(Received for publication, January 30, 1996; and in revised form, March 8, 1996)
From the
In earlier studies the DNA site required for sterol regulation of 3-hydroxy-3-methylglutaryl coenzyme A reductase was shown to be distinct from the classic sterol regulatory element (SRE-1) of the low density lipoprotein receptor gene (Osborne, T. F. (1991) J. Biol. Chem. 266, 13947-13951). However, oxysterol-resistant cells that continuously overproduce one of the sterol regulatory element binding proteins in the nucleus result in high unregulated expression of both genes (Yang, J., Brown, M. S., Ho, Y. K., and Goldstein, J. L.(1995) J. Biol. Chem. 270, 12152-12161) suggesting a direct role for the SREBPs in the activation of the reductase gene. In the present studies we demonstrate that SREBP-1 binds to two adjacent sites within the previously identified sterol regulatory element of the reductase gene even though there is only limited homology with the SRE-1 of the receptor. We also show that SREBP-1 specifically activates the reductase promoter in transient DNA transfection studies in HepG2 cells and that mutations which eliminate sterol regulation and SREBP-1 binding also abolish transient activation by SREBP-1. Although specific, the magnitude of the activation observed is considerably lower than for the low density lipoprotein (LDL) receptor analyzed in parallel, suggesting there is an additional protein required for activation of the reductase promoter that is limiting in the transient assay. SREBP also binds to two additional sites in the reductase promoter which probably play an auxiliary role in expression. When the DNA sequence within the sites are aligned with each other and with the LDL receptor SRE-1, a consensus half-site is revealed 5`-PyCAPy-3`. The LDL receptor element contains two half-sites oriented as a direct repeat spaced by one nucleotide.
The SREBP proteins are special members of the basic-helix-loop-helix-zipper (bHLHZip) family of DNA binding proteins since they bind the classic palindromic E-box site as well as the direct repeat SRE-1 element. The SREBP binding sites in both the reductase and those recently identified in other sterol regulated promoters appear to contain a half-site with considerable divergence in the flanking residues. Here we also show that a 22-amino acid domain located immediately adjacent to the basic domain of the bHLHZip region is required for SREBP to efficiently recognize divergent sites in the reductase and 3-hydroxy-3-methylglutaryl-CoA synthase promoters but, interestingly, this domain is not required for efficient binding to the LDL direct repeat SRE-1 or to a palindromic high-affinity E-box element.
Balanced cholesterol metabolism in mammalian cells is maintained
through the feedback regulation of key proteins involved in its
cellular uptake and biosynthesis. A major control point is at the level
of transcription for genes that encode important proteins of both
processes(1, 2) . DNA sequences required for
cholesterol regulation of important enzymes of cholesterol biosynthesis
and the low density lipoprotein (LDL) ()receptor, the unique
protein required for cholesterol uptake, have been identified by DNA
transfection studies in cultured cells(1, 2) . The
original studies identified a sequence similarity in the target site
for cholesterol regulation (called the SRE-1) in the promoter for the
LDL receptor and HMG-CoA synthase(3, 4, 5) .
Further studies demonstrated that the SRE-1 of the LDL receptor is a
10-base pair site that binds a specific subfamily of
basic-helix-loop-helix-zipper (bHLHZip) DNA binding proteins called the
sterol regulatory element binding proteins or
SREBP's(6, 7, 8) .
When mammalian cells are starved for cholesterol the SREBP proteins are cleaved from the membrane of the endoplasmic reticulum and nuclear envelope by proteolysis and the soluble amino-terminal fragment containing the DNA binding and transcriptional activation functions is translocated to the nucleus where it activates expression of appropriate target genes. The first evidence for this maturation process came from classic pulse-chase and gel electrophoresis studies on cells that were cultured in the presence and absence of sterols(9) . SREBP-1 and -2 proteins were regulated in an identical fashion in tissue culture cells(9) . However, a recent study in animals has suggested that the two SREBPs are regulated independently by cholesterol deprivation in the liver(10) . Nonetheless, the two proteins have a similar domain structure, bind to the same DNA target sites in vitro, and appear to activate the same target genes when overexpressed in transfection studies(8, 11, 12) .
A fundamental role for SREBP processing in cholesterol regulation was uncovered when SREBP expression was analyzed in mutant cell lines that were selected for resistance to the toxic effects of oxysterols(11, 13) . Several independently derived oxysterol-resistant cell lines all resulted from genomic rearrangements that lead to production of an altered form of the SREBP-2 protein. All variant proteins lack the membrane spanning domains which are responsible for localizing the precursor to the endoplasmic reticulum in a hairpin configuration(12, 14) . Thus, SREBP-2 is constitutively targeted to the nucleus resulting in high expression of the LDL receptor and cholesterol synthetic enzymes independent of the cholesterol content of the culture medium.
We have shown previously that the DNA target site for cholesterol regulation of HMG-CoA reductase appeared to be distinct from the direct repeat SRE-1 recognition site for SREBP in the LDL receptor (15) and the palindromic E-box that is the characteristic recognition site of bHLH proteins and was identified for SREBP-1 in a previous study(16) . In addition, we have purified a protein that binds to the reductase regulatory site that is unrelated to SREBP(17) . The involvement of a unique protein in regulation of HMG-CoA reductase would allow the cell to independently regulate cholesterol uptake from its biosynthesis. However, since the oxysterol-resistant mutant cell lines constitutively overproduce HMG-CoA reductase as well as HMG-CoA synthase and the LDL receptor, there is compelling genetic evidence suggesting HMG-CoA reductase is directly activated by SREBP as well.
In the present studies we demonstrate that SREBP-1 directly activates the promoter for HMG-CoA reductase through non-canonical recognition sites in the previously characterized sterol regulatory region of the reductase promoter.
The plasmid with a generic TATA element from the hamster HMG-CoA synthase promoter fused to the luciferase gene has been described previously(18) . The plasmids ``3X Wt'' and ``3xmut'' were constructed in two steps. First, the simple Sp1 site of repeat 3 from the human LDL receptor promoter was inserted upstream of the generic TATA element in the above plasmid. Then oligonucleotides containing three tandem copies of the sequence from the wild type reductase promoter from -161 to -136 or a mutant derivative with two base changes at -156 and -155 in all three otherwise normal copies were inserted upstream of the Sp1 site. Plasmids containing the desired insertions in the wild type orientation were identified and confirmed by restriction enzyme digestion and DNA sequencing before further analyses. Methods for the oligonucleotide insertion procedures have been described previously(18) . Amino-terminal truncation derivatives of SREBP-1 were constructed by polymerase chain reaction with the wild type plasmid as template and appropriately designed oligonucleotides with a 10 to 1 mixture of Taq and Pfu polymerases. The polymerase chain reaction fragments were subcloned into the pRSET expression vector from Invitrogen.
Figure 1:
SREBP-1 binds to the sterol regulatory
region of the HMG-CoA reductase promoter. A, analytical gel
mobility shift analysis. A double stranded DNA probe corresponding to
the wild type reductase promoter sequence from -161 to -136
was end-labeled with P and used in a mobility shift
experiment as described under ``Experimental Procedures.'' In lanes 3-15 a non-labeled competitor DNA corresponding to
the exact same sequence as the probe except for a single nucleotide
substitution at the position indicated above each lane was included in
the DNA binding reaction. The top DNA strand for the reductase promoter
is presented. In lane 16 a competitor DNA corresponding to the
LDL receptor SREBP binding site was included. All competitor DNAs were
included at a 200-fold molar excess to that of the labeled probe. The
DNA probe was loaded by itself in lane 1 and SREBP was
incubated with the probe in the absence of competitor in lanes 2 and 17. B, methylation interference analysis of
SREBP binding to HMG-CoA reductase sterol regulatory region. The same
probe used in A was uniquely labeled on either the upper (lanes 1-3) or lower (lanes 4-6) strand
before treatment with dimethylsulfate followed by a standard gel shift
analysis. The free (F) and bound (B) DNA regions were
excised and the associated DNA was treated with piperidine before
analysis on a 7% denaturing polyacrylamide gel as
described(17) . Guanosine residues where modification inhibited
DNA binding are indicated for the upper or lower DNA strand. C, the data from A and B are summarized on
the DNA sequence of this region shown in C. The symbols correspond to important residues for SREBP binding as determined
by the experiments of A and B. The underlined area corresponds to the original SRE-1 homology that was proposed
previously (4, 5) and clearly does not correspond to
the SREBP-1 binding site as determined
here.
Figure 4: Region outside of bHLH domain increases SREBP-1 DNA binding site repertoire. A, 10 ng of recombinant SREBP corresponding to amino acids 1-490 (lanes 1-5) or an equivalent amount of a mutant expressing amino acids 321-490 (lanes 6-10) were used in analytical gel mobility shift experiments with five different DNA binding site probes. The individual probes are more thoroughly described under ``Experimental Procedures'' and correspond to the high affinity E-box (E, lanes 1 and 6), the LDL receptor SRE-1 (LDL, lanes 2 and 7), the 3` SRE-1 of the hamster HMG-CoA synthase promoter (S1, lanes 3 and 8), the 5` SRE-1 of the hamster synthase promoter (S2, lanes 4 and 9), or the reductase site of Fig. 1C (RED, lanes 5 and 10). B, diagram of SREBP-1 protein derivatives used in these studies. The SREBP-1 coding sequence from amino acid 1 to 490 is shown at the top of the figure. This region contains all of the information required to activate transcription. There is an acidic activation domain at the extreme amino terminus, a proline-rich domain, and the bHLHZip domain as indicated. The filled in vertical bars denote the residues that make up the heptad repeat or leucine zipper. The extended basic domain protruding in the amino-terminal direction from the classic bHLH domain is also indicated. All of the amino-terminal deletion derivatives were made by polymerase chain reaction and expressed as 6X His fusion proteins in pRSET (Invitrogen) and purified by metal chelation chromatography.
Figure 5:
Quantitative comparison of SREBP-1 binding
to different recognition sites. Protein titration assays were performed
as described under ``Experimental Procedures'' with
recombinant SREBP-1 derivatives containing residues 1-490 (left), 234-490 (center), or 321-490 (right) on the five different DNA binding sites. The
identities of the probe DNAs are as follows: the LDL receptor SRE-1 (open circles), HMG-CoA reductase site of Fig. 1(closed circles), the HMG-CoA synthase 3` SRE-1 (open triangles), the synthase 5` SRE-1 (filled
triangles), and the high affinity E-box (open squares).
The fraction of DNA probe bound (ordinate) is plotted versus the log of the active molar protein concentration (abscissa). These data were used to calculate the K values displayed in Table 1.
The equilibrium dissociation constant (K)
is equal to 1/K
.
The strong genetic evidence discussed above suggested the HMG-CoA reductase promoter may be a direct target for activation by SREBPs and since SREBP-1 and -2 have a similar domain structure and seem to bind and activate the same promoters, all of the present studies have been performed with SREBP-1. A DNA fragment corresponding to the well characterized sterol regulatory site from -161 to -136 of the hamster reductase promoter was used as a probe in a gel mobility shift experiment with a recombinant form of SREBP-1 protein corresponding to amino acids 1-490 (Fig. 1A). This includes all of the protein information required for DNA binding and transcriptional activation(9, 12, 18) . SREBP-1 bound with high affinity and specificity to the reductase probe. The specificity was evaluated by using a series of competitor oligonucleotides corresponding to the exact same DNA fragment except for a single base change as indicated above each lane on the gel in Fig. 1A. Competitor DNAs containing a single mutation at each contiguous base from -156 to -152 corresponding to ATGGT (lanes 5-9) did not compete for SREBP binding and a change from a guanosine to a thymidine (lane 12) also competed less effectively than the other DNA probes. All other single point mutant derivatives competed effectively indicating that alterations at these other positions did not perturb specific recognition by SREBP-1. In addition, binding was also abolished by including a competitor DNA containing the classic SRE-1 element from the LDL receptor (lane 16).
To further characterize this reductase SREBP-1 binding site we performed a methylation interference experiment (Fig. 1B). Methylation of the N-7 position of the guanosine residues at -151, -153, -154, and -157 on the top strand and -150 of the bottom strand decreased binding by SREBP-1. The data of Fig. 1, A and B, are summarized on the DNA sequence of this region shown in Fig. 1C. The SREBP binding region corresponds to a focused area with critical residues that span 9 base pairs from -156 to -149. Importantly, the nucleotide mutations that diminish SREBP-1 binding also decreased regulation by sterols in a previous study ( (15) and see Fig. 2B). It should be noted that this site corresponds to the previously identified sterol regulatory site and not the overlapping SRE-1 homology (underlined in Fig. 1C) that was originally proposed(4) .
Figure 2: Three SREBP-1 DNA binding regions in reductase promoter. A, DNA probes end-labeled on the upper strand at position -277 from the wild type reductase (lanes 1-5) or two mutant derivatives each with a single point mutation which abolishes sterol regulation at either -155 (lanes 6-8) or -166 (lanes 9-11), were used in a standard DNase I footprinting experiment. Ten (lane 3) or 25 (lanes 4, 7, and 10) µl of recombinant SREBP-1 (amino acids 1-490) at a concentration of 2 µg/ml were used in the analyses. The wild type probe was also subjected to dimethylsulfate modification and piperidine cleavage (30) and electrophoresed alongside the footprinting reactions to mark the positions of the resulting footprints (lane 1). The wild type DNA was electrophoresed on a separate gel. The horizontal lines between the two autoradiograms serve to line up the corresponding DNase I cleavage products and the footprints (FP1-3) between the two gels. The narrowed regions of SREBP binding at footprint 2 observed with the mutant probes are denoted by the vertical lines at the right and the arrows mark the positions of the single point mutation within each mutant probe. B, the DNA sequence of each footprint region is presented and the limited degree of similarity is underlined. Note that the sequence presented corresponds to the bottom strand relative to Fig. 1to facilitate comparison with the LDL receptor site. The LDL receptor SRE-1 element is also shown and the half-site of each direct repeat is also underlined. ``N'' means that an adenine, cytosine, or guanosine and ``Y'' means that a cytosine or a thymidine can be present without compromising SREBP binding or sterol regulation. On the sequence of the footprint 2 region the single point mutations that abolished sterol regulation in a previous study (15) are denoted by asterisks, the bases in bold correspond to single point mutants that eliminated SREBP binding in Fig. 1A, and the circles are positioned under base -166 and -155 which correspond to the positions of single point mutants analyzed by footprinting in A. The reductase half-site limited consensus is presented at the bottom.
We also evaluated the binding of SREBP-1 to the reductase promoter by the DNase I footprinting procedure. A radioactive probe labeled on the top strand was incubated with recombinant SREBP-1 before treatment with DNase I and electrophoresis through a denaturing polyacrylamide gel. The results demonstrate that SREBP-1 binds specifically to three separated regions on the HMG-CoA reductase promoter fragment which are labeled footprints 1-3 in Fig. 2A. Footprint 2 corresponds to the DNA region used in the gel mobility shift studies of Fig. 1and to the cis-acting site required for sterol regulation defined by our earlier studies. Footprints 1 and 3 define other sites of SREBP binding in the reductase promoter. A mutation of footprint 1 was not directly evaluated in previous studies and its role in sterol regulation is unclear ( (5) and see ``Discussion''). A mutation of the DNA region corresponding to footprint 3 was previously shown to result in a severe drop in promoter activity but sterol regulation appeared to be maintained(5) . This is probably due to the dominant effect of the footprint 2 region on sterol regulation.
The sterol regulatory mutations from our previous single nucleotide mutagenesis study (15) clustered into two areas of SREBP footprint 2: one identified a short four-base site from -166 to -163 and the other from -159 to -150 (see Fig. 2B). This suggested there are two separate protein binding sites in this interval. To evaluate the possibility that SREBP-1 interacted at two separated sites in this region we prepared probes for DNase I protection studies from mutant promoters with a single base mutation in each clustered sterol sensitive region. When a probe with a sterol compromising mutation at -155 was used in the DNase I protection experiment, SREBP bound normally to footprints 1 and 3 but the degree of protection over the footprint 2 region was confined to a smaller area on one side of the wild type footprint and away from the position of the point mutation (Fig. 2A, compare lanes 4 and 7). A similar result was obtained when a probe with a single base mutation at -166 was used, however, the narrowed area of nuclease protection was confined to the opposite side of the wild type footprint which again is in the area away from the location of the single point mutation (compare lanes 4, 7, and 10).
These DNase I footprinting results indicate there are three regions where SREBP-1 binds to the promoter for HMG-CoA reductase and one of them (footprint 2) contains two recognition sites. When the sequence of all three footprint elements are compared with each other and the two sterol sensitive sites of protection in footprint 2 are considered separately there is a limited degree of sequence similarity. Interestingly, this similarity (underlined in Fig. 2B) corresponds to an SREBP-1 ``half-site'' as proposed by Smith (21) and Yokoyama et al.(8) .
The experiments presented so far document that SREBP-1 binds specifically to the HMG-CoA reductase promoter in a manner consistent with it being involved in sterol regulation. Next, we performed transient DNA transfection studies to evaluate activation by SREBP-1. The wild type reductase promoter was fused to the luciferase reporter gene and the resulting plasmid was transfected into HepG2 cells along with a plasmid that expressed the mature transcriptionally active form of SREBP-1 (amino acids 1-490) constitutively from the cytomegalovirus promoter. We reproducibly observed a 10-20-fold activation of the reductase promoter by SREBP-1 which was not observed when a mutant containing a sterol defective block substitution mutation (altering all bases between -161 to -152; mutant D1 of(5) ) was analyzed in parallel (mut. in Fig. 3). This 20-fold specific activation is much less than that observed for the LDL receptor promoter analyzed in parallel (Fig. 3B).
Figure 3: SREBP-1 activates the reductase promoter in HepG2 cells. HepG2 cells were cultured and transfected with the indicated plasmids as detailed under ``Experimental Procedures.'' The identities of the reporter plasmids are as follows: Wt is the wild type reductase promoter from -277 to +20 (filled triangles); mut is a block substitution mutant of the wild type promoter (open triangles); 3XWt is a synthetic construct (shown at the bottom) containing 3 copies of the wild type reductase promoter from -161 to -136 (closed squares); and 3Xmut is a derivative of this where base changes at -156 and -155 that abolish SREBP binding and sterol regulation are substituted in all three copies (open squares). Each of these was linked to the repeat 3 DNA element from the LDL receptor promoter which corresponds to a generic Sp1 site. A, these reporter plasmids were transfected into the cells alone or with 30 or 300 ng (per dish) of a plasmid that expresses SREBP-1 (amino acids 1-490) from the cytomegalovirus promoter as indicated on the abscissa. Fold activation was calculated as described under ``Experimental Procedures'' and represents the corrected luciferase activity expressed from each reporter in the presence of the indicated amount of SREBP-1 expression plasmid in the co-transfection relative to its absence. B, the activation of the LDL receptor promoter (LDLR; filled circles) is plotted with the data of A for the wild type and block-substitution derivative of the full-length promoter. Note the different scales used for the ordinate of A and B.
To determine if SREBP-1 could activate gene expression directly through binding to one of the non-canonical sites present in the sterol regulatory region of footprint 2 we prepared a synthetic promoter containing three tandem copies of one of the reductase SREBP binding site from footprint 2 (the one shown in Fig. 1C) linked to the generic binding site for Sp1 contained in repeat 3 of the LDL receptor promoter (see the bottom of Fig. 3). The inclusion of the generic Sp1 site was required because SREBP is a very weak transcriptional activator by itself and it functions effectively with Sp1 in the LDL receptor promoter(18) . This synthetic construct was activated to a higher level than the wild type reductase promoter (5) (Fig. 3A). To evaluate the specificity of this activation we prepared a double point mutant derivative of this synthetic plasmid with mutations at -156 and -155 in all three copies of the reductase DNA element. The wild type bases at these sites are important for both sterol regulation (15) and recognition by SREBP-1 (Fig. 1). This mutant construct was not activated in the transient assay by SREBP-1 (Fig. 3A). Thus, SREBP binds to non-canonical sites in the reductase promoter and the important sterol regulatory site (footprint 2) is also a target for SREBP-1 activation in a transient DNA transfection assay.
SREBP proteins are members of the bHLHZip family of transcription factors. These proteins typically recognize the ``E-box'' element which is the palindromic sequence 5`-CANNTG-3`(22) . Individual members display unique preferences for both the internal and flanking bases and SREBP-1 prefers 5`-CACGTG-3`(16) . In addition, the SREBPs are unusual members of this family because they also recognize the direct repeat SRE-1 element typified by the human LDL receptor site 5`-ATCACCCCAC-3`. This dual recognition specificity is dependent at least in part on a tyrosine residue in the basic region of the bHLHZip motif(16) . As demonstrated above, the reductase SREBP-1 sites contain only a half-site of the SRE-1 consensus sequence and flanking residues are not conserved.
To study the interaction of SREBP-1 with its different sites in more detail we have analyzed binding of the wild type and mutant forms to the different cis-acting sites. In the above studies, the protein fragment used contained amino acids 1-490 of the human SREBP-1 protein. This corresponds to all of the information required for DNA binding and transcriptional activation. In Fig. 4we analyzed binding of SREBP-1 derivatives that retain different extents of the amino-terminal amino acids that flank the bHLHZip motif (see Fig. 4B) to five different SREBP recognition sites. The full-length protein (amino acids 1-490) recognized all five different cis-acting recognition sites similarly (Fig. 4A, lanes 1-5). However, different results were obtained when we performed DNA binding studies with a protein fragment that lacked all of the amino acids upstream of the bHLHZip motif (Fig. 4A, lanes 6-10). This truncated version recognized the high affinity E-box site and the LDL receptor SRE-1 similar to the full-length protein. In contrast, this shorter protein recognized the 3`-SRE-1 site of the HMG-CoA synthase promoter weakly and failed to appreciably recognize the companion SRE-1 element of the synthase or the SREBP-1 site shown in Fig. 1of the reductase promoter (compare lanes 3-5 with 8-10).
These results were more carefully documented
by performing protein titration assays (Fig. 5) and the data
were used to calculate the apparent dissociation constant (K) for SREBP-1 binding at each site (Table 1). In these studies we also compared the binding of other
SREBP-1 truncation mutants that retain intermediate amounts of the
protein upstream of the bHLHZip region as indicated in Fig. 4B.
Table 1shows the sequence of the
five different sites used and the K values
obtained for several derivatives containing the indicated portion of
the coding sequence of SREBP-1. All of the DNA sites were recognized
similarly by protein derivatives containing amino acids 299 to 320 in
addition to the bHLH domain ( Table 1and data not shown). (
)However, removal of this small interval resulted in a
protein that binds efficiently to the E-box and LDL receptor but not to
the other sites. Thus, there is a small region that extends in the
amino-terminal direction from the basic region that is essential for
SREBP to recognize DNA sites that deviate from the direct repeat of the
LDL receptor SRE-1 or the perfect palindrome of the high affinity
E-box. A deletion of the basic domain of the bHLHZip motif eliminated
binding to all tested site.
Both SREBP-1 and -2 were identified as proteins that recognized the LDL receptor SRE-1 element and the recombinant proteins bound to the same DNA sites and activated the same target genes(6, 7, 8, 9, 12) . In addition, they were both regulated by sterol dependent proteolysis in cultured cells(9) . Oxysterol-resistant mutant cell lines arise from DNA rearrangements at the SREBP-2 locus that result in production of a truncated SREBP-2 protein lacking the carboxyl-terminal domain that anchors the precursor to the membrane of the endoplasmic reticulum(11, 13) . This prevents the normal sequestration of the protein from the nucleus in sterol replete cells and leads to continuous accumulation of nuclear SREBP-2 independent of the level of regulatory oxysterols in the culture medium. Thus, cholesterol regulated genes including the LDL receptor, HMG-CoA synthase, and reductase, are expressed at a high level that is refractory to the sterol needs of the cell. HMG-CoA synthase and the LDL receptor contain similar SRE-1 DNA elements that bind SREBP proteins in vitro and these sites are directly responsible for sterol regulation in the cell(3, 4) . However, the SRE-1 homology originally proposed for the HMG-CoA reductase promoter (5) does not correspond to the sterol regulatory site that was identified by an exhaustive single-nucleotide substitution analysis (15) .
The fact that reductase was activated in the
oxysterol-resistant cells prompted us to re-investigate the possibility
that it was activated directly by SREBP. In the experiments presented
here we show that SREBP-1 binds to the reductase promoter and mutations
that abolish sterol regulation also abolish SREBP-1 binding. In
addition, we show that transient expression of the mature-soluble form
of SREBP-1 activates the reductase promoter through the identified
sterol regulatory SREBP binding sites. We have also demonstrated that
transient expression of the mature-soluble form of SREBP-2 activates
the reductase promoter in a similar manner.
In earlier reports the reductase promoter was not significantly activated by transiently expressed SREBPs in the human 293 cell line (8, 11) . The difference with our results is likely because we used a different cell line (HepG2) and we used a higher level of plasmid DNA expressing the mature form of SREBP-1. When analyzed in parallel, the LDL receptor promoter was activated to a much greater absolute level even in our assays (Fig. 3). Since the magnitude of activation for the reductase promoter by SREBP is lower, it is likely that another factor is required in addition to SREBP for high level activation. This co-regulator may be the Red 25 protein we previously identified which also binds to the sterol regulatory-SREBP site between -159 and -150 of the reductase promoter(17) .
There are three regions of SREBP binding in the reductase promoter defined by DNase I protection studies: footprints 1-3 in Fig. 1and 2. The previously identified sterol regulatory region corresponds to footprint 2(15) . This interval is divided into two mutation sensitive regions that each constitute the core recognition site for SREBP (Fig. 2B). Footprints 1 and 3 share the core element of 5`-PyCAPy-3`. The footprint 1 region has not previously been tested by mutagenesis, however, it is unlikely to be critical for sterol regulation since synthetic constructs that contain the footprint 2 region fused to the herpes TK promoter (and lack the footprint 1 region) are regulated efficiently by sterols(5) . Also, the present studies have been performed with the hamster reductase promoter and the footprint 1 region exhibits extensive sequence divergence when compared to the corresponding region of the human promoter(23) . In fact, the SREBP half-site of footprint 1 is not conserved. The footprint 3 region is conserved with the human promoter and a mutation of this region in the hamster resulted in a severely crippled promoter that was still regulated by sterols(5) . Thus, the footprint 2 region was still able to support transcriptional activation upon sterol deprivation.
An auxiliary role for SREBP binding at footprints 1 and 3 is suggested by the phenotype of one class of sterol regulatory mutants in the footprint 2 region of the reductase promoter that resulted in high constitutive promoter activity independent of the sterol level of the culture medium. These mutants were also expressed at a high level when transfected into the sterol resistant SRD-2 cells that overexpress nuclear SREBP-2(24) . In contrast, all of the sterol regulatory mutations of the LDL receptor and HMG-CoA synthase promoters resulted in low constitutive promoter activity even in the SRD-2 cells(3, 4, 6, 24, 25, 26) . The low unregulated expression is consistent with the inactivation of a simple site for a transcriptional activator such as SREBP. The high constitutive expression of the unusual reductase mutants is likely due to the fact that footprint 2 mutations retain the additional SREBP binding sites of footprints 1 and 3 which could support activation by the high level of nuclear SREBP in the SRD-2 cells. The high activity of the reductase promoter mutants in normal cells cultured in the presence of sterols can only be simply explained by the action of an inhibitory protein that binds to the footprint 2 region and suppresses promoter activity.
Taken together, all of the available data indicate the SREBP footprint 2 region is the key sterol regulatory site for HMG-CoA reductase. This element contains two separate SREBP recognition half-sites ( Fig. 1and 2) and both are required for normal sterol regulation(15) . This region is also directly responsible for activation by SREBP-1 in transient DNA transfection studies (Fig. 3).
The sites that are recognized by SREBP in the reductase promoter do not exhibit a high degree of sequence similarity with the known SREBP recognition sites of the LDL receptor or the high affinity E-box recognition site (Fig. 2). In fact, the original SRE-1 homology region predicted previously by sequence inspection of the reductase promoter (4) does not correspond to the element that is responsible for sterol regulation (15) or SREBP binding ( Fig. 1and Fig. 2). Interestingly, using techniques similar to those described here we have also demonstrated that the PII promoter for acetyl-CoA carboxylase, the rate controlling enzyme of fatty acid biosynthesis, is regulated by sterols and SREBP through non-canonical SREBP recognition sites(27) . In fact, one of the acetyl-CoA carboxylase half-sites is 5`-ACCAT-3` which is identical to one of the critical reductase half-sites in footprint 2.
In a recent report Ericsson et al.(28) have shown that SREBP-1 binds to a non-canonical SREBP recognition site that is responsible for sterol regulation of the promoter for farnesyl diphosphate synthase, another sterol regulated gene of cholesterol biosynthesis. These studies along with our current observations suggest that the DNA binding specificity for SREBP needs to be investigated in more detail. Indeed, the Myc protein, another member of the bHLH family which binds to classic E-box sequences, also recognizes several non-canonical variant sites(29) .
In the current study we also demonstrate that a
small domain of 22 amino acids from SREBP-1 is required for recognition
of the reductase ``non-canonical'' recognition site. This
domain is also required for efficient recognition of the two synthase
elements (Fig. 4) and for recognition of non-canonical sites in
the acetyl-CoA carboxylase promoter. The potential
significance of this observation is that different forms of SREBP-1 may
be expressed with the capacity to recognize only a subset of
recognition elements. The amino acids within this region extend the
basic domain (predicted pI = 8.7) and there is a predicted
-helical stretch as well. These features could provide additional
protein-DNA contacts that are required for recognition of the variant
DNA sites by SREBP-1.