Scientific Overview: Transcriptional Regulation

Research Interests

The genetic information encoded in DNA must first be transcribed 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 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 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 genome of HSV-1 encodes approximately 80 different proteins. However, the virus does not have its own machinery for expressing those genes; instead, it must divert the gene expression machinery of the host cell. That process is triggered by a viral regulatory protein designated VP16, whose function it is to stimulate transcription of the first viral genes to be expressed in the infected cell (the immediate-early, or IE, genes).

VP16 recruitment of host cell transcription machinery

The prevailing model for the mechanism of transcriptional activation is that a portion of an activating protein (such as VP16) called the activation domain (AD) can bind to the host cell RNA polymerase II or to its accessory proteins. In this manner, VP16 recruits or tethers accessory proteins to the genes that are to be activated. Over the years, several accessory proteins (also known as general transcription factors) have been implicated as potential targets for VP16. Of those, the evidence seems to point most directly at TFIID, a multi-protein complex that includes the TATA-binding protein (TBP). TBP itself can bind rather efficiently to the VP16 activation domain, and mutations in VP16 that disrupt transcriptional activation also disrupt the interaction with TBP. We have pursued the structure of the VP16-TBP interaction by methods including X-ray crystallography and nuclear magnetic resonance. We have also tested the hypothesis that VP16 can influence the orientation of TBP on the TATA-box DNA of a target gene promoter. This hypothesis, proposed by other laboratories, is based on the fact that both TBP and the TATA sequence to which it binds are quite symmetric, and yet TBP can effectively support transcription in only a single orientation. We developed a new quantitative method for assessing TBP orientation and using this method have now demonstrated that TBP binds in a well-oriented manner even in the absence of VP16. Moreover, on a TATA site engineered to be completely symmetric, to which TBP binds in both orientations, the VP16 activation domain has no significant influence. This work resolves a long-standing issue regarding TBP orientation and eliminates one hypothesis for the mechanism of transcriptional activation.

Chromatin-modifying coactivators in herpes virus infections

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 packaging creates an impediment to transcription, during which RNA polymerase must separate the two strands of DNA. The impediment can be overcome with the help of chromatin-modifying coactivator proteins, some of which alter the histone proteins by post-translational modifications (e.g., acetylation or methylation) and others of which can slide or remove the histone proteins to permit access by RNA polymerase to the DNA.

Experiments using the VP16 activation domain in artificial contexts (for example, in yeast genetic assays) have indicated that VP16 can recruit various coactivator proteins to target genes. However, the HSV-1 viral DNA is not packaged with histones in the infectious virion, and prior evidence suggested the viral DNA remained largely chromatin-free during infection. Therefore, we wondered whether VP16 would recruit these coactivators to viral IE genes, and if so whether those coactivators would be acting on histone proteins (which didn’t seem to be present) or on some other target. Our results have clearly indicated that VP16 can recruit certain coactivators to IE genes during lytic infection. We have also shown that at least some histone proteins do associate with viral DNA, although perhaps not to the same extent as with cellular DNA. We are currently exploring further which histones associate with viral DNA, how quickly they are put in place, the mechanisms used to put them in place, and what VP16 and other regulatory proteins might do to counteract the repressive effects of chromatin, which could be considered a molecular defense mechanism.

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 has 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, as an inhibitor of p300 or CBP activity, 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 (Fig. 1). We are now trying to determine whether that effect is indeed channeled through the p300 and CBP proteins or whether it arises from another of the biological activities of curcumin.

Figure 1. HSV-1 infection of Vero cell monolayers. HSV-1 infection results in plaques or holes in a monolayer of cultured human cells (left). In the presence of curcumin (right), plaques are generally smaller and the cells within the plaques are not as completely obliterated. Photo by M. Roemer.

Gene activation during cold acclimation in 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 prominent experimental organism Arabidopsis, can sense low (but nonfreezing) temperature in a way that provides protection from subsequent freezing temperatures (Fig. 2). This process is known as cold acclimation. Michael Thomashow, an MSU plant scientist, has explored the genes expressed during this process, and we collaborated with his 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. We are now working to determine whether GCN5 and the ADA2 proteins are partially or fully responsible for this cold-induced acetylation. We are also collaborating with groups in Greece and Pennsylvania to explore the distinct biological activities of the two ADA2 proteins. 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.

Figure 2. Acclimation of Arabidopsis seedlings. Arabidopsis seedlings were grown on agar plates for three weeks at 20 °C. The plants in the right panel were chilled at 4 °C for two days. All plants were then subjected to subfreezing temperatures (–5 °C) for one day and then were returned to warm temperatures to recover. The acclimated plants remain healthy and green; the nonacclimated plants lose much of their color and die. Photo by K. Pavangadkar.

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