A Critical Role for cAMP Response Element-binding Protein (CREB) as a Co-activator in Sterol-regulated Transcription of 3-Hydroxy-3-methylglutaryl Coenzyme A Synthase Promoter

Abstract

3-Hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) synthase, a key regulatory enzyme in the pathway for endogenous cholesterol synthesis, is a target for negative feedback regulation by cholesterol. When cellular sterol levels are low, the sterol regulatory element-binding proteins (SREBPs) are released from the endoplasmic reticulum membrane, allowing them to translocate to the nucleus and activate SREBP target genes. However, in all SREBP-regulated promoters studied to date, additional co-regulatory transcription factors are required for sterol-regulated activation of transcription. We have previously shown that, in addition to SREBPs, NF-Y/CBF is required for sterol-regulated transcription of HMG-CoA synthase. This heterotrimeric transcription factor has recently been shown to function as a co-regulator in several other SREBP-regulated promoters, as well. In addition to cis-acting sites for both SREBP and NF-Y/CBF, the sterol regulatory region of the synthase promoter also contains a consensus cAMP response element (CRE), an element that binds members of the CREB/ATF family of transcription factors. Here, we show that this consensus CRE is essential for sterolregulated transcription of the synthase promoter. Using in vitro binding assays, we also demonstrate that CREB binds to this CRE, and mutations within the CRE that result in a loss of CREB binding also result in a loss of sterol-regulated transcription. We further show that efficient activation of the synthase promoter in Drosophila SL2 cells requires the simultaneous expression of all three factors: SREBPs, NF-Y/CBF, and CREB. To date this is the first promoter shown to require CREB for efficient sterol-regulated transcription, and to require two different co-regulatory factors in addition to SREBPs for maximal activation.3-Hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) 1 synthase is a key rate-limiting enzyme of cholesterol biosynthesis, converting acetoacetyl-CoA and acetyl-CoA into HMG-CoA. In order to achieve cellular cholesterol homeostasis, this and other genes of cholesterol metabolism are regulated by classical feedback repression: they are up-regulated when sterol levels fall and down-regulated when sterol levels rise (1-5). This control is exerted primarily at the transcriptional level through the action of the sterol regulatory element-binding proteins (SREBPs), a unique subfamily of basic helix-loop-helix zipper (bHLHZip) proteins. SREBPs are expressed as 125-kDa precursor proteins that are anchored to the endoplasmic reticulum and nuclear membranes. When cellular sterol levels fall, the precursor is released from the membrane by a two-step proteolytic mechanism, allowing the mature, transcriptionally active SREBPs to translocate to the nucleus (6). Once inside the nucleus, SREBPs activate cholesterogenic target genes through binding to sterol regulatory elements (SREs) present in their promoters. SREBPs also activate key genes of fatty acid metabolism (7-9) thus, they are central transcription factors of mammalian lipid metabolism. SREBPs alone...

Differential Regulation of Human and Mouse Orphan Nuclear Receptor Small Heterodimer Partner Promoter by Sterol Regulatory Element Binding Protein-1

“…Optimal transcriptional activation by SREBPs depends on additional transcription factors such as Sp1, NF-Y, cAMP response element-binding protein, or CCAAT/enhancer-binding protein ␣ (41,42,45,50). Activation of SREBP target genes require NF-Y binding to adjacent CCAAT motifs that are usually located within 21 bp of the SRE (43).…”

Sterol regulation of human fatty acid synthase promoter I requires nuclear factor-Y- and Sp-1-binding sites

“…In addition, the interaction of NF-Y and SREBP in the absence of DNA has been demonstrated in vitro (39). Recent studies, however, indicate that the sterol response of the HMG-CoA synthase promoter, which requires NF-Y and SREBP, also requires an additional coactivator, CREB, the cAMP response element-binding protein (40). CREB-binding protein (CBP) and p300, a CBP-related protein, both bind the activation domain of SREBP in vitro (41).…”

Failure to Cleave Sterol Regulatory Element-binding Proteins (SREBPs) Causes Cholesterol Auxotrophy in Chinese Hamster Ovary Cells with Genetic Absence of SREBP Cleavage-activating Protein

“…As expected, the SRD-13A cells lacked nuclear SREBP-2 under either condition (lanes 3 and 4). All three of the permanently transfected SCAP-expressing SRD-13A cells showed normal amounts of nuclear SREBP-2, and this was abolished when cyclodextrin was omitted (lanes [5][6][7][8][9][10]. The upper panel of Fig.…”

“…The transcriptionally active NH 2 -terminal segment of ϳ480 amino acids projects into the cytosol. This segment contains a basic helix-loop-helixleucine zipper sequence that allows the protein to dimerize, to bind DNA, and to recruit transcriptional coactivators (6,7). The NH 2 -terminal segment is followed by a 90-amino acid membrane attachment segment that consists of two membrane-spanning helices separated by a short hydrophilic loop of 31 amino acids that projects into the lumen of the ER and nuclear envelope.…”