Understanding the biological mechanisms underlying prolificacy is critical for improving prolificacy in sheep. This study utilised RNA-seq to profile the ovarian tissue from high (HP) and low (LP) prolific lines of Dohne Merino (D) and Merino (M) breeds. Differentially expressed genes (DEGs) of the four biological conditions: DHP, DLP, MHP and MLP (n = 3 each) were identified using edgeR package. Only genes with at least 10 trimmed mean of M-values (TMM) counts in all samples proceeded to differential analysis and genes considered as DEGs had a false discovery rate <= 0.05. A total of 3 DEGs were identified in DHP vs. DLP, 115 in MHP vs. MLP, 205 in DHP vs. MHP and 243 in DLP vs. MLP. Functional enrichment analysis revealed more DEGs between breeds than within breeds, suggesting more insights into the biological mechanisms underpinning prolificacy through comparisons between breeds. Merino DEGs enriched biological pathways promoting follicular cyst development in the low prolific group. Moreover, the DEGs between breeds enriched pathways linked to reproduction, cellular, immune, neuronal and metabolic pathways, while low prolific ewes demonstrated an additional downregulated pathway in skeletal development. These results suggest association between low prolificacy and poor skeletal development, which may result in lower body weight or high preweaning mortality. Furthermore, 51 genes overlapped between high and low prolific lines and were enriched in cell processes linked to both follicular growth and follicular cysts, suggesting that similar genes may be involved in follicular proliferation and follicular cysts development. We therefore recommend that breeders consider the reproductive health of high prolific ewes to prevent the development of follicular cysts in the prolific line. Overall, these findings provide insights into the biological processes underlying.

Fescue toxicosis, caused by the ingestion of ergot alkaloids present in endophyte-infected tall fescue (E+), is associated with impaired reproductive performance in beef cattle. Because early embryonic development depends on maternally derived RNAs and proteins accumulated during oocyte maturation, disruption of oocyte contents by ergot alkaloids may affect embryonic development. This study evaluated the effects of maternal exposure to E+ on reproductive performance and blastocyst gene expression in beef heifers. Twenty-four commercial Angus heifers were assigned to either E+ (n = 12) or endophyte-free (E−; n = 12) tall fescue diets for 49 days. At the end of the trial, follicles were aspirated to assess oocyte recovery and quality and to produce embryos by in vitro fertilization. Eight blastocysts (n = 4/group) were subjected to RNA sequencing and aligned to the ARS-UCD1.2 genome. Normalized gene expression counts were compared between groups to identify differentially expressed (DE) genes. Exposure to E+ reduced the number of oocytes recovered, viable oocytes, and cleaved embryos compared with controls (P < 0.05). Although the number of blastocysts did not significantly differ between treatments, embryos derived from E+ heifers exhibited delayed developmental progression. Transcriptomic analysis identified 79 DE genes (FDR < 0.05), all of which were upregulated in E+ embryos and are associated with development, extracellular matrix organization, complement and coagulation cascades, and stress responses. To our knowledge, this is the first study to support the hypothesis that preconception exposure to ergot alkaloids compromises oocyte competence, leading to inherited molecular alterations that impair early embryonic development.

Variants in RNA binding motif protein 20 (RBM20) are causative in a severe form of dilated cardiomyopathy referred to as RBM20 cardiomyopathy, yet the mechanisms are unclear. Moreover, the reason(s) for phenotypic heterogeneity in carriers with different pathogenic variants are similarly opaque. To gain insight, we carried out multi-omics analysis, including the first analysis of gene expression changes at the protein level, of mice carrying two different pathogenic variants in the RBM20 nuclear localization signal (NLS). Direct comparison of the phenotypes confirmed greater premature morality in S639G variant carrying mice compared to mice with the S637A variant despite similar cardiac remodeling and dysfunction. Analysis of differentially spliced genes uncovered alterations in the splicing of both RBM20 target genes and non-target genes, including several genes previously implicated in arrhythmia. Global proteomics analysis found that a greater number of proteins were differentially expressed in the hearts of Rbm20S639G mice relative to WT than in Rbm20S637A versus WT. Gene ontology analysis suggested greater mitochondrial dysfunction in Rbm20S639G mice, although direct comparison of protein expression in the hearts of Rbm20S639G versus Rbm20S637A mice failed to identify any significant differences. Similarly, few differences were found by direct comparison of gene expression at the transcript level in Rbm20S639G and Rbm20S637A despite greater coverage. Our data provide a comprehensive overview of gene splicing and expression differences associated with pathogenic variants in RBM20, as well as insights into the molecular underpinnings of phenotypic heterogeneity associated with different dilated cardiomyopathy-associated variants.

During early pregnancy, glucose is essential for the uterine epithelium and the developing embryo. In cows, progesterone increases the secretion of glucose into the uterine lumen. The uterine epithelium can convert glucose to fructose, but other fates of glucose in the uterine epithelium have been sparsely investigated. Therefore, our objective was to investigate how progesterone influences glucose metabolism in immortalized bovine uterine epithelial (BUTE) cells. BUTE cells were grown to 80

A common goal of the dairy industry is to shorten the calving interval to reap several benefits associated with improved fertility. Early pregnancy detection is crucial to shorten this interval, allowing for prompt reinsemination of cows that failed to conceive after the first service. Currently, the industry lacks a method to accurately predict pregnancy within the first 3 wk. The polypeptide cytokine IFN-tau (IFNT) is the primary signal for maternal recognition of pregnancy in ruminants. As IFNT is released from the early conceptus, it initiates a cascade of effects, including upregulation of IFN-stimulated genes (ISG). Expression of ISG can be detected in the peripheral blood. The present study aimed to characterize peripheral transcriptomic changes, including the ISG, as early as d 7 after embryo transfer. A total of 170 Holstein heifers received in vitro-produced embryos. Whole blood was collected from these heifers within 24 h of the embryo transfer (d 0), d 7, and d 14 after embryo transfer. The heifers were divided into 2 groups, pregnant and nonpregnant, based on pregnancy diagnosis on d 28 via ultrasound. Total RNA was extracted from the peripheral blood of pregnant and nonpregnant heifers, pooled and sequenced. Expression analysis on d 7 heifers resulted in 13 significantly differentially expressed genes mostly related to innate immunity. Differential expression analysis comparing pregnant heifers on d 0 to the same heifers on d 14 showed 51 significantly differentially expressed genes. Eight genes were further quantified through reverse-transcription quantitative real-time PCR for biological validation. On d 7 after embryo transfer, mRNA transcriptions of EDN1 , CXCL3 , CCL4 , and IL1A were significantly upregulated in pregnant heifers (n = 14) compared with nonpregnant heifers (n = 14), with respective fold changes of 8.10, 18.12, 29.60, and 29.97. Although on d 14 after embryo transfer, mRNA transcriptions of ISG15 , MX2 , OASY1 , and IFI6 were significantly upregulated in the blood of pregnant heifers (n = 14) compared with the same heifers on d 0, with respective fold changes of 5.09, 2.59, 3.89, and 3.08. These findings demonstrate that several immunerelated genes and ISG are activated during the first 2 wk after embryo transfer, which may explain how the maternal immune system accommodates the allogenic conceptus. To further investigate the diagnostic potentials of these genes, future studies are warranted to analyze the specificity and sensitivity of these biomarkers to predict early pregnancy.

Abstract Tall fescue was first introduced to the United States over 70 years ago. Since then, it has become the most common forage source for beef cattle in the Southeastern region of the US. Most tall fescue establishes a symbiotic relationship with a toxic fungal endophyte (Epichloë coenophiala), which contributes to the improved vigor of the plant due to acquired resistance to grazing, insects, heat, and drought. However, when consumed by cattle, it may cause fescue toxicosis, a condition characterized by reduced feed intake, body weight (BW) loss, vasoconstriction, and impaired reproductive performance in livestock species. Therefore, the aim of this study was to investigate the effect of endophyte-infected tall fescue consumption on growth performance and health-related traits in beef cattle. For this purpose, 24 Simmental x Angus heifers were divided into two dietary treatments, in which 12 heifers received endophyte-free tall fescue seeds (E-) and 12 heifers were fed with endophyte-infected tall fescue seeds (E+, 6,764 ppb of ergovaline) mixed with the diet for 45 d. The E+ heifers received a daily dose of 20 μg of ergovaline per kg of BW. The E- heifers received the same quantity of seeds as the E+ heifers. For each heifer, phenotypes related to growth (BW), vital signs (respiration rate [RR], and body temperature [TEMP]), and liver functions (serum albumin [ALB], alkaline phosphatase [ALP], aspartate aminotransferase [AST], gamma-glutamyltransferase [GGT], cholesterol [COL], bilirubin [BIL], and triglycerides [TRI]) were analyzed at the end of the experiment. Phenotypes were analyzed using a linear model in R with diet included as a fixed effect. For analysis of BW, the BW at the beginning of the trial was included in the model as a covariate. Results showed that COL and ALP were significantly less (P < 0.05) in the E+ heifers compared with the E- (-32.92 ± 11.16 mg/dL and -32.33 ± 13.95 U/L, respectively). Conversely, E+ heifers had greater (P < 0.05) BW, RR, TEMP, and AST when compared with E- heifers (-17.82 ± 5.12 kg, 25.83 ± 5.52 breaths/min, 0.56 ± 0.12 oC, and 5.83 ± 2.18 U/L, respectively). Measurements of ALB, BIL, GGT, and TRI were not different between E+ and E- heifers. Our results suggest that heifers consuming endophyte-infected tall fescue have compromised vital signs and metabolically altered liver, which may impair growth performance. This study increases our understanding of the biological mechanisms affected by the fescue toxin in beef cattle.

Breeding animals able to effectively respond to stress could be a long-term, sustainable, and affordable strategy to improve resilience and welfare in livestock systems. In the present study, the concentrations of 29 plasma biomarkers were used as candidate endophenotypes for metabolic stress response in single-SNP, gene- and haplotype-based GWAS using 739 healthy lactating Italian Holstein cows and 88,271 variants. Significant genetic associations were found in all the 3 GWAS approaches for plasma gamma-glutamyl transferase concentration on BTA17, for paraoxonase on BTA4, and for alkaline phosphatase and zinc on BTA2. On these chromosomes, single-SNP and gene-based chromosome-wide association studies were performed, confirming GWAS findings. The signals identified for paraoxonase, gamma-glutamyl transferase, and alkaline phosphatase were in proximity to the genes coding for them. The heritability of these 4 biomarkers ranged from moderate to high (from 0.39 to 0.54). Plasma biomarkers are known to undergo large changes in concentration during metabolic stress in the transition period, with an interindividual variability in the rate of change and recovery time. Genetics may account in part for these differences. To assess this, we studied a subset of 139 periparturient cows homozygous at 3 SNPs known to be respectively associated with concentration of plasma ceruloplasmin, paraoxonase, and gamma-glutamyl transferase. We compared the immune-metabolic profile measured in plasma at -7, +5, and +30 d relative to calving between groups of opposite homozygotes. A significant effect of the genotype was found on paraoxonase and gamma-glutamyl transferase plasma concentration at all the 3 time points. No evidence for genotype effect was detected for ceruloplasmin. Understanding the genetic control underlying metabolic stress response may suggest new approaches to foster resilience in dairy cows.

Environmental effects on gene expression and offspring development can be mediated by epigenetic modifications. It is well established that maternal diet influences DNA methylation patterns and phenotypes in the offspring; however, the epigenetic effects of paternal diet on developing offspring warrants further investigation. Here, we examined how a prepubertal methionine-enriched paternal diet affected sperm DNA methylation and its subsequent effects on embryo gene expression. Three treatment and three control rams were bred to seven ewes, and blastocysts were flushed for RNA extraction. Semen was collected from all rams and submitted for reduced representation bisulfite sequencing analysis. In total, 166 differentially methylated cytosines were identified in the sperm from treatment versus control rams. Nine genes were found to be differentially expressed in embryos produced from treatment versus control rams, and seven differentially methylated cytosines in the sperm were found to be highly correlated with gene expression in the embryos. Our results demonstrate that sperm methylation differences induced by diet may influence fetal programming.

Understanding the genomic features of livestock is essential for successful breeding programs and conservation. This information is scarce for local goat breeds in Egypt. In the current study, genomic regions with selection signatures were identified as well as runs of homozygosity (ROH), genomic inbreeding coefficients (FROH) and fixation index (FST) were detected in Egyptian Nubian, Damascus, Barki and Boer goat breeds. A total of 46,268 SNP markers and 337 animals were available for the genomic analyses. On average, 145.44, 42.02, 87.90 and 126.95 ROHs were detected per individual in the autosomal genome of the respective breeds. The mean accumulative ROH lengths ranged from 46.5 Mb in Damascus to 360 Mb in Egyptian Nubian. The short ROH segments (< 2 Mb) were most frequent in all breeds, while the longest ROH segments (> 16 Mb) were exclusively found in the Egyptian Nubian. The highest average FROH was observed in Egyptian Nubian (~ 0.12) followed by Boer (~ 0.11), while the lowest FROH was found in Damascus (~ 0.05) and Barki breed (~ 0.03). The estimated mean FST was 0.14 (Egyptian Nubian and Boer), 0.077 (Egyptian Nubian and Barki), 0.075 (Egyptian Nubian and Damascus), 0.071 (Barki and Boer), 0.064 (Damascus and Boer), and 0.015 (Damascus and Barki), for each pair of breeds. Interestingly, multiple SNPs that accounted for high FST values were observed on chromosome 6 in regions harboring ALPK1 and KCNIP4. Genomic regions overlapping both FST and ROH harbor genes related to immunity (IL4R, PHF23, GABARAP, GPS2, and CD68), reproduction (SPATA2L, TNFSF12, TMEM95, and RNF17), embryonic development (TCF25 and SOX15) and adaptation (MC1R, KDR, and KIT), suggesting potential genetic adaptations to local environmental conditions. Our results contribute to the understanding of the genetic architecture of different goat breeds and may provide valuable information for effective preservation and breeding programs of local goat breeds in Egypt.

The mammalian genome undergoes two global epigenetic reprogramming events during the establishment of primordial germ cells and in the pre-implantation embryo after fertilization. These events involve the erasure and re-establishment of DNA methylation marks. However, imprinted genes and transposable elements (TEs) maintain their DNA methylation signatures to ensure normal embryonic development and genome stability. Despite extensive research in mice and humans, there is limited knowledge regarding environmentally induced epigenetic marks that escape epigenetic reprogramming in other species. Therefore, the objective of this study was to examine the characteristics and locations of genomic regions that evade epigenetic reprogramming in sheep, as well as to explore the biological functions of the genes within these regions. In a previous study, we identified 107 transgenerationally inherited differentially methylated cytosines (DMCs) in the F1 and F2 generations in response to a paternal methionine-supplemented diet. These DMCs were found in TEs, non-repetitive regions, and imprinted and non-imprinted genes. Our findings suggest that genomic regions, rather than TEs and imprinted genes, have the propensity to escape reprogramming and serve as potential candidates for transgenerational epigenetic inheritance. Notably, 34 transgenerational methylated genes influenced by paternal nutrition escaped reprogramming, impacting growth, development, male fertility, cardiac disorders, and neurodevelopment. Intriguingly, among these genes, 21 have been associated with neural development and brain disorders, such as autism, schizophrenia, bipolar disease, and intellectual disability. This suggests a potential genetic overlap between brain and infertility disorders. Overall, our study supports the concept of transgenerational epigenetic inheritance of environmentally induced marks in mammals.

Background and aim: The complex dynamic interplay between different biological pathways involved in atherosclerosis development has rendered the identification of specific therapeutic targets a challenging quest. We aimed to identify specific genes and mechanistic pathways associated with the early development of fibroatheromas in a swine model of atherosclerosis. Methods: The Wisconsin Miniature SwineTM model of Familial Hypercholesterolemia (WMS-FH, n = 11) and genetically related WMS controls (WMS-N, n = 11) were used. The infrarenal aorta was harvested from both groups for histopathologic and transcriptomic profiling at 12 months. Bioinformatic analysis was performed to identify hub genes and pathways central to disease pathophysiology. The expression of ITGB2, the top ranked hub gene, was manipulated in cell culture and the expression of interconnected genes was tested. Results: Fibro-atheromatous lesions were documented in all WMS-FH aortic tissues and displayed internal elastic lamina (IEL) disruption, significant reduction of myofibroblast presence and disorganized collagen deposition. No fibro-atheromas were observed in the control group. A total of 266 differentially expressed genes (DEGs) were upregulated in WMS-FH aortic tissues, while 29 genes were downregulated. Top identified hub genes included ITGB2, C1QA, LCP2, SPI1, CSF1R, C5AR1, CTSS, MPEG1, C1QC, and CSF2RB. Overexpression of ITGB2 resulted in elevated expression of other interconnected genes expressed in porcine endothelial cells. Conclusion: In a swine translational model of atherosclerosis, transcriptomic analysis identified ITGB2 as a central hub gene associated inflammation and early fibroatheroma development making it a potential therapeutic target at this stage of disease.

Human patients carrying genetic mutations in RNA binding motif 20 (RBM20) develop an aggressive and arrhythmogenic form of dilated cardiomyopathy (DCM). Five of over 900 genetic mutations in RBM20 have recently been validated in animal models. Four of these five mutations are located in the arginine/serine-rich (RS) domain with the fifth in the RNA recognition motif (RRM). Interestingly, only mutations in the RS domain cause severe DCM, which suggests that disruption of RS domain function is crucial for the pathogenesis of DCM. To test this hypothesis, we generated mice expressing RBM20 with an in-frame deletion of the RS domain ( Rbm20 ΔRS ). We show that Rbm20 ΔRS mice manifest DCM with RBM20 mis-localization and granule formation, similar to that in RS domain mutation knock-in (KI) animals. Conversely, mice expressing RBM20 lacking the RRM do not exhibit RBM20 nucleocytoplasmic transport or granule formation, and, similar to RRM mutation KI mice, do not develop DCM. These data suggest that the critical nuclear localization signal (NLS) in RBM20 is located within the RS domain and that disruption of this sequence leads to RBM20 mis-localization and granule formation. In vitro experiments employing sequence deletion plasmids and immunocytochemical staining revealed that the first nine amino acids in the RS domain constitute the core NLS in RBM20, which is further supported by the fact that all three validated DCM-causing mutations are located in this segment. Analysis of plasmids containing DCM-associated mutations in other regions of RBM20, as well as in the non-NLS RS domain sequence, further confirmed that only mutations in the NLS facilitate re-localization and granule formation. Phosphorylation of residues within the NLS has been shown play an important role in protein nucleocytoplasmic transport. To test this, we mutated all phosphorylatable serine residues in the NLS to unphosphorylatable alanine or phosphomimetic aspartate and provide evidence that phosphorylation is dispensable for nucleocytoplasmic transport. Collectively, our findings identify the critical NLS in RBM20 and demonstrate that mutations in this NLS cause severe DCM through disruption of RS domain-mediated nuclear localization and sarcoplasmic granule formation.

Babesiosis and anaplasmosis are tick-borne diseases that substantially affect the economic outcomes of livestock production in tropical countries. This study aimed to evaluate the genetics of resistance to infection caused by these parasites through the estimation of heritabilities and genetic correlations of infection levels among Babesia bigemina (BigIL), B. bovis (BovIL), and Anaplasma marginale (AmIL), and tick counts (TC). The predictive ability of single and multi-trait genomic prediction models was evaluated through various combinations of these traits. To our knowledge, this is the first genomic study to examine BigIL and AmIL. Infection levels of BigIL (n = 1,882), BovIL (n =1,858), and AmIL (n =1,523) were estimated from blood samples using real-time PCR. TC phenotypes (n = 5,867) were obtained by counting the number of parasites larger than 4.5 mm from the right-hand side of each animal. Genotypic data were available for 3,977 animals which were then imputed up to similar to 777,000 SNP and, after quality control, 502,398 SNP remained for downstream analyses. Variance components for BigIL and AmIL and the genetic correlations between traits were estimated using a Bayesian approach. The single-step best linear unbiased prediction was used to estimate genomic breeding values (GBV). The heritability estimates for BigIL and AmIL were low at 0.094 and 0.090, respectively, suggesting high environmental influence levels for both traits. The genetic correlations between tick count and infection levels for BigIL (0.239), BovIL (0.160), and AmIL (-0.019) were low, as well as the correlation between AmIL and BovIL (0.043). The genetic correlations between BigIL and BovIL (0.524) and BigIL and AmIL (0.793) were high, which contributed to improved GBV accuracies when these traits were combined in multi-trait models in comparison to single-trait models. These results suggested that multi-trait genomic prediction models of infection levels for tick-borne diseases are preferable to single-trait models. Additionally, our results indicated that the TC data and the GBV based on them are not useful for predicting infection levels of BigIL, BovIL, and AmIL.

Human patients carrying genetic mutations in RNA binding motif 20 (RBM20) develop a clinically aggressive dilated cardiomyopathy (DCM). Genetic mutation knockin (KI) animal models imply that altered function of the arginine-serine-rich (RS) domain is crucial for severe DCM. To test this hypothesis, we generated an RS domain deletion mouse model (Rbm20ΔRS). We showed that Rbm20ΔRS mice manifested DCM with mis-splicing of RBM20 target transcripts. We found that RBM20 was mis-localized to the sarcoplasm in Rbm20ΔRS mouse hearts and formed RBM20 granules similar to those detected in mutation KI animals. In contrast, mice lacking the RNA recognition motif showed similar mis-splicing of major RBM20 target genes but did not develop DCM or exhibit RBM20 granule formation. Using in vitro studies with immunocytochemical staining, we demonstrated that only DCM-associated mutations in the RS domain facilitated RBM20 nucleocytoplasmic transport and promoted granule assembly. Further, we defined the core nuclear localization signal (NLS) within the RS domain of RBM20. Mutation analysis of phosphorylation sites in the RS domain suggested that this modification may be dispensable for RBM20 nucleocytoplasmic transport. Collectively, our findings revealed that disruption of RS domain-mediated nuclear localization is crucial for severe DCM caused by NLS mutations.

Transgenerational epigenetic inheritance (TEI) requires transmission of environmentally induced epigenetic changes and associated phenotypes to subsequent generations without continued exposure to the environmental factor that originated the change. TEI is well-established in plants and Caenorhabditis elegans; however, occurrence in mammals is debated and poorly understood. Here, we examined whether paternal diet from weaning to puberty-induced changes in sperm DNA methylation that were transmitted to subsequent generations. Over 100 methylated cytosines, environmentally altered in the F0 generation, were inherited by the F1 and F2 generations. Furthermore, the F0 paternal diet was associated with growth and male fertility phenotypes in subsequent generations. Differentially methylated cytosines were correlated with gene expression. Our results demonstrate that some sperm methylation sites may escape DNA methylation erasure and are transmitted to subsequent generations despite the 2 waves of epigenetic programming: in primordial germ cells and in embryos after fertilization. These results advance our understanding of the complex relationships between nature and nurture.

Dilated cardiomyopathy (DCM) is a heritable and genetically heterogenous disease often idiopathic and a leading cause of heart failure with high morbidity and mortality. DCM caused by RNA binding motif protein 20 (RBM20) mutations is diverse and needs a more complete mechanistic understanding. RBM20 mutation S637G (S639G in mice) is linked to severe DCM and early death in human patients. In this study, we generated a RBM20 S639G mutation knock-in (KI) mouse model to validate the function of S639G mutation and examine the underlying mechanisms. KI mice exhibited severe DCM and premature death with a ~ 50% mortality in two months old homozygous (HM) mice. KI mice had enlarged atria and increased ANP and BNP biomarkers. The S639G mutation promoted RBM20 trafficking and ribonucleoprotein (RNP) granules in the sarcoplasm. RNA Seq data revealed differentially expressed and spliced genes were associated with arrhythmia, cardiomyopathy, and sudden death. KI mice also showed a reduction of diastolic stiffness and impaired contractility at both the left ventricular (LV) chamber and cardiomyocyte levels. Our results indicate that the RBM20 S639G mutation leads to RNP granules causing severe heart failure and early death and this finding strengthens the novel concept that RBM20 cardiomyopathy is a RNP granule disease.

The objective of this study was to investigate potential causal relationships among hot carcass weight (HCW), longissimus muscle area (LMA), backfat thickness (BF), Warner-Bratzler shear force (WBSF), and marbling score (MB) traits in Nellore cattle using structural equation models (SEM). The SEM fitted comprises the following links between traits: WBSF -> LMA, WBSF -> HCW, HCW -> LMA, BF -> HCW, and BF -> MB, where the arrows indicate the causal direction between traits, with structural coefficients posterior means (posterior standard deviation) equal to 0.29 cm2/kg (0.09), 0.43 kg/kg (0.29), 0.10 cm2/kg (0.006), 1.92 kg/mm (0.28), and 0.03 scoregrade/mm (0.006), respectively. The final SEM revealed some important putative causal relationships among the traits studied here. The implied causal effects suggest that interventions on meat tenderness and fat content would affect overall growth and muscle deposition. Knowledge regarding potential causal relationships inferred among the traits studied here can have important implications for the genetic selection and management of Nellore cattle for improvement of carcass and meat quality.

Arginine-serine (RS) domain(s) in splicing factors are critical for protein-protein interaction in pre-mRNA splicing. Phosphorylation of RS domain is important for splicing control and nucleocytoplasmic transport in the cell. RNA-binding motif 20 (RBM20) is a splicing factor primarily expressed in the heart. A previous study using phospho-antibody against RS domain showed that RS domain can be phosphorylated. However, its actual phosphorylation sites and function have not been characterized. Using middle-down mass spectrometry, we identified 16 phosphorylation sites, two of which (S638 and S640 in rats, or S637 and S639 in mice) were located in the RSRSP stretch in the RS domain. Mutations on S638 and S640 regulated splicing, promoted nucleocytoplasmic transport and protein-RNA condensates. Phosphomimetic mutations on S638 and S640 indicated that phosphorylation was not the major cause for RBM20 nucleocytoplasmic transport and condensation in vitro. We generated a S637A knock-in (KI) mouse model (Rbm20S637A ) and observed the reduced RBM20 phosphorylation. The KI mice exhibited aberrant gene splicing, protein condensates, and a dilated cardiomyopathy (DCM)-like phenotype. Transcriptomic profiling demonstrated that KI mice had altered expression and splicing of genes involving cardiac dysfunction, protein localization, and condensation. Our in vitro data showed that phosphorylation was not a direct cause for nucleocytoplasmic transport and protein condensation. Subsequently, the in vivo results reveal that RBM20 mutations led to cardiac pathogenesis. However, the role of phosphorylation in vivo needs further investigation.

DNA methyltransferases (DNMT) and histone deacetylases (HDAC) inhibitors are used as cancer epigenome drugs. However, these epigenetic drugs lack targeting specificity and could risk inducing genome instability and the expression of oncogenes. Therefore, there is a need to develop new therapeutic strategies where specific cancer genes can be targeted for silencing or activation. The CRISPR/dCas9 system represents a promising, powerful therapeutic tool because of its simplicity and specificity. Protamine 1 (PRM1) is exclusively expressed in sperm and has a vital role in the tight packaging of DNA, thus inducing transcriptional silencing in sperm cells. We hypothesized that the activation of the PRM1 gene in tumorigenic cells would lead to DNA condensation and reduce the proliferation of these cells. To test our hypothesis, we transfected human embryonic kidney cells 293T with a dCas9-P300 plasmid that adds acetyl groups to the promoter region of PRM1 via specific gRNAs plasmids. RNA-Seq analysis of transfected cells revealed high specificity of targeted gene activation. PRM1 expression resulted in a significant decrease in cell proliferation as measured by the BrdU ELISA assay. To confirm that the activation of PRM1 was due to acetyl groups deposited to H3K27, a ChIP-qPCR was performed. The acetylation of the PRM1 promoter region targeted by dCas9-p300 in transfected cells was higher than that of the control cells. Interestingly, the targeted promoter region for acetylation showed reduced DNA methylation. These findings demonstrate the efficacy of epigenome editing in activating PRM1 in non-expressing tumorigenic cells, which could be used as a promising therapeutic strategy in cancer treatment.

homeostasis. The enrichment analysis by DAVID tool revealed 14 significant (P< 0.05) terms related to fatty acids such as cellular response to retinoic acid, glycerol transport, regulation of Notch signaling pathway, and Ras signaling pathway. These results would contribute to the improvement of nutritional and health value of beef lipids in indicine cattle by including information from causal mutations in genetic evaluation through weighted The objective of this study was to identify genomic regions associated with beef fatty acid (FA) profile in Nellore cattle finished in feedlot, using haplotype based analyzes. Also, the metabolic pathway enrichment analysis was performed to seek for genes with known functions, close to the haplotype blocks, associated with the evaluated traits, providing subsidies for a better understanding of how the genes affect this trait. A total of 963 Nellore bulls with phenotype for FA, were genotyped using the Illumina BovineHD BeadChip. Samples from the Longissimus thoracis muscle were taken for FAs profile measurement by gas chromatography using a SP-2560 capillary column. Haplotype blocks were defined based on the linkage disequilibrium (LD), where pairs of SNPs are in strong LD if the confidence interval is equal to or greater than 95% with D' equal to 0.98 and the lower limit is above 0.70. The model used to estimate the haplotypes effects for the sum of beef fatty acid groups included the fixed effects of contemporary group, animal age at slaughter (as linear covariable), haplotypes (as linear regression over the number of haplotype copies: 0, 1 e 2), and the additive genetic effect as random (polygenic effect). Genome-wide association analyzes were performed for each haplotype block (single marker association) by a Restricted Maximum Likelihood model (REML) using ASREML v. 4.3 software. A total of 15, 15, 4, 3, 6, 5, and 10 haplotypes were significantly associated (FDR< 0.10) with monounsaturated fatty acids (MUFA), polyunsaturated fatty acids (PUFA), total amount of saturated fatty acids (SFA), PUFA:SFA ratio, omega 3 (n3), omega 6 (n6), and n6:n3 ratio, respectively. The statistically significant haplotypes harbored 38 genes, among which we highlighted those involved in lipid biosynthesis and metabolism, low-density lipoprotein oxidation, transport of fatty acids and cholesterol, adipocyte functions, biosynthesis of glycoprotein hormones, regulation of serum triglyceride and high-density lipoprotein-cholesterol levels, and adipogenesis. In addition, several novel regions were identified, including a putative quantitative trait locus for lipid metabolism and energy homeostasis. The enrichment analysis by DAVID tool revealed 14 significant (P< 0.05) terms related to fatty acids such as cellular response to retinoic acid, glycerol transport, regulation of Notch signaling pathway, and Ras signaling pathway. These results would contribute to the improvement of nutritional and health value of beef lipids in indicine cattle by including information from causal mutations in genetic evaluation through weighted their effects differently in genomic predictions.