Cloning and characterization of the promoter regions from the parent and paralogous creatine transporter genes

Abstract

Interconversion between phosphocreatine and creatine, catalyzed by creatine kinase is crucial in the supply of ATP to tissues with high energy demand. Creatine's importance has been established by its use as an ergogenic aid in sport, as well as the development of intellectual disability in patients with congenital creatine deficiency. Creatine biosynthesis is complemented by dietary creatine uptake. Intracellular transport of creatine is carried out by a creatine transporter protein (CT1/CRT/CRTR) encoded by the SLC6A8 gene. Most tissues express this gene, with highest levels detected in skeletal muscle and kidney. There are lower levels of the gene detected in colon, brain, heart, testis and prostate. The mechanism(s) by which this regulation occurs is still poorly understood. A duplicated unprocessed pseudogene of SLC6A8-SLC6A10P has been mapped to chromosome 16p11.2 (contains the entire SLC6A8 gene, plus 2293 bp of 5'flanking sequence and its entire 3'UTR). Expression of SLC6A10P has so far only been shown in human testis and brain. It is still unclear as to what is the function of SLC6A10P. In a patient with autism, a chromosomal breakpoint that intersects the 5'flanking region of SLC6A10P was identified; suggesting that SLC6A10P is a non-coding RNA involved in autism. Our aim was to investigate the presence of cis-acting factor(s) that regulate expression of the creatine transporter, as well as to determine if these factors are functionally conserved upstream of the creatine transporter pseudogene. Via gene-specific PCR, cloning and functional luciferase assays we identified a 1104 bp sequence proximal to the mRNA start site of the SLC6A8 gene with promoter activity in five cell types. The corresponding 5'flanking sequence (1050 bp) on the pseudogene also had promoter activity in all 5 cell lines. Surprisingly the pseudogene promoter was stronger than that of its parent gene in 4 of the cell lines tested. To the best of our knowledge, this is the first experimental evidence of a pseudogene with stronger promoter activity than its parental gene.

Keywords: 3T3 Swiss; ATP; Creatine transport; DNA complementary to RNA; EGFP; GAPDH; Glyceraldehyde 3-phosphate dehydrogenase; HEK293; MEFs; ORF; PCR; Promoter; Pseudogene; RTPCR; SK-N-SH; SLC6A10P; SLC6A8; SV40; Simian virus 40; Transcriptional regulation; UTR; adenosine triphosphate; base pair; bp; cDNA; chr; chromosome; enhanced green fluorescent protein; gDNA; genomic DNA; human embryonic kidney cell line; human neuroblastoma cell line; kb; kilo base pair; mRNA; messenger RNA; miRNA; microRNA; mouse embryo fibroblasts; mouse fibroblast cell line; open reading frame; polymerase chain reaction; reverse transcriptase-polymerase chain reaction; solute carrier family 6, member 10 pseudogene; solute carrier family 6, member 8; untranslated region.