John J. Dunn Member of the Brookhaven Council Biology Department, 463 Brookhaven National Laboratory Upton, NY 11973-5000 tel: (631) 344-3012 fax: (631) 344-3407 jdunn@bnl.gov The Group: John J. Dunn 344-3012 Barbara N. Lade 344-4960 Laura-Li Loffredo 344-4960 Sean McCorkle 344-4270 Beth Mulligan 344-4960 Judith A. Romeo 344-7430

Research Interests: Genome Signature Tags:      It is well established that interactions between transcription factors and their cognate DNA binding sites form fundamental combinatorial networks within cells that control critical steps in gene expression. Currently, our understanding of how cells encode the diversity of information about where and when genes will be expressed is very limited. Cracking this 'regulatory code' by computational analysis and functional assays is a major problem in biology. The Dunn lab has combined Genomic Signature Tags (GSTs) technology, a method for identifying and quantitatively analyzing genomic DNAs originally devised in our lab, with chromatin immunoprecipitation (ChIP) assays to develop several whole genome, high-throughput, sequence-based approaches for delimiting the positions of bound regulatory proteins in living cells. In one protocol, pairs of 20-21 bp long GST's, derived from both ends of ChIP precipitated DNA fragments, are sequenced en masse to identify the genomic segments originally present in these complexes. Prior to sequencing, short bar-coding oligonucleotides are inserted between the GST pairs from each ChIP sample. The bar-codes allow different samples to be mixed and then sequenced in a multiplex format. We are also developing methods that will use antibodies biotinylated in vitro for ChIP to permit more stringent washing of the pulled-down samples. This should result in a higher signal to noise ration which would be advantageous for direct Solexa-based sequencing of the recovered ChIP DNA fragments. Epigenetics:      Vertebrate DNA is heavily methylated at cytosine residues in CpG dinucleotide sequences to form 5mCpG except for short stretches of CpG-rich DNA, CpG islands, about 0.5 to 2 kb long which are normally free of cytosine methylation. Methylation within CpG islands is a common way to regulate nearby gene activity without altering the DNA code. Recent studies have suggested that aberrant methylation within CpG islands which is referred to as an epigenetic modification may play an important role in mediating stable changes in nervous system function including the behavioral changes seen in psychiatric disorders such as schizophrenia. Our hypothesis is that similar changes are probably associated with methamphetamine (MA) associated drug-induced dependence and psychosis. Our goal is to develop methods to detect methylated CpG dinucleotides near genes with clinical relevance following MA treatment of rodents using DNA samples isolated from different brain regions. The technology is relevant to developing systems to understand drug addiction, cancer and ageing as well as the cellular responses of human cells to low doses of ionizing radiation for DOE’s Low Dose Radiation Program. The methodology also is applicable to understanding how cytosine methylation regulates gene expression in plants and is therefore potentially important for future research as part of BNL’s initiative to develop sustainable biofuels.      The major principle behind our work is that largely intact CpG islands with methylated residues can be isolated by exploiting the differential affinity of these DNA fragments for recombinant proteins containing methyl-CpG binding domains. The fragments can then be identified by hybridization or by cloning and sequencing using our GST-based methods. All these approaches are being used in our present studies. In particular we are investigating whether the E. coli McrA protein, a putative mC-specific nuclease, can be used to cleave DNA at mC residues. To this end we have cloned, expressed and purified the recombinant protein to homogeneity. Current efforts are aimed at determining whether McrA is a nuclease or if it exerts its biological effect by binding to mC containing DNA. We also plan to test whether the DNA glycosylase/lyase activity of the Arabidopsis ROSI protein can be used to cleave vertebrate and plant DNA which contains mCpG and mCpNpG sequences at the mCs. Lyme Disease:      In an ongoing collaborative effort, we are conducting genomic and proteomic analyses of the Lyme disease spirochete, Borrelia burgdorferi to understand how specific gene products participate in immune evasion and pathogenicity. Our laboratory is part of a large group effort to determine the whole genome sequences of several pathogenic and non-pathogenic Borrelia strains. One aspect of our work involves developing multiple displacement amplification or MDA-based approaches to amplify specific plasmids for further analysis from the very complicated mixture of plasmids present in early Borrelia isolates. These same amplification techniques are being exploited as part of a collaborative project with Eli Hatchwell (Stony Brook University) to amplify human DNA and still retain its epigenetic profile. One problem with standard PCR-based DNA amplification methodology is that all methylated CpG dinucleotides in a DNA template are converted to unmodified CpGs.

Becker M., Bunikis J., Lade B.D., Dunn J.J., Barbour A.G. and Lawson C.L. Structural investigation of Borrelia burgdorferi OspB, a bactericidal Fab target. J Biol Chem.280(17), 17363-17370 (2005). PubMed PDB files: 1P4P, 1RJL; Jmol viewer

Kumaran D., Eswaramoorthy S., Luft B., Koide S., Dunn J.J., Lawson C.L. and Swaminathan S. Crystal structure of outer surface protein C (OspC) from the Lyme disease spirochete, Borrelia burgdorferi. EMBO J. 20, 971-978 (2001). PubMed PDB files: 1F1M, 1GGQ; Jmol viewer

Ding W., Huang X., Yang X., Dunn J.J., Luft B.J., Koide S. and Lawson C.L. Structural identification of a key protective B-cell epitope in Lyme disease antigen OspA. J Mol Biol. 302, 1153-1164 (2000). PubMed PDB file 1FJ1; Jmol viewer

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