Single Molecule Fluorescence In Situ Hybridization (smFISH) Analysis in Budding Yeast Vegetative Growth and Meiosis.

Single molecule fluorescence in situ hybridization (smFISH) is a powerful technique to study gene expression in single cells due to its ability to detect and count individual RNA molecules. Complementary to deep sequencing-based methods, smFISH provides information about the cell-to-cell variation in transcript abundance and the subcellular localization of a given RNA. Recently, we have used smFISH to study the expression of the gene NDC80 during meiosis in budding yeast, in which two transcript isoforms exist and the short transcript isoform has its entire sequence shared with the long isoform. To confidently identify each transcript isoform, we optimized known smFISH protocols and obtained high consistency and quality of smFISH data for the samples acquired during budding yeast meiosis. Here, we describe this optimized protocol, the criteria that we use to determine whether high quality of smFISH data is obtained, and some tips for implementing this protocol in other yeast strains and growth conditions

Transcription of a 5' extended mRNA isoform directs dynamic chromatin changes and interference of a downstream promoter.

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

Kinetochore inactivation by expression of a repressive mRNA.

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

Meiotic regulation of kinetochore composition and function

The survival and fitness of an organism rely on faithful transmission of the genome. In sexually reproducing organisms, genome transmission requires a specialized cell division named meiosis, in which the parental genome is shuffled and halved. The kinetochore, a conserved protein complex that mediates chromosome segregation, is a key player in meiosis. Here we investigated how the budding yeast kinetochore undergoes dynamic changes in this specialized cell division. The microtubule-binding part of the kinetochore (outer kinetochore) dissociates from the chromosomes in meiotic prophase and reassembles before the meiotic divisions. This dynamic behavior is crucial to establish the meiosis-specific chromosome segregation. We have discovered a multi-level network that regulates the dynamic kinetochore behavior by controlling the abundance of one single kinetochore subunit, Ndc80. Ndc80 is downregulated in meiotic prophase and later re-synthesized during the meiotic divisions. This fluctuation in the Ndc80 level requires regulation on Ndc80 synthesis and turnover. Central to the Ndc80 synthesis regulation is the toggling of two NDC80 mRNAs: (1) a coding mRNA that is translated into Ndc80 protein, and (2) a 5’-extended mRNA termed LUTI. Rather than coding for Ndc80, LUTI expression impedes the expression of the coding mRNA via chromatin modifications, leading to Ndc80 synthesis repression. Two meiotic master transcription factors, Ime1 and Ndt80, regulate the expression of LUTI and the coding mRNA, respectively, and integrate Ndc80 regulation into the larger meiotic program. Besides synthesis, Ndc80 degradation is temporally controlled: Ndc80 is degraded in meiotic prophase and not in metaphase I. Ndc80 degradation requires active proteasomes, a degron sequence at Ndc80’s N-terminus, and Aurora B phosphorylation on Ndc80, which is known to correct erroneous microtubule-kinetochore attachments and is rewired to degrade Ndc80 in meiotic prophase. Crucially, mis-regulation of Ndc80 abundance disrupts chromosome segregation, gamete formation, and progeny fitness, highlighting the importance of Ndc80 modulation. Altogether, this work reveals a beautiful case in evolution in which the gene expression, turnover, and activity of the kinetochore are tuned to accommodate the specialized chromosome segregation program meiosis

Meiotic regulation of kinetochore composition and function

The survival and fitness of an organism rely on faithful transmission of the genome. In sexually reproducing organisms, genome transmission requires a specialized cell division named meiosis, in which the parental genome is shuffled and halved. The kinetochore, a conserved protein complex that mediates chromosome segregation, is a key player in meiosis. Here we investigated how the budding yeast kinetochore undergoes dynamic changes in this specialized cell division. The microtubule-binding part of the kinetochore (outer kinetochore) dissociates from the chromosomes in meiotic prophase and reassembles before the meiotic divisions. This dynamic behavior is crucial to establish the meiosis-specific chromosome segregation. We have discovered a multi-level network that regulates the dynamic kinetochore behavior by controlling the abundance of one single kinetochore subunit, Ndc80. Ndc80 is downregulated in meiotic prophase and later re-synthesized during the meiotic divisions. This fluctuation in the Ndc80 level requires regulation on Ndc80 synthesis and turnover. Central to the Ndc80 synthesis regulation is the toggling of two NDC80 mRNAs: (1) a coding mRNA that is translated into Ndc80 protein, and (2) a 5’-extended mRNA termed LUTI. Rather than coding for Ndc80, LUTI expression impedes the expression of the coding mRNA via chromatin modifications, leading to Ndc80 synthesis repression. Two meiotic master transcription factors, Ime1 and Ndt80, regulate the expression of LUTI and the coding mRNA, respectively, and integrate Ndc80 regulation into the larger meiotic program. Besides synthesis, Ndc80 degradation is temporally controlled: Ndc80 is degraded in meiotic prophase and not in metaphase I. Ndc80 degradation requires active proteasomes, a degron sequence at Ndc80’s N-terminus, and Aurora B phosphorylation on Ndc80, which is known to correct erroneous microtubule-kinetochore attachments and is rewired to degrade Ndc80 in meiotic prophase. Crucially, mis-regulation of Ndc80 abundance disrupts chromosome segregation, gamete formation, and progeny fitness, highlighting the importance of Ndc80 modulation. Altogether, this work reveals a beautiful case in evolution in which the gene expression, turnover, and activity of the kinetochore are tuned to accommodate the specialized chromosome segregation program meiosis

Combination cyclin-dependent kinase 4/6 inhibitors and endocrine therapy versus endocrine monotherapy for hormonal receptor-positive, human epidermal growth factor receptor 2-negative advanced breast cancer: A systematic review and meta-analysis.

PURPOSE:This meta-analysis aimed to assess the efficacy and safety of cyclin-dependent kinase (CDK) 4/6 inhibitors plus endocrine therapy (ET) in hormonal receptor-positive (HR+), human epidermal growth factor receptor 2-negative (HER2-) advanced breast cancer (ABC). METHODS:We searched PubMed, Embase, Cochrane, ClinicalTrials.gov., ASCO, ESMO and AACR databases from inception to October 10, 2019 for randomized controlled trials (RCTs) that compared CDK 4/6 inhibitors plus ET to single-agent ET with no treatment-line restriction. The main outcomes analyzed were progression-free survival (PFS), overall survival (OS), objective response rate (ORR), clinical benefit rate (CBR), and adverse events (AEs). RESULTS:Of 938 identified studies, 9 RCTs with 5043 women were eligible and included. Compared with ET alone, CDK 4/6 inhibitors and ET combination improved in PFS (hazard ratio (HR) 0.54, 95% confidence interval (CI) 0.50-0.59, p< 0.00001) and OS (HR 0.77, 95% CI 0.69-0.85, p< 0.00001), regardless of ET strategies (HR 0.54, 95% CI 0.50-0.59 in PFS; HR 0.77, 95% CI 0.69-0.85 in OS), treatment line of advanced disease (HR 0.52, 95% CI 0.46-0.59 in PFS; HR 0.75, 95% CI 0.66-0.85 in OS) and menopausal status (HR 0.54, 95% CI 0.50-0.58 in PFS; HR 0.76, 95% CI 0.68-0.84 in OS). Higher risk of grade 3/4 AEs (RR 2.66, 95% CI 2.44-2.90, p < 0.00001) were observed in the combination group than in the ET group. CONCLUSIONS:Combination therapy with CDK 4/6 inhibitors and ET prolongs survival in HR+/ HER2- ABC. This combination is a better therapeutic strategy than endocrine monotherapy in HR+/HER2- ABC, regardless of treatment line, menopausal status and other individual characteristics

Clinicopathological and prognostic significance of chemokine receptor CXCR4 overexpression in patients with esophageal cancer: a meta-analysis

The prognostic significance of CXC chemokine receptor type 4 (CXCR4) for survival of patients with esophageal cancer remains controversial. To investigate its expression impact on clinicopathological features and survival outcome, a meta-analysis was performed. A comprehensive search in the PubMed, Embase, and Web of Science (up to October 8, 2013) was performed for relevant studies using multiple search strategies. Correlation between CXCR4 expression and clinicopathological features and overall survival (OS) was analyzed. A total of 1,055 patients with esophageal cancer from seven studies were included. The pooled odds ratios (ORs) which indicated CXCR4 expression was associated with tumor depth (OR = 0.35, confidence interval (CI) = 0.27-0.47, P < 0.00001), status of lymph node (OR = 0.36, CI = 0.21-0.61, P < 0.0002), TNM (tumor, node, metastasis) stage (OR = 0.38, CI = 0.25-0.56, P < 0.00001), and histological type (OR = 1.81, CI = 1.07-3.05, P = 0.03). Poor overall survival of esophageal cancer was found to be significantly related to CXCR4 overexpression (hazard ratio (HR) 1.49, 95 % CI = 1.24-1.80, P < 0.0001), whereas combined ORs exhibited that CXCR4 expression has no correlation with gender or tumor differentiation. Based on the published studies, CXCR4 overexpression in patients with esophageal cancer indicated worse survival outcome and was associated with common clinicopathological poor prognostic factors

An automated feeding system for the African killifish reveals the impact of diet on lifespan and allows scalable assessment of associative learning.

The African turquoise killifish is an exciting new vertebrate model for aging studies. A significant challenge for any model organism is the control over its diet in space and time. To address this challenge, we created an automated and networked fish feeding system. Our automated feeder is designed to be open-source, easily transferable, and built from widely available components. Compared to manual feeding, our automated system is highly precise and flexible. As a proof of concept for the feeding flexibility of these automated feeders, we define a favorable regimen for growth and fertility for the African killifish and a dietary restriction regimen where both feeding time and quantity are reduced. We show that this dietary restriction regimen extends lifespan in males (but not in females) and impacts the transcriptomes of killifish livers in a sex-specific manner. Moreover, combining our automated feeding system with a video camera, we establish a quantitative associative learning assay to provide an integrative measure of cognitive performance for the killifish. The ability to precisely control food delivery in the killifish opens new areas to assess lifespan and cognitive behavior dynamics and to screen for dietary interventions and drugs in a scalable manner previously impossible with traditional vertebrate model organisms