Promoter Specific Hypermethylation Sensors for Early Cancer Detection

Summary

Principal Investigator: Indraneel Ghosh
Abstract: DESCRIPTION (provided by applicant): The long-term objective of the proposed research project is to provide a robust, sensitive, and rapid method for the direct detection of CpG island methylation in the promoter region of specific genes implicated in cancer. Cytosine methylation occurs at CpG dinucleotides in 70-80% of the human genome, most often in repetitive genomic regions. On the other hand CpG islands, defined as short sequences with statistically high CpG content, present in the promoter region of many genes (60%) are primarily protected from methylation in normal tissues. These CpG islands have been found to be methylated in cancer leading to transcriptional repression. Recent experiments provide strong correlation between CpG hypermethylation at promoter sites of numerous genes and the incidence of cancer, thus making specific promoter hypermethylation a valuable marker for early detection. Current methods for detection of specific CpG island methylation rely on extensive bisulfite treatment of methylated DNA followed by PCR based amplification, sequencing, or microarray techniques. These current methods, though powerful are also laborious, time-intensive and expensive for characterizing known sites of hypermethylation. Towards the goal of rapidly determining promoter CpG hypermethylation we will apply our newly developed technology called SEquence Enabled Reassembly (SEER) of proteins. The SEER system allows for the recognition of specific sequences of double-stranded DNA that result in the concomitant assembly of functional protein reporters (green fluorescent protein, 2- Lactamase, and firefly luciferase). Promoter specific hypermethylation will be detected by methyl-CpG binding domains (MBDs), while the correct promoter sequence will recognized by designed zinc-fingers. Our approach has the potential to provide sensitive turn-on sensors for directly reporting upon CpG methylation at known promoter sites. This approach if successful will rapidly distinguish between normal and cancerous tissues in a clinical setting without the requirement for bisulfite treatment, PCR amplification, and sequencing. We will provide proof of concept by 1) designing and optimizing turn-on biosensors for detecting specific methylation events in model DNA constructs;and 2) designing and testing biosensors that target promoter regions of genes (BRCA1, CDH1, p15, p16, MGMT, GSTp1) implicated in cancer. PUBLIC HEALTH RELEVANCE: Promoter specific hypermethylation will be detected by methyl-CpG binding domains (MBDs), while the correct promoter sequence will recognized by designed zinc-fingers. Our approach has the potential to provide sensitive turn-on sensors for directly reporting upon CpG methylation at known promoter sites. This approach if successful will rapidly distinguish between normal and cancerous tissues in a clinical setting without the requirement for bisulfite treatment, PCR amplification, and sequencing.
Funding Period: ----------------2009 - ---------------2011-
more information: NIH RePORT

Top Publications

  1. pmc Direct DNA methylation profiling using methyl binding domain proteins
    Yinni Yu
    Department of Bioengineering, University of Utah, 72 South Central Campus Drive, Room 2750, Salt Lake City, Utah 84112, USA
    Anal Chem 82:5012-9. 2010
  2. pmc Toward a general approach for RNA-templated hierarchical assembly of split-proteins
    Jennifer L Furman
    Department of Chemistry and Biochemistry, University of Arizona, 1306 East University Boulevard, Tucson, Arizona 85721, USA
    J Am Chem Soc 132:11692-701. 2010
  3. ncbi A turn-on split-luciferase sensor for the direct detection of poly(ADP-ribose) as a marker for DNA repair and cell death
    Jennifer L Furman
    Department of Chemistry and Biochemistry, University of Arizona, 1306 East University Blvd, Tucson, AZ 85721, USA
    Chem Commun (Camb) 47:397-9. 2011
  4. pmc Evaluating the global CpG methylation status of native DNA utilizing a bipartite split-luciferase sensor
    Ahmed H Badran
    Department of Chemistry and Biochemistry, University of Arizona, Tucson, Arizona 85721, United States
    Anal Chem 83:7151-7. 2011
  5. pmc Split-protein systems: beyond binary protein-protein interactions
    Sujan S Shekhawat
    Department of Chemistry and Biochemistry, University of Arizona, 1306 East University Blvd, Tucson, AZ 85721, USA
    Curr Opin Chem Biol 15:789-97. 2011

Detail Information

Publications5

  1. pmc Direct DNA methylation profiling using methyl binding domain proteins
    Yinni Yu
    Department of Bioengineering, University of Utah, 72 South Central Campus Drive, Room 2750, Salt Lake City, Utah 84112, USA
    Anal Chem 82:5012-9. 2010
    ..Fitting skew normal probability density functions to our data, we estimate an accuracy of 97.5% for our method in identifying methylated CpG loci, which can be improved through optimization of probe design and surface density...
  2. pmc Toward a general approach for RNA-templated hierarchical assembly of split-proteins
    Jennifer L Furman
    Department of Chemistry and Biochemistry, University of Arizona, 1306 East University Boulevard, Tucson, Arizona 85721, USA
    J Am Chem Soc 132:11692-701. 2010
    ..These approaches provide potentially general design paradigms for the conditional reassembly of fragmented proteins in the presence of any desired ssRNA target...
  3. ncbi A turn-on split-luciferase sensor for the direct detection of poly(ADP-ribose) as a marker for DNA repair and cell death
    Jennifer L Furman
    Department of Chemistry and Biochemistry, University of Arizona, 1306 East University Blvd, Tucson, AZ 85721, USA
    Chem Commun (Camb) 47:397-9. 2011
    ....
  4. pmc Evaluating the global CpG methylation status of native DNA utilizing a bipartite split-luciferase sensor
    Ahmed H Badran
    Department of Chemistry and Biochemistry, University of Arizona, Tucson, Arizona 85721, United States
    Anal Chem 83:7151-7. 2011
    ..Finally, we demonstrated that this bipartite sensor can be utilized for monitoring dose-dependent changes in global levels of methylation in DNA from HeLa cells challenged with 5-aza-2'-deoxycytidine, a DNA methyltransferase inhibitor...
  5. pmc Split-protein systems: beyond binary protein-protein interactions
    Sujan S Shekhawat
    Department of Chemistry and Biochemistry, University of Arizona, 1306 East University Blvd, Tucson, AZ 85721, USA
    Curr Opin Chem Biol 15:789-97. 2011
    ..Finally, we discuss autoinhibition strategies leading to turn-on sensors as well as future directions in split-protein methodology including possible therapeutic approaches...