A number of new information has emerged from this work. First, rat mtGPAT has two promoters: distal and proximal. Second, the distal promoter is ~30 kb upstream of the first initiation codon. Third, there are two transcripts synthesized - one from each promoter. Finally, only the distal promoter, not the proximal promoter, responds to starvation and refeeding in rat liver cells.
The promoter inspector analysis shows the presence of hepatocyte nuclear factor (HNF-1 and HNF-4), NFκB, NFY binding reverse CAAT box (32
), activating protein binding site (AP1 and AP4), myocyte enhancer factor (MEF2), upstream stimulatory factor (USF), and matrix attachment regions (MARS) in the proximal promoter and SREBP, ChREBP, SP1 and CTF1 binding sites in distal promoter (). The presence of MARS sequence shows that the proximal region may have a significant role in transcription of mtGPAT gene. The MARS sequences link chromatin loops to protein scaffold known as the nuclear matrix and they have been found in repeat sequences (27
). They are the nucleation site for unwinding DNA as seen in human 5′ beta-interferon gene and immunoglobulin heavy chain gene enhancer (28
). Distal promoter region has ChREBP binding site which directly promotes lipogenic enzymes like ACC, FAS and LPK gene transcription (31
). The 30 kb intron has three inverted loops that might be responsible in bringing the distal promoter region close to the first initiation codon thus enabling the transcription of the distal promoter product to occur. Inverted repeats have been shown to be involved in transcription, replication, and gene amplification (21
Comparison of 5′UTR regions of mouse mtGPAT and rat mtGPAT revealed that there was a deletion of 167 nucleotides in rat making the rat 5′UTR region shorter than that of the mouse (8
). This 167 nucleotide sequence is actually present in the rat genomic sequence. So, its absence in the rat mtGPAT cDNA suggests that this length of 5′UTR is spliced out in the rat. If so, the splicing of 5′ UTR in rat mtGPAT may generate more than one type of transcripts. This consideration suggested the presence of another promoter (22
). So, we examined the genomic region just upstream of first initiation codon. The luciferase assay with transfected rat liver cells shows about 4.8-fold increase in luciferase activity with the proximal 500 bp () and a 2.5-fold increase with the proximal 1050 bp () as compared to the cells transfected with the promoterless vector. The distal promoter’s luciferase activity is about 10.8 fold meaning that the promoter activity is more in case of the distal promoter. Highest promoter activity could be localized to the 500 bp region of the proximal promoter which contains consensus binding sites for transcription factors. The promoter activity of outside this region (1050 bp) is significantly lower. Portions of the 1050 bp region of proximal promoter contain repeat sequences () that might cause a reduction in transcription as seen with luciferase assay (). The general view about multiple promoters is that when a single gene is transcribed from multiple promoters, an organism gains additional flexibility in the control of expression of that gene (35
). In the case of the α-amylase gene, there are two promoters: weak downstream promoter which is active in liver while a strong upstream promoter is active in the parotid gland. This difference in the two promoters account for almost 100-fold difference in expression levels of the gene in the two above mentioned tissues (36
). This could be the case with rat mtGPAT gene where the two promoters function differentially in a tissue specific way or nutritionally or hormonally.
In the preliminary EMSA (), 200 bp probes were used for both distal and proximal promoters to check for binding of a number of transcription factors in the probe region. The binding of ChREBP and NFκB to distal and proximal regions was further confirmed by ChIP assays (). This result shows that these factors bind specifically to the predicted sites and hence are major determinants of activity of the promoters.
The two transcripts of rat mtGPAT that we obtained after RACE PCR show the presence of alternative exons. The longer transcript has 176 bp from the first exon that is ~30 kb upstream of the second exon while the proximal promoter transcript starts from exon 3 (). This situation is very similar to that of ACC gene that has three promoters and three different transcripts which are nutritionally and hormonally regulated (25
It has been shown that ChREBP is expressed at high levels in the liver, adipose, kidney and small intestine (32
). Distal promoter region has consensus binding sites for key transcription factors like SREBP and ChREBP which are responsible for the transcriptional regulation of lipogenic enzymes. It has been shown that distal promoter can recruit ChREBP () implying that this promoter could be responsible for the transcriptional regulation of mtGPAT (54).
From the combined data of computer analysis (), luciferase assay (), EMSA (), ChIP assay () and 5′RACE PCR (), we conclude that mtGPAT has two promoters (distal and proximal) that are responsible for generating two transcripts.
It is known that activities of critical lipogenic enzymes like mtGPAT and FAS are tightly controlled nutritionally and hormonally (20
). So, when cells were starved and refed, luciferase activity regulated by the distal promoter almost tripled although that by the proximal promoter remained unchanged (). The results suggest that the distal but not the proximal promoter is the inducible one. Thus, the two promoters can be functionally differentiated.
It is interesting to note that, like GPAT, human ACC gene has more than one promoter (32
). Both enzymes are positively affected by starvation and refeeding (20
) and also by insulin and EGF (30
) and negatively by glucagon and adrenaline (29
). Our preliminary work with EGF shows that EGF increases luciferase expression by two fold in rat liver cells transfected with rat mtGPAT distal promoter (data not shown). A question arises why both ACC and GPAT, the first step in fatty acid and glycerolipid synthesis, respectively, need to be similarly regulated in the overall synthesis of the glycerolipids? Probably, without GPAT being regulated similar to ACC, the fatty acids produced may be used either for synthesis of glycerolipids or for β-oxidation.
Since promoters are tissue specific and function differently at different developmental levels (29
), the two regulatory regions might be functioning differently in other cell lines. Our results suggest that distal promoter and not the proximal promoter is the one that is inducible by nutritional regulation. The elucidation of the function of these transcription factors for these promoters and the role of ~30 kb intron are expected to help in further understanding the mechanism of transcription and regulation of the mtGPAT gene.