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Modern approaches in studying gene expression
Methods 26 (2002) 215–216 www.academicpress.com
Editorial
Modern approaches in studying gene expression The regulation of eukaryotic gene expression is an extremely complicated process, which involves multiple protein complexes that act at different levels to control the conversion of genetic information into functional RNA or protein products. Arguably, the most important point of regulation is at the transcriptional level. Transcriptional activation requires both chromatin remodeling and promotion of RNA polymerase recruitment and initiation. Each stage in this process is controlled by a different set of regulatory complexes. Furthermore, the whole process is often initiated in response to extra- or intracellular signals. Posttranslational modification is one of the common methods of converting these signals into a change in the activity of the transcriptional regulatory machinery. In this issue, a series of recently developed methods and approaches to studying eukaryotic gene regulation are presented. These permit investigation of posttranslational modifications, and also the composition of regulatory complexes. In addition, techniques are presented that permit the study of the global consequences of changes in gene expression and the mechanisms underlying these changes. These techniques are particularly important in the postgenomic era, where we can now interrogate the complete transcriptome of eukaryotic cells. Signaling pathways play key roles in regulating transcriptional programs. In the presence of multiple signaling pathways, often with similar components, it is important to be able to determine the specificity of signaling to a particular transcription factor or coregulatory protein. Using the MAP kinase pathways as an example, Whitmarsh presents a series of approaches that can be used to demonstrate the specificity of signaling to a particular target protein. In addition to phosphorylation, a number of other posttranslational modifications, including acetylation, methylation, and ubiquitination, play important roles in regulating transcription factor activity. Methylation is important in the study of histone modifications in chromatin and of the regulatory proteins themselves. Schneider and Bannister provide a comprehensive series of assays that can be used to study protein methylation in vitro and in vivo. Ubiquitination regulates both protein stability and the transactivation capacity of eukaryotic transcription factors. Using the coactivator protein TAFII 250 as an example, Sauer and colleagues demonstrate how novel enzymes that can add
ubiquitin groups to proteins (ubiquitin ligases) can be identified using solution and membrane-based assays. Acetylation of both histones and the transcriptional regulators themselves is of critical importance in regulating gene expression. Reagents such as inhibitors that can be used in vitro and in vivo to study the role of histone acetyltransferases (HATs) therefore have important applications in studying the mechanisms of transcriptional regulation. Moreover, due to the deregulation of HATs in cancer, such reagents might have important therapeutic benefits. Aherne and colleagues present a high-throughput screening approach to identify high-affinity HAT inhibitors. This approach can easily be modified to identify inhibitors of other important enzymatic activities involved in gene regulation. Eukaryotic transcription factors and coregulatory proteins act in concert with additional proteins that exist in either stable or transient complexes. Recently, there has been a drive to isolate such interaction partners. Norton and colleagues describe the use of immunoprecipitation techniques for studying transcription factor interactions and demonstrate the importance of extraction and binding conditions in obtaining reliable data. In an extension of this approach, affinity chromatography can be used to isolate protein complexes from cells in which one component is coupled to a matrix. Svejstrup and colleagues illustrate how such approaches can be coupled with modern mass spectrometry to identify components of multifactor complexes. With the completion of several genome sequencing projects and ongoing EST projects, this is a powerful approach to identifying novel complex components and initiating downstream studies on their mechanisms of action. Due to the complexity of regulation in eukaryotes, it is difficult to study the function of a single transcription factor in eukaryotic cells. Genetically, this can be achieved by creating knockout cells/animals in which a single transcriptional regulatory protein is deleted. However, such studies can suffer from functional redundancy in multifamily members or indirect consequences of action of the transcription factor. Vickers and Sharrocks describe an approach that uses potent dominant-negative transcription factors and an inducible expression system to specifically ablate gene regulation by a particular transcription factor. This can be coupled with micro-/macroarray technology to identify
1046-2023/02/$ - see front matter Ó 2002 Elsevier Science (USA). All rights reserved. PII: S 1 0 4 6 - 2 0 2 3 ( 0 2 ) 0 0 0 2 4 - 5
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Editorial / Methods 26 (2002) 215–216
downstream targets. Oliver and colleagues have identified some of the limitations of microarray analyses and have suggested methods to overcome them. One major problem in using microarray technology to study the effect on a single parameter is that the cellular environment or mutated gene often gives rise to a change in growth rate. This change in growth rate subsequently leads to indirect effects that mask the direct effects of the mutation or an experimental variable. Through the use of chemostat yeast cell cultures, growth rate variables can be removed, thereby unmasking gene expression changes. The next decade promises to be an exciting time in studying gene expression mechanisms. With the intro-
duction and refinement of more global technologies such as microarray studies, we will be able to map complete regulatory networks. In addition, evolving techniques to study the regulatory consequences of posttranslational modifications on individual transcription factors and coactivators/corepressors will play a key role in furthering our understanding of the intricacies of gene regulation. By combining research based on global and ‘‘single-molecule’’ studies, significant advances are eagerly anticipated.
Andrew D. Sharrocks Guest Editor
Editorial
Modern approaches in studying gene expression The regulation of eukaryotic gene expression is an extremely complicated process, which involves multiple protein complexes that act at different levels to control the conversion of genetic information into functional RNA or protein products. Arguably, the most important point of regulation is at the transcriptional level. Transcriptional activation requires both chromatin remodeling and promotion of RNA polymerase recruitment and initiation. Each stage in this process is controlled by a different set of regulatory complexes. Furthermore, the whole process is often initiated in response to extra- or intracellular signals. Posttranslational modification is one of the common methods of converting these signals into a change in the activity of the transcriptional regulatory machinery. In this issue, a series of recently developed methods and approaches to studying eukaryotic gene regulation are presented. These permit investigation of posttranslational modifications, and also the composition of regulatory complexes. In addition, techniques are presented that permit the study of the global consequences of changes in gene expression and the mechanisms underlying these changes. These techniques are particularly important in the postgenomic era, where we can now interrogate the complete transcriptome of eukaryotic cells. Signaling pathways play key roles in regulating transcriptional programs. In the presence of multiple signaling pathways, often with similar components, it is important to be able to determine the specificity of signaling to a particular transcription factor or coregulatory protein. Using the MAP kinase pathways as an example, Whitmarsh presents a series of approaches that can be used to demonstrate the specificity of signaling to a particular target protein. In addition to phosphorylation, a number of other posttranslational modifications, including acetylation, methylation, and ubiquitination, play important roles in regulating transcription factor activity. Methylation is important in the study of histone modifications in chromatin and of the regulatory proteins themselves. Schneider and Bannister provide a comprehensive series of assays that can be used to study protein methylation in vitro and in vivo. Ubiquitination regulates both protein stability and the transactivation capacity of eukaryotic transcription factors. Using the coactivator protein TAFII 250 as an example, Sauer and colleagues demonstrate how novel enzymes that can add
ubiquitin groups to proteins (ubiquitin ligases) can be identified using solution and membrane-based assays. Acetylation of both histones and the transcriptional regulators themselves is of critical importance in regulating gene expression. Reagents such as inhibitors that can be used in vitro and in vivo to study the role of histone acetyltransferases (HATs) therefore have important applications in studying the mechanisms of transcriptional regulation. Moreover, due to the deregulation of HATs in cancer, such reagents might have important therapeutic benefits. Aherne and colleagues present a high-throughput screening approach to identify high-affinity HAT inhibitors. This approach can easily be modified to identify inhibitors of other important enzymatic activities involved in gene regulation. Eukaryotic transcription factors and coregulatory proteins act in concert with additional proteins that exist in either stable or transient complexes. Recently, there has been a drive to isolate such interaction partners. Norton and colleagues describe the use of immunoprecipitation techniques for studying transcription factor interactions and demonstrate the importance of extraction and binding conditions in obtaining reliable data. In an extension of this approach, affinity chromatography can be used to isolate protein complexes from cells in which one component is coupled to a matrix. Svejstrup and colleagues illustrate how such approaches can be coupled with modern mass spectrometry to identify components of multifactor complexes. With the completion of several genome sequencing projects and ongoing EST projects, this is a powerful approach to identifying novel complex components and initiating downstream studies on their mechanisms of action. Due to the complexity of regulation in eukaryotes, it is difficult to study the function of a single transcription factor in eukaryotic cells. Genetically, this can be achieved by creating knockout cells/animals in which a single transcriptional regulatory protein is deleted. However, such studies can suffer from functional redundancy in multifamily members or indirect consequences of action of the transcription factor. Vickers and Sharrocks describe an approach that uses potent dominant-negative transcription factors and an inducible expression system to specifically ablate gene regulation by a particular transcription factor. This can be coupled with micro-/macroarray technology to identify
1046-2023/02/$ - see front matter Ó 2002 Elsevier Science (USA). All rights reserved. PII: S 1 0 4 6 - 2 0 2 3 ( 0 2 ) 0 0 0 2 4 - 5
216
Editorial / Methods 26 (2002) 215–216
downstream targets. Oliver and colleagues have identified some of the limitations of microarray analyses and have suggested methods to overcome them. One major problem in using microarray technology to study the effect on a single parameter is that the cellular environment or mutated gene often gives rise to a change in growth rate. This change in growth rate subsequently leads to indirect effects that mask the direct effects of the mutation or an experimental variable. Through the use of chemostat yeast cell cultures, growth rate variables can be removed, thereby unmasking gene expression changes. The next decade promises to be an exciting time in studying gene expression mechanisms. With the intro-
duction and refinement of more global technologies such as microarray studies, we will be able to map complete regulatory networks. In addition, evolving techniques to study the regulatory consequences of posttranslational modifications on individual transcription factors and coactivators/corepressors will play a key role in furthering our understanding of the intricacies of gene regulation. By combining research based on global and ‘‘single-molecule’’ studies, significant advances are eagerly anticipated.
Andrew D. Sharrocks Guest Editor