ZO-1 regulates VE-cadherin–dependent endothelial junctions and actomyosin organization, thereby influencing cell–cell tension, migration, angiogenesis, and barrier formation

Differentiation programs such as meiosis depend on extensive gene regulation to mediate cellular morphogenesis. Meiosis requires transient removal of the outer kinetochore, the complex that connects microtubules to chromosomes. How the meiotic gene expression program temporally restricts kinetochore function is unknown. We discovered that in budding yeast, kinetochore inactivation occurs by reducing the abundance of a limiting subunit, Ndc80. Furthermore, we uncovered an integrated mechanism that acts at the transcriptional and translational level to repress NDC80 expression. Central to this mechanism is the developmentally controlled transcription of an alternate NDC80 mRNA isoform, which itself cannot produce protein due to regulatory upstream ORFs in its extended 5’ leader. Instead, transcription of this isoform represses the canonical NDC80 mRNA expression in cis, thereby inhibiting Ndc80 protein synthesis. This model of gene regulation raises the intriguing notion that transcription of an mRNA, despite carrying a canonical coding sequence, can directly cause gene repression.

Cell differentiation programs require dynamic regulation of gene expression. During meiotic prophase in Saccharomyces cerevisiae, expression of the kinetochore complex subunit Ndc80 is downregulated by a 5’ extended long undecoded NDC80 transcript isoform. Here we demonstrate a transcriptional interference mechanism that is responsible for inhibiting expression of the coding NDC80 mRNA isoform. Transcription from a distal NDC80 promoter directs Set1-dependent histone H3K4 dimethylation and Set2-dependent H3K36 trimethylation to establish a repressive chromatin state in the downstream canonical NDC80 promoter. As a consequence, NDC80 expression is repressed during meiotic prophase. The transcriptional mechanism described here is rapidly reversible, adaptable to fine-tune gene expression, and relies on Set2 and the Set3 histone deacetylase complex. Thus, expression of a 5’ extended mRNA isoform causes transcriptional interference at the downstream promoter. We demonstrate that this is an effective mechanism to promote dynamic changes in gene expression during cell differentiation.

SummaryMany active eukaryotic gene promoters exhibit divergent noncoding transcription, but the mechanisms restricting expression of these transcripts are not well understood. Here, we demonstrate how a sequence-specific transcription factor represses divergent noncoding transcription at highly expressed genes in yeast. We find that depletion of the transcription factor Rap1 induces noncoding transcription in a large fraction of Rap1-regulated gene promoters. Specifically, Rap1 prevents transcription initiation at cryptic promoters near its binding sites, which is uncoupled from transcription regulation in the protein-coding direction. We further provide evidence that Rap1 acts independently of previously described chromatin-based mechanisms to repress cryptic or divergent transcription. Finally, we show that divergent transcription in the absence of Rap1 is elicited by the RSC chromatin remodeler. We propose that a sequence-specific transcription factor limits access of basal transcription machinery to regulatory elements and adjacent sequences that act as divergent cryptic promoters, thereby providing directionality toward productive transcription.

Summary Long undecoded transcript isoforms (LUTIs) represent a class of non-canonical mRNAs that downregulate gene expression through the combined act of transcriptional and translational repression. While single gene studies revealed important aspects of LUTI-based repression, how these features affect gene regulation on a global scale is unknown. Using transcript leader and direct RNA sequencing, here, we identify 74 LUTI candidates that are specifically induced in meiotic prophase. Translational repression of these candidates appears to be ubiquitous and is dependent on upstream open reading frames. However, LUTI-based transcriptional repression is variable. In only 50% of the cases, LUTI transcription causes downregulation of the protein-coding transcript isoform. Higher LUTI expression, enrichment of histone 3 lysine 36 trimethylation, and changes in nucleosome position are the strongest predictors of LUTI-based transcriptional repression. We conclude that LUTIs downregulate gene expression in a manner that integrates translational repression, chromatin state changes, and the magnitude of LUTI expression.

Yeast cells enter and undergo gametogenesis relatively asynchronously, making it technically challenging to perform stage-specific genomic and biochemical analyses. Cell-to-cell variation in the expression of the master regulator of entry into sporulation, IME1, has been implicated to be the underlying cause of asynchronous sporulation. Here, we find that timing of IME1 expression is of critical importance for inducing cells to undergo sporulation synchronously. When we force expression of IME1 from an inducible promoter in cells incubated in sporulation medium for 2 hr, the vast majority of cells exhibit synchrony during premeiotic DNA replication and meiotic divisions. Inducing IME1 expression too early or too late affects the synchrony of sporulation. Surprisingly, our approach for synchronous sporulation does not require growth in acetate-containing medium, but can be achieved in cells grown in rich medium until saturation. Our system requires solely IME1, because the expression of the N6-methyladenosine methyltransferase IME4, another key regulator of early sporulation, is controlled by IME1 itself. The approach described here can be combined easily with other stage-specific synchronization methods, and thereby applied to study specific stages of sporulation, or the complete sporulation program.

Background The start and end sites of messenger RNAs (TSSs and TESs) are highly regulated, often in a cell-type-specific manner. Yet the contribution of transcript diversity in regulating gene expression remains largely elusive. We perform an integrative analysis of multiple highly synchronized cell-fate transitions and quantitative genomic techniques in Saccharomyces cerevisiae to identify regulatory functions associated with transcribing alternative isoforms. Results Cell-fate transitions feature widespread elevated expression of alternative TSS and, to a lesser degree, TES usage. These dynamically regulated alternative TSSs are located mostly upstream of canonical TSSs, but also within gene bodies possibly encoding for protein isoforms. Increased upstream alternative TSS usage is linked to various effects on canonical TSS levels, which range from co-activation to repression. We identified two key features linked to these outcomes: an interplay between alternative and canonical promoter strengths, and distance between alternative and canonical TSSs. These two regulatory properties give a plausible explanation of how locally transcribed alternative TSSs control gene transcription. Additionally, we find that specific chromatin modifiers Set2, Set3, and FACT play an important role in mediating gene repression via alternative TSSs, further supporting that the act of upstream transcription drives the local changes in gene transcription. Conclusions The integrative analysis of multiple cell-fate transitions suggests the presence of a regulatory control system of alternative TSSs that is important for dynamic tuning of gene expression. Our work provides a framework for understanding how TSS heterogeneity governs eukaryotic gene expression, particularly during cell-fate changes.

Despite extensive efforts to address it, the vastness of uncharacterized ‘dark matter’ microbial genetic diversity can impact short-read sequencing based metagenomic studies. Population-specific biases in genomic reference databases can further compound this problem. Leveraging advances in hybrid assembly (using short and long reads) and Hi-C technologies in a cross-sectional survey, we deeply characterized 109 gut microbiomes from three ethnicities in Singapore to comprehensively reconstruct 4497 medium and high-quality metagenome assembled genomes, 1708 of which were missing in short-read only analysis and with >28× N50 improvement. Species-level clustering identified 70 (>10% of total) novel gut species out of 685, improved reference genomes for 363 species (53% of total), and discovered 3413 strains unique to these populations. Among the top 10 most abundant gut bacteria in our study, one of the species and >80% of strains were unrepresented in existing databases. Annotation of biosynthetic gene clusters (BGCs) uncovered more than 27,000 BGCs with a large fraction (36–88%) unrepresented in current databases, and with several unique clusters predicted to produce bacteriocins that could significantly alter microbiome community structure. These results reveal significant uncharacterized gut microbial diversity in Southeast Asian populations and highlight the utility of hybrid metagenomic references for bioprospecting and disease-focused studies.