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2009 Scientific Report

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Van Andel Research

Van Andel Research Institute | Scientific Report Research Interests The genetic information encoded in DNA must first be copied, in the form of RNA, before it can be translated into the proteins that do most of the work in a cell. Some genes must be expressed more or less constantly throughout the life of any eukaryotic cell. Others must be turned on (or turned off) in particular cells either at specific times or in response to a specific signal or event. Thus, regulation of gene expression is a key determinant of cell function. Our laboratory explores the mechanisms that regulate the first step in that flow, the process termed transcription. Over the past 20 years, my laboratory has used infection by herpes simplex virus as an experimental context for exploring the mechanisms of transcriptional activation. In the past 10 years, we have also asked similar questions in a very different biological context, the acclimation of plants to cold temperature. Transcriptional activation during herpes simplex virus infection Herpes simplex virus type 1 (HSV-1) causes the common cold sore or fever blister. The initial lytic (or productive) infection by HSV-1 results in the obvious symptoms in the skin and mucosa, typically in or around the mouth. After the initial infection resolves, HSV-1 finds its way into nerve cells, where the virus can hide in a latent mode for long times—essentially for the lifetime of the host organism. Occasionally, some trigger event (such as emotional stress, damage to the nerve from a sunburn, or a root canal operation) will cause the latent virus to reactivate, producing new viruses in the nerve cell and sending those viruses back to the skin to cause a recurrence of the cold sore. The DNA of HSV-1 encodes approximately 80 different proteins. However, the virus does not have its own machinery for expressing those genes; instead, the virus must divert the gene expression machinery of the host cell. That process is triggered by a viral regulatory protein designated VP16, whose function is to stimulate transcription of the first viral genes to be expressed in the infected cell (the immediate-early, or IE, genes). Chromatin-modifying coactivators in herpes virus infection and a paradox The strands of DNA in which the human genome is encoded are much longer than the diameter of a typical human cell. To help fit the DNA into the space of a cell, eukaryotic DNA is typically packaged as chromatin, in which the DNA is wrapped around “spools” of histone proteins, and these spools are then further arranged into higher-order structures. This elaborate packaging creates a problem when access is needed to the information carried in the DNA, such as when particular genes need to be expressed. This problem is solved in part by chromatin-modifying coactivator proteins, which either chemically change the histone proteins or else slide or remove them. Transcriptional activator proteins such as VP16 can recruit these chromatin-modifying coactivator proteins to target genes. We have shown this to be true for artificial reporter genes in human cells or in yeast, and it’s also true for the viral genes that VP16 activates during an active infection. Curiously, however, the DNA of herpes simplex virus is not wrapped around histones inside the viral particle, and it also seems to stay free of histones inside the infected cell. That observation leads to a paradox: why would VP16 recruit chromatin-modifying coactivators to the viral DNA if the viral DNA doesn’t have any chromatin to modify? 52

VARI | 2009 We took several approaches to test whether the coactivators that are recruited to viral DNA by the VP16 activation domain really play a significant role in transcriptional activation. In some experiments, we knocked down expression of given coactivators using short interfering RNAs (siRNAs) and then measured viral gene expression during subsequent infection by HSV-1. In other experiments, we used cell lines that have mutations disrupting the expression or activity of a given coactivator. A third set of experiments used curcumin, derived from the curry spice turmeric, which is thought to inhibit the enzymatic activity of certain coactivators. In each of these situations, we expected to find that viral gene expression was inhibited, but the experiments yielded unexpected results: in each case, expression of the viral genes was essentially unaffected. We were forced to conclude that our initial hypothesis was wrong; the coactivators, although present, are not required for viral gene expression during lytic infection. The death of one hypothesis, however, gives life to new ideas. After the initial infection of a cold sore subsides, herpes simplex virus establishes a lifelong latent infection in sensory neurons. In the latent state, the viral genome is essentially quiet: very few viral genes are expressed. Moreover, the viral genome becomes packaged in chromatin much like the silent genes of the host cell. So our new hypothesis is that the coactivators recruited by VP16 are required to reactivate the viral genes from the latent or quiescent state. We’ve begun to test that hypothesis in quiescent infections in cultured cells, but the key tests will be in whole organisms with genuinely latent herpesvirus infections. Gene activation during cold acclimation of plants Although plants and their cells obviously have very different forms and functions than animals and their cells, the mechanisms used for expressing genetic information are quite similar. For the past decade, we have explored the role of chromatin-modifying coactivators in regulating genes that are turned on in low-temperature conditions. Some plants, including the prominent experimental organism Arabidopsis, can sense low (but nonfreezing) temperature in a way that provides protection from subsequent freezing temperatures, a process known as cold acclimation. We have collaborated with Michael Thomashow, a plant scientist at Michigan State University, to explore the mechanisms involved in activating genes during cold acclimation. To this point, we have focused on one particular histone acetyltransferase, termed GCN5, and two of its accessory proteins, ADA2a and ADA2b. Mutations in the genes encoding these coactivator proteins result in diminished expression of cold-regulated genes. Moreover, histones located at these cold-regulated genes become more highly acetylated during initial stages of cold acclimation. However, contrary to our expectations, the GCN5 and ADA2 proteins are not responsible for this cold-induced acetylation. In fact, we’ve tested several other Arabidopsis histone acetyltransferases, and none (on their own) seem solely responsible for this acetylation. It seems likely that redundant mechanisms are at work, such that when we disrupt one pathway, another pathway compensates. We also collaborated with groups in Greece and Pennsylvania to explore the distinct biological activities of the two ADA2 proteins. Although the two proteins have very similar sequences and both are expressed throughout the plant, mutations in the genes encoding these two proteins have very different phenotypes. The ada2b mutants are very short, have smaller cells than normal, and are sterile. In contrast, the ada2a mutants seem quite normal in most attributes. Plants with mutations in both ADA2a and ADA2b are strikingly similar to plants with mutations in GCN5. We suspect that GCN5 can partner with either ADA2a or ADA2b and that these two distinct complexes affect different sets of genes and thus different developmental and stress response pathways. This work may help us understand whether the mechanisms by which plants express their genes can be modulated so as to protect crops from loss in yield or viability due to environmental stresses such as low temperature. 53

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