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, transcription, using infection by herpes simplex virus and the acclimation of plants to cold temperature as experimental contexts.
Transcriptional Activation in Herpes simplex Virus Infection
Herpes simplex virus type 1 (HSV-1) causes the common cold sore or fever blister. The initial lytic infection by HSV-1 results in the obvious symptoms, typically in or around the mouth. After the initial infection, 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. Occasionally, some event (such as emotional stress or damage to the nerve from a sunburn or a root canal operation) will cause the latent virus to reactivate, producing new viruses and recurrence of the cold sore.
The DNA genome of HSV-1 encodes approximately 80 different proteins. The virus does not have its own machinery for expressing those genes, so it diverts 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 (the immediate-early, or IE, genes). In the prevailing model for the mechanism of transcriptional activation, the activation domain of an activating protein (such as VP16) can bind to the host cell RNA polymerase II or to its accessory proteins. In this manner, VP16 recruits or tethers these accessory proteins to the genes that are to be activated.
Chromatin-Modifying Coactivators
Eukaryotic DNA is typically packaged as chromatin, in which the DNA is wrapped around “spools” of histone proteins, and these spools are arranged into higher-order structures. This packaging creates an impediment to transcription, during which RNA polymerase must separate the two strands of DNA. The impediment is overcome with the help of chromatin-modifying coactivator proteins; some chemically alter the histones and others remove the histones so that RNA polymerase can access the DNA. VP16 can recruit various coactivator proteins to target genes, and results from our lab have clearly indicated that VP16 can recruit certain coactivators to IE genes during lytic infection. We have also shown that some histone proteins do associate with viral DNA, although perhaps not to the same extent as with cellular DNA.
Yet, the fact that coactivators are present on viral DNA is not sufficient evidence that they play a significant role in transcriptional activation. We have tested whether particular coactivators are necessary for effective expression of HSV-1 IE genes during lytic infection, using siRNA knockdown of certain coactivators or using mutant cell lines having disrupted expression or activity of a coactivator. We were surprised to find that viral genes were expressed efficiently regardless of what we did to diminish the coactivator activity. These results indicate that our initial hypothesis was wrong; the coactivators, although present, are not required for viral gene expression during lytic infection. Another possibility is that the coactivators are required to reactivate the viral genes from the latent or quiescent state, and we will test that hypothesis during the coming year.
Can a Curry Spice Block Herpes Infections?
Curcumin, the bright yellow component of the curry spice turmeric, affects eukaryotic cells in several ways. Another laboratory had reported that curcumin could block the histone acetyltransferase activity of two coactivator proteins, p300 and CBP. Because we had shown that VP16 can recruit p300 and CBP to viral IE gene promoters, we tested whether curcumin would block viral IE gene expression and thus block HSV infection. Indeed, curcumin has dramatic effects on IE gene expression and substantial effects on virus infection; however, subsequent experiments indicate that this effect is not mediated by the p300 and CBP proteins. For example, the effects on viral infection were observed using lower curcumin concentrations than those required to substantially inhibit global histone H3 acetylation. Moreover, we detected no effect of curcumin on the presence of H3 at viral gene promoters or on the acetylation of H3 at those promoters. These results suggest that curcumin affects VP16-mediated recruitment of RNA polymerase II to IE gene promoters by a mechanism independent of p300/CBP histone acetyltransferase activity. We conclude that curcumin does block herpes infections, but we don’t yet know the mechanism by which it does so.
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. About ten years ago, we applied our emerging interest in chromatin-modifying coactivators to an interesting question in plant biology. Some plants, including the popular experimental organism Arabidopsis, can sense low but nonfreezing temperature in a way that provides protection from subsequent freezing temperatures. This process is known as cold acclimation. Michael Thomashow, an MSU plant scientist, identified genes that are expressed during this process and a transcription factor that activates these genes in response to low temperature. We have collaborated with the Thomashow laboratory to explore the mechanisms involved. We have characterized 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 are also collaborating 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 (Figure 1). 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 effectively modulated so as to protect crop plants from loss in yield or viability due to environmental stresses such as low temperature.
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Figure 1. Growth of Arabidopsis plants, wild-type and mutants. |
External Collaborators
- Kanchan Pavangadkar and Michael F. Thomashow, Michigan State University, East Lansing
- Amy S. Hark, Muhlenberg College, Allentown, Pennsylvania
- Kostas Vlachonasios, Aristotle University of Thessaloniki, Greece
