Sex as a Biological Variable in Nutrition Research: From Human Studies to Animal Models.

Biological sex is a fundamental source of phenotypic variability across species. Males and females have different nutritional needs and exhibit differences in nutrient digestion and utilization, leading to different health outcomes throughout life. With personalized nutrition gaining popularity in scientific research and clinical practice, it is important to understand the fundamentals of sex differences in nutrition research. Here, we review key studies that investigate sex dimorphism in nutrition research: sex differences in nutrient intake and metabolism, sex-dimorphic response in nutrient-restricted conditions, and sex differences in diet and gut microbiome interactions. Within each area above, factors from sex chromosomes, sex hormones, and sex-specific loci are highlighted.

The 19S proteasome is directly involved in the regulation of heterochromatin spreading in fission yeast.

Cumulative evidence suggests that non-proteolytic functions of the proteasome are involved in transcriptional regulation, mRNA export, and ubiquitin-dependent histone modification and thereby modulate the intracellular levels of regulatory proteins implicated in controlling key cellular functions. To date, the non-proteolytic roles of the proteasome have been mainly investigated in euchromatin; their effects on heterochromatin are largely unknown. Here, using fission yeast as a model, we randomly mutagenized the subunits of the 19S proteasome subcomplex and sought to uncover a direct role of the proteasome in heterochromatin regulation. We identified a mutant allele, rpt4-1, that disrupts a non-proteolytic function of the proteasome, also known as a non-proteolytic allele. Experiments performed using rpt4-1 cells revealed that the proteasome is involved in the regulation of heterochromatin spreading to prevent its uncontrolled invasion into neighboring euchromatin regions. Intriguingly, the phenotype of the non-proteolytic rpt4-1 mutant resembled that of epe1Δ cells, which lack the Epe1 protein that counteracts heterochromatin spreading. Both mutants exhibited variegated gene-silencing phenotypes across yeast colonies, spreading of heterochromatin, bypassing of the requirement for RNAi in heterochromatin formation at the outer repeat region (otr), and up-regulation of RNA polymerase II. Further analysis revealed Mst2, another factor that antagonizes heterochromatin spreading, may function redundantly with Rpt4. These observations suggest that the 19S proteasome may be involved in modulating the activities of Epe1 and Mst2. In conclusion, our findings indicate that the proteasome appears to have a heterochromatin-regulating function that is independent of its canonical function in proteolysis.

Lack of accelerated ovarian aging in a follicle-stimulating hormone receptor haploinsufficiency model.

Follicle-stimulation hormone (FSH) and FSH receptor (FSHR) signaling is essential for lifelong ovarian and endocrine functions in females. Previous studies have reported that Fshr haploinsufficiency in female mice led to accelerated ovarian aging, including anticipated progressive fertility decline, irregular estrus cycles, increased follicular atresia and premature ovarian failure at 7 to 9 months of age. Interestingly, these phenotypes resemble key characteristics of human menopause and thus Fshr haploinsufficiency was proposed as a promising research mouse model of menopause. However, the Fshr haploinsufficiency model had not been fully explored, especially at the molecular level. In this study, we characterized the ovarian and endocrine functions of a Fshr heterozygous knockout allele that was generated on the C57BL/6 genetic background as part of the Knockout Mouse Project (KOMP). Based on our analyses of these mice using a breeding assay, ovarian tissue histology and serum hormone quantifications (i.e. FSH, AMH, INHA) analyses, the KOMP Fshr heterozygous knockout female mice do not show the anticipated phenotypes of ovarian aging in terms of fertility and endocrine function. We further confirmed that the expression of Fshr is unaltered in the ovaries of the KOMP Fshr heterozygous knockout animals compared to wild-type. Together, our data suggests that the KOMP Fshr heterozygous knockout strain does not recapitulate the previously reported ovarian aging phenotypes associated to another model of Fshr haploinsufficiency.

Single-cell RNA-seq of primary bone marrow neutrophils from female and male adult mice.

Widespread sex-dimorphism is observed in the mammalian immune system. Consistently, studies have reported sex differences in the transcriptome of immune cells at the bulk level, including neutrophils. Neutrophils are the most abundant cell type in human blood, and they are key components of the innate immune system as they form a first line of defense against pathogens. Neutrophils are produced in the bone marrow, and differentiation and maturation produce distinct neutrophil subpopulations. Thus, single-cell resolution studies are crucial to decipher the biological significance of neutrophil heterogeneity. However, since neutrophils are very RNA-poor, single-cell profiling of these cells has been technically challenging. Here, we generated a single-cell RNA-seq dataset of primary neutrophils from adult female and male mouse bone marrow. After stringent quality control, we found that previously characterized neutrophil subpopulations can be detected in both sexes. Additionally, we confirmed that canonical sex-linked markers are differentially expressed between female and male cells across neutrophil subpopulations. This dataset provides a groundwork for comparative studies on the lifelong transcriptional sexual dimorphism of neutrophils.

Protocol for isolation of adult mouse ear pinnae-derived primary fibroblasts.

Researchers need in vitro models that mirror the biology of organisms. Primary fibroblasts play essential roles in wound healing and are present in many tissues. They are widely used in studies of cell cycle control, reprogramming, and aging. Though extraction protocols exist, alternatives that maximize use of available resources are useful. Here, we present our protocol for extracting primary fibroblasts from adult mouse ear pinnae, an often-discarded source of primary cells, which consistently yield large, pure numbers of primary fibroblasts.

Multi-omic profiling of primary mouse neutrophils predicts a pattern of sex and age-related functional regulation.

Neutrophils are the most abundant human white blood cell and constitute a first line of defense in the innate immune response. Neutrophils are short-lived cells, and thus the impact of organismal aging on neutrophil biology, especially as a function of biological sex, remains poorly understood. Here, we describe a multi-omic resource of mouse primary bone marrow neutrophils from young and old female and male mice, at the transcriptomic, metabolomic and lipidomic levels. We identify widespread regulation of neutrophil 'omics' landscapes with organismal aging and biological sex. In addition, we leverage our resource to predict functional differences, including changes in neutrophil responses to activation signals. To date, this dataset represents the largest multi-omics resource for neutrophils across sex and ages. This resource identifies neutrophil characteristics which could be targeted to improve immune responses as a function of sex and/or age.

The microbiome: an emerging key player in aging and longevity.

Revolutionary advancements of high-throughput sequencing and metagenomic tools have provided new insights to microbiome function, including a bidirectional relationship between the microbiome and host aging. The intestinal tract is the largest surface in the human body that directly interacts with foreign antigens - it is covered with extremely complex and diverse community of microorganisms, known as the gut microbiome. In a healthy gut, microbial communities maintain a homeostatic metabolism and reside within the host in a state of immune tolerance. Abnormal shifts in the gut microbiome, however, have been implicated in the pathogenesis of age-related chronic diseases, including obesity, cardiovascular diseases and neurodegenerative diseases. The gut microbiome is emerging as a key factor in the aging process. In this review, we describe studies of humans and model organisms that suggest a direct causal role of the gut microbiome on host aging. Additionally, we also discuss sex-dimorphism in the gut microbiome and its possible roles in age-related sex-dimorphic phenotypes. We also provide an overview of widely used microbiome analysis methods and tools which could be used to explore the impact of microbiome remodeling on aging.

Dynamic modules of the coactivator SAGA in eukaryotic transcription.

SAGA (Spt-Ada-Gcn5 acetyltransferase) is a highly conserved transcriptional coactivator that consists of four functionally independent modules. Its two distinct enzymatic activities, histone acetylation and deubiquitylation, establish specific epigenetic patterns on chromatin and thereby regulate gene expression. Whereas earlier studies emphasized the importance of SAGA in regulating global transcription, more recent reports have indicated that SAGA is involved in other aspects of gene expression and thus plays a more comprehensive role in regulating the overall process. Here, we discuss recent structural and functional studies of each SAGA module and compare the subunit compositions of SAGA with related complexes in yeast and metazoans. We discuss the regulatory role of the SAGA deubiquitylating module (DUBm) in mRNA surveillance and export, and in transcription initiation and elongation. The findings suggest that SAGA plays numerous roles in multiple stages of transcription. Further, we describe how SAGA is related to human disease. Overall, in this report, we illustrate the newly revealed understanding of SAGA in transcription regulation and disease implications for fine-tuning gene expression.

SAGA DUBm-mediated surveillance regulates prompt export of stress-inducible transcripts for proteostasis.

During stress, prompt export of stress-inducible transcripts is critical for cell survival. Here, we characterize a function of the SAGA (Spt-Ada-Gcn5 acetyltransferase) deubiquitylating module (DUBm) in monitoring messenger ribonucleoprotein (mRNP) biogenesis to regulate non-canonical mRNA export of stress-inducible transcripts. Our genetic and biochemical analyses suggest that there is a functional relationship between Sgf73p of DUBm and the essential mRNA export factor, Yra1p. Under physiological conditions, Sgf73p is critical for the proper chromatin localization and RNA binding of Yra1p, while also quality controlling the biogenesis of mRNPs in conjunction with the nuclear exosome exonuclease, Rrp6p. Under environmental stress, when immediate transport of stress-inducible transcripts is imperative, Sgf73p facilitates the bypass of canonical surveillance and promotes the timely export of necessary transcripts. Overall, our results show that the Sgf73p-mediated plasticity of gene expression is important for the ability of cells to tolerate stress and regulate proteostasis to survive under environmental uncertainty.

Dot1 regulates nucleosome dynamics by its inherent histone chaperone activity in yeast.

Dot1 (disruptor of telomeric silencing-1, DOT1L in humans) is the only known enzyme responsible for histone H3 lysine 79 methylation (H3K79me) and is evolutionarily conserved in most eukaryotes. Yeast Dot1p lacks a SET domain and does not methylate free histones and thus may have different actions with respect to other histone methyltransferases. Here we show that Dot1p displays histone chaperone activity and regulates nucleosome dynamics via histone exchange in yeast. We show that a methylation-independent function of Dot1p is required for the cryptic transcription within transcribed regions seen following disruption of the Set2-Rpd3S pathway. Dot1p can assemble core histones to nucleosomes and facilitate ATP-dependent chromatin-remodeling activity through its nucleosome-binding domain, in vitro. Global analysis indicates that Dot1p appears to be particularly important for histone exchange and chromatin accessibility on the transcribed regions of long-length genes. Our findings collectively suggest that Dot1p-mediated histone chaperone activity controls nucleosome dynamics in transcribed regions.

Separation of a functional deubiquitylating module from the SAGA complex by the proteasome regulatory particle.

Gene expression is an intricate process tightly linked from gene activation to the nuclear export of mRNA. Recent studies have indicated that the proteasome is essential for gene expression regulation. The proteasome regulatory particle binds to the SAGA complex and affects transcription in an ATP-dependent manner. Here we report that a specific interaction between the proteasomal ATPase, Rpt2p and Sgf73p of the SAGA complex leads to the dissociation of the H2Bub1-deubiquitylating module (herein designated the Sgf73-DUBm) from SAGA both in vitro and in vivo. We show that the localization of the Sgf73-DUBm on chromatin is perturbed in rpt2-1, a strain of Saccharomyces cerevisiae that is specifically defective in the Rpt2p-Sgf73p interaction. The rpt2-1 mutant also exhibits impaired localization of the TREX-2 and MEX67-MTR2 complexes and is defective in mRNA export. Our findings collectively demonstrate that the proteasome-mediated remodelling of the SAGA complex is a prerequisite for proper mRNA export.