Final Report Summary - CHROMATRANSCRIPT (Role of the chromatin architecture in the regulation of gene transcription)
Objectives of the project
The chromatin fiber contained in eukaryotic cells is characterized by a complex multi-scale architecture which allows efficient packing of the DNA double helix into the nucleus. Our understanding of the complex 3-dimensional folding of the genome in the nucleus and its dynamics is still incomplete, yet such folding probably plays an important regulatory role in several key cellular processes such as the regulation of gene expression, DNA replication and DNA repair. In particular, it is now well established that the modulation of gene transcription leads to dramatic changes in the nuclear architecture. Nevertheless, these modifications affecting the chromatin architecture are still poorly described and their exact role in the regulation of transcription remains unclear.
The overall objective of this research project is to investigate these different aspects of the process of nuclear restructuring in association to modulation of gene transcription using the estrogen-regulated genes as a “model” system. To reach this goal, the first step of the project, achieved at the end of the first reporting period, was to develop methodologies to characterize quantitatively the dynamics of the chromatin architecture. The second step of the project was consisting in applying these different approaches for the fine characterization of the chromatin remodeling processes associated with the modulation of transcription
Results
Methodological developments for the quantitative analysis of the dynamic chromatin architecture
The initial objective of this project was to develop tools to image the dynamic chromatin architecture at multiple spatio-temporal scales. First, we established a photolabeling approach which allows highlighting in fluorescence specific sub-nuclear regions. This method is based on the conversion of photoconvertible fluorophores fused to core histones. The subsequent timelapse imaging and automatic segmentation of the labeled regions allow characterizing at the micron scale the remodeling of the chromatin architecture. In order to reach smaller space-scales, we used an approach based on the incorporation of fluorescent nucleotides at the DNA replication foci to characterize the local chromatin movements. Our results indicate that this approach is suitable to quantitatively analyze local chromatin movements over a wide range of time-scales going from tens of milliseconds to minutes.
The two methods mentioned above allows characterizing the higher organization scales of the chromatin. However, to fully characterize this structure, it is also necessary to analyze the behavior of the first structuring scale: the chromatin fiber. One key element of this fiber is the nucleosome. We setup a custom imaging system to analyze the dynamics of single fluorescently labeled nucleosomes in living cells. The original setup that we have established allowed us to characterize the nucleosome motions at unprecedented spatio-temporal resolution. The second important characteristic of the chromatin fiber is its packing state. In order to assess the dynamics of this packing-state in living cells, we have initiated the development of an approach based on the monitoring of the Förster Resonance Energy Transfert (FRET) between incorporated fluorescent nucleotides. By measuring quantitatively the FRET signal using fluorescence lifetime imaging, we found that this approach is indeed suitable to assess chromatin packing state in living cells.
Altogether, the different methodological developments mentioned above allow characterizing the chromatin architecture and its dynamics at multiple spatio-temporal scales.
Progresses in the characterization of the dynamic chromatin architecture in lving cells
To study the modifications in the chromatin organization in response to the modulation of gene transcription, we analyzed chromatin movements in MCF-7 cells (human breast cancer cell line) labeled with fluorescent nucleotides. Our results indicate that the chromatin fiber is highly flexible in living cells. Next we analyzed whether the modulation of transcription by estradiol stimulation could affect the chromatin steady-state dynamics. Surprisingly, we did not find significant differences in chromatin motions between MCF-7 cells treated or not with estradiol.
Using our single molecule imaging system, we characterized the motions of single nucleosomes in living cells. Our results reveal that histones display constrained diffusive motion in agreement with recent observations. However, similar to the analysis performed at larger scales in the previous section, we did not observe a significant change in nucleosome motions upon estradiol stimulation.
Considering the absence of changes in chromatin dynamics observed in the context of the modulation of transcription by estradiol stimulation, we decided to invest more in the analysis of chromatin remodeling at DNA breaks. Such analysis was not planned in the original project. Our results clearly identify the chromatin remodeler Alc1 as a major mediator of the rapid chromatin relaxation observed at DNA breaks through its ATP-dependent chromatin remodeling activity.
Perspectives
In terms of methodologies to characterize chromatin dynamics, the methods that were developed in the course of this project have allowed us to analyze quantitatively the chromatin motions in living cells at difference space scales. In particular, using DNA labeling with fluorescent nucleotides, we could show that chromatin movements in mammalian cells are compatible with the classical Rouse polymer model, similar to what was shown in bacteria or yeast cells. Using this model, we could predict that the chromatin fiber is highly flexible in the living cells, in agreement with recent images obtained by electron microscopy.
Concerning the question of the interplay between chromatin dynamics and the modulation of gene transcription by estrogen, our data suggest that even though estradiol stimulation is though to affect the global organization of the nucleus, the local motions displayed by the chromatin at the megabase scale (probed by the fluorescent nucleotides) or the single nucleosome level are not significantly affected.
Our data concerning the chromatin relaxation at DNA lesions have allowed us to precisely decipher the molecular mechanisms responsible for this chromatin remodeling process. The next step will be to understand the exact functional role of this mechanism in the context of the DNA repair processes.
The chromatin fiber contained in eukaryotic cells is characterized by a complex multi-scale architecture which allows efficient packing of the DNA double helix into the nucleus. Our understanding of the complex 3-dimensional folding of the genome in the nucleus and its dynamics is still incomplete, yet such folding probably plays an important regulatory role in several key cellular processes such as the regulation of gene expression, DNA replication and DNA repair. In particular, it is now well established that the modulation of gene transcription leads to dramatic changes in the nuclear architecture. Nevertheless, these modifications affecting the chromatin architecture are still poorly described and their exact role in the regulation of transcription remains unclear.
The overall objective of this research project is to investigate these different aspects of the process of nuclear restructuring in association to modulation of gene transcription using the estrogen-regulated genes as a “model” system. To reach this goal, the first step of the project, achieved at the end of the first reporting period, was to develop methodologies to characterize quantitatively the dynamics of the chromatin architecture. The second step of the project was consisting in applying these different approaches for the fine characterization of the chromatin remodeling processes associated with the modulation of transcription
Results
Methodological developments for the quantitative analysis of the dynamic chromatin architecture
The initial objective of this project was to develop tools to image the dynamic chromatin architecture at multiple spatio-temporal scales. First, we established a photolabeling approach which allows highlighting in fluorescence specific sub-nuclear regions. This method is based on the conversion of photoconvertible fluorophores fused to core histones. The subsequent timelapse imaging and automatic segmentation of the labeled regions allow characterizing at the micron scale the remodeling of the chromatin architecture. In order to reach smaller space-scales, we used an approach based on the incorporation of fluorescent nucleotides at the DNA replication foci to characterize the local chromatin movements. Our results indicate that this approach is suitable to quantitatively analyze local chromatin movements over a wide range of time-scales going from tens of milliseconds to minutes.
The two methods mentioned above allows characterizing the higher organization scales of the chromatin. However, to fully characterize this structure, it is also necessary to analyze the behavior of the first structuring scale: the chromatin fiber. One key element of this fiber is the nucleosome. We setup a custom imaging system to analyze the dynamics of single fluorescently labeled nucleosomes in living cells. The original setup that we have established allowed us to characterize the nucleosome motions at unprecedented spatio-temporal resolution. The second important characteristic of the chromatin fiber is its packing state. In order to assess the dynamics of this packing-state in living cells, we have initiated the development of an approach based on the monitoring of the Förster Resonance Energy Transfert (FRET) between incorporated fluorescent nucleotides. By measuring quantitatively the FRET signal using fluorescence lifetime imaging, we found that this approach is indeed suitable to assess chromatin packing state in living cells.
Altogether, the different methodological developments mentioned above allow characterizing the chromatin architecture and its dynamics at multiple spatio-temporal scales.
Progresses in the characterization of the dynamic chromatin architecture in lving cells
To study the modifications in the chromatin organization in response to the modulation of gene transcription, we analyzed chromatin movements in MCF-7 cells (human breast cancer cell line) labeled with fluorescent nucleotides. Our results indicate that the chromatin fiber is highly flexible in living cells. Next we analyzed whether the modulation of transcription by estradiol stimulation could affect the chromatin steady-state dynamics. Surprisingly, we did not find significant differences in chromatin motions between MCF-7 cells treated or not with estradiol.
Using our single molecule imaging system, we characterized the motions of single nucleosomes in living cells. Our results reveal that histones display constrained diffusive motion in agreement with recent observations. However, similar to the analysis performed at larger scales in the previous section, we did not observe a significant change in nucleosome motions upon estradiol stimulation.
Considering the absence of changes in chromatin dynamics observed in the context of the modulation of transcription by estradiol stimulation, we decided to invest more in the analysis of chromatin remodeling at DNA breaks. Such analysis was not planned in the original project. Our results clearly identify the chromatin remodeler Alc1 as a major mediator of the rapid chromatin relaxation observed at DNA breaks through its ATP-dependent chromatin remodeling activity.
Perspectives
In terms of methodologies to characterize chromatin dynamics, the methods that were developed in the course of this project have allowed us to analyze quantitatively the chromatin motions in living cells at difference space scales. In particular, using DNA labeling with fluorescent nucleotides, we could show that chromatin movements in mammalian cells are compatible with the classical Rouse polymer model, similar to what was shown in bacteria or yeast cells. Using this model, we could predict that the chromatin fiber is highly flexible in the living cells, in agreement with recent images obtained by electron microscopy.
Concerning the question of the interplay between chromatin dynamics and the modulation of gene transcription by estrogen, our data suggest that even though estradiol stimulation is though to affect the global organization of the nucleus, the local motions displayed by the chromatin at the megabase scale (probed by the fluorescent nucleotides) or the single nucleosome level are not significantly affected.
Our data concerning the chromatin relaxation at DNA lesions have allowed us to precisely decipher the molecular mechanisms responsible for this chromatin remodeling process. The next step will be to understand the exact functional role of this mechanism in the context of the DNA repair processes.