independent trans-CpGs were retained defining 31 hotpots. For each hotspot, a local sequence-level GWAS was conducted using the first principal component derived from the associated trans-CpGs. Out of the 31 detected hotspots, three were located close to transcription factor genes (RUNX1, NFIC and FOXA3) for which the associated trans-CpGs were enriched for the corresponding binding motif. Two other hotspots were located within KDM5A and KDM5B, and their corresponding trans-CpGs were strongly overrepresented in H3K4me3 narrow peaks in blood as well as in other tissues. Conclusions By identifying functional candidate genes associated with blood DNA methylation in cattle, these findings provide new insights into the regulatory architecture of DNA methylation in mammals, highlighting the value of large-scale molecular data from livestock populations.

Primary cilium-dependent Hedgehog signalling is essential for embryonic development, tissue patterning, and organ homeostasis, and its disruption causes a spectrum of developmental disorders collectively termed ciliopathies. Whether environmental toxicants can chemically induce ciliopathy-like states by targeting this pathway, however, remains poorly understood. Here we show that endosulfan, a banned organochlorine pesticide epidemiologically linked to severe congenital and reproductive defects in exposed human populations, disrupts Hedgehog signalling by driving GLI transcription factor processing into repressor forms and suppressing target gene expression at both transcriptional and protein levels. Having excluded direct effects on core ciliary receptors and GLI-DNA binding, we identify the pathway kinases PKA and GSK3{beta} as direct targets of endosulfan: endosulfan increases PKA activity through allosteric fine-tuning, and -- in a pharmacologically rare finding -- acts as the first reported small-molecule activator of GSK3{beta}, shifting the kinase toward a catalytically active conformation. We further identify Cetn3 and Cep250 as novel GLI-regulated genes required for centriole cohesion, both of which are repressed upon endosulfan exposure, providing a mechanistic link to the reproductive defects reported in exposed populations and animal models. These findings identify endosulfan as a candidate chemical inducer of ciliopathy and reveal how an environmental toxicant can hijack core kinase signalling to disrupt Hedgehog-dependent development.

Abstract Background. Chronic low-level exposure to lead, cadmium, mercury, and arsenic remains a determinant of premature mortality in the U.S. general population, but previous hazard-ratio analyses do not characterize how exposure shifts the lower tail of the survival distribution, where premature mortality is concentrated. Objectives. We estimated the association of whole-blood lead, whole-blood total mercury, urinary cadmium, and the sum of urinary inorganic and methylated arsenic species with the 10th, 25th, and 50th conditional quantiles of follow-up time to all-cause mortality among U.S. adults aged 40 years and older. Methods. NHANES Continuous 1999 to 2018 was linked to the National Death Index through December 31, 2019 (n = 29,652). Censored quantile regression was fit per metal on the log2 scale at quantiles {tau}{0.10, 0.25, 0.50}. A restricted-cubic-spline (RCS) censored-quantile-regression was fit for blood lead and urinary cadmium to investigate the threshold effect. Results. Over a median follow-up of 9.1 years, 7,215 deaths were ascertained. A doubling of urinary cadmium was associated with -1.57 years of follow-up (95% CI: -2.08, -1.07) at the 10th conditional quantile, -1.50 (-2.04, -0.96) at the 25th, and -1.49 (-1.93, -1.04) at the median (Benjamini Hochberg q < 0.001 throughout). A doubling of whole-blood lead was associated with -0.70 years (95% CI: -0.99, -0.40) at the 10th conditional quantile, -0.62 (-0.92,-0.31) at the 25th, and -0.61 years (-0.89, -0.34) at the median; the absolute loss was largest at {tau} = 0.10 for both metals. Urinary arsenic-metabolite sum was not associated with conditional follow-up at the estimable quantiles. Despite adjustment for dark and fatty-fish intake or DHA/EPA, whole-blood total mercury was associated with longer follow-up (i.e., negatively associated with mortality risk), possibly due to residual confounding by broader dietary or socioeconomic factors, rather than a true protective effect. The cadmium association was additionally robust to the mutual adjustment of lead. Discussion. Low-to-moderate urinary cadmium and whole-blood lead were associated with fewer years of follow-up survival at the lower-tail and median conditional quantiles of survival, with the largest absolute losses at the lower tail of the conditional survival distribution, where premature mortality is concentrated. These findings support continued reductions in U.S. cadmium exposure and lead with particular benefit for adults most vulnerable to premature death.

The leading cause of mortality and morbidity in children under the age of 5 is preterm birth. The timing of birth is influenced by both genetic and environmental factors, but the underlying mechanisms remain poorly understood, making its prediction difficult. In this study, we investigated the potential of using machine learning models to predict preterm birth based on genetic data from the Norwegian Mother, Father and Child Cohort Study (MoBa). We trained and evaluated several classification algorithms on individual-level genetic data from over 15,000 mothers and children. Our results indicate that the predictive capacity of maternal gestational duration-associated loci for preterm birth is limited, with the highest AUC values around 0.57. Additionally, incorporating more SNPs within the associated loci did not improve prediction performance. As expected, the contribution of the maternal genome to preterm birth prediction was found to be larger than that of the fetal genome. Overall, our findings suggest that while genetic testing provides some information about an individual's risk for preterm birth, further research incorporating additional factors is necessary to enhance predictability.

Significance Missense mutations can lead to pathological effects in human cells. Predictive methods that account for structural context, such as AlphaMissense, can provide pathogenicity scores. The accumulation of pathogenicity hotspots can reveal important structural features within individual proteins of protein families, such as GLUT transporters. Mapping pathogenicity scores onto the structure can thus provide a mechanistic explanation of the protein function necessary for its role in the cell. Abstract Non-synonymous amino acid substitutions (missense mutations) are common in the general population; some are causative of serious disease. Depending on their structural context, they can disrupt protein function, folding, or dynamics. Computational predictive methods developed in recent years, such as AlphaMissense, provide new insights into how missense mutations affect protein structure by predicting and mapping their pathogenicity across each amino acid in the human proteome. In this study, we identify recurring patterns of pathogenicity prediction across the GLUT family membrane transporters encoded by genes slc2a1-14. Within the GLUT transporter family, we observe higher pathogenicity profiles in the transmembrane domains, particularly in pore-lining and binding-site residues. Predicted missense pathogenicity is elevated throughout residues assigned to the central cavity, suggesting sensitivity of the transport pathway. Another finding shows higher pathogenicity in specific transmembrane helices of the protein, with the same pattern across all proteins. On the other hand, we observed lower pathogenicity values in some representatives of the GLUT family. These findings show that the pathogenicity of glucose transport within the GLUT family may be shaped by functional redundancy and physiological essentiality across GLUT groups.

BackgroundHeterochromatic long arm of mouse Y chromosome harbors the multicopy species-specific sequences Ssty, Sly, Asty and Orly that are transcribed in testis and have known functions in male fertility. Of these Ssty and Sly encode proteins - yet all the transcripts are not translated. To investigate the roles of these Y-heterochromatic transcripts further, we analyzed them. MethodsMice with 2/3rd deletion of the Y-chromosome (XYRIIIqdel) and its wild type (XYRIII) were used in this study. Bioinformatic approaches, small RNA northern blots, Electrophoretic Mobility Shift Assays, Luciferase reporter assays, dPCR analysis, RT-qPCR assays and western blotting techniques were used to identify piRNAs that regulate autosomal genes. ResultsWe demonstrate that the multicopy gene families from mouse Y-long arm generate piRNAs predominantly in testis. We observed sequences homologous to these piRNAs in the UTRs of a few autosomal genes, which are differentially expressed in the sperms of XYRIIIqdel mice. Furthermore, the Endogenous Retrovirus Element (ERV) LTR, found in the Orly1 transcript identified piRNAs in the database, showed homology to UTRs and associated genomic regions of a few autosomal genes. Orly1 showed a reduction in genomic copy number by digital PCR in XYRIIIqdel mice. One of the four autosomal genes containing the ERV segment in their UTRs, showed a differential testicular protein expression in the mutant mice. ConclusionsThus, we further elucidate that different classes of repeats from Y-chromosome regulate autosomal gene expression via piRNAs. Besides, this study also identified novel roles for a Y-derived ERV in autosomal gene regulation in testis.

Most cyanobacterial genomes are rich in the GCGATCGC octamer, also known as Highly Iterated Palindrome 1 (HIP1). Despite its description over three decades ago, the biological function of this highly abundant sequence is only beginning to be elucidated. HIP1 is recognized by two DNA methylases, DmtA and DmtC, and is characterized by its evolutionary conservation and a quasi-periodic distribution within genomes. However, whether the phylogenetic distribution of HIP1 correlates with the presence of functional categories of protein families remains unknown. Here we investigated whether certain protein families share a phylogenetic distribution with this abundant palindromic sequence across cyanobacterial genomes. Our analysis revealed a strong phylogenetic correlation between several proteins of the Type IV secretion system (T4SS) and the abundance of HIP1. This finding aligns with recent discoveries demonstrating that HIP1 enhances DNA transformation in a methylation-dependent manner in two distinct cyanobacterial species. Consequently, we hypothesize that HIP1 function as a conserved adaptation for horizontal gene transfer (HGT) at the phylum level, potentially by serving as a DNA-uptake recognition sequence in cyanobacteria. Significance statementScientists have long been baffled by the HIP1 sequence, a short, highly common, repetitive DNA pattern found across almost all cyanobacterial genomes. Our study used a whole-genome evolutionary approach and found that the presence of this repetitive pattern is tightly linked to the presence of a cells external DNA uptake system. This tight co-evolutionary relationship suggests that HIP1 isnt just random genomic feature, but a conserved evolutionary adaptation used by the entire cyanobacterial phylum to specifically enhance their ability to acquire new genes from one another.

BACKGROUND: Despite effectiveness as a once-weekly antimalarial prophylaxis, mefloquine has fallen out of favor due to its neuropsychiatric side effects. While possible genetic susceptibilities have been identified in preliminary studies, pharmacogenomic testing guidance is not available for mefloquine. METHODS: Volunteers with a history of mefloquine exposure were recruited to a cross-sectional case-control study. Pharmacogenomic analysis was performed on 7 candidate genes of interest with 16 missense variants including ORM1, MTHFR, MDR1, PYK2, HT2A, ADA, and ADORA2A. RESULTS: Fifty participants enrolled including those who had mefloquine exposure and chronic adverse effects (AEs) lasting 6 months or longer (n = 23); with subsequent AEs less than 6 months (n = 12); no AEs (n = 8); and a control group with a history of post-traumatic stress disorder (PTSD) but no mefloquine exposure (n = 7). Psychometric testing showed that mefloquine users with AEs lasting 6 months or more and PTSD patients who had not used mefloquine reported more evidence of sleep impairment, balance and equilibrium disorders, and lower levels of psychological well-being than mefloquine users who reported without AEs or with AEs but lasted less than 6 months. The ADORA2A gene was found to carry a higher burden of variation among volunteers exposed to mefloquine with AEs compared to those who did not. The variant rs141942830 within ADORA2A was observed to be higher among cases compared to the reference allele frequency listed in the gnomAD database but was found to not be significantly enriched. In addition, MTFHR gene was found to be enriched for variation in volunteers with long-term side effects compared to those with short-term or no side effects. CONCLUSIONS: Volunteers who reported long-term adverse events after exposure to mefloquine had excess rare variation within the ADORA2A gene compared to those without adverse events and those with short term adverse events. The ADORA2A rs141942830 was identified as a new variant of interest, as it was elevated but not significantly enriched among cases of long-term AEs, compared to the population frequency reported by gnomAD. These non-silent variants may serve as mediators to alternate pathways for signal transduction or drug metabolism.

Phenylketonuria (PKU) is a genetic metabolic disorder caused by the lack of functional phenylalanine hydroxylase (PAH). Elevated levels of phenylalanine (Phe) are known to be neurotoxic; however, the molecular mechanisms underlying Phe's effects remain elusive. This study investigates the impact of PKU on proteostasis, redox balance, and metabolism in BTBR PAHenu2 mice, a severe disease animal model. Combined proteomics and metabolomics revealed impaired redox homeostasis in the brain and disrupted mitochondrial energy metabolism (ATP and TCA intermediates). The dysregulation was further supported by decreased levels of ATP, reduced glutathione (GSH), cysteine, and reduced catalase activity. Western blot analyses revealed substantial remodeling of protein degradation systems: the 19S regulatory (Rpt1) subunit and 26S proteasome content and activity were significantly increased, and ubiquitinated protein levels were elevated, indicating protein turnover and activation of the ubiquitin-proteasome system. Autophagy was also activated, as evidenced by a reduced LC3-II/LC3-I ratio, decreased p62 levels, unchanged ATG5 levels, and increased HSPA8 protein expression. By contrast, UPR markers remained stable despite an increase in the oxidized-to-reduced PDI ratio, suggesting a localized shift without activation of a full ER stress response. In parallel, systemic alterations were assessed in whole blood. Indeed, GSH, cysteine, ATP and ADP were decreased in PKU, whereas NADPH increased. These changes were accompanied by reduced activities of GSH reductase and GSH peroxidase, thereby confirming metabolic and redox disruption. Collectively, these findings indicate that PKU is associated with activation of protein degradation pathways as an adaptive response to cellular stress combined with redox imbalance and energy dysregulation.

The AMY2B gene encodes pancreatic amylase, a critical enzyme for starch digestion. While previous studies have examined AMY2B copy number variation (CNV) in domestic and some wild animals, less is known about wild carnivores inhabiting regions with limited anthropogenic starch exposure. We analyzed blood samples for serum amylase activity and copy number variation in AMY2B gene from 8 wolves (Canis lupus), 11 brown bears (Ursus arctos), and 3 red foxes (Vulpes vulpes) from Bosnia and Herzegovina. AMY2B gene copy number was assessed using droplet digital PCR (ddPCR), and serum amylase activity and glucose levels were quantified. Although the number of fox samples was limited, foxes and wolves consistently harbored two copies of AMY2B, while brown bears exhibited higher CNV (3.67-8.40, mean 5.88). Serum amylase activity was highest in foxes, moderate in wolves, and variable but lower in bears. Despite differences in AMY2B copy number and serum amylase activity, circulating glucose concentrations did not differ significantly among species. Our findings suggest that variation in AMY2B copy number among wild carnivores may be associated with species-specific evolutionary histories and dietary adaptations, providing insight into genomic mechanisms underlying carbohydrate utilization in natural populations.

Congenital disorders of glycosylation and deglycosylation are rare, serious, and lethal disorders afflicting humans. CDGs and CDDGs result in loss of function enzymes which fail to build or break down oligosaccharides on proteins. This can produce protein aggregates and, in turn, reactive oxygen species that harm the cell eventually leading to autophagy and apoptosis. Because sperm contain high concentrations of polyunsaturated fatty acids, they are especially sensitive to these effects, which is understood as one of the leading factors in human male infertility. Sperm are developed in zebrafish similarly to humans and are useful models to examine human reproductive health, as well as genetic disorders. The combination of these advantages makes the analysis of sperm from zebrafish with heterozygous ALG1 or DPAGT1 CDGs or the NGLY1 CDDG suitable. Analysis of sperm concentration, motility, status, viability, and hypoosmotic swelling demonstrated the effects of these disorders on sperm quality. Results showed a significant decrease in sperm concentration, motility, and hypoosmotic swelling for all mutant zebrafish compared to the wild type. This suggests that CDGs and CDDGs influence the amount of sperm produced, the percentage of sperm cells that are mobile, and the integrity of the plasma membrane.

Herpes simplex virus type 1 (HSV-1) is a common human pathogen that undergoes lytic replication in epithelial and other permissive cell types and can establish latency in peripheral neurons. ICP22 is a multifunctional HSV-1 immediate-early protein that localizes to the nucleus of infected cells; however, its interactions with host cellular factors remain incompletely understood. Here, ICP22 was demonstrated to interact with the human antisense function 1 protein (ASF1), including both ASF1a and ASF1b, in transfected cells and HSV-1-infected cells, respectively. ICP22 also colocalized with ASF1 in the nucleus. ICP22 amino acids 213 to 340 are important for the interaction of ICP22 with ASF1, whereas amino acids 37 to 153 of ASF1a and ASF1b are critical for their interactions with ICP22. Furthermore, ICP22 expression was associated with reduced ASF1-H3.1 co-immunoprecipitation under the tested conditions. ASF1 knockdown also reduced HSV-1-BAC-Luc luciferase output, indicating that ASF1 contributes to efficient infection-associated reporter activity in this study. Collectively, these results indicate that the interaction of HSV-1 ICP22 with ASF1 might help regulate the transcription of viral or cellular genes during HSV-1 infection. Keywords: HSV-1, ICP22, ASF1, histone H3.

Chromatin immunoprecipitation-sequencing (ChIP-seq) has wide use in identifying transcription factor binding sites. DNA sequence motifs specific to a targeted transcription factor occur more frequently near ChIP-seq peak centres. The most common approach to quantifying relative motif enrichment ranks motifs by p-value. Because sample sizes can vary substantially across examined motifs, p-value magnitudes may reflect this heterogeneity rather than the biological effect of interest. As alternatives, we considered four ranking methods based on effect sizes: (a) a modified Cliffs delta, (b) the lower bound of a frequentist asymptotic confidence interval, (c) the lower bound of a frequentist finite-sample confidence interval, and (d) the lower bound of a Bayesian credible region. Through extensive simulations, the four alternatives better recovered the simulated central-enrichment ordering under heterogeneous sample sizes. Using published ChIP-seq data for GATA3, the effect size methods ranked the known targeted motif highest, even compared to highly similar motifs for other GATA family members, while p-value ranking did not. In a separate SRF application, all four alternative methods also consistently ranked the known motif highest. We recommend the asymptotic confidence interval lower bound for its simplicity, ease of implementation, and intuitive interpretation. The software is freely available (https://github.com/ScottMastro/motif-ranking).

Classical swine fever (CSF) is a highly contagious disease caused by Classical swine fever virus (CSFV), posing a serious threat to the global swine industry. This study aimed to investigate the effect of CSFV on differential genes of histone lactylation at the H3K18 site in the PI3K-AKT signaling pathway. The site with the most significant change in histone lactylation antibody level was screened by Western blot. Omics analysis was performed using CUT&Tag technology to identify differential genes in the PI3K-AKT pathway between the CSFV-infected group and the mock group, followed by validation using RT-qPCR. Functional analysis of significantly differential proteins was conducted, and the protein expression level of THBS4 was detected by Western blot. The results showed that after CSFV infection of 3D4/21 cells, the H3K18la site exhibited the most significant difference in antibody level. A total of 8,859 differential genes at the H3K18la site were identified by CUT&Tag analysis, including 6,349 up-regulated genes and 2,510 down-regulated genes. Further focusing on the PI3K-AKT signaling pathway, 10 differential genes were identified, comprising 6 up-regulated genes and 4 down-regulated genes. Compared with the control group, the mRNA expression levels of CD19, LAMA1, PDGFRA, BDNF, ANGPT4, and THBS4 were up-regulated in the CSFV-infected group, while FOXO3 and NRTN were down-regulated. Western blot results showed that the protein expression level of THBS4 increased after CSFV infection. These findings lay an important foundation for understanding the molecular mechanisms regulating viral replication and immune evasion, and have significant scientific implications and potential application value.

Precise regulation of Drosophila Myc is essential for growth and homeostasis, yet regulation of its transcriptional control remains incompletely understood. We investigated the Myc cis-regulatory landscape using in vivo reporter assays, EMSA, and LC-MS/MS-based identification of DNA-associated proteins. By truncating Myc cis-regulatory modules (CRMs), we delineated the activity of conserved non-coding elements across adult female tissues and larval stages. Specific DNA-protein interactions were confirmed by EMSA using nuclear embryonic extracts. We developed a Solid Surface Magnetic Enrichment protocol (SSMEP) to pull down DNA-protein complexes formed on Myc cis-elements. Affinity purification followed by LC-MS/MS enabled the identification of candidate transcriptional regulators associated with Myc-CRMs. This integrative approach provides new insights into promoter structure and trans-regulatory architecture of Myc and their roles in developmental gene expression programs. Our study identifies a distal enhancer required for larval and pupal patterning, with activation dependent on specific spacing relative to the TATA-box core promoter, a strong enhancer cluster within 5'-UTR cis-elements active in ovaries and embryos, and a DPE-core promoter requiring nearby enhancer action. The DPE-linked enhancer can function with both Inr-DPE and TATA-box promoters.The identified Myc-CRMs interact with conserved signaling pathways to tightly control Myc during development. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=77 SRC="FIGDIR/small/736081v1_ufig1.gif" ALT="Figure 1"> View larger version (32K): org.highwire.dtl.DTLVardef@fa98a9org.highwire.dtl.DTLVardef@3c82d3org.highwire.dtl.DTLVardef@b1025corg.highwire.dtl.DTLVardef@111e972_HPS_FORMAT_FIGEXP M_FIG C_FIG HighlightsO_LIMyc Oocyte Element: an eRNA-producing enhancer in ovaries and early embryos C_LIO_LIDPE promoter synergizes with nearby enhancer to drive Drosophila Myc transcription C_LIO_LILate enhancer licenses TATA promoter for larval tissue-specific Myc transcription C_LIO_LIPromoter-enhancer dynamics differentially drive Myc during development C_LIO_LISSMEP protocol helps purify and enrich low-abundance DNA-protein complexes C_LI

Background: Sequence-to-function (S2F) deep learning models are increasingly used to prioritize non-coding regulatory variants, but their behavior across ancestrally diverse populations remains unclear. Because both training data and reference resources are heavily European-centered, multi-ancestry benchmarks are needed to determine whether S2F scores capture regulatory effects consistently across populations with different allele-frequency and LD patterns. Methods: We evaluated Borzoi and AlphaGenome using whole blood eQTL data from the MAGENTA cohort, including African American (AA; N=224), Caribbean Hispanic (CH; N=209), and Non-Hispanic White (NHW; N=235) participants. Model predictions were benchmarked against sampled nominal eQTLs and ancestry-stratified SuSiE fine-mapped variants using Spearman correlation, direction concordance, inter-model convergence, and distance-matched AUROC, with sensitivity analyses for minor allele frequency and comparison-set definition. We also compared FILER functional annotation overlap among high-Posterior Inclusion Probability (PIP) variants across ancestries. Results: Both models showed weak agreement with nominal eQTL effect sizes across ancestries and TSS-distance bins ({rho}[&le;]0.138), with direction concordance only marginally above chance. Agreement and discrimination improved for high-confidence fine-mapped variants, and Borzoi and AlphaGenome showed stronger inter-model convergence on fine-mapped variants than on nominal eQTLs, consistent with enrichment for regulatory variants whose effects are more apparent to sequence-based models. In distance-matched AUROC analyses at PIP [&ge;]0.9 using PIP <0.01 variants as low-PIP comparison variants, the AA high-PIP variant set yielded the highest discrimination for both Borzoi (0.837 [95% CI: 0.790-0.870]) and AlphaGenome (0.820 [0.793-0.845]). The CH-versus-NHW ordering was model-dependent: Borzoi yielded higher AUROC in NHW than CH, whereas AlphaGenome produced nearly identical CH and NHW estimates. AUROC values were lower when intermediate-PIP variants were used as comparison variants, but the AA set retained the highest discrimination. MAF-stratified sensitivity analyses attenuated some ancestry contrasts but did not eliminate the higher AA discrimination pattern. Functional annotation analysis showed that AA high-PIP variants more often overlapped chromatin accessibility and chromatin-contact annotations than NHW variants, despite lower overlap with prior eQTL and sQTL annotation catalogs. Conclusions: Borzoi and AlphaGenome showed limited agreement with nominal eQTL effect sizes, but better distinguished high-confidence fine-mapped eQTLs from low-PIP variants. These results support using S2F scores as prioritization evidence for fine-mapped regulatory variants, especially promoter-proximal high-PIP variants, rather than as standalone predictors of eQTL effect size. The strongest discrimination was observed for the AA high-PIP variant set. Overall, the AA result is best interpreted as stronger separation of high-PIP variants from lower-PIP comparison variants, shaped by fine-mapping resolution, LD, the choice of comparison variants, and annotation composition.

BackgroundPrenatal cannabis use is becoming increasingly more commonplace. However, cannabis exposure is linked to adverse pregnancy outcomes, including gestational hypertension, preeclampsia, and preterm birth. The aim of this study was to determine the morphological and molecular effects of prenatal cannabinoid exposure on the placenta. MethodsPregnant Sprague-Dawley rats were exposed daily to vaporized THC (100 mg/mL) starting at gestational day (GD)5 until GD19 when dams were sacrificed and fetuses and placentas collected. Fetuses were genotyped for genetic sex and transcriptomic analysis was performed on male and female THC-exposed and control placentas. ResultsOn GD19, both the fetuses and placentas from the THC group were significantly larger than the control. When separated by sex, both male and female THC fetuses were significantly larger; however, only male THC placentas were significantly larger than male control placentas with no significant difference in placental weight between female control and THC placentas. RNA-sequencing revealed enriched biological processes related to nutrient transport and lipid catabolism, protein-lipid complex formation, and lipoprotein particle remodeling and organization. Further transcriptomic analysis determined that the differentially expressed genes and enriched biological processes related to lipid metabolism were preferentially enriched in the female THC placentas compared to the male, suggesting a sex-specific effect. DiscussionCollectively, these data present sex-specific effects of prenatal cannabinoid exposure on placental growth and global gene expression. These data also suggest that sex influences gene expression of genes related to lipid metabolism in the THC-exposed placentas.