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University of Groningen Post-Transcriptional Control of C/Ebpα and C/Ebpβ Proteins Zaini, Mohamad University of Groningen Post-transcriptional control of C/EBPα and C/EBPβ proteins Zaini, Mohamad Amr IMPORTANT NOTE: You are advised to consult the publisher's version (publisher's PDF) if you wish to cite from it. Please check the document version below. Document Version Publisher's PDF, also known as Version of record Publication date: 2017 Link to publication in University of Groningen/UMCG research database Citation for published version (APA): Zaini, M. A. (2017). Post-transcriptional control of C/EBPα and C/EBPβ proteins: Insights into their role in energy homeostasis and diseases. University of Groningen. Copyright Other than for strictly personal use, it is not permitted to download or to forward/distribute the text or part of it without the consent of the author(s) and/or copyright holder(s), unless the work is under an open content license (like Creative Commons). The publication may also be distributed here under the terms of Article 25fa of the Dutch Copyright Act, indicated by the “Taverne” license. More information can be found on the University of Groningen website: https://www.rug.nl/library/open-access/self-archiving-pure/taverne- amendment. Take-down policy If you believe that this document breaches copyright please contact us providing details, and we will remove access to the work immediately and investigate your claim. Downloaded from the University of Groningen/UMCG research database (Pure): http://www.rug.nl/research/portal. For technical reasons the number of authors shown on this cover page is limited to 10 maximum. Download date: 04-10-2021 Post-transcriptional Control of C/EBPα and C/EBPβ Proteins Insights into their role in energy homeostasis and diseases PhD thesis by Mohamad Amr Zaini Post-transcriptional Control of C/EBPα and C/EBPβ Proteins. Insights into their role in energy homeostasis and diseases M.A. Zaini The research described in this thesis was mostly conducted at the European Research Institute for the Biology of Ageing, University Medical Center Groningen, University of Groningen, The Netherlands. And also at Leibniz Institute for Age Research - Fritz Lipmann Institute (FLI), Jena, Germany. The printing of this thesis was financially supported by the University of Groningen and the Graduate School for Drug Exploration (GUIDE). Cover design and layout: Mohamad Amr Zaini Printing: ISBN (print): 978-94-034-0282-6 ISBN (electronic): 978-94-034-0281-9 Copyright © 2017 by Mohamad Amr Zaini. All rights reserved. No part of this thesis may be reproduced, stored in a retrieval system, or transmitted in any form or by any means without permission of the author. Post-transcriptional Control of C/EBPα and C/EBPβ Proteins Insights into their role in energy homeostasis and diseases PhD thesis to obtain the degree of PhD at the University of Groningen on the authority of the Rector Magnificus Prof. E. Sterken and in accordance with the decision by the College of Deans. This thesis will be defended in public on Monday 11 December 2017 at 12:45 hours by Mohamad Amr Zaini born on 20 June 1986 in Homs, Syria Supervisor Prof. C.F. Calkhoven Co-supervisor Dr. C. Müller Assessment Committee Prof. G. de Haan Prof. T. Heinzel Prof. J.P. Tuckermann Paranymphs: Tobias Ackermann Gertrud Kortman ﺳﻮرةﻃﻪاﻵﻳﺔ﴿١١٤﴾ “My LORD increase me in knowledge.’’ The Holy Quran (20:114) Dedication: This work is dedicated to the souls of all the Syrian martyrs in the last seven years. Chapter I Aim and outlines of the thesis 11 Chapter II C/EBPα and C/EBPβ transcription factors: Their post- 17 transcriptional control and roles in various tissues Chapter III A p300 and SIRT1 regulated acetylation switch of C/EBPα 47 controls mitochondrial function Chapter IV A screening strategy for the discovery of drugs that reduce 97 C/EBPβ-LIP translation with potential calorie restriction mimetic properties Chapter V Shwachman–Bodian–Diamond syndrome (SBDS) protein 127 deficiency impairs translation re-initiation from C/EBPα and C/EBPβ mRNAs Chapter VI Discussion and Perspectives 159 Appendices Nederlands Samenvatting 181 Acknowledgments 187 Curriculum Vitae 197 Aim and outlines of the thesis Aim and outlines of the thesis Aim C/EBPα and C/EBPβ proteins are transcription factors playing important roles in the control of cellular proliferation, differentiation and metabolism of different tissues. Their functions are strictly regulated at the translational and post-translational levels. One objective of this thesis is to investigate whether C/EBPα is acetylated, which lysine acetyl-transferases and de-acetylases are involved in this process and how acetylation can regulate C/EBPα function in cell metabolism and energy homeostasis. A second objective is to convert the upstream open reading frame (uORF)-dependent translation control mechanism of C/EBPβ into a high-throughput screening system to look for compounds that decrease C/EBPβ-LIP levels and by that act as calorie restriction mimetics based on the calorie restriction like phenotype found in C/EBPβ'uORF mice. As a third objective, we aimed to investigate whether C/EBPα and C/EBPβ translation is regulated by the SBDS ribosomal maturation protein and whether their translational deregulation is involved in the Shwachman Diamond Syndrome (SDS) which is caused by mutations in SBDS. 11 Chapter I Outlines of the thesis Chapter II I start this thesis with a general introduction about C/EBPα and C/EBPβ transcription factors highlighting their expression, structure, dimerization and DNA binding ability. I explain how three protein isoforms are translated from their mRNA by a uORF dependent translation initiation/re-initiation mechanism and how this translation is regulated. I describe how the truncated isoforms (C/EBPα-p30 and C/EBPβ-LIP) negatively regulate the function of the full-length isoforms (C/EBPα- p42 and C/EBPβ-LAP) pointing to the importance of the isoform ratio in determining their biological functions. Then, I discuss how the function of C/EBPα and C/EBPβ is regulated by different post-translational modifications including methylation, phosphorylation, acetylation and sumoylation. Finally, I demonstrate how C/EBPα and C/EBPβ regulate the differentiation in adipocytes and hematopoietic cells and how their function and expression are modulated during cellular proliferation and in different types of cancers. Chapter III We investigate if C/EBPα is acetylated and whether the acetylation of C/EBPα regulates its function in cell metabolism and energy homeostasis. We found that C/EBPα is acetylated at several lysine residues by the p300 lysine acetyl- transferase and deacetylated by the SIRT1 lysine deacetylase. We observed that acetylation has no effect on C/EBPα subcellular localization or DNA binding ability but modulates its transcriptional activity in a cell reporter assay. Transcriptome analysis performed with the Hepa1-6 mouse hepatoma cell line expressing C/EBPα mutants mimicking acetylation or deacetylation showed that 43% of the differentially expressed genes are genes involved in metabolic processes. Further analysis showed that Hepa1-6 cells expressing a C/EBPα deacetylation mimicking mutant have a higher mitochondrial mass and an increased oxygen consumption rate comparing to cells expressing the wt and acetylation mutant C/EBPα. This finding is in agreement with the results of the transcriptome analysis revealing mitochondrial genes to be significantly upregulated in cells expressing the mutant that mimics deacetylated C/EBPα. In this study, we show the importance of C/EBPα acetylation in the regulation of cell metabolism and uncover SIRT1 as critical modulator of the transcriptional function of C/EBPα. 12 Aim and outlines of the thesis Chapter IV We develop a reporter system that is applicable for a screening to find new compounds that downregulate the C/EBPβ-LIP isoform and thereby might function as anti-cancer and calorie restriction mimetic drugs. This project was based on the phenotype observed in the C/EBPβ'uORF knockin mice that have constitutively low C/EBPβ-LIP levels due to a mutation in a cis-regulatory upstream open reading frame and show increased health- and lifespans combined with reduced tumor incidence upon aging. For this goal we converted the uORF dependent translation control mechanism of C/EBPβ into a dual luciferase-based reporter system to screen for compounds that translationally decrease the C/EBPβ-LIP/LAP ratio. The reporter system was validated following genetic and pharmacological approaches and then used to screen an FDA approved drugs library following a high throughput screening strategy. Drugs that decrease the translation reinitiation index as readout of the reporter were identified and selected ones were further validated in cell culture experiments for the effect on the C/EBPβ-LIP/LAP isoform ratio and downstream effects on cellular metabolism. In this study we produced a tool to screen for compounds that specifically affect the uORF dependent translation process and might have calorie restriction and anticancer effects. Chapter V We investigate whether deregulated C/EBPα and C/EBPβ isoform expression might play a role in the Shwachman–Diamond syndrome (SDS) which is a disorder caused by mutations in the SBDS gene and which is characterized by bone marrow failure with neutropenia, exocrine pancreatic insufficiency and skeletal abnormalities. We ask the question if the failure of the ribosomal maturation protein SBDS is affecting the translation of C/EBPα and C/EBPβ mRNAs and to which extent this impaired translation is contributing to SDS phenotypes. We found that the SBDS protein plays an important role specifically in translation re-initiation and its loss of function by mutations or knockdown resulted in downregulation of the truncated isoforms, C/EBPα-p30 and C/EBPβ-LIP, while the translation of the full length isoforms, C/EBPα-p42 and C/EBPβ-LAP, was not affected. Furthermore, we found that deregulation of C/EBPα and C/EBPβ translation upon SBDS malfunction resulted in lower levels of MYC expression leading to decreased proliferation of progenitor cells which potentially contributes to the hematological phenotype of SDS.
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