UvA-DARE is a service provided by the library of the University of Amsterdam (https://dare.uva.nl)
UvA-DARE (Digital Academic Repository)
Gene expression in chromosomal Ridge domains : influence on transcription,
mRNA stability, codon usage, and evolution
Gierman, H.J.
Publication date
2010
Link to publication
Citation for published version (APA):
Gierman, H. J. (2010). Gene expression in chromosomal Ridge domains : influence on
transcription, mRNA stability, codon usage, and evolution.
General rights
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), other than for strictly personal, individual use, unless the work is under an open content license (like Creative Commons).
Disclaimer/Complaints regulations
If you believe that digital publication of certain material infringes any of your rights or (privacy) interests, please let the Library know, stating your reasons. In case of a legitimate complaint, the Library will make the material inaccessible and/or remove it from the website. Please Ask the Library: https://uba.uva.nl/en/contact, or a letter to: Library of the University of Amsterdam, Secretariat, Singel 425, 1012 WP Amsterdam, The Netherlands. You will be contacted as soon as possible.
7
149
Summary
7
Summary
Chapter 1 explains that the integration of the human genome sequence and
high-throughput expression data, allowed the construction of the Human Transcriptome Map. The map showed that highly expressed genes cluster into Regions of Increased Gene Expression (Ridges) in the human genome. Conversely, poorly expressed genes cluster in AT-rich, gene-poor domains called Ridges. Ridges and anti-Ridges are large chromosomal domains of several Mb that typically span 80–90 genes. GC content and gene density are high in Ridges and low in anti-Ridges. In this thesis, we investigate gene expression in Ridges by looking at the influence of Ridges on transcription, mRNA stability, codon usage and evolution.
In Chapter 2 we compare the expression of a green fluorescent protein (GFP)
reporter gene between 90 different clones of the human embryonal kidney cell line HEK293. Each clone had a single integration of the lentiviral GFP reporter construct. Our experiment provides direct evidence that chromosomal Ridge domains up-regulate expression 4-fold compared to anti-Ridges. When comparing clones with integrations in the 10 most active Ridges with the 10 most inactive anti-Ridges, the difference in GFP expression is even 8-fold. We show that this domain-wide effect is present throughout the entire Ridge domain, but breaks down directly outside Ridges. Microarray analysis of HEK293 cells demonstrates that the high GFP expression in Ridges is not caused by highly expressed neighboring genes. These findings demonstrate that Ridges provide a domain-wide up-regulation of transcription in the human genome.
Chapter 3 investigates whether the high GC content of Ridge genes further augments
their expression by increasing the thermodynamic stability of Ridge mRNAs. We find that GC content determines 90% of mRNA folding stability as predicted by calculating the free minimal energy of mRNA secondary structure. Microarray analysis of human cells treated with two transcriptional inhibitors shows that Ridge mRNAs have 1.5–2.1 hour longer half-lives than anti-Ridge mRNAs. We show that this effect is independent of mRNAs with destabilizing AU-rich element (ARE). We show that the difference in half-life is dependent on GC content and folding stability, but not on expression. We find that long-lived mRNAs are 3-fold enriched for Ridge genes compared to anti-Ridge genes. We propose that the high GC content and increased half-lives of Ridge genes, make up a system of superimposed mechanisms within Ridges that amplify gene expression.
Chapter 4 analyzes the effect of GC content on codon usage and translation initiation
in the human genome. Protein levels range from a few to millions per cell per gene, but mRNA levels only reach up to several hundreds of thousands copies per cell per gene. We hypothesize that differences in translation efficiency help establish the amplified range of protein levels. There is ample experimental evidence that the use of preferred codons (which are GC-rich), strongly increases translation efficiency. We show that Ridge genes have GC-rich coding sequences and are biased for preferred codons, compared to anti-Ridge genes. Also, optimal translation initiation sites are
7
GC-rich and more frequent in Ridge genes. We propose a model that explains how the human genome can achieve extreme levels of protein expression, by the translocation of genes to Ridges. This immediately increases their transcription, allowing for natural selection to fix such an event. Over time, the GC content of the translocated gene increases, causing an increase in the use of preferred codons and optimal translation initiation sites. Such a system would greatly enhance the range of protein expression levels in the human genome.
In Chapter 5, we investigate the role of the Polycomb-group protein enhancer of
zeste homolog 2 (EZH2) in the childhood cancer neuroblastoma. EZH2 is a known oncogene, which has been implicated in the domain-wide silencing of tumor suppressor genes in cancer. We show that EZH2 overexpression is associated with gain of chromosome arm 7q, which occurs in 57% of neuroblastoma patients. High levels of EZH2 mark a poor prognosis, independent of MYCN amplification. We show that knockdown of EZH2 induces a G1 arrest and down-regulates genes highly enriched for E2F binding sites. Pathway analysis shows that down-regulated genes are involved in DNA replication and cell cycle progression. Microarray analysis of 88 primary tumors shows that EZH2 is correlated with the same DNA replication and cell cycle genes in vivo. We propose that EZH2 is a potential oncogene that regulates cell cycle progression in neuroblastoma by interfering with the pRB/E2F pathway.
Chapter 6 discusses potential mechanisms that might underlie the domain-wide
transcriptional up-regulation by Ridges. We conclude that spreading of activating histone modifications, nuclear localization, gene density or GC content can not explain Ridges. Ridges are highly enriched for CpG islands, which are associated with the promoters of 70–90% of all genes. CpG islands recruit general transcription factors and histone acetyltransferases and can do so independently of the transcriptional status of the gene they are associated with. We propose that the high gene density of CpG islands in Ridges facilitates the transcription initiation and/or transcription of nearby genes. We discuss how during evolution, the interplay of specialized chromatin domains with CpG islands has possibly lead to the emergence of Ridges. We discuss the potential impact of Ridges on vertebrate evolution and explain how the emergence of Ridges might have contributed to the genomic organization of warm-blooded vertebrates. Warm-blooded vertebrates have very small population sizes, which strongly reduces the effectiveness of natural selection to drive changes in gene expression, especially up-regulation. We propose that Ridges compensate for the paucity of natural selection in warm-blooded vertebrates, and have accelerated their evolution by facilitating changes in the tissue-specificity and up-regulation of gene expression.
