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Transcription factor regulation.
In many cases, cells need to rapidly express certain genes, for example when exposed to environmental changes or certain molecules. Often these cases require a very quick transcriptional response. However, expressing these genes continuously in the absence of stimuli is very costly to the cell. Continuous expression of such genes might also hamper other essential processes of the cell. This requires a switching mechanism by which the cell will express the required gene only when the proper stimulus is present.
It has been shown that specific transcription factors are the key to this control. There are transcription factors that are present at all times in the cell, albeit in an inactive form. It is only upon stimulation of the cell that these factors gain their activity.
The types of stimuli that the cell may encounter can be very different, ranging from physical stresses such as heat shocks to a change of energy source, and comprising defensive responses to toxic molecules such as antibiotics or heavy metals. The means by which transcription factors switch from an inactive form to an active one are just as different.
Some transcription factors require the binding of a ligand in order to become active. At this time, this is by far the most common control mechanism encountered in cells, and ligands can be anything from toxic or unwanted molecules like heavy metals or antibiotics to energy sources such as galactose in yeast, or hormones in multicellular organisms. The interaction between the ligand and the transcription factor can have varying effects from one transcription factor to the next. All imply a change of conformation, but for example the binding of Ace1 to copper ions in yeasts results in binding of Ace1 to the promoter of the metallothionein gene, and its consequent transcription (Thiele 1992). In a totally different scenario, when the thyroid hormone receptor binds its ligand, its DNA binding capability remains unchanged. The presence or absence of the ligand doesn’t change the fact that this receptor remains bound to its target sequences. What the ligand does do is modify the receptor so that instead of binding co-repressors, it binds co-activators, resulting in activation of the target gene (Brzozowski et al. 1997; Wurtz et al. 1996).
This mechanism is very much related to that of other transcription factors of the nuclear receptor family. These may bind specific molecules that have little or no impact on their DNA binding capability, but modifies the receptor so that it may bind a co-activator (Brzozowski et al. 1997; Willmann & Beato 1986). Interestingly, some of these nuclear receptors, of the steroid receptor family, apparently have another control mechanism. In the absence of their ligands, these receptors are sequestered in the cytoplasm, in complex with a chaperone protein, hsp90 (Pratt 1997). When the cell is subjected to hormone treatment, the interaction between the receptor and hsp90 is disrupted, and the receptors translocate to the nucleus where they may bind their target sequences.
The regulation of a transcription factor activity by relocating it to a different compartment than the nucleus is a mechanism shared with some other transcription factors, for example Yap1, which locates to the nucleus only when levels of oxidative stress are high (Wood et al. 2003; Wood et al. 2004). However, although Yap1 is exported out of the nucleus when oxygen levels lower, the same does not happen to steroid hormone receptors when hormone levels are depleted. In fact, when these receptors bind to their ligands and translocate to the nucleus, they remain there even after removal of the hormones, although they are in an inactive form. It is only upon the next cell division that these return to the cytoplasm.
The use of fluorescence microscopy.
Recent advances in fluorescence microscopy techniques offer the opportunity to study transcription of individual genes in individual cells. The use of these techniques has provided valuable insight into the dynamics of transcription and related processes. Furthermore, whereas biochemical methods only provide information on the situation at a certain time point, microscopy is possible in live cells, and it is therefore possible to establish techniques that will recover information from the very same cells throughout time. Live cell imaging of transcriptional activation has helped the understanding of chromatin decondensation (Janicki et al. 2004; Rafalska-Metcalf et al. 2010) and the activation dynamics of heat shock genes (Yao et al. 2007; Yao et al. 2006; Zobeck et al. 2010). Using FRAP techniques on gene arrays, various kinetics of RNA Polymerase 2 could be established (Darzacq et al. 2007). FRAP has also been the essential tool in understanding the dynamics of nuclear proteins, including all sorts of transcription factors. Fixed cell techniques such as immunostaining usually offer high sensitivity, and have also provided information on the function of various members of the splicing machinery (Spiluttini et al. 2010). Another fixed cell method, fluorescent in situ hybridization (FISH) has yielded important data essential for establishing transcriptional activity models in baker’s yeast (Zenklusen et al. 2008; Gandhi et al. 2011). Latest advances in fluorescent microscopy, and notably the advent of single particle tracking methodologies and hyperresolution capable systems offer even greater opportunities in the study of transcription.
The object of my work is to study the dynamics of transcription in mammalian cells, and more precisely the dynamics of transcriptional activation, by combining various microscopy techniques in order to study a single gene array in single cells. I will approach this study from a RNA Polymerase 2 processivity and transcript production point of view. In particular, I will study the dynamics, the processivity and the role of the very first RNA Polymerases to be loaded on the gene, the pioneering polymerases.
Dynamic changes in RNA Polymerase 2 processivity during transcriptional activation.
Transcription is the first step of genetic expression, and as an essential function of living cells, it is a tightly regulated phenomenon. In this study we monitored the changes in the dynamics of RNA polymerase 2 on a gene array that switches from a non transcribing state to an actively transcribing one. The purpose of such a study is to understand how the various regulatory steps are lifted as transcriptional activators bind to the promoter of a gene. The study’s aim is also to provide information on the processivity of the pionneering RNA polymerases. For this purpose, we took advantage of the high degree of control the Tet-on/Tet-off system offers to the experimentator (Baron, Gossen, and Bujard 1997; Gossen and Bujard 1992). We created an artificial reporter gene array, inserted in the genome of human osteosarcoma cells (U2OS) [Fig 1]. This gene array was designed in such a way that the locus, its transcriptional activity and the protein product of the gene could be tracked at all time in live cells using fluorescence microscopy. Furthermore, the reporter gene was designed so that it would only respond to tetracycline-repressor based transcription factors. Its’ activation or repression would therefore not interfere with the transcriptional activity of other genes, and transcription factors activating other genes would not affect our reporter gene. This high degree of control allowed us to specifically study the activation of a gene from a totally inactive (but not literally repressed) status to an actively transcribing status.
We combined this biological model with different microscopy techniques in order to study cells independently, thus avoiding averaging bias, and to identify any particular behavior during transcriptional activation that would have gone unnoticed using sample-wise techniques. We were successful in isolating one peculiar type of phenomenon, which seemingly is a transitional state between the inactive gene and the actively transcribing gene. In this state, RNA Polymerases are loaded on the gene array, but do nothing more than initiate transcription in a repetitive manner, leading to an accumulation in the cell of short incomplete transcripts. This transitional state is apparently not linked to any defect of phosphorylation of serine 2 on the CTD of Rpb1.
In order to have complete control over the transcriptional activity on our reporter gene, we decided to use an artificial gene [Fig 1]. The promoter of this gene is composed of 7 Tetracyclin operons, followed by a cytomegalovirus (CMV) minimal promoter. The open reading frame comprises a portion of the beta-globin gene (the first two exons and introns), the luciferase coding sequence, the Cyan Fluorescent Protein (CFP) coding sequence, and ends with the peroxysomal targetting signal Serine-Lysine-Leucine (SKL). In the 3′ UTR, 24 sequences from the MS2 bacteriophage were inserted to allow tagging the mRNA with the MS2 coat protein (MS2cp). The total size of the reporter gene is 5.5kb. The reporter gene was inserted in a single locus of the genome of osteosarcoma (U2OS) cells, along with multiple repeats of lactose operons (LacO).
These repeats allows us to track the locus in the genome using the lactose repressor (LacI). Clonal cell lines were established, and the number of genes present in the gene array was quantified using quantitative PCR. The proper co-insertion of LacO repeats and the gene arrays was verified by colocalization of LacI and MS2cp when the gene was active. This system therefore allows us to monitor the gene array locus, its transcriptional output and the correct expression of the protein encoded. Furthermore, microscopy observations and luciferase assays confirm that the expression of the gene array is normally silent and is activated by tet-repressor based transcription factors [Fig 1]. Amongst the multiple clonal cell-lines, we chose one (the 4A cell line) that had no background expression, had a strong luciferase response upon induction, but with a low number of copies of the reporter gene in the array (34 genes). In this cell line, newly formed accumulation of fluorescently tagged proteins of interest, Rpb1 or MS2 for example, dedicated to the transcription of the gene array are large enough to be studied using usual microscopy techniques, yet small enough so that the studied dynamics will not be biased by a mass effect.
mRNA degradation by Xrn1 stimulates transcription.
This study stemmed from perspectives developed by Professor Mordechai Choder, from the University of Haifa. Prof. Choder’s team have recently presented very strong evidence that some actors of transcription also facilitate the cytoplasmic decay of the RNA produced. They have shown that in yeast, the Rpb4 and Rpb7 heterodimer, two components of RNA Polymerase II, actively shuttle between the nucleus and the cytoplasm. Furthermore, they have shown that nuclear export of Rpb4 and Rpb7 is dependent on transcription, and that this export facilitates the degradation of mRNAs in the cytoplasm (Goler-Baron et al. 2008; Lotan et al. 2007; Selitrennik et al. 2006). Their study thus showed that there exists at least a one-way signal from the transcription machinery that partially controls the mRNA decay machinery.
The question that arose was that there might also be a feedback message from the mRNA decay machinery to the transcriptional machinery. The implications of such a discovery would change the current vision that the control of mRNA levels in a cell is due to two uncoupled processes: transcription and mRNA decay. The study in which we participated demonstrates that the mRNA decay machinery modulates transcription to some extent, and that the very last process in the life of an mRNA stimulates the very first process in the life of an mRNA. Studies have previously shown that the Xrn1 protein, the major 3’-5’ exonuclease could be inactivated in yeast without loss of viability; furthermore, microarray assays have shown that this inactivation also had no significant effect on the mRNA levels of a large number of genes (F. He et al. 2003). This is unexpected as the Xrn1 pathway is the major mRNA decay pathway in yeast. Our study has shown that in fact, yeast in which Xrn1 is mutated to an exonuclease deficient form or yeast where Xrn1 is simply deleted do have impaired mRNA degradation, and that most mRNAs have extended life-times compared to their counterparts in wild type strains. However, the mRNA levels of tested genes remained unchanged whether Xrn1 was mutated, deleted, or present and functional.
Similar results were obtained when other members of the mRNA degradation machinery were tested. The study shows, using nuclear export deficient strains, that some components of the mRNA decay machinery shuttled between the nucleus and the cytoplasm and, by ChIP, that these components interact with genes in a fashion similar to RNA Polymerase II. Furthermore, the nuclear import of the decay factors was impaired when Xrn1 was mutated. Strangely, these decay factors did not have impaired nuclear import when Xrn1 was deleted. Finally, this study also shows that the mRNA production of genes upon activation was much slower in yeast strains where the mRNA decay machinery was deficient.
Table of contents :
Chapter 1 Introduction
Gene expression and RNA Polymerase
Controling gene expression: the role of transcription factors.
Mechanisms of gene repression.
Transcription factor regulation
Transcriptional dynamics.
The use of fluorescence microscopy.
Chapter 2 Results
Dynamic changes in RNA Polymerase 2 processivity during transcriptional activation.
Discussion
Chapter 3 Other publications
mRNA degradation by Xrn1 stimulates transcription.
Short exposure to the DNA intercalator DRAQ5 dislocates the transcription machinery and induces cell death.
The In Vivo Kinetics of RNA Polymerase II Elongation During Co-Transcriptional Splicing.
Imaging transcription in living cells
Mouaikel-Libri
Chapter 4 Methodology Improvements
Live Cell Imaging
FISH.
Hyperresolution work in progress.
Chapter 5 New types of data require novel analysis methodologies
Chapter 6 Materials and methods
Cell Culture.
Transfection
Generation of derived cell-lines.
Table 1 : Generated cell lines.
Luciferase.
FISH.
Chemical coupling of FISH probes
Immunofluorescence.
Fixed cell imaging
Live cell imaging
Chapter 7 Bibliography
