Menu

Close

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

Ina Hollerer, Juliet C Barker, Victoria Jorgensen, Amy Tresenrider, Claire Dugast-Darzacq, Xavier Darzacq, Leon Y Chan, Robert Tjian, Elcin Unal, Gloria A Brar

Preprint posted on October 05, 2018 https://www.biorxiv.org/content/early/2018/10/05/264721

Toggling promoters and open reading frames to regulate gene expression

Selected by Clarice Hong

Categories: genetics

Background

Gene regulation is generally thought to be a linear process where transcriptional and translational regulation play independent roles in modulating gene expression. In the canonical model, mRNAs transcribed from a given gene always carry the same set of information and are later modified by other, independent mechanisms such as splicing or RNA editing. However, widespread alternative promoter usage, as shown by high-throughput transcription start site (TSS) mapping, suggests that mRNAs (from the same gene) generated by different TSSs can carry different information in their 5’ leaders, which in turn instructs translation. For example, expression can be altered by simply having one isoform contain an upstream open reading frame (uORF) that prevents proper translation of the correct ORF. The authors have first dissected this mechanism in the context of a single, essential gene and later have shown it to be pervasive in yeast meiosis. However, whether or not mammalian cells use such an integrated mechanism remains unknown. This preprint highlights a specific oncogene in humans, MDM2, where such a mechanism occurs and show that it might play an important role in regulating stem cell differentiation.

Key findings

The primary claim of the preprint is that MDM2 is regulated by an integrated transcriptional and translational mechanism. By toggling between two separate promoters, two mRNA transcripts with different translational efficiencies are produced (Figure 1). To test this hypothesis, the authors defined 3 key features of integrated regulation from their previous observations in yeast and proceeded to test whether these mechanisms also apply to MDM2 regulation.

Figure 1. Summary of MDM2 regulation by an integrated mechanism (Figure 5 of the preprint).

One, the isoform from the distal promoter contains an uORF that represses proper translation. MDM2 has previously been shown to express two different isoforms, a longer isoform called MDM2LUTI and a shorter isoform, MDM2PROX, from the distal (P1) and proximal (P2) promoters respectively. MDM2LUTI contains 2 additional uORFs in its 5’ leader, and is therefore translated less efficiently than MDM2PROX, which only carries the main ORF (and no uORF).

Two, transcription from the distal promoter disrupts transcription from the proximal promoter, allowing for a complete switch of promoter usage. This step is extremely crucial to ensure that only the desired transcript is produced. To test this, the authors used a CRISPR/dCas9-based transcription inhibition technique using a catalytically dead Cas9  (CRISPRi) to inhibit expression at the P1 promoter, and showed that expression of MDM2PROX was indeed increased. The same trend was observed despite inhibition of p53 expression, a known activator of the P2 promoter, suggesting that the upregulation of MDM2PROX was independent of p53 activation. These results imply that P1 most likely inhibits P2 expression directly through transcriptional inhibition rather than some secondary pathway. To understand how this inhibition is achieved, the authors then measured the levels of H3K36me3 in the promoters and gene body of MDM2. H3K36me3 is a co-transcriptional modification, and is thus a mark of active transcription. It has also been implicated in repression of spurious transcription. Indeed, when P1 is inhibited, there is a decrease in H3K36me3 signal at the P1 and P2 promoters. This suggests that when P1 is transcribed, H3K36me3 is deposited at P2, which then represses it activity.

Finally, usage of the two promoters should be actively regulated by a developmental switch of some sort. In this preprint, the authors assessed this using an in vitro differentiation model, where they induced human embryonic stem cell (hESC) differentiation into neuronal and endodermal lineages and measured promoter usage throughout the differentiation process. They found that neuronal precursor cells and endodermal cells appear to prefer MDM2PROX, despite higher expression of MDM2LUTI than MDM2PROX in hESCs. This suggests that promoter usage for MDM2 is developmentally regulated, and the integrated regulation could represent a mechanism by which proper gene expression is achieved during development.

What I liked about the preprint

Gene regulation is generally thought of as a complex series of steps. However, the idea that transcriptional and translational regulation can be integrated actually simplifies the model. If the different steps are independent, we would have had to evolve mechanisms of controlling each step for optimal expression. Using this mechanism, the regulation is at the level of the promoter toggle, and translational regulation is simply a consequence of transcription. Personally, I find this idea quite comforting, because it means there are less steps to conquer in our understanding of gene expression. This mechanism also highlights how non-coding DNA can be used to regulate translation instead of transcription, which is something that we can use to understand the role of non-coding variants in development and disease. Finally, I think that the authors did a really good job laying out the hypothesis and its predictions and then systematically testing each prediction, something that is too often lacking in many scientific papers.

Future directions and questions

The obvious next step from this finding is to figure out how ubiquitous this regulation mechanism is in human cells, especially for developmentally important genes. I would also be very interested to understand exactly what signals determine which promoter is used, since it seems like the MDM2LUTI is preferred in neuronal precursors and endodermal cells, but not in differentiated neurons. Specifically, since the expression of MDM2 was only measured up to day 4 of endodermal differentiation, if they were allowed to grow further, would those cells also switch back to MDM2LUTI or continue to prefer MDM2PROX ?

Furthermore, H3K36me3 is postulated to be the mechanism by which the P1 transcript represses transcription from P2. It would be important to see what happens when the H3K36me3 writer enzyme in the cell types used is downregulated. Is P1 and P2 transcription now able to occur simultaneously, or does active transcription from P1 simply limit RNA polymerase access to P2 and thereby physically block transcription? Understanding how promoter usage is regulated could lead to many insights into basic transcriptional mechanisms and disease, such as the switch of the MDM2 promoter observed in cancer.

 

 

Posted on: 30th October 2018 , updated on: 31st October 2018

Read preprint (No Ratings Yet)




  • Have your say

    Your email address will not be published. Required fields are marked *

    This site uses Akismet to reduce spam. Learn how your comment data is processed.

    Sign up to customise the site to your preferences and to receive alerts

    Register here

    Also in the genetics category:

    Super-resolution Molecular Map of Basal Foot Reveals Novel Cilium in Airway Multiciliated Cells

    Quynh Nguyen, Zhen Liu, Rashmi Nanjundappa, et al.



    Selected by Robert Mahen

    Single cell RNA-Seq reveals distinct stem cell populations that drive sensory hair cell regeneration in response to loss of Fgf and Notch signaling

    Mark E. Lush, Daniel C. Diaz, Nina Koenecke, et al.

    AND

    Distinct progenitor populations mediate regeneration in the zebrafish lateral line.

    Eric D Thomas, David Raible



    Selected by Rudra Nayan Das

    1

    The coordination of terminal differentiation and cell cycle exit is mediated through the regulation of chromatin accessibility

    Yiqin Ma, Daniel J McKay, Laura Buttitta



    Selected by Gabriel Aughey

    1

    Ribosomal DNA and the rDNA-binding protein Indra mediate non-random sister chromatid segregation in Drosophila male germline stem cells

    George Watase, Yukiko Yamashita



    Selected by Maiko Kitaoka

    A non-canonical arm of UPRER mediates longevity through ER remodeling and lipophagy.

    Joseph R Daniele, Ryo Higuchi-Sanabria, Vidhya Ramachandran, et al.



    Selected by Sandra Malmgren Hill

    Psychiatric risk gene NT5C2 regulates protein translation in human neural progenitor cells

    Rodrigo R.R. Duarte, Nathaniel D. Bachtel, Marie-Caroline Cotel, et al.



    Selected by Joanna Cross

    The Toll pathway inhibits tissue growth and regulates cell fitness in an infection-dependent manner

    Federico Germani, Daniel Hain, Denise Sternlicht, et al.



    Selected by Rohan Khadilkar

    Rapid embryonic cell cycles defer the establishment of heterochromatin by Eggless/SetDB1 in Drosophila

    Charles A Seller, Chun-Yi Cho, Patrick H O'Farrell



    Selected by Gabriel Aughey

    Signaling dynamics control cell fate in the early Drosophila embryo

    Heath E Johnson, Stanislav Y Shvartsman, Jared E Toettcher



    Selected by Yara E. Sánchez Corrales

    1

    PUMILIO hyperactivity drives premature aging of Norad-deficient mice

    Florian Kopp, Mehmet Yalvac, Beibei Chen, et al.



    Selected by Carmen Adriaens

    Arterio-Venous Remodeling in the Zebrafish Trunk Is Controlled by Genetic Programming and Flow-Mediated Fine-Tuning

    Ilse Geudens, Baptiste Coxam, Silvanus Alt, et al.



    Selected by Andreas van Impel

    CRISPR/Cas9-mediated gene deletion of the ompA gene in an Enterobacter gut symbiont impairs biofilm formation and reduces gut colonization of Aedes aegypti mosquitoes

    Shivanand Hegde, Pornjarim Nilyanimit, Elena Kozlova, et al.



    Selected by Snehal Kadam

    millepattes micropeptides are an ancient developmental switch required for embryonic patterning

    Suparna Ray, Miriam I Rosenberg, Hélène Chanut-Delalande, et al.



    Selected by Erik Clark

    Neural crest cells regulate optic cup morphogenesis by promoting extracellular matrix assembly

    Chase Dallas Bryan, Rebecca Lynne Pfeiffer, Bryan William Jones, et al.



    Selected by Ashrifia Adomako-Ankomah

    1

    The cis-regulatory logic underlying abdominal Hox-mediated repression versus activation of regulatory elements in Drosophila

    Arya Zandvakili, Juli Uhl, Ian Campbell, et al.



    Selected by Clarice Hong

    1

    Dynamic control of proinflammatory cytokines Il-1β and Tnf-α by macrophages is necessary for functional spinal cord regeneration in zebrafish

    Themistoklis M. Tsarouchas, Daniel Wehner, Leonardo Cavone, et al.



    Selected by Shikha Nayar

    1

    Close