Cent. Eur. J. Biol. • 3(1) • 2008 • 11-18 DOI: 10.2478/s11535-008-0001-1

Central European Journal of Biology

Visualizing transcription in real-time

Mini-Review Yehuda Brody, Yaron Shav-Tal*

The Mina & Everard Goodman Faculty of Life Sciences, Bar-Ilan University, Ramat-Gan 52900, Israel

Received 31 October 2007; Accepted 10 December 2007

Abstract: The transcriptional process is at the center of the gene expression pathway. In eukaryotes, the transcription of protein-coding genes into messenger RNAs is performed by RNA polymerase II. This enzyme is directed to bind at upstream gene sequences by the aid of transcription factors that assemble transcription-competent complexes. A series of biochemical and structural modifications render the polymerase transcriptionally active so that it can proceed from an initiation state into a functional elongating phase. Recent experi- mental efforts have attempted to visualize these processes as they take place on genes in living cells and to quantify the kinetics of in vivo transcription. Keywords: mRNA transcription • Nucleus • Live-cell imaging • Cellular dynamics

© Versita Warsaw and Springer-Verlag Berlin Heidelberg.

1. Introduction review will describe the developments in the field of live-cell imaging that have allowed the analysis of mammalian RNA polymerase II transcription kinetics as The central dogma of molecular biology describes the they unfold in real-time. flow of information in the living cell from DNA to RNA to protein [1]. Information can also flow back from protein to nucleic acids. The fundamental process within this pathway that enables the mobility of genetic information 2. Lighting-up the nucleus from the stagnant DNA molecules contained within the cell nucleus to the ribosomal centers of protein Fluorescence microscopy provides the capability of translation and production in the cytoplasm is the following specific molecules as they are produced and process of messenger RNA (mRNA) transcription. Much while they travel throughout the cell. Molecules are scientific attention has been devoted to the unraveling labeled with a fluorescent marker [7] and the dynamic of the mechanisms involved in mRNA transcription and properties of a molecule in time and space can be the regulation of RNA polymerase. Indeed, we have tracked. The most common label is the green fluorescent before us a detailed picture of the atomic structure of the protein (GFP) [8] and its variants of many colors (e.g. polymerase complex [2], the various protein players [3], cyan, yellow, red fluorescent proteins). GFP is a protein, the biochemical modifications involved and the intricate and therefore its fusion to a protein of interest renders mechanistic properties of the molecules that control that protein fluorescent and traceable by fluorescence transcription [4,5]. While much of this information was microscopy. Fusing of different proteins, each with extracted using biochemical and biophysical in vitro a different fluorescent version of GFP, permits the procedures, in recent years live-cell experimentation simultaneous visualization of the proteins in the same has allowed molecular pathways to be viewed within the cell using different fluorescent channels. This approach living context of the cell [6]. These in vivo approaches opened up a plethora of biological questions that relate now add new levels of molecular dynamics and to the intra-cellular dynamics of proteins, as well as sub- quantifiable kinetics to our view of transcription. This cellular organelles or bodies that can be GFP-labeled with their specific protein components [9]. For instance,

* E-mail: [email protected] 11 Visualizing transcription in real-time

GFP labeling of nuclear proteins revolutionized the way structure that is specifically and stably bound by the we perceive the nucleus [10]. We now understand that MS2 protein. As with the DNA labeling system, here the interactions of proteins identified by biochemical too the MS2 protein is fused to GFP thus providing an assays as part of nuclear protein complexes, or of the indirect method of nucleic acid GFP labeling. In fact, proteins detected in nuclear bodies of fixed cells, are in many MS2 sequences per gene are added thereby reality single molecules that are in constant movement providing a decoration of many GFP molecules per one in and out of their complexes and structures [11]. Only mRNA molecule. This label provides a good signal-to- certain proteins, such as histone proteins, remain bound noise ratio that is crucial for live-cell image acquisition of to the DNA for very long periods [12-14]. a particular subset of molecules that are surrounded by Since GFP is a protein, it is not suitable for the a larger population of similar molecules, and is therefore direct labeling of specific nucleic acid sequences (DNA, preferable to other labeling methods that produce RNA). Therefore, indirect methods for GFP-labeling of significant fluorescent backgrounds that might mask the nucleic acids were devised. These methods are based desired molecule [28]. The MS2 labeling technique set on the use of unique DNA or RNA sequences that are the grounds for experiments focused at the detection of attached to the particular nucleic acid under study. The real-time mRNA transcription in a variety of organisms unique sequences are then recognized and bound by [29-31] and for following the travels of single molecules specific protein factors that are fused to GFP, thereby of mRNA within living cells and organisms [27,32-34]. achieving indirect GFP labeling. These unique nucleic We now turn to see how these techniques were used for acid sequences originate from prokaryotic organisms the analysis of real-time transcription kinetics. and therefore do not serve as binding sites for intra- cellular eukaryotic proteins, but only for their cognate proteins. 3. Dynamics of transcription For the labeling of specific DNA regions thelac O-lacI machinery assembly system is utilized [15,16]. Multiple copies of the bacterial lac operator (lacO) sequence are inserted adjacent to The act of transcription by RNA polymerase (Pol) II can the gene of interest. The lac repressor protein (lacI) not proceed without the appropriate quorum of proteins binds specifically to the lacO sequence, and since the first assembling on the promoter region. Transcription is lacI protein is also fused to GFP, the system provides a a multi-step process requiring initiation of transcription means of fluorescently labeling the gene locus with many followed by a transition to a productive elongation GFP molecules. The result of this approach in living complex that will generate full-length mRNAs. These cells is that the gene of interest (which was integrated steps involve several structural changes in the into the genome together with the lacO sequences) polymerase together with the recruitment of a multitude appears as a condensed dot within the nuclear volume of accessory factors, namely transcription and elongation [17]. This approach has allowed the examination of factors. The interactions of transcription factors with a general questions relating to DNA dynamics in living gene sequence have been analyzed in vivo. cells. For instance, to monitor and quantify the mobility Multi-photon imaging in live Drosophila salivary gland of gene loci in the nuclei of living cells [18-20], to cells that contain polytene chromosomes (many copies visualize the change of chromatin structure due to gene of the whole genome in the same cell) has allowed the activation [21,22], or to specifically monitor proteins that detection and analysis of transcription factor binding associate with a gene in vivo and which are part of the dynamics to an endogenous set of genes [35]. The heat transcriptional or mRNA processing machineries [22- shock transcription factor (HSF) is recruited to heat- 25]. Recently, this approach allowed the detection of shock genes on polytene chromosomes during induction long-range movements of a DNA locus after targeting of heat-shock. Expression of a GFP-HSF fusion protein by a transcriptional inducer [26]. This type of movement in these cells demonstrated that HSF moves diffusely may be dependent on nuclear actin and myosin proteins throughout the nucleoplasm prior to heat shock, but since specific inhibitors reduced these translocations. binds tightly to heat shock genes when this response is The in vivo labeling of a specific mRNA transcript activated. This study also demonstrated the recruitment employs a similar approach. A bacteriophage derived of GFP-RNA polymerase to these genes after heat shock sequence termed MS2 is typically inserted at the 3’ end induction. Intuitively, these results seem reasonable as of the gene of interest, so that it does not disrupt the we envisage transcription factors binding to promoter coding region [27]. When this sequence is transcribed it sequences and forming stable transcriptional complexes, becomes an integral part of the mRNA. The MS2 mRNA thereby recruiting RNA Pol. However, these findings are sequence folds and produces a stem-loop secondary actually surprising in view of several previous studies on

12 Y. Brody, Y. Shav-Tal

exogenous genes that have drawn a different picture of instance, a 90 Mb integration of a lacO gene was labeled transcription factor dynamics. For example, the binding with LacI fused to the transcriptional activator VP16 kinetics of the well known NF-κB factor to DNA are highly [16]. VP16 is a transcription factor for the immediate transient [36]. Other live-cell studies have used hormone early genes of the herpes simplex virus, and contains signaling systems. These elegant systems utilize a highly acidic C-terminus that is an extremely potent biological interactions in which steroid receptors interact transcriptional activator in mammalian cells. VP16 with their hormone ligands, that then translocate from is thought to act in part through the recruitment of a the cytoplasm to the nucleus (therefore termed nuclear histone acetyltransferase (HAT), which is involved in receptors) and bind to promoter sequences to facilitate transcriptional activation, to the promoter region. The gene activation. An integration of mouse mammary presence of VP16 on this gene array brought about tumor virus (MMTV) promoter elements were used as the striking unfolding of the gene locus, and a string- a binding site for the glucocorticoid receptor (GR) [37]. like DNA structure could be observed. Further studies Uninduced GR resides in the cytoplasm complexed with in living cells showed that the open DNA structure is a variety of proteins including heat shock protein 90 and stable for extended time periods [41]. A cellular system the heat shock protein 70 that act as chaperones. When designed for following gene expression in vivo has the endogenous glucocorticoid hormone cortisol diffuses shown that such exogenous gene integrations behave through the cell membrane into the cytoplasm it binds to as would be expected from an endogenous gene [22]. GR resulting in the release of the heat shock proteins, In this system the integrated gene was under induction and its translocation to the nucleus. Kinetic analysis of control and therefore different elements involved in the GFP-GR binding to the MMTV array after activation with transition from an inactive to an active transcriptional dexamethasone, a synthetic activator of the receptor, state could be tested in vivo. The observed exchange of showed that GR molecules exchanged rapidly on the GFP-labeled histone proteins on the gene locus yielded binding elements implying that transcription factors do an open activated transcriptional status, followed by the not form static stable holoenzyme complexes at the recruitment of GFP-labeled RNA Pol II, the synthesis of promoter [23,37]. Another system based on the estrogen mRNA, and the appearance of splicing factors and 3’ end receptor exhibited similar kinetic properties [38], as did processing factors, thereby indicating that the active site other basal transcription factors such as TATA-binding was functioning as a bona fide transcription site. The protein (TBP) and TFIIB [39]. This transcription factor labeled mRNA traveled through the nucleoplasm [34], binding model was coined the „hit and run” model, were exported to the cytoplasm and were translated into meaning that numerous transient binding interactions protein. By fluorescently labeling the DNA, mRNA and occur on an activated gene and that gene activation protein as described above (lacO/LacI, MS2 and GFP is the sum of these stochastic interactions [40]. In light respectively), the whole “dogma” could be visualized in of the HSF data presented above, it stands to reason a single living cell [22]. that gene activation complexity is two-fold: the first level pertains to the abundance of specific protein-protein interactions that are required for the assembly of a 5. Dynamics of RNA polymerase functional complex, while another level of complexity activity depends on the kinetic probabilities for these proteins to actually meet and interact. From the stand point of the polymerase, the transcription process includes pre-initiation complex formation at the promoter, transcription initiation, elongation, termination, 4. Dynamics of the activated genomic and polymerase dissociation from the DNA template. locus Using GFP-fused RNA Pol II the kinetic properties of polymerase populations in the nucleus were measured. Structural changes in the chromatin environment must While GFP-tagged transcription factors acting on the take place in order for transcription to proceed. In MMTV array showed rapid exchange (seconds) with order to observe the transitions occurring in chromatin the surrounding nucleoplasm, GFP-RNA Pol II showed structure in vivo using light microscopy, it is required slower exchange kinetics (minutes) implying a stable that large-scale structural changes transpire. Use of interaction of the polymerase as it elongates through genomic integrations of multiple lacO repeats, thereby genes [23]. Further quantitative analysis of the gene- forming exogenous genomic loci that can be labeled bound and unbound fractions of RNA Pol II in the nucleus with GFP-lacI, have provided an exquisite look at of living cells showed that ~75% of the polymerases act chromatin opening and unfolding in real-time. For as a “free” diffusing fraction that can bind and dissociate

13 Visualizing transcription in real-time

rapidly from the promoter regions. The remaining Pol II, DNA, and RNA form a stable tertiary complex ~25% of the polymerases were found to be immobile, while Pol II moves processively along the DNA template suggesting that this population is composed mainly of incorporating NTPs to the 3’ end of the nascent RNA. engaged polymerases [42]. In a further attempt to obtain Utilizing the cell system described above [22] in which a better resolution of the engaged Pol II populations, three functional, actively transcribing gene can be detected in populations were detected [43]. Apart from the diffusive a live cell, it was possible to extract kinetic data on RNA inactive fraction, two other fractions were defined via the Pol II activity as it moves along a specific gene in vivo. use of transcriptional inhibitors. An elongation inhibitor This gene contains a combination of both lacO and MS2 that inhibits the positive transcription elongation factor nucleic acid labeling elements. This system enabled pTEFb and prevents the transition of the polymerase the detection of the gene locus with red fluorescent to its active elongating form is 5,6-dichloro-1-β-D- protein (RFP) fused to LacI, simultaneously with the ribofuranosylbenzimidazole (DRB) [44]. Applying of mRNA made from the gene, since the gene contains 24 DRB to living cells led to the detection of polymerases MS2 repeats that are labeled in the mRNA by multiple incorporated at the pre-initiation stage involved in GFP-MS2 proteins (Figure 1). Kinetic analysis of GFP- attempts to load onto the gene. Still, a third fraction labeled proteins is performed by photobleaching of the remained engaged with long transcription units after fluorescent signal at a point of interest and the subsequent DRB treatment, but was sensitive to heat shock, and was measuring of the recovery of the green fluorescent therefore considered as the ‘elongating’ fraction. These signal in the bleached spot [30]. This technique is called experiments were performed on random chromosomal fluorescence recovery after photobleaching (FRAP) and regions in the nucleus and allowed a general view at is usually used for diffusion measurements [46]. Since the dynamics of GFP-Pol II populations in the cell. In a the gene (red signal) and the mRNA (green signal) could following step, the precise kinetics were measured on a be followed at the same time, it was possible to bleach specific gene in living cells. the mRNA signal without loosing the exact location of the During the initiation step, RNA Pol II binds to the gene, and therefore to monitor the recovery of the signal promoter, the DNA is unzipped, and ribonucleotides are on the gene in real-time (Figure 1). In biological terms, added to produce an mRNA molecule. Early in initiation, this FRAP recovery is in fact the act of transcription of abortive transcripts can be produced by the polymerase MS2 stem-loops in the nascent mRNAs produced by the [45]. When the nascent transcript RNA reaches a size gene, and is therefore a means for quantifying the speed of ~13 nts, the transcription complex escapes the of the polymerase as it produces new mRNAs. This promoter and elongation begins. During elongation, type of analysis yielded some interesting observations

Figure 1. Quantifying mRNA synthesis in vivo: FRAP at a transcription site that is actively synthesizing mRNA molecules, which are then labeled with MS2-GFP. (a) Differential interference contrast images of a live cell. (b) Marking of the gene locus with RFP-LacI (red dot). (c) The green dot shows the active transcription site at which mRNA molecules are labeled with MS2-GFP. The dotted circle indicates the pho- tobleached region. (d-i) After photobleaching the mRNA signal at the site is mostly reduced, and is followed by recovery of the signal over time as new mRNAs are transcribed. (j) Normalized locus recovery as an average recovery curve from 10 cells (black dots). Curves show best-fit solutions for the mathematical model (see equation) with single exponential (red) or two exponentials (blue). The latter fit the data best and was used for modeling of Pol II elongation. The data enabled direct measurements of elongation kinetics and the detection of polymerase pausing. The figure was adapted from [30].

14 Y. Brody, Y. Shav-Tal

regarding in vivo transcription [30]. Transcription rates assemble into a functional initiation complex, and only for Pol II transcription that were measured biochemically 1 would manage to make it all the way through to the are commonly noted in the range of 1.0 – 2.0 kilobases/ end of the gene. These sophisticated modeling methods min [47-50]. However, the above in vivo analysis shows were used for another transcription unit, the nucleolus that elongation speeds of up to 4.3 kb/min are possible. that transcribes ribosomal RNA [59]. There too measured This finding is corroborated by one of the first estimations transcription rates for RNA Pol I skyrocketed to speeds made for RNA Pol II elongation rates, which concluded by of up to 5.7 kb/min. The advantage of the nucleolus is radioactive labeling assays that transcription rates can that it is an endogenous array of repeated genes and be found in the 3.0 – 6.0 kb/min range [51]. Moreover, that there is no need for stable gene integration as with this live-cell approach provided high resolution analysis Pol II transcribed genes. Future technical developments of elongation kinetics, and demonstrated that these fast will be needed to allow the examination of Pol II elongation speeds coalesced with long Pol II pausing kinetics on particular endogenous genes and to extract times, therefore resulting in the lower speed values information at the resolution of a single gene. These measured in biochemical assays. Indeed, transcription kinetic approaches will allow us to probe unanswered elongation is a remarkably discontinuous process questions relating to transcription dynamics. For with many potential blocks being encountered as the instance, two models describe the organization of polymerase proceeds. Pausing is interpreted as the nuclear mRNA transcription sites. The common theory switching of a fraction of the elongating polymerases was described above, namely binding of RNA Pol into an offline conformation 52[ ], and it is likely to have a II to the promoter, and then its sliding along the DNA biologically relevant role in regulating the expression of template while incorporating complementary NTPs. The certain genes [53-55]. Also, reverse translocation in the other model is termed the transcription factory model absence of synthesis (backtracking) can lead to certain [60]. According to this model, Pol II molecules are classes of transcriptional arrest [56]. Transcriptional immobilized within transcription sites while the DNA is pausing is studied mostly in vitro in prokaryotic reeled in and out of the site. This theory is based on the systems, and has also been observed for the eukaryotic fact that mRNA molecules were found concentrated in transcriptional machinery [54], although how these ~2,000 discrete transcription sites. The concentration of processes occur in vivo remains unclear. Using the factors in transcription factories allows them to carry out above live-cell analysis, it was possible to extract from the processing events necessary for producing a mature the kinetic data both phases of active elongation and transcript such as 5’ capping, pre-mRNA splicing, pausing, and to demonstrate that the RNA Pol II enzyme polyadenylation and proofreading [61]. Studies in HeLa is a processive motor, probably more potent than cells suggest that each transcription factory can contain anticipated. Still, RNA Pol II elongation rates may differ ~30 active polymerases and associated transcripts [62]. from gene to gene. A study following the transcription of The ability to analyze the dynamic activity of RNA Pol HIV reporters measured transcription rates of about 2 II on specific genes will allow the testing of these and kb/min, and showed that the polymerase stays longer other models. Ultimately, such approaches will assist in on the gene than the mRNA, and that the mRNAs were reaching an in depth understanding of the transcriptional released immediately after the start of polyadenylation process. [57]. Future studies will illuminate what are the points of regulation that define elongation rates, such as promoter sequences, transcription factors or even co- Acknowledgements transcriptional processing events. This work was supported by grants to Yaron Shav-Tal by the Israel Science Foundation, France-Israel Biomedical 6. Dynamics – where are we going? Imaging program, the Israel Cancer Research Fund and the Israel Cancer Association. Yaron Shav-Tal is the The above mentioned type of kinetic analysis requires Jane Stern Lebell Family Fellow in Life Sciences at Bar- the integration of mathematical modeling approaches Ilan University. into routine biological assays [58]. For instance, the kinetic model used in the above study could position the major point of Pol II regulation at the step of initiation, and could quantify this regulation in molecule numbers [30]. If 90 polymerases were to attempt to interact with the promoter region, only 11 of these would manage to

15 Visualizing transcription in real-time

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