טכניון מכון טכנולוגי לישראל
הטכניון מכון טכנולוגי לישראל - בית הספר ללימודי מוסמכים  
Ph.D Thesis
Ph.D StudentDrucker-Gonen Nitzan
SubjectThe Key Role of the Proton-Coupled Folate Transporter
(PCFT/SLC46A1) in Folate Homeostasis and
Antifolate Resistance
DepartmentDepartment of Biology
Supervisor Professor Yehuda Assaraf
Full Thesis textFull thesis text - English Version


Abstract

Folates are essential B9-vitamins that play a critical role as one-carbon donors in a multitude of biosynthetic pathways including RNA synthesis and DNA replication, mitochondrial protein synthesis, amino acid metabolism and methyl group biogenesis. Folates are negatively charged at physiological pH and thus cannot cross the plasma membrane by passive diffusion. The proton-coupled folate transporter (PCFT/SLC46A1) is one of three transport systems known to accommodate folate transport. PCFT functions as a folate-proton co-transporter in the acidic microclimate of the upper small intestine. As nothing was known about the regulation of PCFT gene expression under physiological and pathological conditions, we initiated studies to decipher the cis- and trans-acting regulatory elements that regulate PCFT gene expression. We identified the promoter of the human PCFT gene and demonstrated that it harbors a 1085bp CpG island, which plays a key role in regulation of PCFT gene expression. This CpG island was densely methylated in human leukemia cells and the PCFT gene was consequently silenced. Using a sequential deletion analysis of the PCFT promoter we found that a 271bp fragment upstream to the first ATG achieved the same transcriptional transactivation as does the entire 3.1kb fragment. We also found that the basal promoter is rich in functional GC-box sites that play a major role in regulation of PCFT gene expression. We next studied unique trans-acting elements that may be involved in the regulation of PCFT gene expression. We discovered that NRF-1, the dominant transcription factor regulating mitochondrial biogenesis and respiration, binds to, and transactivates the human PCFT promoter. This finding constitutes one of the first functional linkages between mitochondria biogenesis and folate metabolism, thereby revealing that PCFT is an NRF1-responsive gene that is regulated as a mitochondrial gene. These findings enhance our understanding of the regulation of PCFT gene expression in normal and malignant tissues.