Final Report Summary - CHROMATINSYS (Systematic Approach to Dissect the Interplay between Chromatin and Transcription)
We are all familiar with the analogy of DNA as the “blueprint” of life. One of the greatest challenges in biology is to understand how living cells read these complex instructions. In particular, how do cells turn the proper genes to “ON” or “OFF” at the right timing? This transcriptional regulation is critical to all forms of life from microorganisms to humans, and is established by several levels of regulatory mechanisms. A key integration point in transcriptional regulation is at level of chromatin -- the packaging of DNA inside the cells.
The chromatin template is composed of repeating subunits – nucleosomes – each of which comprises ~150bp of DNA wrapped roughly twice around an octamer of small basic histone proteins. There are two major ways in which cells modulate this template to influence gene expression. Chromatin remodeling machines can disrupt histone-DNA contacts, resulting in nucleosome eviction, changes in nucleosome location, or an altered nucleosome composition. These changes can expose or hide parts of the DNA sequence, thus affecting binding of regulatory proteins and transcriptional machinery to the specific location. In addition, the histone proteins are subject to many covalent modifications, or marks. These marks can be recognized by other proteins, which in turn can affect how other regulatory proteins access the DNA at the specific locality. Thus, similar to the way marking on boxes can influence where and how they are shipped, the marking on chromatin can influence how the DNA at that particular location is accessed and used. The discovery of multiple different marks and numerous “writer” and “reader” proteins, suggested that cells can deposit and revise annotations over the DNA sequence, which by itself is unmodified during the life of a cell or an organism, as local memory of past decisions.
The extreme evolutionary conservation of histone modifications and their related proteins from yeast to humans indicates that these processes are central for proper cellular function. Indeed, dysregulation of chromatins structure is implicated in a wide range of diseases including human malignancies. The basic mechanisms of transcription are also conserved from yeast to human, and insights gained into the interaction of chromatin and transcription in yeast have proven to have direct ramifications to our understanding of disease relevant pathways.
In this project we developed new experimental strategies to observe positions of nucleosomes, and how they are marked in a genome-wide manner. We combined these assays with novel methods for fast acting conditional shutdown of protein activity, to examine how the chromatin structure changes once a crucial protein is “removed” from the system. This strategy allowed us to examine how key proteins, such chromatin remodelers and “writers” of histone marks, maintain the chromatin structure. Unlike previous approaches, we could disentangle direct effects (that appear immediately after the shutdown) from longer term indirect effects (as the cell shift to stress mode due to the missing function).
Another experimental method we employed is to assay the system as it responds to an external cue. As an analogy, observing two cars driving on a straight and open highway does not allow us to determine the quality of their breaks or the engine. However, when they hit a sharp curve differences between the cars become apparent both in slowing down before the turn and in speeding up after it. Thus, observing how cells that underwent different genetic perturbations, such as the conditional shutdown of a protein, are respond to environmental changes that require dramatic changes to the transcription program provide rich information on the functions that they are missing due to the intervention.
The chromatin template is composed of repeating subunits – nucleosomes – each of which comprises ~150bp of DNA wrapped roughly twice around an octamer of small basic histone proteins. There are two major ways in which cells modulate this template to influence gene expression. Chromatin remodeling machines can disrupt histone-DNA contacts, resulting in nucleosome eviction, changes in nucleosome location, or an altered nucleosome composition. These changes can expose or hide parts of the DNA sequence, thus affecting binding of regulatory proteins and transcriptional machinery to the specific location. In addition, the histone proteins are subject to many covalent modifications, or marks. These marks can be recognized by other proteins, which in turn can affect how other regulatory proteins access the DNA at the specific locality. Thus, similar to the way marking on boxes can influence where and how they are shipped, the marking on chromatin can influence how the DNA at that particular location is accessed and used. The discovery of multiple different marks and numerous “writer” and “reader” proteins, suggested that cells can deposit and revise annotations over the DNA sequence, which by itself is unmodified during the life of a cell or an organism, as local memory of past decisions.
The extreme evolutionary conservation of histone modifications and their related proteins from yeast to humans indicates that these processes are central for proper cellular function. Indeed, dysregulation of chromatins structure is implicated in a wide range of diseases including human malignancies. The basic mechanisms of transcription are also conserved from yeast to human, and insights gained into the interaction of chromatin and transcription in yeast have proven to have direct ramifications to our understanding of disease relevant pathways.
In this project we developed new experimental strategies to observe positions of nucleosomes, and how they are marked in a genome-wide manner. We combined these assays with novel methods for fast acting conditional shutdown of protein activity, to examine how the chromatin structure changes once a crucial protein is “removed” from the system. This strategy allowed us to examine how key proteins, such chromatin remodelers and “writers” of histone marks, maintain the chromatin structure. Unlike previous approaches, we could disentangle direct effects (that appear immediately after the shutdown) from longer term indirect effects (as the cell shift to stress mode due to the missing function).
Another experimental method we employed is to assay the system as it responds to an external cue. As an analogy, observing two cars driving on a straight and open highway does not allow us to determine the quality of their breaks or the engine. However, when they hit a sharp curve differences between the cars become apparent both in slowing down before the turn and in speeding up after it. Thus, observing how cells that underwent different genetic perturbations, such as the conditional shutdown of a protein, are respond to environmental changes that require dramatic changes to the transcription program provide rich information on the functions that they are missing due to the intervention.