Rotation Projects:  WE LOVE ROTATING GRAD STUDENTS!!!!!!

 

1-Fanconi anemia (FA) and replication:  We have found that the central FA protein FANCD2 is monoubiquitinated in response to DNA damage that causes replication fork collapse and that FA mutant cells are hypersensitive to DNA crosslinks.  FANCD2 activates downstream repair proteins in the homologous recombination and translesion synthesis pathway.  FANCD2 also interacts with the MCM2-7 helicase, which is responsible for replication licensing and restart after pause.  This project will focus on a-functional importance of MCM2-7 helicase in the FA pathway by measuring the helicase’s role in DNA damage tolerance (cell survival assays), FANCD2 ubiquitination (immunoblotting), and homologous recombination (cell based HR substrate assays).

 

2-RNA-FA connection:  in addition to triggering DNA repair pathways, we have determined that the FA pathway prevents excess RNA production leading to competition with DNA repair intermediates.  R loops, the RNA:DNA hybrid counterpart to D loops, form in the excess of RNA and DNA damage. The rotation student will measure R loop formation in FA mutant cells using DRIP to immunoprecipitate R loops from mutant cells and wild type controls in the presence of absence of DNA damage. 

 

3-Functionally analyze FA mutations found in tumors:  it has been noted that mutations in FA genes have been found in tumors.  In our personalized medicine program at Yale, we have found a significant percentage of FA mutations as well.  However, little work has been performed to determine if these mutations are deleterious or not.  The student will mutagenize wild type cDNAs by PCR and transduce into FA mutant cells. The resulting cells will be assessed for cellular sensitivity and ability to activate the FA pathway by FANCD2 monoubiquitination as well as presence of chromosomal instability upon metaphase spread.

 

4-FA Synthetic lethal screen hit and suppressor screen hit validation:  Since FA mutations are present somatically in tumors, we hypothesize that identifying synthetic lethal genes which when targeted result in enhanced cell kill will present an opportunity to improve cancer therapy.  Our high throughput screen has identified several interesting hits in the PLK1, Ras, and proteasome pathways.  The rotation project will use cell based assays to assess how targeting these pathways will synergize with underlying FA mutant DNA damage hypersensitivity.  Conversely, the same methods will measure how targeting suppressor pathways will counteract the FA mutant phenotype in an effort to develop therapies to spare patients with FA bone marrow failure and cancer risk.

 

5-HTLV I Tax effect in different p53 mutant backgrounds:  We have observed that expression of Tax in a p53 deleted background results in DNA damage hypersensitivity.  This is significant because many resistant tumors become so due to p53 mutations, and p53 is the commonly mutated gene in cancers.  Our goal is to develop Tax as a chemosensitization tool in order to treat cancer more effectively.  This rotation project will test which p53 mutant backgrounds are amenable to this approach, as deletion of p53 accounts for only 10% of aberrant p53 expression, and gain of function or dominant negative p53 mutants are an important but as of yet untested means of chemoresistance that we can target with the Tax protein.

 

6-Histone epigenetic state of Congenital Dyserythropoietic Anemia (CDA):  we study CDA I and the protein whose gene is mutated, codanin.  The function of codanin is unknown.  Recently, we have identified binding proteins of codanin that are transcription factors and histone modifying enzymes.  In addition, cells mutant for codanin display altered chromatin structure, implying that histone modification may be altered.  This project will assess the epigenetic state of histones, focusing on methylation of lysine 4 of histone 3.  We will also test methyltransferase inhibition on erythropoiesis in K562 and CD34 primary cells stimulated down the erythroid lineage and mutated or not for codanin to determine if manipulation of histone modification can rescue the CDA phenotype.