Ünal Lab Publications


2018

Tunable Transcriptional Interference at the Endogenous Alcohol Dehydrogenase Gene Locus in Drosophila melanogaster. bioRxiv

Victoria Jorgensen*, Jingxun Chen*, Helen Vander Wende*, Devon Harris, Siu Wah Wong-Deyrup, Yuzhang Chen, Prashanth Rangan, Gloria Ann Brar, Eric M Sawyer#, Leon Chan# and Elçin Ünal# (*equal contribution, #corresponding author)

Neighboring sequences of a gene can influence its expression. In the phenomenon known as transcriptional interference, transcription at one region in the genome can repress transcription at a nearby region in cis. Transcriptional interference occurs at a number of eukaryotic loci, including the alcohol dehydrogenase (Adh) gene in Drosophila melanogaster. Adh is regulated by two promoters, which are distinct in their developmental timing of activation. It has been shown using transgene insertion that when the promoter distal from the Adh start codon is deleted, transcription from the proximal promoter becomes de-regulated. As a result, the Adh proximal promoter, which is normally active only during the early larval stages, becomes abnormally activated in adults. Whether this type of regulation occurs in the endogenous Adh context, however, remains unclear. Here, we employed the CRISPR/Cas9 system to edit the endogenous Adh locus and found that removal of the distal promoter does also result in the untimely expression of the proximal promoter-driven mRNA isoform in adults, albeit at lower levels than previously reported. Importantly, we show that transcription from the distal promoter is sufficient to repress proximal transcription in larvae and that the degree of this repression depends on the degree of distal promoter activity. Finally, repression of the endogenous Adh proximal promoter is associated with the enrichment of histone 3 lysine 36 trimethylation (H3K36me3), a chromatin mark necessary for transcription-coupled gene repression in yeast. We conclude that the endogenous Adh locus is developmentally regulated by transcriptional interference in a tunable manner.

Developmental Regulation of an Organelle Tether Coordinates Mitochondrial Remodeling in Meiosis. bioRxiv

Eric M Sawyer, Pallavi Joshi, Luke E Berchowitz, Elçin Ünal

Cellular differentiation involves remodeling cellular architecture to transform one cell type to another. By investigating mitochondrial dynamics during meiotic differentiation in budding yeast, we sought to understand how organelle morphogenesis is developmentally controlled in a system where regulators of differentiation as well as organelle architecture are known, but the interface between them remains unexplored. We found that mitochondria abruptly detach from the cell cortex shortly before segregating into gametes. Mitochondrial detachment is enabled by the programmed destruction of the mitochondria-endoplasmic reticulum-cortex anchor (MECA), an organelle tether that forms contact sites between mitochondria and the plasma membrane. MECA regulation is governed by a meiotic transcription factor, Ndt80, which promotes the activation of a conserved kinase, Ime2. We found that MECA undergoes Ime2-dependent phosphorylation. Furthermore, Ime2 promotes MECA degradation in a temporally controlled manner. Our study defines a key mechanism that coordinates mitochondrial morphogenesis with the landmark events of meiosis and demonstrates that cells can developmentally regulate tethering to induce organelle remodeling.

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Evidence for an Integrated Gene Repression Mechanism based on mRNA Isoform Toggling in Human Cells. bioRxiv

Ina Hollerer, Juliet C. Barker*, Victoria Jorgensen*, Amy Tresenrider, Claire Dugast-Darzacq, Xavier Darzacq, Leon Chan, Robert Tjian R, Elçin Ünal E# and Gloria Ann Brar# (* equal contribution, # corresponding author)

We recently discovered a common mode of gene regulation in budding yeast, by which mRNA production represses protein expression. Whether this regulatory mechanism is conserved was unknown. Here we find that a similar mechanism regulates the human oncogene MDM2, which is transcribed from two promoters. Transcription from the distal MDM2 promoter produces a poorly translated mRNA isoform and transcription from the proximal promoter produces a well-translated transcript. Remarkably, we find that down-regulation of transcription from the distal promoter markedly up-regulates expression from the proximal promoter and results in the loss of histone H3K36 trimethylation marks. Moreover, we observe transcript toggling between the two different MDM2 isoforms as a natural part of two distinct human embryonic stem cell differentiation programs. We conclude that the integrated gene repression mechanism recently identified in yeast is conserved in human cells.

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Single Molecule Fluorescence In Situ Hybridization (smFISH) Analysis in Budding Yeast Vegetative Growth and Meiosis. J. Vis. Exp.  (2018)

Jingxun Chen, David T McSwiggen, Elçin Ünal

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.

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Ensuring the Fidelity of Chromosome Segregation. Mol Biol Cell. (2018)

Elçin Ünal, Jorge Z. Torres

At the 2017 first joint ASCB EMBO Meeting, the fields of cell division, cell cycle, and cell death took the center stage in three separate Minisymposia. In this review, we highlight some of the topics presented in the Minisymposium entitled “Ensuring Fidelity of Chromosome Segregation.”

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2017

One-two punch mechanism of gene repression: a fresh perspective on gene regulation. Current Genetics (2017)

Amy Tresenrider and Elçin Ünal 

Cellular differentiation depends on temporally controlled waves of gene activation and inactivation that ultimately transform one cell type into another. It is well established that transcription factor cascades coordinate the timely activation of gene expression clusters during development. In comparison, much less is understood about how gene repression events are coordinated with the transcription factor-driven waves of gene activation and how this repression is achieved at a mechanistic level. Using budding yeast as a model, we recently discovered a new gene regulatory event, whereby a central meiotic transcription factor induces the expression of an mRNA isoform to repress gene expression through an integrated transcriptional and translational mechanism. This new model could explain how gene activation and inactivation waves can be temporally coordinated. In this review, we discuss our findings and their potential implications.

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Kinetochore inactivation by expression of a repressive mRNA. eLife (2017).

Jingxun Chen*, Amy Tresenrider*, Minghao Chia, David T McSwiggen, Gianpiero Spedale, Victoria Jorgensen, Hanna Liao, Folkert Jacobus van Werven#, Elçin Ünal E (* equal contribution, # corresponding author)

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.

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Transcription of a 5' extended mRNA isoform directs dynamic chromatin changes and interference of a downstream promoter. eLife (2017)

Minghao Chia, Amy Tresenrider, Jingxun Chen, Gianpiero Spedale, Victoria Jorgensen, Elçin Ünal#, Folkert Jacobus van Werven# (# corresponding author)

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 NDC80promoter. 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.

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