"USING EPIDRUGS TO INVESTIGATE GENE ACTIVATION MECHANISMS: TBX1 GENE MODEL."

ABSTRACT THE DISCOVERY OF THE CONTRIBUTION OF ALTERATIONS IN THE EPIGENOME TO GENETIC DISEASES HAS DRIVEN RESEARCH INTO EPIDRUGS THAT TARGET SUCH ALTERATIONS. 22Q11.2 DELETION SYNDROME, ALSO CALLED DIGEORGE SYNDROME, IS A RARE GENETIC DISEASE THAT IS PRIMARILY A DISEASE OF TBX1 HAPLOINSUFFICIENCY. STUDIES CONDUCTED IN ANIMAL MODELS OF 22Q11.2DS, AND ESPECIALLY IN TBX1 MUTANT MICE, SUGGEST THAT EPIGENETIC CHANGES AT THE TBX1 LOCUS MIGHT CONTRIBUTE TO DISEASE PATHOGENESIS. THEREFORE, EPIDRUGS CAPABLE OF MODULATING TBX1 EXPRESSION ARE OF PARTICULAR INTEREST BECAUSE OF THEIR POTENTIAL TO INCREASE OUR UNDERSTANDING OF THE MECHANISMS BY WHICH TBX1 IS REGULATED DURING EMBRYOGENESIS, WHICH ARE LARGELY UNKNOWN. THEY MAY ALSO HAVE THERAPEUTIC VALUE FOR TREATING SOME OF THE SIGNS AND SYMPTOMS OF 22Q11.2DS, ALTHOUGH MANY OF THESE ARE CONGENITAL ANOMALIES AND THEREFORE NOT AMENABLE TO PHARMACOLOGICAL INTERVENTION. THE MAJOR GOAL OF THIS RESEARCH PROJECT WAS TO IDENTIFY EPIGENETIC CHANGES IN THE FORM OF HISTONE MODIFICATIONS THAT ARE TBX1-ACTIVATING IN CELL CULTURES AND IN EMBRYONIC TISSUES. FOR THIS, I USED TWO DIFFERENT APPROACHES; THE FIRST WAS A CANDIDATE DRUG APPROACH FOR WHICH I SELECTED EPIDRUGS THAT TARGET HISTONE MODIFICATIONS THAT ARE GENERALLY ASSOCIATED WITH GENE ACTIVATION. I THEN TESTED THE ABILITY OF SINGLE AND COMBINED EPIDRUG TREATMENTS TO INCREASE TBX1 EXPRESSION IN TWO CELL CULTURE SYSTEMS. THE SECOND WAS A BIOINFORMATICS-BASED APPROACH, IN WHICH A NEWLY DEVELOPED ALGORITHM WAS APPLIED TO EXTRACT INFORMATION ABOUT HISTONE MODIFICATIONS ACROSS THE MOUSE GENOME FROM LARGE DATASETS OF GENE EXPRESSION AND CHROMATIN ORGANIZATION DEPOSITED IN PUBLIC DATABASES AND INTEGRATE IT WITH THE SEQUENCE OF NEWLY IDENTIFIED PUTATIVE TBX1 ENHANCERS. THIS NOVEL APPROACH ALLOWED ME TO IDENTIFY THE MOST HIGHLY REPRESENTED HISTONE MODIFICATIONS ASSOCIATED WITH ACTIVATION OF THE TBX1 LOCUS. THE GOALS OF THE PROJECT WERE LARGELY MET, AS EACH APPROACH IDENTIFIED TWO PUTATIVE TBX1-ACTIVATING HISTONE MODIFICATIONS. IN ADDITION, I IDENTIFIED A COMBINATION OF EPIDRUG TREATMENT THAT TARGETS THESE MODIFICATIONS AND INCREASES TBX1 EXPRESSION IN CELLS. IN FUTURE EXPERIMENTS, THE COMBINED EPIDRUG TREATMENT REGIME WILL BE USED TO DETERMINE THE IN VIVO RELEVANCE OF THESE TBX1-ACTIVATING HISTONE MODIFICATIONS THROUGH PHENOTYPIC RESCUE EXPERIMENTS IN TBX1 MUTANT MICE.