👤 Eva Parisi

This study investigated the hepatic expression of genes involved in stress response (CASP6, CAT, SOD1, HSPA2) and lipid metabolism (LPL, SREBF, FABP1, ACOX1, FADS2) in a local slow-growing chicken breed, the Bionda Piemontese (BP), following two isonitrogenous diets (crude protein, 170 g/kg as fed): a control diet (L) and a high-fat diet (H) obtained by supplementing 6 % palm kernel oil, during the finisher period. Sixty (male and female) chickens were included in the study and slaughtered at five months of age. RNA was extracted from 36 liver samples (18 male and 18 female) and sequenced using the targeted RNA-seq method followed by bioinformatic analysis using DESeq2 to detect differences in gene expression. Overall, the high-fat dietary supplementation did not significantly alter the expression of most stress-related genes, indicating that the high-fat diet did not elicit a hepatic stress response in BP chickens. However, within each sex, the high-fat diet tended to upregulate FADS2 and FABP1 in females, and slightly downregulate ACOX1 in males. Considering sex as an independent factor, FABP1 expression was higher in females, whereas males exhibited significantly higher LPL expression. These findings highlight a clear sexual dimorphism in hepatic lipid gene expression in BP chickens. While dietary fat supplementation had limited impact on differentially expressed genes, the study underscores the importance of sex in shaping metabolic gene expression. It also provides evidence supporting the possible metabolic resilience of the BP breed to tolerate changes in dietary fat content. Further studies are needed to substantiate this claim, and to investigate the long-term effects and the tissue-specific responses of dietary fat supplementation in other organs. Show less

Congenital heart disease (CHD) is a prevalent condition characterized by defective heart development, causing premature death and stillbirths among infants. Genome-wide association studies (GWASs) have provided insights into the role of genetic variants in CHD pathogenesis through the identification of a comprehensive set of single-nucleotide polymorphisms (SNPs). Notably, 90-95% of these variants reside in the noncoding genome, complicating the understanding of their underlying mechanisms. Here, we developed a systematic computational pipeline for the identification and analysis of CHD-associated SNPs spanning both coding and noncoding regions of the genome. Initially, we curated a thorough dataset of SNPs from GWAS-catalog and ClinVar database and filtered them based on CHD-related traits. Subsequently, these CHD-SNPs were annotated and categorized into noncoding and coding regions based on their location. To study the functional implications of noncoding CHD-SNPs, we cross-validated them with enhancer-specific histone modification marks from developing human heart across 9 Carnegie stages and identified potential cardiac enhancers. This approach led to the identification of 2,056 CHD-associated putative enhancers (CHD-enhancers), 38.9% of them overlapping with known enhancers catalogued in human enhancer disease database. We identified heart-related transcription factor binding sites within these CHD-enhancers, offering insights into the impact of SNPs on TF binding. Conservation analysis further revealed that many of these CHD-enhancers were highly conserved across vertebrates, suggesting their evolutionary significance. Utilizing heart-specific expression quantitative trait loci data, we further identified a subset of 63 CHD-SNPs with regulatory potential distributed across various cardiac tissues. Concurrently, coding CHD-SNPs were represented as a protein interaction network and its subsequent binding energy analysis focused on a pair of proteins within this network, pinpointed a deleterious coding CHD-SNP, rs770030288, located in C2 domain of MYBPC3 protein. Overall, our findings demonstrate that SNPs have the potential to disrupt gene regulatory systems, either by affecting enhancer sequences or modulating protein-protein interactions, which can lead to abnormal developmental processes contributing to CHD pathogenesis. Show less

Stress granules (SGs) are conserved biomolecular condensates that originate in response to many stress conditions. These membraneless organelles contain nontranslating mRNAs and a diverse subproteome, but our knowledge of their regulation and functional relevance is still incipient. Here, we describe a mutual-inhibition interplay between SGs and Cdc28, the budding yeast Cdk. Among Cdc28 interactors acting as negative modulators of Start, we have identified Whi8, an RNA-binding protein that localizes to SGs and recruits the mRNA of CLN3, the most upstream G1 cyclin, for efficient translation inhibition and Cdk inactivation under stress. However, Whi8 also contributes to recruiting Cdc28 to SGs, where it acts to promote their dissolution. As predicted by a mutual-inhibition framework, the SG constitutes a bistable system that is modulated by Cdk. Since mammalian cells display a homologous mechanism, we propose that the opposing functions of specific mRNA-binding proteins and Cdk's subjugate SG dynamics to a conserved hysteretic switch. Show less

The precise coordination of growth and proliferation has a universal prevalence in cell homeostasis. As a prominent property, cell size is modulated by the coordination between these processes in bacterial, yeast, and mammalian cells, but the underlying molecular mechanisms are largely unknown. Here, we show that multifunctional chaperone systems play a concerted and limiting role in cell-cycle entry, specifically driving nuclear accumulation of the G1 Cdk-cyclin complex. Based on these findings, we establish and test a molecular competition model that recapitulates cell-cycle-entry dependence on growth rate. As key predictions at a single-cell level, we show that availability of the Ydj1 chaperone and nuclear accumulation of the G1 cyclin Cln3 are inversely dependent on growth rate and readily respond to changes in protein synthesis and stress conditions that alter protein folding requirements. Thus, chaperone workload would subordinate Start to the biosynthetic machinery and dynamically adjust proliferation to the growth potential of the cell. Show less

Cell size scales with ploidy in a great range of eukaryotes, but the underlying mechanisms remain unknown. Using various orthogonal single-cell approaches, we show that cell size increases linearly with centromere (CEN) copy number in budding yeast. This effect is due to a G1 delay mediated by increased degradation of Cln3, the most upstream G1 cyclin acting at Start, and specific centromeric signaling proteins, namely Mad3 and Bub3. Mad3 binds both Cln3 and Cdc4, the adaptor component of the Skp1/Cul1/F-box (SCF) complex that targets Cln3 for degradation, these interactions being essential for the CEN-dosage dependent effects on cell size. Our results reveal a pathway that modulates cell size as a function of CEN number, and we speculate that, in cooperation with other CEN-independent mechanisms, it could assist the cell to attain efficient mass/ploidy ratios. Show less

Cells sense myriad signals during G1, and a rapid response to prevent cell cycle entry is of crucial importance for proper development and adaptation. Cln3, the most upstream G1 cyclin in budding yeast, is an extremely short-lived protein subject to ubiquitination and proteasomal degradation. On the other hand, nuclear accumulation of Cln3 depends on chaperones that are also important for its degradation. However, how these processes are intertwined to control G1-cyclin fate is not well understood. Here, we show that Cln3 undergoes a challenging ubiquitination step required for both degradation and full activation. Segregase Cdc48/p97 prevents degradation of ubiquitinated Cln3, and concurrently stimulates its ER release and nuclear accumulation to trigger Start. Cdc48/p97 phosphorylation at conserved Cdk-target sites is important for recruitment of specific cofactors and, in both yeast and mammalian cells, to attain proper G1-cyclin levels and activity. Cdk-dependent modulation of Cdc48 would subjugate G1 cyclins to fast and reversible state switching, thus arresting cells promptly in G1 at developmental or environmental checkpoints, but also resuming G1 progression immediately after proliferative signals reappear. Show less

Cells commit to a new cell cycle at Start by activation of the G1 Cdk-cyclin complex which, in turn, triggers a genome-wide transcriptional wave that executes the G1/S transition. In budding yeast, the Cdc28-Cln3 complex is regulated by an ER-retention mechanism that is important for proper cell size control. We have isolated small-cell-size CDC28 mutants showing impaired retention at the ER and premature accumulation of the Cln3 cyclin in the nucleus. The differential interactome of a quintuple Cdc28(wee) mutant pinpointed Whi7, a Whi5 paralog targeted by Cdc28 that associates to the ER in a phosphorylation-dependent manner. Our results demonstrate that the Cln3 cyclin and Whi7 act in a positive feedback loop to release the G1 Cdk-cyclin complex and trigger Start once a critical size has been reached, thus uncovering a key nonlinear mechanism at the earliest known events of cell-cycle entry. Show less