Joseph Reese Associate Professor of Biochemistry and Molecular Biology 203 Althouse Laboratory, University Park, PA 16802 Phone: (814) 865-1976 Fax: (814) 863-7024 Email: jcr8@psu.edu B.A. Boston University M.S. University of Illinois, Urbana Ph.D. University of Illinois, Urbana Postdoctoral Fellow, University of Massachusetts Medical Center Member of Center for Eukaryotic Gene Regulation

UV Resistance Pathways in Eukaryotes

Cancer results from of an accumulation of genetic damage and the ensuing uncontrolled cell proliferation. Two common initiators of DNA damage are chemical mutagens and viral pathogens. Eukaryotic cells respond to these challenges by activating a well-known DNA damage sensing, signaling and repair pathway that is stimulated by damage to DNA. Activation of this pathway causes the execution of cell cycle delays, referred to as checkpoints, and the expression of DNA repair genes. Alterations in these functions are known to predispose people to cancer and other diseases. Our laboratory uses a combination of biochemistry, genetics and molecular biology to study the regulation of DNA damage resistance in the simple eukaryote Saccharomyces cerevisiae and viral and mutagen resistance in mammalian cells. The two main focus areas of the lab are the role of chromatin, and modifiers of its structure and function, in regulating the expression of DNA damage inducible genes and the importance of a RNA helicase in DNA damage resistance and viral pathogenesis.

Regulation of DNA damage inducible genes by chromatin and the mechanism of targeting transcription factors to sites within chromatin

Interaction of transcription factors with chromatin

The genome of eukaryotes is packaged into a histone and nonhistone protein complex called chromatin. The basic structural unit of chromatin is the nucleosome which contains 2 copies of four histone proteins that form an octamer and wrap ~146 bp of DNA around the outside of its cylindrical structure. The extraordinary tight association of DNA with histones, and the distortion of DNA caused by its wrapping around the histone octamer, precludes the binding of most DNA binding proteins, and thus, chromatin is generally considered to be a barrier to nuclear functions such as transcription. However, it is becoming increasing clear that a number of transcription factors bind to sites within chromatin and some can interact with sites embedded in a nucleosome. A potential mechanism is that the transcription factor contains a domain that recognizes the structural features of the nucleosome, and chromatin in general. Examples of such domains are starting to emerge, and most chromatin binding domains recognize the N-terminal tails of histones.

We are using the repressor of yeast DNA damage inducible genes, Crt1, as a model for how transcription factors recognize their sites within a chromatin environment. Crt1 is bound to the upstream regulatory region of its target genes when the chromatin is in a repressive configuration and tightly positioned nucleosomes occupy the promoter (Li and Reese, 2001; Zhang and Reese, 2004). The primary amino acid sequence of Crt1 predicts that it binds DNA through a modified winged-helix motif that is most similar to the globular domain of histone H5. Histone H5 is known to preferentially bind to nucleosomal DNA over free DNA due to the structure imparted on the DNA by its wrapping around the histone octamer. Furthermore, we discovered a novel nucleosome-interaction domain (NID) within Crt1, adjacent to its DNA binding domain. These data suggest, and we propose, that Crt1 binds to its sequence elements within the context of repressive chromatin by making contacts with structural features of the nucleosome (Figure 1).

Figure 1. Three models of how transcription factors interact within chromatin. (A) The binding site is located between nucleosomes and the NID binds to an adjacent nucleosome (B) site is located within a nucleosomes  (C) The NID stabilizes the interaction by making contact with an adjacent array of nucleosomes within chromatin

We are carrying out biochemical and genetic analysis of Crt1 to identify how it interacts with sites within chromatin. We are also identifying other transcription factors in yeast with chromatin binding domains and characterizing their ability to interact with sites within chromatin.

Chromatin structure and the regulation of DNA repair genes

UV radiation and other mutagens activate a nuclear DNA damage checkpoint kinase pathway, which in turn induces the expression of DNA repair genes. We use yeast DNA damage repair genes as a model system to understand how cellular signals are translated into changes in chromatin structure and gene expression. The genes encoding the subunits of the enzyme catalyzing the rate limiting step in dNTP synthesis, ribonucleotide reductase (RNR), are strongly induced upon DNA damage. The RNR genes are primarily regulated by a transcriptional repression mechanism mediated by damage-response elements (DREs) in their promoters, which are binding sites for the sequence-specific DNA binding protein Crt1.

Figure 2. DNA damage signals favors the actions of chromatin disruption factors to activate transcription

DNA damage signals cause dramatic changes in chromatin structure over the RNR3 gene (Li and Reese, 2001). In the inactive state, a number of redundant corepressors and enzymes that compact chromatin are bound to the promoter. Some of these complexes are recruited by Crt1. The checkpoint signaling pathway converts Crt1 into an activator, resulting in the release of corepressors and the recruitment of coactivators and chromatin remodeling enzymes (Zhang and Reese, 2005). Crt1 then is released from the promoter, leaving the general transcription machinery to maintain the recruitment of the coactivators (Sharma et al., 2003). Crt1 plays a central role in this process and we are using it, and the RNR genes, as a model for how cellular signals modulate the activities of transcription factors and cause the exchange of chromatin remodeling and modifying activities.

Role of a highly conserved RNA helicase in DNA damage resistance and viral pathogenesis

Dhh1 is a predominantly cytoplasmic DEAD-box RNA helicase, implicated in the processes of RNA turnover and translational control. A crystal structure of the core domain has been solved and we have studied its RNA and ATP binding in vitro. Dhh1 is widely distributed throughout the cytoplasm, but is concentrated in discrete foci called processing bodies and stress granules in yeast. Dhh1, and its associated factors, are believed to play a role in controlling the shuttling of mRNAs through these structures to regulate the expression of specific messages under conditions of cellular stress. We have shown that yeast cells containing a deletion of DHH1 are sensitive to DNA damage and other cell cycle perturbations, and that the mutants are specifically defective for the recovery from the G1/S DNA damage checkpoint (Bergkessel and Reese, 2004). Importantly, overexpression of its human orthologue, DDX6, can complement the phenotypes of the dhh1 mutant, indicating that yeast is an excellent model system to study the function of DDX6.

Figure 3.  X-ray crystallography structure of the core domain.

DDX6, like its yeast orthologue, it is widely distributed throughout the cytoplasm and a fraction of the protein can be found concentrated in foci. Interestingly, we found that exposing cells to ultraviolet irradiation (UV) or other DNA mutagens cause the rapid redistribution of DDX6 in the cytoplasm, leading to an increase in the size and abundance of DDX6-containing cytoplasmic foci (Figure 4).

Figure 4. Huh7 cells (liver carcinoma) were irradiated with 3 J/M2 ultraviolet irradiation. Indirect immunofluorescence using a DDX6 antibody was used to detect DDX6, which appears as red in the figure. UV increases the size and number of foci within the cell.

We are extending our observations in yeast (Dhh1) and mammalian tissue culture cells (DDX6) to identify their functions in DNA damage resistance by:

• Conducting structure/function analysis of the helicase to determine which regions of the protein are required for mRNA trafficking and the ability of cells to respond to DNA damage.
• Using biochemical and proteomics approaches to study the changes in the association of Dhh1/DDX6 with cellular proteins as cells respond to DNA damage and other stresses.
• Developing biochemical and genomics-based approaches to identify the specific mRNAs that are regulated by the helicase and its associated proteins.
• Determining which signaling pathways are involved in the redistribution of the helicase under stress conditions.

We propose that the helicase's ability to affect the expression and trafficking of specific mRNAs in and out of the stress granules are essential for cell cycle control and resistance to mutagens (Figure 5).

Figure 5. Model for how DDX6/Dhh1 controls cell cycle functions during stress.

DDX6 in the pathology of HCV and progression of hepatocellular carcinoma

DDX6 is strongly overexpressed in liver tissue of patients infected with the hepatitis C virus (HCV), and its expression correlates with progression to hepatocellular carcinoma. HCV is a positive strand RNA virus that chronically infects liver cells, and infection often leads to serve liver diseases such as cirrhosis and hapatocellular carcinoma. Replication of the RNA occurs in the cytoplasm on membraneous structures. In collaboration with Drs. Craig Cameron (BMB, Penn State University) and Harriet Isom (Hershey Medical Center) we are exploring the role of DDX6 in HCV pathology and liver disease. We propose that DDX6 is involved in viral pathogenesis by regulating the expression of specific mRNAs that allows cells to respond to and cope with the chronic infection, and/or may regulate the stability/replication of the genome of HCV in the cytoplasm of cells. We are in the process of testing these models, and are interested in determining if HCV replication or expression of viral proteins cause the redistribution of DDX6 in the cell, if gain or loss of DDX6 function affects HCV replication and identifying which genes in HCV and regions of DDX6 are involved in these processes.

Representative Publications:

• Li, B. and Reese, J.C. (2000) Derepression of DNA Damage-Regulated Genes requires Yeast TBP-Associated Factors. EMBO J. 19:4091-4100. Download PDF file from publisher's site)

• Stitzel, M.L. Durso, R. and Reese, J.C. (2001)The Proteasome Regulates the UV-induced Activation of the AP-1 Like Transcription Factor Gcn4. Genes and Development15 128-133. (Download PDF file from publisher's site)

• Li, B. and Reese, J.C. (2001). Ssn6-Tup1 Regulates RNR3 by Positioning Nucleosomes and Affecting the Chromatin Structure at the Upstream repression Sequences. J. Biological Chemistry 276:33788-97. (Download PDF from publisher's site)

• Durso, R.J., Fisher, A. K., Albright-Frey, T.J and Reese, J.C. (2001). Analysis of TAF90 Mutants Displaying Allele-Specific and Global Defects in Transcription. Molecular and Cellular Biology 21:7331-7344. (Download PDF from publisher's site)

• Sharma, V.M, Li, B. and J.C. Reese (2003). SWI/SNF-dependent chromatin remodeling of RNR3 requires TAFIIs and the general transcription machinery. Genes and Development 17:502-515. (Download PDF from publisher's site)

• Bergkessel, M. and Reese, J.C. (2004). The Yeast ortholog of proto-oncogene p54/RCK regulates G1/S checkpoint recovery by a novel mechanism, Genetics, 167:21-33. (Download PDF from publisher's site)

• Zhang, Z. and Reese, J.C. (2004). Ssn6-Tup1 requires the ISW2 Complex to position nucleosomes in Saccharomyces cerevisiae. EMBO J.23:2246-2257. (Download PDF from publisher's site)

• Zhang, Z and Reese, J.C. (2005). Molecular genetic analysis of the Yeast Repressor Rfx1/Crt1 reveals a novel two-step regulatory mechanism. Molecular and Cellular Biology 25:7399-7411. (Download PDF from publisher's site)

• Sharma, V.M., Tomar, R.S., Dempsey, A.E. and Reese, J.C. (2007). Histone deacetylases RPD3 and HOS2 regulate transcriptional activation of DNA damage inducible genes. Molecular and Cellular Biology 27, 3199-3210. (Download PDF from publisher's site)

• Zhang, H. and Reese, J.C. (2007) Exposing the core promoter is sufficient to activate transcription and alter coactivator requirement at RNR3. Proc. Natl. Acad. Sci. U.S.A. 104, 8833-8838. (Download PDF from publisher's site)

• Tomar, R.S., Zhang, S., Brunke-Reese, D.L., Wolcott, H.N. and Reese, J.C. (2008) Yeast Rap1 regulates genomic integrity by activating DNA damage repair genes. EMBO J. (in press)
  

Complete List of Publications and Training 

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