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A Role for CREB But Not The Highly Similar ATF-2 Protein inSterol Regulation Of The Promoter for 3-Hyroxy-3-Methlyglutaryl

Coenzyme A Reductase

Tawny T. Ngo*, Mary K. Bennett*, Andrew L. Bourgeois, Julia I. Toth& Timothy F. Osborne**

Department of Molecular Biology and BiochemistryUniversity of California, Irvine

Running Title: Resolving the functions of individual members of a transcription factorfamily

*Equal Contributors

**send proofs and reprint requests to:Tim OsborneDepartment of Molecular Biology & BiochemistryUniversity of California, IrvineIrvine, California 92697-3900Telephone #: (949) 824-2979Fax# (949) 824-8551

Copyright 2002 by The American Society for Biochemistry and Molecular Biology, Inc.

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ABSTRACT

Sterol regulatory element binding proteins (SREBPs) activate promoters for key genes

of metabolism to keep pace with the cellular demand for lipids. In each SREBP

regulated promoter, at least one ubiquitous co-regulatory factor that binds to a

neighboring recognition site is also required for efficient gene induction. Some of these

putative co-regulatory proteins are members of transcription factor families that all bind

to the same DNA sequence elements in vitro and are often expressed in the same cells.

These two observations have made it difficult to assign specific and redundant functions

to the unique members of a specific gene family. We have used the chromatin

immunoprecipitation (ChIP) technique coupled with a transient complementation assay

in Drosophila SL2 cells to directly compare the ability of two members of the

CREB/ATF family to function as co-regulatory proteins for SREBP dependent

activation of the HMG CoA reductase promoter. Results from both of these

experimental systems demonstrate that CREB is an efficient SREBP co-regulator but

ATF-2 is not.

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INTRODUCTION

The sterol regulatory element binding proteins ( SREBPs)1 are key metabolically

regulated transcription factors. They are translated as large precursors, inserted into the

membrane of the ER, and their amino terminal domains are released into the cytosol

when the cellular lipid level falls. The signaling pathway that results in SREBP release

is not completely understood but requires two sequential proteolytic events, the first of

which is actively regulated by sterols and fatty acids (1). The soluble amino terminal

fragment contains the DNA binding and transcriptional stimulation functions and once it

is released from the membrane it enters the nucleus to increase expression of various

genes that are important for cellular lipid homeostasis (2,3).

SREBPs are weak activators of transcription by themselves and they require co-

regulatory transcription factors that bind nearby DNA sequences to efficiently stimulate

gene expression (3). The identity and combinations of co-regulatory factors and the

number and arrangements of the SREBP sites are promoter specific. These differences

are likely to provide a framework for gene specific regulatory responses to the different

SREBP isoforms and to specific cellular regulatory cues. For example, in the promoter

for the low density lipoprotein (LDL) receptor, there is a single SREBP site and Sp1 is

the lone SREBP co-regulatory factor and binds to two separate sites (4). In the

promoters for 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) synthase, farnesyl

diphosphate (FPP) synthase, and squalene synthase there are multiple SREBP sites and

at least one of the co-regulators is the CCAAT-binding factor/nuclear factor-Y

(CBF/NF-Y) (5-7). HMG CoA synthase additionally requires a member of the

CREB/ATF family (8).

In previous studies, we have shown separate DNA sites that bind CBF/NF-Y and

members of the CREB/ATF family are both required for expression from the HMG

CoA reductase promoter (9). CREB sites were originally identified as cis-acting

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elements that confer transcriptional regulation in response to elevated cAMP levels (10-

12). In the somatostatin promoter, it was shown that cAMP responsiveness was

mediated through the basic-leucine zipper containing transcription factor termed cAMP

response element binding protein or CREB (13). Protein kinase A phosphorylates

CREB in response to increased cellular cAMP which allows it to interact efficiently

with the transcriptional co-activator protein called CREB binding protein (CBP) to

stimulate transcription of cAMP target genes (14,15). Several related CRE binding

proteins have been identified and cloned (16) and together they comprise the

CREB/ATF family of transcription factors. Individual members of this family bind to

CREs present in numerous eukaryotic promoters, and activate transcription in response

to various cellular signals (17). Major questions concerning this and any other related

"families" of genes are to determine how much overlap there is in function and to

identify specific physiological roles for the individual proteins.

In addition to mutagenesis studies that showed there is a CRE-like element in the

HMG CoA reductase promoter, we have used the chromatin immunoprecipitation

technique (ChIP) to demonstrate that both CREB and CBF/NF-Y are both recruited to

the HMG CoA reductase promoter by SREBP when cells are deprived of exogenous

cholesterol (18). Taken together, our previous studies indicate that both CBF/NF-Y and

CREB are important SREBP co-regulators for HMG CoA reductase. However, since

CREB is a member of the CREB/ATF family, it was important to determine whether

individual members of this family can substitute for CREB in the sterol regulatory

response mediated by SREBP. In the current report we utilize the ChIP method to

provide evidence that while CREB is recruited to the HMG CoA reductase promoter

efficiently by SREBP activation, binding of another member of the family, ATF-2, is

not altered. We also provide evidence from direct promoter activation studies in

Drosophila SL2 cells that CREB is an efficient SREBP co-regulator and efficiently

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stimulates the HMG CoA reductase promoter along with SREBP NF-Y. In contrast,

ATF-2 is unable to substitute for CREB in this independent assay system as well.

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MATERIALS AND METHODS

Cells and Media : The CHO-7 and SL2 cell lines were cultured as described

previously (4,18). Lipoprotein deficient serum was prepared by ultracentrifugation of

newborn bovine serum as described previously (19). Cholesterol and 25-OH cholesterol

were obtained from Steraloids Inc., and stock solutions were dissolved in absolute

ethanol.

Cell Culture: Stock flasks of CHO-7 cells (20) were grown in a 50/50 mixture of

Hams F12 and Dulbecco’s modified essential medium (DMEM) (Irvine Scientific)

containing 10% (v/v) fetal bovine serum (FBS) at 37oC and 5% CO2. Tissue culture

dishes (15 cm) were plated at 500,000 cells/dish on day 0 in the above medium. On day

1 the dishes were rinsed twice with 1X phosphate buffered saline (PBS) and half of the

dishes were fed with either induced media (HamsF12/DMEM containing lipoprotein-

depleted serum instead of FBS) or suppressed media (Hams F12/DMEM containing

lipoprotein-depleted serum with 10 ug/ml cholesterol and 1 ug/ml of 25-OH-

cholesterol). Cells were processed for the CHIP procedure after an additional 24 hr.

incubation.

Chromatin Immunoprecipitation assay: We used a modification of the procedure of

Farnham and colleagues (21) as described previously (18). Dishes of CHO-7 cells were

placed in a fume hood and treated with formaldehyde (final concentration of 1% v/v)

followed by a room temperature incubation for 8 minutes. The reaction was quenched

by the addition of glycine (final concentration of 125 mM) and the dishes were

incubated for an additional 5 minutes at room temperature, medium was removed

followed by 3 rinses with cold 1X PBS. Samples were then subjected to the protocol

described in our previous report (18). The CREB and ATF-2 antibodies were from

Santa Cruz (sc-186 and sc-187 respectively). After immunoprecipitation, DNA was

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extracted and samples were ultimately resuspended in 50 ul sterile H2O and 2-4 ul were

used in each polymerase chain reaction (PCR).

Standard PCR reactions for hamster HMG CoA reductase promoter were performed

with 32-P kinased oligonucleotides and amplitaq gold (Perkin Elmer). The primers for

HMG CoA reductase were designed to hybridize and amplify a ~230 bp product

encompassing the region displayed in Fig. 1A. To provide reactions that were in the

linear dose response for the individual samples, we performed test PCR reactions and

varied the number of cycles to obtain conditions where the signal intensity was linear

with respect to amount of input as described previously (18).

Transient Transfection Assay in Drosophila SL2 cells: Drosophila SL2 cells were

cultured in Shields and Sang insect medium (Sigma) containing 10% heat inactivated

fetal bovine serum and were seeded at 480,000 cells/well in six well dishes on day 0.

On day 1 cells were transfected by the calcium phosphate co-precipitation method with

each dish receiving 2 υg of each test plasmid, 10.75 υg of salmon sperm DNA, and 1 υg

of the control plasmid pPAC ß-gal containing the coding region of the E. coli ß-

galactosidase gene driven by the Drosophila actin 5C promoter. The pPAC SREBP-1a

constructs used for activation studies in SL2 cells contain the coding regions of the Sp1

or SREBP-1a (aa 1-490) gene under the control of the Drosophila actin 5C promoter

and was described before (4). The pPAC NF-Y constructs containing the coding

regions for the 3 individual CBF/NF-Y subunits (A, B, and C) were described

previously (22). The coding sequence for an epitope (Gln Pro Glu Leu Ala Pro Glu

Asp Pro Glu Asp) from the herpes virus type I glycoprotein D protein was inserted at

the amino terminus of pPAC vectors that encode the full coding sequence of human

ATF-2 or CREB.

On day 3 cells were harvested and Luciferase and ß- galactosidase activity were

measured in cell extracts as described previously (22). The expression levels for CREB

and ATF-2 protein were normalized using the common HSV epitope for comparison.

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Briefly, transfection experiments were performed as above with differing amounts of

the HSV-CREB and HSV-ATF-2 vectors and a constant amount of the pPAC-ß-gal

control plasmid. Protein extracts from the transfected cells were first normalized for

transfection efficiency by measuring the ß -galactosidase activity of individual extracts

and normalized amounts were analyzed by immunoblotting as described below.

SDS-PAGE and immunoblot analysis: SL-2 nuclear extracts, CHO-7 total chromatin

extracts (equivalent amounts normalized for A260) or equivalent amounts of material

precipitated by the ATF-2 antibody were analyzed by SDS-PAGE and immunoblotting

with the antibodies indicated in the figure legends. The HSV antibody was from

Novagen (catalogue # 69171) and the IGG 7D4 monoclonal antibody directed against

hamster SREBP-2 (obtained from ATCC) were used. The blots were developed with

the ECL kit from Pierce.

Protein-Protein Interaction Assays: The coding regions for CREB or ATF-2 were

inserted into pGEX2 (Pharmacia) and expressed in and purified from E. coli as

described (8). Recombinant SREBP-1a (amino acids 1-490) was incubated with

purified GST-CREB or GST-ATF-2 and the mixtures were bound to glutathione agarose

beads that were subsequently washed and analyzed for specifically bound material by an

immunoblotting protocol as described (8).

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RESULTS

CREB is a member of a transcription factor family where individual proteins are all

highly similar in their basic and leucine zipper DNA binding/dimerization domains.

Even though they all bind the same cis-acting consensus sequence, the affinities of the

different homo- and heterodimeric combinations vary for different CRE elements

(23,24). Additionally, they do not all respond identically to cellular signaling pathways.

For example, ATF-2 activates transcription along with the Adenovirus E1a protein (25),

whereas both ATF-1 and CREB stimulate genes in response to changes in cAMP levels

(14,26). As more is understood about the functions of the various CREB/ATF proteins,

the reasons for these differences will become better understood.

Using the chromatin immunoprecipitation technique ( ChIP) we previously

demonstrated that CREB was recruited to the HMG CoA reductase CRE site when

SREBP nuclear localization was induced by sterol depletion (18). We wanted to

determine if other members of the family could participate in this key nutritional

response. In the current studies we used an antibody to the ATF-2 protein to evaluate its

binding to the HMG CoA reductase CRE site in response to sterol deprivation and

SREBP activation. Chromatin extracts were prepared from two sets of dishes of CHO-7

cells. One set was cultured in medium containing lipoprotein depleted serum (LPDS) to

stimulate SREBP nuclear localization and the other set received LPDS with cholesterol

and 25 OH cholesterol added back to keep SREBPs tethered to the ER membrane and

sequestered away from their target genes.

The chromatin was then processed by our standard ChIP protocol followed by a PCR

reaction with primers that amplify the HMG CoA reductase promoter region

encompassing the CRE site (Fig. 1A). As a control, we showed there was equivalent

levels of HMG CoA reductase promoter DNA in the starting chromatin samples (Fig.

1B lanes 1 and 2). When equal amounts of chromatin from the two sets of dishes were

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incubated with an antibody against ATF-2 prior to the immunoprecipitation and PCR

reaction, there were also equal levels of HMG CoA reductase promoter DNA present in

both samples (lanes 3 and 4). However, when the CREB antibody was used there was a

significantly higher level of HMG CoA reductase promoter DNA present in the sample

prepared from cells cultured under sterol depleted conditions versus the sterol treated set

(lanes 7 and 8).

An immunoblotting analysis demonstrated that the mature SREBP-2 transcription

factor was properly regulated by the sterol depletion protocol (Fig. 2A). Additional

immunoblotting experiments presented in Figs. 2B and C demonstrated that equal

amounts of protein for both CREB and ATF-2 were present in the starting chromatin

preparations (lanes 1 and 2 of Figs. 2B and C). Also, the ATF-2 protein was

quantitatively removed and equal amounts were recovered by the immunoprecipitation

protocol (Fig. 2C compare lanes 3-6). We could not perform an immunoblot to

determine if CREB was quantitatively precipitated because the CREB protein migrates

too close to an immunoglobulin protein subunit from the immunoprecipitation reaction,

which reacts with the secondary antibody and obscures the CREB band on the resulting

gel.

These ChIP results along with the experiments from our previous study strongly

suggest that CREB is an efficient co-regulatory factor for SREBP in the HMG CoA

reductase promoter and that the ATF-2 protein does not participate in this response. In

order to evaluate whether there is a difference in the ability of CREB and ATF-2 to

directly stimulate transcription from the HMG CoA reductase promoter, we used the

transfection-complementation system in Drosophila SL2 cells that we have used

extensively in previous reports. These cells do not express functional equivalents of

several mammalian transcriptional regulatory proteins, including Sp1 (27). However,

expression from mammalian promoters can be evaluated when expression plasmids for

a critical missing regulatory protein(s) are included in the transfection protocol.

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Therefore, the SL2 transfection assay provides a background for evaluating mammalian

promoters and their missing trans-acting regulatory proteins in an intact cell system

(27). In fact, we have used SL2 cells to demonstrate that SREBP activation of the HMG

CoA synthase promoter requires both NF-Y and CREB (8).

When we transfected SL2 cells with the HMG CoA reductase promoter reporter

construct alone or with an SREBP expression construct, a low level of promoter activity

was observed (Fig. 3, filled triangle at abscissa origin). This is consistent with previous

studies indicating that SREBP is a very weak activator by itself. When increasing

amounts of either ATF or CREB expression vectors were included in addition to

SREBP, a similar low level of activation was still observed (Fig. 3 open symbols).

When expression constructs for the three subunits of NF-Y were included along with the

SREBP expression construct the promoter was activated about 7 fold. When the CREB

or ATF-2 constructs were included on top of the NF-Y plasmids, a robust activation was

observed for CREB (Fig. 3 filled squares) but ATF-2 failed to induce expression above

the level achieved by SREBP and NF-Y alone (Fig. 3 filled circles). When SREBP was

omitted from the transfection experiment, there was no activation by NF-Y and CREB

alone2.

To evaluate whether the low activation mediated by ATF-2 could be explained by a

lower level of protein accumulation relative to CREB, we evaluated expression of the

two proteins after transfection into SL2 cells. We had inserted the coding sequence for

an HSV glycprotein D epitope at the extreme amino-terminus of the two expression

vectors so that we could compare protein expression levels using the same antibody.

When protein extracts from the transfected SL2 cells were analyzed, both CREB and

ATF-2 were expressed at similar levels (Fig. 4).

Taken together with the results from the ChIP experiments of Fig. 1 these

transfection results provide strong support for the conclusion that CREB is recruited to

the native HMG CoA reductase promoter and efficiently activates the isolated promoter

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in SL2 cells and both are dependent on SREBP. However, ATF-2 is neither recruited to

the native HMG CoA reductase promoter by SREP nor is it an efficient co-regulator for

SREBP activation of the cloned promoter in the SL2 cell system.

In previous studies, we showed that CREB interacts with SREBP in solution and this

interaction is likely part of the mechanism for the synergistic activation of transcription

of the HMG CoA reductase promoter by these two proteins. To evaluate whether ATF-

2 was also capable of interacting with SREBP we compared the ability of GST fusion

proteins of CREB and ATF-2 to bind to SREBP in solution (Fig. 5). The results

demonstrate that under conditions where CREB binds SREBP efficiently, ATF-2

binding was minimal (compare lanes 3 and 4). Thus, the lack of efficient interaction

between SREBP and ATF-2 is likely part of the reason why it is not recruited to the

HMG CoA reductase promoter by SREBP activation.

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DISCUSSION

All CREB/ATF family proteins are highly similar in their basic and leucine zipper

regions and they bind the same cis-acting consensus sequence. Therefore, it is possible

that individual members can have both overlapping and unique roles in specific and

diverse biological processes. In the current studies, we tested the ability of ATF-2 to

substitute for CREB in activation of the HMG CoA reductase promoter by SREBPs.

Using antibodies to each protein in chromatin immunoprecipitation studies, we showed

that SREBP activation by sterol depletion resulted in efficient recruitment of CREB to

the HMG CoA reductase promoter but ATF-2 binding was unaltered by this nutritional

challenge. We did detect the HMG CoA reductase promoter DNA in the chromatin

samples that were precipitated with the ATF-2 antibody however, the level was

unaltered by the sterol manipulation protocol. These results indicate that ATF-2 may

bind and activate the HMG CoA reductase promoter to basal levels but it is not recruited

to the promoter by SREBP and thus, it cannot substitute for CREB as an important

SREBP co-regulatory protein.

The result of our co- transfection studies in Drosophila SL2 cells support and

significantly extend this conclusion as well. The data in Fig. 3 shows that SREBP

activates the HMG CoA reductase promoter in cooperation with NF-Y and CREB but

when expressed at similar levels, ATF-2 cannot substitute for CREB.

Since CREB and ATF-2 bind to the same DNA sequence in vitro, it was important to

investigate the mechanism for the differential recruitment of CREB to the HMG CoA

reductase promter measured in the Chromatin Immunoprecipitation analysis. In Fig. 5,

we show that CREB but not ATF-2 was capable of interacting with SREBP in solution

in the absence of DNA. Thus, consistent results from three separate experimental

approaches support our conclusions and provide at least a partial mechanistic

understanding for the selectivity. It was previously shown that the somatostatin

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promoter is stimulated in a cell type specific manner by cAMP through the action of

CREB (28). Importantly, these authors showed that ATF-2 was also unable to substitute

for CREB in this response.

Our earlier mutational studies of the HMG CoA reductase promoter showed that

both the CRE and NF-Y sites were simultaneously required for normal sterol dependent

regulation (9). The data from Fig. 3 are also consistent with this conclusion since both

CREB and NF-Y were required along with SREBP for efficient activation. These

observations are similar to our previous findings for the HMG CoA synthase promoter

where both CREB and CBF/NF-Y were required for efficient activation by SREBP.

Thus, two early genes that control simple carbon flux into the cholesterol/isoprenoid

biosynthetic pathway require a similar set of SREBP co-regulatory proteins. This

provides a molecular strategy to ensure the common early steps of the multivalent

cholesterol/isoprenoid pathway are tightly co-regulated (29).

The chromatin immunoprecipitation method is a useful procedure for analyzing

changes in the binding of specific regulatory proteins to their putative target elements in

native chromatin in response to change in the intracellular environment. With the

availability of antibodies with suitable specificity ChIP can be used to effectively

analyze the functional roles of highly similar proteins or even differentially modified

versions of the same transcriptional regulatory protein that bind to very similar or

identical DNA sites in vitro.

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FOOTNOTES

1). Abbreviations used: SREBP, sterol regulatory element binding protein; LDL, low

density lipoprotein; HMG-CoA, 3-hydroxy-3-methylglutaryl coenzyme A; CREB,

cyclic AMP response element binding protein; ATF-2, activating transcription factor-2;

CBF, CAAT-binding factor; NF-Y, nuclear factor-Y; 25-OH cholesterol, 25-

hydroxycholesterol; ChIP, chromatin immunoprecipitation

2). T. N. and T.F. Osborne unpublished data

3). Acknowledgments: This work was supported in part by grants from the National

Institutes of Health (HL48044) and the American Heart Association (0150231N). TN

and AB were recipients of undergraduate fellowships from the Undergraduate Research

Opportunities Program at UC Irvine.

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FIGURE LEGENDS

Figure 1. Chromatin immunoprecipitation analysis of CREB and ATF-2 binding

to HMG CoA reductase promoter in CHO cells cultured in the absence and

presence of regulatory sterols.

A). A schematic representation of the native HMG CoA reductase promoter with the

heterogeneous transcription initiation sites, TATA element and binding sites for

SREBPs and NF-Y and CREB/ATF is shown. B). An autoradiogram of a

polyacrylamide gel that displays the results of the PCR for the HMG CoA reductase

promoter is shown. The primers were designed to hybridize just upstream of the NF-Y

site and just downstream of the ATF/CREB site as shown in A. The input chromatin

was analyzed in lanes 1 and 2 (1 ul of a 1:300 dilution), and 3 ul of each resultant

immunoprecipitation with the indicated antibodies were also analyzed as indicated. No

primary antibody was used for the reactions in lanes 5-6.

Figure. 2. Immunoblot characterizatin of chromatin extracts.

Equivalent amounts (A260) of chromatin Extracts from CHO-7 cells cultured in the

absence (I) or presence (S) of cholesterol and 25 OH cholesterol were processed for

immunoblotting using the indicated antibodies. The chromatin samples before

immunoprecipitation (input) were analyzed for SREBP-2, CREB and ATF-2 (lanes 1-2

of each panel). The chromatin was subjected to immunoprecipitation with an antibody

to ATF-2 and the material remaining in the supernatant ("sup." C, lanes 3-4) and equal

aliquots of the total immunoprecipitation pellets from both samples ("IP" C, lanes 5-6)

were analyzed. The migration positions for the precursor (P) and mature (M) forms of

SREBP-2 are indicated in A. The migration positions for CREB and ATF-2 are

indicated by arrows at the right in panels B and C respectively. The dark staining band

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lower in the gel in C, lanes 5 and 6 corresponds to immunotreaction with a subunit of

the antibody used for the immunoprecipitation reaction.

Figure 3. Activation of the HMG CoA reductase promoter in Drosophila SL2

cells. SL2 cells were transfected with the wild-type HMG CoA reductase reporter

construct along with the pPAC β-galactosidase construct as an internal control for

specificity and transfection efficiency. A pPAC vector expressing amino acids 1-490 of

SREBP-1a was included at 1 ng and it resulted in a basal level of activation that was set

at 1.0. The pPAC HSV-ATF-2 (circles) or pPAC HSV-CREB (squares) plasmids were

included at increasing concentrations as indicated on the abscissa. Where indicated

(closed symbols), 3 ng of pPAC constructs encoding each of the three NF-Y/CBF

subunits: A, B, and C were also added to the transfection precipitate. DNA

transfections, luciferase and β-galactosidase assays were performed as described

previously (22) and in the methods section. Data represent average values from a

typical experiment performed in duplicate for each sample.

Figure 4. Expression of HSV tagged ATF-2 and CREB in SL2 cells.

SL2 cells were transfected with the HSV tagged CREB and ATF-2 expression vectors

and after 48 hrs. total nuclear extracts (50 ug) were analyzed by immunoblotting with an

antibody against the HSV epitope tag. An equivalent amount of extract from mock-

transfected cells was analyzed in lane 1.

Figure 5. SREBP-1a physically interacts with CREB in solution. An immunoblot

from an SDS-PAGE gel using an antibody to SREBP-1 is shown. Recombinant

SREBP-1a was added in lane 1 as control (Con.). Eluates from a GST control column

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Ngo et. al.7/1/02

20

(lane 2), GST-ATF-2 column (lane 3), or GST-CREB column (lane 4) were analyzed as

indicated and described in Materials and Methods.

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Ngo et al 2002 Fig. 1

CREB

I S

Input

1 2 7 8

ATF2 none

Antibody

I S I S I S

5 63 4

SREBP ATF/CREBNF-Y

+1-100 -50

TATT

-150

A.

B.

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Ngo et al. 2002 Fig. 2

I S I S I S

ATF-2Input Sup. IP

1 2 3 4 5 6

C.

SREBP-2

I SP

M

1 2

A.

I S

CREB

1 2

B.Input

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0

5

10

15

20

25

Fo

ld A

ctiv

atio

n

0 250 500 750 10001250

DNA (ng)

CREB+

NF-Y

ATF+NF-Y

ATF

CREB

Ngo et al. 2002 Fig. 3

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-

ATF-2

CREB

ATF CREB

1 2 3

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SREBP-1

1 2 3

Ngo et al. Fig. 5

4

CREBATFGSTCon.

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OsborneTawny T. Ngo, Mary K. Bennett, Andrew L. Bourgeois, Julia I. Toth and Timothy F.

promoter for 3-hyroxy-3-methlyglutaryl coenzyme A reductaseA role for CREB but not the highly similar ATF-2 protein in sterol regulation of the

published online July 10, 2002J. Biol. Chem. 

  10.1074/jbc.M202135200Access the most updated version of this article at doi:

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