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During embryonic development of multicellular organisms, groups of genes are temporally and spatially regulated. Cellular differentiation is the consequence of constitutive or inducible activation of some gene loci, whereas others remain silent or become repressed. The ability for expressing tissue-specific genes is determined during development and can be transmitted to daughter cells for a long time after adequate inductive events. How is this achieved? How is the induction event performed? And how is the maintenance of the cell fate accomplished? Here we intend to study particularly the inductive step, and the role of chromatin organization and of specific promoters' relocation inside the nucleus in this process.
Chromatin organization in the nucleus has recently gained considerable interest. One of the most interesting questions is the correlation between interphase chromatin structure, location, dynamics and gene expression. There are striking correlations between chromatin compaction and localization within the nucleus, and the expression levels of genes during differentiation or response to stress [1, 2]. However, causal or temporal relationships remain unclear.
Chromatin structure and compaction, which determine to a large extent whether a gene can be expressed due to an "open state" that is accessible to the transcriptional machinery, has been extensively investigated in the last decades. Notably, an ever-increasing number of multicomponent enzyme complexes (such as chromatin modifiers or remodeling complexes) that regulate chromatin structure and function are identified and analyzed . The structural changes of chromatin, also known as chromatin remodeling events, play determinant roles in establishing tissue-specific gene expression and therefore, also regulate cellular differentiation [4-6]. However, differentiation processes are very complex and the presence of specific transcription factors and chromatin compaction changes do not seem to be the only requirements for proper differentiation. We hypothesize that relocation of specific genes ââ‚¬" the reshaping of nuclear architecture - will also participate in proper cellular differentiation.
In comparison to chromatin compaction, little is known about the mechanism underlying chromatin localization and movement within the nucleus and even less is understood about how it impacts transcription or differentiation. In some species, active euchromatic genes are either localized internally or close to nuclear pores, while silent genes within heterochromatin are often clustered adjacent to the nuclear envelope [7, 8]. Nevertheless, in mammalian cells no strict correlation has been established, perhaps due to the limited number and types of loci analyzed [9, 10].
Recently, in the C. elegans nematode model, a robust correlation has been demonstrated between the nuclear position of tissue-specific promoters and their activation . This study an adapted the GFP-lacI /lacO recognition system [12-14] in order to visualize the chromatin compaction state and the localization of specific chromatin arrays in C. elegans, in the living organism during development . The arrays are composed of several copies of a transgene which are stably integrated into the genome. One copy of the transgene contains a tissue-specific promoter with a reporter gene and importantly a lacO sequence which is detected and bound by a constitutively expressed GFP-lacI protein. Therefore, the locus of the array is detected visually as a bright GFP spot .
The results of this study led to the main conclusion that the nuclear interior is a transcriptionally active domain for genes activated during differentiation. Following cell differentiation, in cells of L1 larvae stage, tissue-specific promoters within the integrated array are induced and relocalize significantly to the center of the nucleus instead of having a random distributionm which is found in early embryonic nuclei prior to the differentiation event . The inward relocalization of developmentally activated promoters is valid for endogenous loci, for arrays carrying a small number of transgenes, as well as for arrays that carry hundreds of copies of the same transgene. These 'big' arrays have heterochromatin characteristics; they are enriched in the repressive epigenetic marks - H3K27me3, H3K9me3- and prior to the cell differentiation, unlike the small arrays, they are found at the nuclear periphery, like heterochromatin. These findings were shown for tissue-specific promoters from the three germ layers, of three distinct worm tissues: muscle, gut and hypodermis .
This newly developed system by Peter Meister and colleagues in the laboratory of Prof. Susan Gasser allows the monitoring the subnuclear position and expression of chromatin arrays in their native multicellular environment. It is also an exceptionally well suited system for assessing other important biological questions described below, which are technically almost impossible to probe elsewhere. Moreover, the choice of C. elegans as an organism model is essential, since the history of each cell is known and invariant, the timing of cell fate acquisition is precisely known, and finally, the worms are transparent and amenable to microscopy, genetics and biochemical manipulations.
In this proposed project we make use of all the advantages that C. elegans and this chromatin monitoring system provide, and we intend to set up an additional technique on this basis and to address the two following biological questions.
We aim to identify components that contribute to chromatin movement, especially of certain tissue-specific promoters repositioning inside the nucleus upon differentiation, and to explore the functional implications of this movement in transcription and cellular differentiation.
Using the same research methodology that the one we will use for the objective 1, we are also interested in the mechanism which relocates stress -response promoters, such as heat-shock promoters to the nuclear periphery when they are induced (Gasser personal communication). Does the mechanism of this movement use similar molecules or distinct device than in the relocation of developmentally regulated promoters?
In order to achieve these objectives we must go through several steps:
Step 1. Define a precise interval of time during which the subnuclear relocation of the promoter of interest takes place, and define minimal sequences that confer relocation.
Step 2. Identify the chromatin factors (proteins and potentially RNA molecules), which interact with the chromatin array before, during and after the promoter relocation.
Step 3. Evaluate the best candidates involved in the promoter repositioning and validate their role in chromatin movement, and their effects on transcription and differentiation.
Step 1. Define the precise promoter elements needed and interval of time during which the subnuclear relocation of the muscle-specific promoter takes place.
Defining the precise window of time during which the array relocate inside the 3D nuclear space is a very important step because the movements of the genomic loci we intend to study are not arbitrary. We assume that a driving force move the appropriate promoters to the appropriate subnuclear localization, with an appropriate timing. Strikingly, the subset of components that is susceptible to act as a driving force may have a transient and short interaction with the chromatin array, and that is why we will be able to enrich our samples in molecules that are responsible for the array active movements if we establish the time period during which the movement occurs. Yet, it is also possible that the array loci 'passively' relocate to the appropriate nuclear compartment, maybe by a diffusion-based chromatin movement follow by a strong association with components of a certain subnuclear compartment. In both cases, the results obtain in step 2 will provide new understanding on the promoters' movement during differentiation and stress response. In the case driving forces exist, we will expect to "pull-down" with the array at the relocation time period, some ATP dependent proteins, such as chromatin remodelers, or/and some structural nuclear components such as actin and microtubule filaments, etc.
For objective 1: We will focus on two body wall muscle-specific promoters that are induced at different developmental stages. We will take advantage of one of the C. elegans strain which has been already generated in the Gasser's lab. This strain carries an array (gwls4) which possesses the myo-3 promoter with a mCherry reporter gene, lacO sequences and a GFP-LacI fusion protein expressed under baf-1 promoter which is constitutively expressed. The myo-3 gene encodes for a muscle structural protein MYO-3 (myosin heavy chain). Also, myo-3 expression begins relatively early, in the 'pre-comma' stage embryo, and remains active in all the body wall muscle cells of the developing and adult worm . The regulatory cascade of transcription factors that led to muscle cell differentiation is partially known, e.g.PAL-1> HLH-1> UNC-120; HND-1> tnt-3; myo-3; acr-13 and many others . According to the current knowledge, and according to the availability of existing strains or constructs from the worm international community at the time I will begin the project, another muscle-specific promoter which is induced prior to myo-3 will be chosen. If an additional strain will have to be generated from zero, the technical knowledge and material needed for it are very easily available in the Gasser's lab.
A first, fairly simple goal will be to define the minimal promoter elements needed for the relocation. A series of integrated transgenes will be made with various deleted forms of the myo-3 promoter driving GFP. We will determine by microscopic evaluation of muscle-specific GFP expression and the position of the array, what are the minimal elements of the promoter that confer both relocation and expression in a muscle-specific manner. We know that the master regulator HLH-1 (MyoD in mammals) is required and its induction can relocate the large array containing the full myo-3 promoter (P. Meister, pers. Communication). But there may be sequences and factors in addition to HLH-1 that are important. By defining the minimal sequence element necessary, we can focus on only the essential promoter binding factors.
In order to define the time period during which the relocation of the tissue-specific promoters occurs we will concentrate especially around the initiation time of transcription  of the promoters of interest in relationship to the time spent since the two-cell embryo stage (the whole embryogenesis takes around 800 minutes). Then we will follow and assess through time-lapse microscopy the location of the arrays in the developing muscle cells of the embryo. If determining the exact timing of this event turns out to be technically challenging (for example because of the motility of the embryos after the 1.5-fold stage, or because of the smaller size of nuclei in older embryos) two alternative approaches can be employed. First, we can use immunofluorescence against GFP of the arrays on embryos fixed in a series of developmental time points. Second, we can ectopically express HLH-1/Ce-MyoD (HS::hlh-1) by heat shock induction, as described in Meister et al., 2010. It has been shown to have the power to induce the relocation of the array carrying myo-3, which is a downstream target of hlh-1. The ectopic expression will have to be performed before the 8E stage (~100 cells), where the nuclei are still clear and measurable, and because after the 8E-12E stages, cells fail to adopt alternate fates when challenged with a heterologous regulator. Within 40-80 minutes after the heat shock, the array relocates internally, thus if necessary, during this period we will perform our adapted 'reverse-ChIP' and 'RIP-seq' analyses described in step 2.
For objective 2: In order to investigate the mechanism by which promoters when induced by a stress-signal are relocated to the nuclear periphery (Gasser lab communication;, I will benefit from several transgenic strains that have been engineered in the lab of Prof. Gasser. The strains of interest have genomic integrated arrays carrying a heat-shock promoter followed by a mCherry reporter gene and are observable with the lacO/lacI-GFP system. Some of these strains have a known genomic integration site -at MOS insertion site-. Therefore, the effect of the genomic compaction state in which the array is integrated on the identity of the factors needed to relocate the array after heat-shock is also testable. The time period during which the arrays move to the nuclear periphery is already known, therefore we will use this knowledge to carry out step 2.
Step 2. Identify the chromatin factors (proteins and potentially RNA molecules) which interact with the chromatin array before, during and after its relocation.
The identification of chromatin factors bound to the tested arrays prior, during and after their relocalization following cellular differentiation or stress response is the most significant step towards the understanding of the 3D nuclear gene relocation mechanism. In order to characterize these chromatin factors, we will set up a new method by exploiting the lacO/LacI-GFP system which allows to observe the chromatin arrays but also to target them. This last fact will provide us a unique opportunity to be able relatively easily to target and isolate the tested arrays along with the chromatin factors bound to it in vivo. The idea is to perform a reverse-ChIP (Chromatin Immuno Precipitation) analysis adapted to our system in order to identify the proteins bound to the promoters of the arrays, and an adapted RIP-seq (RIP stands for RNA Immuno Precipitation) analysis in order to identify the potential RNA molecules involved in the mechanism . The reverse-ChIP methodology published last year , is based on the combination of pull-down by DNA probes and mass spectrometric analysis and it enables identification of chromatin-associated proteins.
The adapted techniques we will set up will take advantages of the LacO sites present in the transgenes, which are bound by the lacI-GFP fused proteins. LacI-GFP can serve as an anchor to immunoprecipitates the chromatin of the locus and identify the proteins and RNA molecules bound to it. Notably, the chromatin array we will examine are composed of several copies of the transgene -up to 50- , which should greatly facilitate our approach in comparison to the standard reverse-ChIP and RIP-seq methods. The RIP-seq is a method used for finding which RNA species interact with a particular protein. In our adapted method it will actually reveal if and which RNA molecules are bound to the endogenously formed complexes of chromatin factors and chromatin segments which include the promoter of interest before, during and after its 3D nuclear relocation. The idea to search for RNA molecules, potentially mRNA and non-coding RNAs, involved in gene relocation during cell differentiation or stress-response is due to the increasing revealed roles of RNAs in the control of gene expression. As an example, even some large intergenic non-coding (LINC) RNAs have been shown to associate with the chromatin-modifying complex PRC2 during differentiation and to affect gene expression .
The method we will develop will combine an adapted reverse-ChIP and adapted RIP-seq techniques that will be perform on the same samples. Briefly, in this process intact cells will be subjected to cross-linking with formaldehyde which cross-links nucleic acids (DNA and RNA) to amino acids, and sonicated to shear chromatin fragments into fragments of suitable size. The chromatin fragments carrying the promoter of interest, found on the array, will be immuno-precipitated with a GFP specific antibody and with agarose or magnetic beads. Then, the cross-links will be reversed. Proteinase K digestion and/or DNAse and/or RNAse will be use; thus, the proteins, DNA, and RNA can be purified separately to enable subsequent analyses. The above steps will be technically very challenging and will need to be calibrated; therefore small-scale trials with different parameters will be performed to evaluate the optimal conditions.
Subsequently, the proteins will be analyzed and identified by mass spectrophotometry, or potentially by MudPIT analyses. The RNA molecules associated will be subjected to high-throughput deep sequencing (Solexa) and bioinformatics analyses . DNAs will be recovered and characterized by RT-PCR, as a control. Moreover, other obvious controls that must be done are the same system without LacO sequences in the arrays, and alternatively without LacI-GFP expression.
For the objective 1 and 2 the research methodology is the same, but the only difference is that for the objective1 we have to target only the arrays in a cell-specific manner, only in developing muscle cells. Several technical options are available for this purpose; one of them is the generation of C. elegans strains that will express the LacI-GFP fused protein under a promoter which is not expressed constitutively as baf-1 but only in muscle cells and prior to the promoter we track for relocation (e.g hlh-1 promoter).
Otherwise, an additional interesting point to assess is the correlation between the transcriptional state and the 3D nuclear location of the promoters we will test. In few studies, the transcription has been shown to be able to precede a genomic loci relocation [11, 24], however it is still unclear whether transcription promotes chromatin movement in general, or whether chromatin movement promotes transcription initiation and/or elongation. Therefore, in a straightforward and classical approach, knowing the relocation time period, we will be able to co-immuno-stain some initiation and elongation factors of transcription, along with the GFP of the arrays from developing muscle cells, prior, pending and posterior to the relocation of the array.
Similarly, histone modifications typical for open or closed promoters, and the phosphorylation status of the RNA pol II C terminus, will be characterized in these three situations, for each promoter of interest.
Step 3. Evaluate the best candidates involved in the promoter repositioning and validate their role in chromatin movement, and their effects on transcription and differentiation.
Comparison of the proteins and possible RNAs bound to arrays before, during and after their relocation should allow us to identify factors responsible for this genomic loci repositioning. Expected proteins revealed by our analysis are transcription factors but hopefully we will focus on possible chromatin-modifier enzymes, chromatin-remodelers, or other proteins stimulated by ATP or GTP hydrolysis, and on structural proteins of the nucleus. In addition, we also expect that components of the transcriptional machinery will be found, at least in the active state of the array. Whether RNA molecules are themselves directly involved in process of relocation is unclear. There is very little is known about the impact of RNA on gene positioning, and one must take care in the analysis of recovered RNAs to exclude false positive results. The analysis of the output data for proteins and RNAs identification will require a wide use of bioinformatics tools, in order to evaluate their relevance, in order to classify the candidates by known or predicted function, to define their evolutionary conservation etc.
Once chromatin factors bound to the arrays have been identified, their relevance for chromatin organization and specifically for promoter repositioning following cellular differentiation or stress-response has to be validated. I plan to test this in two ways. First, bound factors will be depleted by RNAi to confirm whether a given factor is necessary for gene positioning. A C. elegans RNAi feeding library is available in the host lab. Second, the best candidates will be exogenously overexpressed and targeted to the LacO sequences of the array, and we will test if this 'gain of function' influences the relocation of the array in general and its timing in particular. Two alternative experiments are theoretically do-able. In one hand, the chosen factor (X) will be fused to the LacI-GFP protein, creating LacI-X-GFP, and we will replace the previous LacI-GFP expressing transgene with this in the strains we wish to track. On the other hand, we could also add simultaneously to the LacI-GFP a LacI-X-mCherry transgene, and visualize both of them through different filters.
Originality and innovative nature of the project
The contribution of this proposed project is expected to advance considerably the understanding in specific genomic loci movement observed in differentiating cells and in cells subsequent to stress-response. Nuclear and chromatin organization began to be much better understood, but the sub-field which we intend to explore in this study is still one of the black boxes. The originality and innovative nature of this project resides both in the goal of the study and in the novel approach we will set up. Using C. elegans to study changes in chromatin organization linked to developmental transitions is a new and exciting progress. By combining classical methods, with the state of the art in microscopy, and with the cutting edge techniques in molecular biology adapted to the lacO/LacI system, this project can provide unique insights into the 3D chromatin positioning, its mechanism and test in a straightforward manner for the first time its function, in vivo, in a multicellular organism. This research may also generate discoveries of novel transcription factors involved in myogenesis. Otherwise, the innovative method we propose to set up here combine in an elegant manner, a kind of 'reverse-ChIP' and 'RIP-seq' procedures thanks to the integrated lacO sequences in the genomic loci/arrays we intend to track and target. This method allows isolating and identifying protein and RNA molecules bound to a specific genomic locus, at different time points, in an efficient and cell-specific manner. Once this new approach will be established, it will be available to the C. elegans research, easily applicable and useful for a multitude of other important biological questions related to chromatin, transcription and development.
Timeliness and relevance of the project
The proposed research is feasible only in the laboratory of Prof. Susan Gasser. Her laboratory expertise and experience in nuclear organization, in advanced microscopy and in the C. elegans model makes it the ideal host laboratory to elucidate the mechanism and function of chromatin movement in differentiating cells and in cells under external stimuli, such as stress. This is one of the world leading laboratories dealing with chromatin organization, and the belonging of this laboratory to the FMI institute contributes also greatly to the appropriate scientific and technical benefit that this project will gain. The Gasser's lab and the FMI institute possess all the cutting edge biological technologies and knowledge required for this project and they possess many collaborators at the European Union level. Moreover, their world-wide reputation will facilitate to start new collaborations for this project, if needed. This project has good chance to generate high impact papers and hopefully will be able to contribute to the scientific excellence of the EU through its innovative and ambitious characteristic.