Project description:We knocked out different exons corresponding to different sections of GABP, a transcription factor known to bind to the telomerase reverse transcriptase (TERT) promoter with the G228 mutation. We investigated the effects of the knockouts in the regulation of TERT expression and other subunits of GABP. GABP genomic binding sites were determined through chromatin immunoprecipitation sequencing (ChIP-seq), and gene expression was determined through total RNA sequencing (RNA-seq). Combining data pertaining to GABP binding sites and gene expression provided insight into the molecular mechanisms of maintaining the length of telomeres in cancers.
Project description:We knocked out different exons corresponding to different sections of GABP, a transcription factor known to bind to the telomerase reverse transcriptase (TERT) promoter with the G228 mutation. We investigated the effects of the knockouts in the regulation of TERT expression and other subunits of GABP. GABP genomic binding sites were determined through chromatin immunoprecipitation sequencing (ChIP-seq), and gene expression was determined through total RNA sequencing (RNA-seq). Combining data pertaining to GABP binding sites and gene expression provided insight into the molecular mechanisms of maintaining the length of telomeres in cancers.
Project description:Within steroid receptor heterocomplexes the large tetratricopeptide repeat-containing immunophilins, cyclophilin 40 (CyP40), FKBP51, and FKBP52, target a common interaction site in heat shock protein 90 (Hsp90) and act coordinately with Hsp90 to modulate receptor activity. The reversible nature of the interaction between the immunophilins and Hsp90 suggests that relative cellular abundance might be a key determinant of the immunophilin component within steroid receptor complexes. To investigate CyP40 gene regulation, we have isolated a 5-kilobase (kb) 5'-flanking region of the human gene and demonstrated that a approximately 50 base pair (bp) sequence adjacent to the transcription start site is essential for CyP40 basal expression. Three tandemly arranged Ets sites within this critical region were identified as binding elements for the multimeric Ets-related transcription factor, GA binding protein (GABP). Functional studies of this proximal promoter sequence, in combination with mutational analysis, confirmed these sites to be crucial for basal promoter function. Furthermore, overexpression of both GABP alpha and GABP beta subunits in Cos1 cells resulted in increased endogenous CyP40 mRNA levels. Significantly, a parallel increase in FKBP52 mRNA expression was not observed, highlighting an important difference in the mode of regulation of the CyP40 and FKBP52 genes. Our results identify GABP as a key regulator of CyP40 expression. GABP is a common target of mitogen and stress-activated pathways and may integrate these diverse extracellular signals to regulate CyP40 gene expression.
