The present study provides insight into the mechanism of the transcriptional regulation of rat mtGPAT gene. We have demonstrated here that the distal promoter is the regulatory promoter under the influence of hormones and growth factor while the proximal promoter acts constitutively. We have further investigated the mechanism of the transcriptional regulation by the distal promoter and have determined that the distal promoter controls the transcriptional regulation by lipogenic transcription factors such as ChREBP and SREBP-1c.
We have previously established the presence of two promoter sequences controlling the transcription of rat mtGPAT gene [13
]. The distal promoter is ~30 kb upstream (first intron) of the first translational codon while the proximal promoter is 63 bp upstream of second translational codon (). The significance of these two promoter regions remains unclear. Previous study showed that when cells were starved and refed, luciferase activity regulated by the distal promoter was about three fold as compared to the cells grown in complete media. Conversely, the proximal promoter’s luciferase activity remained unchanged [13
shows two initiating codons. Probably two proteins are produced under different conditions. We know, from our previous experiments (Onorato et al, 2005, Ref. 23
; Balija and Haldar, Unpublished results) that the whole or a partially truncated (165 bp) mitochondrial GPAT cDNA can be used to express the active acyltransferase. However, these two proteins, even if they are produced simultaneously, cannot be detected as separate entities by Western blot. However, when expressed separately, each of them react to the same antibody.
In this study we found that the levels of expression of the distal transcript were more variable than that of the proximal transcript () after treating the cells with the agents and were more closely correlated with the expression and specific activity of mtGPAT protein (). This was consistent with the idea that the distal promoter is the regulatory promoter which causes inducible/repressible level of expression of the distal transcript while the proximal promoter maintains a basal level of expression as shown by the semiquantitative RTPCR (). The distal transcript is synthesized from TSS1 present in exon 1 while the proximal transcript is synthesized from TSS2 present in exon 3. When usual range of concentrations of the four agents was used similar to previous studies [19
], we found similar dose dependent changes caused in the luciferase activity of the distal promoter. However, it must be mentioned here that the higher concentrations of insulin might be working in part through the IGF-1(insulin-like growth factor I) receptor pathway [30
]. Previous studies have shown that high-dose (1μM) of insulin activate IGF-1 receptor and induce an increase in SREBP-1 mRNA, protein and total lipid production in human SEB-1 sebocytes [31
]. Thus, it seems possible that the high concentrations of insulin could also activate IGF-1 receptors and up-regulate SREBP-1 protein which then bind to the distal promoter of mtGPAT, thereby causing an increase in lipogenesis. In obesity, there is a development of insulin resistance causing an increase in insulin levels and cause an increase in bioavailable IGF-1. These two promote cell growth and inhibit apoptosis thereby contributing to tumorigenesis. However, the differential behavior of the two promoters under the influence of the four agents is obvious (). There is a growing body of evidence that suggests that a number of mammalian genes are regulated by multiple promoters and have transcripts with different TSSs [32
]. These multiple promoters behave differentially depending on the cellular conditions [36
]. For example, proximal promoter of the rat pyruvate carboxylase gene plays a major role in gluconeogenesis and lipogenesis, whereas the distal promoter is necessary for anaplerosis [38
]. 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 the expression of that gene [39
]. This could be the case with rat mtGPAT gene where the two promoters function differentially under the influence of nutritional or hormonal changes. Hence it is important to determine which of the two promoters is responsible for the transcriptional regulation of rat mtGPAT gene under the influence of the hormones and EGF. Although studies show that SREBP-1c and ChREBP act in synergy to induce lipogenic enzymes like ACC and FAS [18
], the role of these transcription factors in the transcriptional regulation of the regulatory distal promoter have not been identified. In this study we have demonstrated that the up- or down-regulation of the distal promoter occurs due to these transcription factors (). Previous studies have shown that exposure of insulin to hepatocytes causes an increase in lipogenic enzyme transcription mediated by an increase in SREBP-1c transcription.
Glucagon, cAMP and fatty acid lead to the phosphorylation of ChREBP and its subsequent exclusion from nucleus, whereas glucose causes dephosphorylation and subsequent nuclear localization of ChREBP. It is known that ChREBP is regulated at two levels, nuclear localization brought about by phosphorylation-dependent mechanism in response to glucagon (or hormones), cAMP, fatty acids and glucose and subsequent DNA binding. Hence in the presence of glucagon and leptin, ChREBP and SREBP-1c are expressed less and are probably in their inactive forms localized in the cytosol. It is known that in the presence of cAMP, a site near the nuclear localization signal is phosphorylated by PKA and ChREBP being unable to enter the nucleus, remains in the cytosol [11
]. Also, the expression of SREBP-1c was also found to be regulated by insulin [40
], and SREBP-1c abundance is tightly related to the nutritional state in liver and adipose tissue. SREBPs get activated by undergoing a sequential two-step cleavage process to release their NH2-terminal segments that can then translocate to the nucleus. This process is tightly regulated [40
]. Hence when cells were treated with insulin or EGF, higher nuclear translocation of the active ChREBP and SREBP-1c was expected and observed (). This might be responsible in the observed up-regulation of their bindings to the distal promoter region. Though EGF caused an increase in the nuclear translocation of ChREBP yet it failed to cause an increase in binding in the distal promoter. In contrast, glucagon and leptin caused a decrease in the nuclear translocation of both the transcription factors thereby causing a decreased binding to the distal promoter region. These observations were reversed when cells were treated with H7 or wortmannin ().
Previous studies have shown that ChREBP and SREBP-1c function coordinately to regulate lipogenesis. The purpose of having two factors to control transcriptional regulation is that the metabolic intermediates cannot be stored until all conditions such as glucose and insulin signals which work via ChREBP and SREBP-1c, respectively, are optimally set. Another explanation is that there is an interplay between ChREBP and SREBP-1c thereby giving a better means of integrated transcriptional regulation [41
]. For example, mice with a deletion of the SREBP-1
gene have an impaired ability to fully respond to a high-carbohydrate diet. However, these mice do retain some significant dietary response, which is caused by ChREBP gene [41
]. Hence the synergistic effect of the two transcription factors would provide an effective means to regulate transcription of lipogenic enzymes by integrating multiple nutritional or hormonal inputs. This integration is important to control the energy status in mammals [41
]. Most lipogenic enzyme genes (e.g., fatty acid synthase) have response elements for binding ChREBP (ChoRE) and SREBP (SRE). These two factors work synergistically to induce transcription of the lipogenic enzyme genes in the presence of glucose and insulin. Glucagon, through its intracellular mediator cAMP, and polyunsaturated fatty acids (PUFAs) act to inhibit the activity of ChREBP and SREBP, respectively [44
]. In this manner, the output of lipogenic enzyme gene production is integrated to multiple nutrient and hormonal signals [41
So, the next question arises why rat mtGPAT gene requires two promoters to regulate its transcriptional activity? It is known that some eukaryotic genes such as rat ACC gene [45
] and rat cAMP phosphodiesterase gene have more than one promoter, usually one that has a TATA box and one that does not [46
]. Rat ACC promoter I has a TATA box and CAAT box similar to rat mtGPAT distal promoter – both are induced under lipogenic conditions. Rat ACC promoter II, like rat mtGPAT proximal promoter, is a TATA less promoter [46
]. Promoter II of rat ACC is expressed constitutively in several tissues while promoter I product is expressed in a tissue specific manner [47
]. This could be the case with rat mtGPAT gene where the distal promoter is the regulatory promoter, whose regulation is brought about by the lipogenic transcription factors SREBP-1c and ChREBP. Furthermore, the proximal promoter of mtGPAT gene has the characteristics of the promoter of “housekeeping”-type genes [48
]. Why? First, there is no TATA box within the promoter region. Secondly, the GC content of the promoter region is high (about 60%) and has potential Sp1 binding sites. This is similar to cystic fibrosis gene which too has a “housekeeping” promoter and the gene is expressed at low levels like the mtGPAT proximal promoter transcript [49
The novel feature of the work presented here is that it further reveals a remarkable similarity how ACC and mtGPAT are regulated. These are two important enzymes strategically located in the overall biosynthetic pathway of glycerolipids including triacylglycerols. Both enzymes are stimulated by ATP, citrate [3
], insulin and EGF, but inhibited by AMPK, glucagon, leptin, adrenaline and casein kinase II [23
]. Moreover, multiple promoters control the expression of ACC (45) and mtGPAT [13
]. Only one of the promoters, that contains TATA box, appears to be affected by the hormones and growth factor, the other promoter(s) act(s) constitutively. Why is this similarity important? Increasing the rate of fatty acid synthesis by stimulating ACC may not guarantee that the fatty acids will be diverted towards triacylglycerol biosynthesis. Without the mtGPAT being similarly regulated as ACC, the fatty acids may be used either for biosynthesis of glycerolipids or for degradation via β-oxidation in the mitochondrial matrix.
In summary, we have investigated the role of the two promoters in the transcriptional regulation of rat mtGPAT gene. Our results indicate that the distal promoter is the regulatory promoter and is capable of up- or down- regulating its transcript (distal transcript) via key lipogenic transcription factors such as ChREBP and SREBP-1c. The proximal promoter, on the other hand, acts like a “housekeeping”-type promoter and maintains a low basal level of transcription of its product (proximal transcript). Knowledge of the ways in which these promoters control the transcription may prove useful in targeting mtGPAT gene for obesity related drugs.