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dc.contributor.advisorRodríguez-Martínez, José A.
dc.contributor.authorCarrasquillo-Dones, Emanuel A.
dc.description.abstractCongenital Heart Diseases (CHD) are the most common disease found in neonates, with over 100,000 new reported cases each year. CHDs are characterized by malformations in the heart’s chambers, walls, and great vessels, sometimes leading to embryonic death. Cardiac transcription factors (cardiac TFs) such as NKX2-5 and TBX5 play a critical role in the heart's developmental stages and physiological processes through DNA-protein interactions by regulating gene expression. Genetic analyses have identified various missense (missense) mutations within the DNA-binding domain (DBD) of NKX2-5 (Homeodomain, HD) and TBX5 (T-box domain) associated with CHDs. We hypothesize that missense mutations in the cardiac TF DNA-binding domain (DBD) alter their DNA-binding properties, ultimately affecting transcriptional pathways necessary for proper heart development. To address this hypothesis, we successfully cloned, over-expressed in E.coli, and purified the DNA binding domains (DBD) of NKX2-5 (HD) and TBX5 (T-box). Functional analysis protocols using Electrophoretic Mobility Shift Assay (EMSA) confirmed binding to their DNA cognate binding sites. In addition, via site-directed mutagenesis, we generated seven missense mutations (A148E, E154G, R161P, T178M, Q181H, R190C, Y191C) of the NKX2-5 HD and seven missense mutations (I54T, M74V, G80R, I101F, R113K, R237Q) of the TBX5 T-box identified in CHD patients. DNA-binding assays show decreased DNA activity in the case of specific mutants like NKX2-5 HD mutants A148E, R161P, T178M, Q181H, R189P, and Y191C, and in the cases of TBX5 T-box mutants I54T, M74V, G80R, I101F, R113K, R237Q. We used high-throughput Systematic Evolution of Ligands and Exponential Enrichment (SELEX-seq) and bioinformatics tools to determine the DNA-binding specificity of wild-type NKX2-5 and TBX5 and their respective mutants. Mutants A148E, R161P, and Y191C display similar DNA-binding specificity as NKX2-5 WT. Interestingly, mutant Y191C shows altered specificity to Hox transcription factors binding site. TBX5 mutants I54T and R113K bind to similar binding sites as TBX5 WT but also to a secondary binding site exclusively. NKX2-5 mutants E154G, T178M, Q181H, R190C, and TBX5 mutants M74V, G80R, I101F, R237Q, and R237W showed no significant enrichment for specific DNA binding sites. Flanking sequence analysis of NKX2-5 WT and its mutants showed similar preferences to 5’ and 3’ flanking sequences. Y191C was an exception indicating a change in preference for the 3’ flanking sequences by its Hox binding site. TBX5, with its mutants, showed similar preferences to 5’ and 3’ flanking sequences. However, the flanking site analysis of TBX5 mutants I54T and R113K altered binding site showed higher affinity for similar flanking sequences in the altered binding site compared to the TBX5 consensus binding site. Like other homeodomains, DNA shape analysis of NKX2-5 showed a higher preference for binding sites with a narrower minor groove width and an enhanced electrostatic potential. NKX2-5 mutant A148E showed a marked increase in transcriptional activation, while mutant R161P showed similar activity compared to NKX2-5. We characterized specific altered functions in NKX2-5 and TBX5 essential for DNA binding and NKX2-5 in gene transactivation. All this indicates that high-throughput and higher order analysis of DNA binding sites can shed light on the effects of disease causing-mutations effects in transcriptional functions by determining TF affinity and specificity. Consequently, loss in TF DNA binding activity and recognition suggests a link of missense mutations with deleterious effects on TF functions, leading to congenital heart defects.en_US
dc.description.sponsorshipInvestigation subsidized with funds from: National Institute of Health grant award SC1GM127231 IQ-Bio-REU Dr. Juan Ramirez-Lugo Award number 1852259 MARC Grant 5T34GM007821-40S1 Research Initiative for Scientific Enhancement (RISE) Grant 5R25GM061151-19en_US
dc.rightsAttribution-NonCommercial-NoDerivs 3.0 United States*
dc.subjectCongenital heart defectsen_US
dc.subjectDNA altered binding sitesen_US
dc.subjectDNA shapeen_US
dc.subject.lcshCongenital heart diseaseen_US
dc.subject.lcshDNA Analysisen_US
dc.subject.lcshDNA-binding proteinsen_US
dc.subject.lcshTranscription factorsen_US
dc.titleProtein-DNA interactomes of NKX2-5 and TBX5 mutations associated to Congenital Heart Defectsen_US
dc.rights.holder© 2022 Emanuel A. Carrasquillo-Donesen_US
dc.contributor.committeeCadilla Vázquez, Carmen L.
dc.contributor.committeePeterson-Peguero, Esther A.
dc.contributor.committeeGonzález Vargas, Carlos I.
dc.contributor.committeeGarcía-Blanco, Mariano
dc.contributor.representativeGhezzi, Alfredo
dc.contributor.campusUniversity of Puerto Rico, Río Piedras Campusen_US
dc.description.graduationSemesterFall (1st Semester)en_US
dc.description.graduationYear2022en_US Intercampus Doctoral Program University of Puerto Rico Rio Piedras Campus and Medical Sciences Campusen_US

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