Site-specific Phosphorylation of Histone H3K36 Methyltransferase Set2p and Demethylase Jhd1p is Required for Stress Responses in Saccharomyces cerevisiae

Histone lysine methylation is a key epigenetic modification that regulates eukaryotic transcription. In Saccharomyces cerevisiae, it is controlled by a reduced but evolutionarily conserved suite of methyltransferase (Set1p, Set2p, Dot1p, and Set5p) and demethylase (Jhd1p, Jhd2p, Rph1p, and Gis1p) enzymes. Many of these enzymes are extensively phosphorylated in vivo; however, the functions of almost all phosphosites remain unknown. Here, we comprehensively analyse the phosphoregulation of the yeast histone methylation network by functionally investigating 40 phosphosites on six enzymes. A total of 82 genomically-edited S. cerevisiae strains were generated through mutagenesis of sites to aspartate as a phosphomimetic or alanine as a phosphonull. These phosphosite mutants were screened for changes in native H3K4, H3K36, and H3K79 methylation levels, and for sensitivity to environmental stress conditions. For methyltransferase Set2p, we found that phosphorylation at threonine 127 significantly decreased H3K36 methylation in vivo, and that an N-terminal phosphorylation cluster at serine residues 6, 8, and 10 is required for the diamide stress response. Proteomic analysis of Set2p phosphosite mutants revealed a specific downregulation of membrane-associated proteins and processes, consistent with changes brought about by SET2 deletion and the sensitivity of mutants to diamide. For demethylase Jhd1p, we found that its sole phosphorylation site at serine 44 is required for the cold stress response. This study represents the first systematic investigation into the phosphoregulation of the epigenetic network in any eukaryote, and shows that phosphosites on histone methylation enzymes are required for a normal cellular response to stress in S. cerevisiae

RNA sequencing of identical twins discordant for autism reveals blood-based signatures implicating immune and transcriptional dysregulation

Background: A gap exists in our mechanistic understanding of how genetic and environmental risk factors converge at the molecular level to result in the emergence of autism symptoms. We compared blood-based gene expression signatures in identical twins concordant and discordant for autism spectrum condition (ASC) to differentiate genetic and environmentally driven transcription differences, and establish convergent evidence for biological mechanisms involved in ASC. Methods: Genome-wide gene expression data were generated using RNA-seq on whole blood samples taken from 16 pairs of monozygotic (MZ) twins and seven twin pair members (39 individuals in total), who had been assessed for ASC and autism traits at age 12. Differential expression (DE) analyses were performed between (a) affected and unaffected subjects (N = 36) and (b) within discordant ASC MZ twin pairs (total N = 11) to identify environmental-driven DE. Gene set enrichment and pathway testing was performed on DE gene lists. Finally, an integrative analysis using DNA methylation data aimed to identify genes with consistent evidence for altered regulation in cis. Results: In the discordant twin analysis, three genes showed evidence for DE at FDR < 10%: IGHG4, EVI2A and SNORD15B. In the case-control analysis, four DE genes were identified at FDR<10% including IGHG4, PRR13P5, DEPDC1B, and ZNF501. We find enrichment for DE of genes curated in the SFARI human gene database. Pathways showing evidence of enrichment included those related to immune cell signalling and immune response, transcriptional control and cell cycle/proliferation. Integrative methylomic and transcriptomic analysis identified a number of genes showing suggestive evidence for cis dysregulation. Limitations: Identical twins stably discordant for ASC are rare, and as such the sample size was limited and constrained to the use of peripheral blood tissue for transcriptomic and methylomic profiling. Given these primary limitations, we focused on transcript-level analysis. Conclusions: Using a cohort of ASC discordant and concordant MZ twins, we add to the growing body of transcriptomic-based evidence for an immune-based component in the molecular aetiology of ASC. Whilst the sample size was limited, the study demonstrates the utility of the discordant MZ twin design combined with multi-omics integration for maximising the potential to identify disease-associated molecular signals

14-3-3 and its binding partners are regulators of protein-protein interactions during spermatogenesis

During spermatogenesis, spermiation takes place at the adluminal edge of the seminiferous epithelium at stage VIII of the epithelial cycle during which fully developed spermatids (i.e. spermatozoa) detach from the epithelium in adult rat testes. This event coincides with the migration of preleptotene/leptotene spermatocytes across the blood-testis barrier from the basal to the apical (or adluminal) compartment. At stage XIV of the epithelial cycle, Pachytene spermatocytes (diploid, 2n) differentiate into diplotene spermatocytes (tetraploid, 4n) in the apical compartment of the epithelium, which begin meiosis I to be followed by meiosis II to form spermatids (haploid, 1n) at stage XIV of the epithelial cycle. These spermatids, in turn, undergo extensive morphological changes and traverse the seminiferous epithelium until they differentiate into elongated spermatids. Thus, there are extensive changes at the Sertoli-Sertoli and Sertoli-germ cell interface via protein 'coupling' and 'uncoupling' between cell adhesion protein complexes, as well as changes in interactions between integral membrane proteins and their peripheral adaptors, regulatory protein kinases and phosphatases, and the cytoskeletal proteins. These precisely coordinated protein-protein interactions affect cell adhesion and cell movement. In this review, we focus on the 14-3-3 protein family, whose members have different binding partners in the seminiferous epithelium. Recent studies have illustrated that 14-3-3 affects protein-protein interactions in the seminiferous epithelium, and regulates cell adhesion possibly via its effects on intracellular protein trafficking and cell-polarity proteins. This review provides a summary on the latest findings regarding the role of 14-3-3 family of proteins and their potential implications on spermatogenesis. We also highlight research areas that deserve attentions by investigators. © 2009 Society for Endocrinology.link_to_subscribed_fulltex

An In Vitro System to Study Sertoli Cell Blood-Testis Barrier Dynamics

The use of an in vitro system based on primary cultures of Sertoli cells isolated from rat testes has greatly facilitated the study of the blood-testis barrier in recent years. Herein, we summarize the detailed procedures on the isolation of undifferentiated Sertoli cells from 20-day-old rat testes, the culture of these cells as a monolayer on Matrigel-coated bicameral units, the characterization of these cultured cells, and the use of the Sertoli cell epithelium for monitoring the integrity of the Sertoli cell blood-testis barrier. This information is based on the routine use of this system in our laboratory to study the Sertoli cell blood-testis barrier in the past two decades, which should be helpful for investigators in the field

Sertoli cells are the target of environmental toxicants in the testis – a mechanistic and therapeutic insight

INTRODUCTION: Sertoli cells support germ cell development in the testis via an elaborate network of cell junctions that confers structural, communicating, and signaling support. However, Sertoli cell junctions and cytoskeletons are the target of environmental toxicants. Because germ cells rely on Sertoli cells for the provision of structural/functional/nutritional support, exposure of males to toxicants leads to germ cell exfoliation due to Sertoli cell injuries. Interestingly, the molecular mechanism(s) by which toxicants induce cytoskeletal disruption that leads to germ cell exfoliation is unclear, until recent years, which are discussed herein. This information can possibly be used to therapeutically manage toxicant-induced infertility/subfertility in human males. AREAS COVERED: In this review, we provide a brief update on the use of Sertoli cell system developed for rodents and humans in vitro, which can be deployed in any research laboratory with minimal upfront setup costs. These systems can be used to collect reliable data applicable to studies in vivo. We also discuss the latest findings on the mechanisms by which toxicants induce Sertoli cell injury, in particular cytoskeletal disruption. We also identify candidate molecules that are likely targets of toxicants. EXPERT OPINION: We provide two hypothetical models delineating the mechanism by which toxicants induce germ cell exfoliation and blood–testis barrier disruption. We also discuss molecules that are the targets of toxicants as therapeutic candidates

Polarity protein Crumbs homolog-3 (CRB3) regulates ectoplasmic specialization dynamics through its action on F-actin organization in Sertoli cells

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