Rahul Kohli, M.D., Ph.D.
University of Pennsylvania
502B Johnson Pavilion
3610 Hamilton Walk
Philadelphia, PA 19104-6073
In mammalian cells, DNA modifications are centered to the largest extent around cytosine bases, which are targeted by three different DNA modifying processes: methylation, oxidation and deamination. Research in the Kohli laboratory focused on the biochemistry and chemical biology of the enzymes that make cytosine such a dynamic base in the genome.
Cytosine methylation by DNA Methyltransferases (DNMTs) generates 5-methylcytosine (5mC), an epigenetic modification associated with silencing, while TET family enzymes can catalyze step-wise oxidation of 5mC to generate three new oxidized 5mC bases (ox-mCs) – 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC) and 5-carboxylcytosine (5caC). These bases that are critical intermediates in the cycle of DNA demethylation and can also potentially serve as independent epigenetic marks. Deamination of either cytosine or modified cytosine bases by AID/APOBEC family enzymes yields targeted transition mutations in the genome. ‘Purposeful’ mutation by AID/APOBECs is used to garble foreign genomes, is exploited by the immune system to mature antibody responses, and has been posited to play roles in DNA demethylation. Such activity also carries risks and, accordingly, the deamination signatures of AID/APOBECs have been prominently left on cancer genomes.
In the Kohli laboratory, we utilize a broad array of approaches, which include: 1) biochemical characterization of enzyme mechanisms, 2) chemical synthesis of enzyme probes, and 3) biological assays spanning epigenetics and immunology to study DNA modifying enzymes.
Contribution to Science
Resolving the enigmatic mechanisms of DNA demethylation. Despite the wealth of studies on the importance of 5-methylcytosine (mC) in mammalian genomes, the mechanism by which DNA can be demethylated has remained elusive. While high profile studies had suggested that deamination of mC or related analogs could be involved in DNA demethylation, the biochemical feasibility of this reaction had not been established. Further, with the discovery of TET family enzymes that can oxidize mC, new avenues for demethylation have been recently proposed. We were the first to show that enzymatic deamination of oxidized analogs of mC is a disfavored route for demethylation and also the first to show that TDG results in specific depletion of 5-formylcytosine from genomes. We have also recently demonstrated that TET2 shows catalytic processivity, providing a mechanism for the generation of highly oxidized mC species despite the relative dearth of their precursors. We have also found key active site determinants that control step-wise oxidation and fund TET enzymes that stall at hmC and provide a means to dissociate the different activities of hmC, fC and caC. We continue to probe the mechanism of TET family enzymes, aiming to unravel the many permutations of modifications with five cytosine states (C, mC, hmC, fC, cac), on two opposite strand (CpG pairs) that can be generated by three different TET enzymes (TET1, TET2, TET3).
- Nabel CS, Jia H, Ye Y, Shen L, Goldschmidt HL, Stivers JT, Zhang Y, Kohli RM (2012) AID/APOBEC deaminases disfavor modified cytosines implicated in DNA demethylation, Nature Chem Biol 8:751-8. (PMC3427411)
- Kohli RM, Zhang Y (2013) TET enzymes, TDG and the Dynamics of DNA Demethylation, Nature 502: 472-9. [Peer-reviewed Review] (PMC4046508)
- Crawford DJ, Liu MY, Nabel CS, Cao XJ, Garcia BA, Kohli RM. (2016) Tet2 catalyzes stepwise 5-methylcytosine oxidation by an iterative and de novo mechanism. J Am Chem Soc 138:730-3.
- Liu MY, Torabifard H, Crawford DJ, DeNizio JE, Cao XJ, Garcia BA, Cisneros GA, Kohli RM (2017) Mutations along a TET2 active site scaffold stall oxidation at 5-hydroxymethylcytosine, Nature Chem Biol, 13:181-187.
Explaining how targeted mutagenesis drives antibody maturation and genomic modification. Our lab has made great strides in understanding how targeted and purposeful mutation is used to improve our immune defenses. The enzyme Activation Induced Deaminase (AID) is the key driver of antibody maturation, catalyzing the targeted deamination of cytosine to generate uracil within the immunoglobulin locus. This targeted mutation is the initiating step in somatic hypermutation and class switch recombination. We have helped to decipher how targeting takes place at the molecular level, demonstrating how particular hotspots in the genome are targeted, and how the enzyme can discriminate DNA from RNA. These cytosine deaminase enzymes have been proposed to play a role in DNA demethylation. Furthermore, they offer new and unexploited tools to understand cytosine modification states in the genome.
- Kohli RM, Abrams SR, Gajula KS, Maul RW, Gearhart PJ, Stivers JT (2009) A portable hotspot recognition loop transfers sequence preferences from APOBEC family members to activation-induced cytidine deaminase, J Biol Chem 284: 22898-22904 (PMC2755697)
- Nabel CS, Lee JW ,Wang LC Kohli RM (2013) Nucleic acid determinants for selective deamination of DNA over RNA by activation-induced deaminase, Proc Natl Acad Sci USA 110: 14225–14230 (PMC3761612)
- Gajula KS, Huwe PJ, Mo CY, Crawford DJ, Stivers JT, Radhakrishnan R, Kohli RM (2014) High-throughput mutagenesis reveals functional determinants for DNA targeting by activation-induced deaminase, Nucleic Acids Res 42: 9964-75 (PMC4150791)
- Schutsky EK, Nabel CS, Davis AKF, DeNizio JE, Kohli RM (2017) APOBEC3A efficiently deaminates methylated, but not TET-oxidized, cytosine bases in DNA, Nucleic Acids Res. doi: 10.1093/nar/gkx345 (PMID 28472485)
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