Regulation of Expression of Branched-Chain α-Keto Acid Dehydrogenase Subunits in Permanent Cell Lines

[43]

BCKAD

GENE EXPRESSION IN HEPATOMA CELL LINES

479

[43] R e g u l a t i o n o f E x p r e s s i o n o f B r a n c h e d - C h a i n a - K e t o Acid D e h y d r o g e n a s e S u b u n i t s in P e r m a n e n t Cell L i n e s By JEFFREY M. CHINSKY and PAUL A. COSTEAS

A large number of studies have demonstrated that there are many influences on the activity of the mammalian mitochondrial enzyme complex, branched-chain ct-keto acid dehydrogenase (BCKAD, EC 1.2.4.4). Changes in BCKAD activity may be produced in response to endocrine factors, exercise, and nutritional state through posttranslational mechanisms of regulation, such as kinase-mediated phosphorylation (inactivation), as well as through pretranslational mechanisms that lead to increases in the RNAs that encode the BCKAD subunit proteins. 1 Examination of steady state levels of RNAs encoding BCKAD subunits (Elot, El//, E2, and kinase) in rodent tissues obtained from animals at different postnatal developmental ages or fed diets with various protein and/or caloric contents suggested that regulation of BCKAD subunit gene expression plays an important role in the response of tissues to varying physiologic conditions.2-5 To study the mechanisms by which these tissues regulate BCKAD promoter activity, cell lines amenable to both the influences of external agents and the expression of transfected BCKAD promoter minigenes needed to be identified. The first step was to identify cell lines that demonstrate altered levels of BCKAD subunit RNAs in response to glucocorticoids, insulin, acidosis, or state of differentiation, known effectors of BCKAD tissue activity in vivo. The cell lines demonstrated to satisfy these conditions included those demonstrating hepatic (H4IIEC3, Hepa 1), renal (LLC-PK1), and fibroblast inducible adipocyte (3T3-L1) characteristics or origin.4-6 Of note is that lack of glucocorticoid effect on BCKAD expression in some cell lines may be due to lack of expression of sufficient glucocorticoid receptor, a condition rectified with stably transfected minigenes (LLC-PK1-GR101). 6 We have focused on using hepatic cell lines because mammalian liver demonstrates the highest activity per organ and the majority of the total body BCKAD 1 M. S. Patel and R. A. Harris, FASEB J. 9, 1164 (1995). 2 y . Zhao, S. C. D e n n e , and R. A. Harris, Biochem. J. 290, 395 (1993). a y . Zhao, K. M. Popov, Y. Shimomura, N. Y. Kedishvili, J. Jaskiewicz, M. J. Kuntz, M. J. Kain, B. Zhang, and R. A. Harris, Arch. Biochem. Biophys. 308, 446 (1994). 4 j. M. Chinsky, L. M. Bohlen, and P. A. Costeas, FASEB J. 8, 114 (1994). 5 p. A. Costeas and J. M. Chinsky, Biochem. J. 318, 85 (1996). 6 X. W a n g , C. Jurkovitz, and S. R. Price, Miner Electolyte Metab. 23, 206 (1997).

METHODS IN ENZYMOLOGY,VOL. 324

Copyright © 2000by AcademicPress All rights of reproductionin any form reserved. 0076-6879/00$30.00

480

REGULATIONAND EXPRESSION

[43]

capacity in laboratory rats. 7 A large number of studies have confirmed the response of hepatic BCKAD subunit RNA levels to physiologic states associated with known changes in levels of circulating hormones. 5,8 Our most consistent results have been obtained with the rat hepatic cell line H4IIEC3. Previous studies had demonstrated that the presence of insulin decreases levels of E l a RNA with no significant effect on E2 RNA levels observed at 24 hr, while glucocorticoids appear to demonstrate the reverse effect.5 The effects of both glucocorticoids and insulin on BCKAD E2 gene expression are described below. General Methodology Cell Culture Rat hepatoma H4IIEC3 cells are obtained from the American Type Culture Collection (Manassas, VA) and maintained in Dulbecco's modified Eagle's medium (DMEM) containing 10% (v/v) fetal calf serum (DMEMFCS) at 37° in a humidified 5% CO2,95% air atmosphere. Specific conditions are indicated in the figure legends, but experiments are performed in an identical manner by plating cells in serum containing medium for 24 hr, washing them with phosphate-buffered saline (PBS) twice, feeding them with serum-free medium for 14-16 hr, and then adding dexamethasone (0.001-1.0/zM), insulin (0.01-0.1 /zM), and/or dibutyryl-cAMP (0.1-1.0 mM) in fresh serum-free medium for the indicated time interval prior to harvest for BCKAD subunit RNA or reporter gene analysis. Cells are prepared for RNA analysis as previously described. 5 For transfections, cells are multiply plated from a single batch of freshly trypsinized cells at 3.5 × 105 cells per 20-mm well into six-well cell culture dishes (Falcon; Becton Dickinson Labware, Lincoln Park, N J). Murine Hepa 1 cells (ATCC) demonstrate similar responses but not to the extent observed with H4IIEC3. Human HepG2 cells (ATCC) do not demonstrate statistically significant changes in BCKAD gene expression, perhaps because of their lack of sufficient expression of the glucocorticoid receptor. Isolation and Blot Hybridization Analysis of Steady State R N A Total cellular RNA is isolated by the guanidine thiocyanate-phenol extraction method, separated by electrophoresis in 1% (w/v) agarose7S. Soemitro,K. P. Block,P. L. Crowell,and A. E. Harper, J. Nutr. 119, 1203 (1989). s A. G. Chico, S. A. Adibi, W.-Q. Liu, S. M. Morris, and H. S. Paul, J. Biol. Chem. 269, 19427 (1994).

[43]

BCKAD

GENE EXPRESSION IN HEPATOMA CELL LINES

481

formaldehyde gels, transferred to nylon membranes (MagnaGraph; Micron Separations, Westboro, MA) and hybridized with randomly primed 32p-labeled cDNA as previously described. 4'9 Ethidium bromide staining of 28S and 18S ribosomal R N A confirms equivalent loading in all lanes. Rehybridization of all blots with several cDNA probes (see below) is performed to ensure relative equality of different R N A preparations. All individual R N A preparations are retested several times to confirm any results presented.

DNA Probes The following 32p-labeled probes are used for Northern blot hybridization experiments: 1.7-kb EcoRI fragment from murine BCKAD Elo~ cDNA (GenBank accession no. 47335), 5 the 1.4-kb EcoRI-ClaI fragment from murine BCKAD E2 cDNA (GenBank accession no. IA2996) containing sequences encoding only the mature preprotein, 1° and the 0.6-kb EcoRIBamHI fragment of CHOB (GenBank accession no. L22552), which detects a single R N A species in rodents and is considered to be the m R N A for ribosomal protein $2. m12 Other useful probes included cDNAs for actin and glyceraldehyde-3-phosphate dehydrogenase (GAPDH).

Minigene Plasmids The isolation, cloning, and sequencing of the murine BCKAD E2 promoter region containing a 7.0-kb 5' upstream genomic sequence and construction of minigene plasmids containing a downstream luciferase reporter sequence (derived from the pGL-Basic luciferase vector; Promega, Madison, WI) are described elsewhere. I3 Available restriction sites in the promoter and 5' upstream genomic region are used in a series of restriction endonuclease and religation steps to obtain sequentially smaller BCKAD E2 gene sequences ranging from 7.0 kb to 300 bp from the full-sized 7.0-kb pGLE2-7.0 minigene plasmid. The restriction enzymes used in their creation include pGLE2-4.0, KpnI; pGLE2-2.3, SpeI; pGLE2-0.9, SacI; pGLE2-0.3, PstI. The promoter activities of these minigenes demonstrate a 10-fold decrease with the loss of 5' upstream genomic material down to 300 bp. 13 The end sequences of the larger minigenes as well as the entire sequence of the smaller minigenes are confirmed by use of the double-stranded D N A 9 p. Chomczynski and N. Sacchi, Anal Biochem. 162, 156 (1987). 10p. A. Costeas, L. A. Tonelli, and J. M. Chinsky, Biochim. Biophys. Acta 1305, 25 (1996). 11 M. M. Harpold, R. M. Evans, M. Salditt-Georgieff, and J. E. Darnell, Cell 17, 1025 (1979). 12L. T. Putowski, D. Choi, J. Mordacq, W. J. Seherzer, K. E. Mayo, E. Y. Adashi, and R. M. Rohan, J. Soc. Gynecol. Invest. 2, 735 (1995). 13 p. A. Costeas and J. M. Chinsky, Biochim. Biophys. Acta 1399, 111 (1998).

482

REGULATION AND EXPRESSION

[43]

(dsDNA) cycle sequencing system (BRL Life Technologies, Gaithersburg, MD) as well as cycle sequencing on an Applied Biosystems (Foster City, CA) automated sequencer (model 373A). All the BCKAD E2 sequences terminate at the BspE2 sequence (TCCGGA) 6-11 bp upstream from the initiation ATG codon in exon 1, and 3-8 bp downstream from the determined BCKAD E2 transcription initiation site. I3 pRSV-SEAP, a minigene plasmid that expresses a secretory form of alkaline phosphatase, is obtained from Tropix (Bedford, MA).

Transient Transfection of H4IIEC3 Cells H4IIEC3 cells are plated in DMEM containing 10% (v/v) FCS, incubated overnight, washed with PBS, and replaced with DMEM without FCS. DMEM (100 lzl) containing plasmid DNA (2/zg: 0.4/zg of RSV-SEAP and 1.6/zg of test BCKAD minigene) is mixed with 100 txl of medium containing LipofectAMINE (8/zl; GIBCO-BRL, Bethesda, MD) and incubated at room temperature for 30 min. The optimal amounts of LipofectAMINE and plasmid DNA needed to obtain highest transfection efficiency are determined by systematic titration of both parameters and determination of transfection efficiency on the basis of levels of secreted alkaline phosphatase (SEAP) in the medium (data not shown). After 800/zl of DMEM is added to the suspension, it is added to the cells and incubated for 4 hr (37 °, 5% CO2). The plates are washed twice with PBS and D M E M 10% (v/v) FCS is added. After overnight incubation, the cells are washed twice with PBS to remove dead cells and medium is replaced with fresh DMEM-10% (v/v) FCS. The cells are incubated for 8-10 hr, at which time medium is collected for SEAP analysis to assess relative transfection efficiency. The cells are washed twice with PBS and FCS-free DMEM is added. After overnight incubation, the medium is replaced with fresh FCSfree DMEM containing insulin (I), dexamethasone (DEX), and/or dibutyryl-cAMP (dBcAMP); the cells are then incubated for the indicated times (usually 24 hr) and harvested for determination of luciferase activity.

Production and Analyses of Stably Transfected Cell Lines H4IIEC3 cells are transfected with DNA at a 1 : 4 ratio of a neomycin 'expression plasmid under the control of the Rous sarcoma virus (RSV) promoter (RSV-neo) to a cloned luciferase expression plasmid (pGL2 Basic; Promega) under the control of an inserted BCKAD E2 genomic promoter sequence (minigene pGLE2-7.0 or pGLE2-0.3). Cells are plated at 2 × 105 cells per 60-mm dish, incubated overnight in DMEM-10% (v/v) FCS, washed with PBS, and then covered with 5 ml of DMEM containing 8% (v/v) modified bovine serum (MBS; Stratagene, La Jolla, CA). Ten micro-

[43]

B C K A D GENE EXPRESSION IN HEPATOMA CELL LINES

483

grams of plasmid DNA is then mixed in 450/zl of water with 50/zl of solution I (2.5 M CaPO4) and 500/zl of solution II [2× N,N-bis-(2-hydroxyethyl)-2aminoethanesulfonic acid (BES)-buffered saline] and the suspension is incubated at room temperature for 10-20 min and added to the cells dropwise. After 3 hr of incubation at 35°, 3% CO2, the cells are washed three times with PBS, covered with DMEM-10% (v/v) FCS, and incubated at 37 °, 5% CO2.14 After overnight incubation, the cells are washed to remove dead cells and the medium replaced with flesh DMEM-10% (v/v) FCS. The following day, the medium is supplemented with G418 (Geneticin, 400 /xg/ml; GIBCO-BRL) and maintained under this selection for 3-4 weeks, with the medium replaced every 4 days. Colonies formed by neomycinresistant cells are individually trypsinized and plated separately or in pools. On expansion, the cells are assayed for luciferase activity and frozen under liquid nitrogen in 10% (v/v) dimethyl sulfoxide (DMSO)-20% (v/v) FCS. Previously frozen cultures of cloned transfected cells are thawed, passaged several times to ensure high viability, and then tested as a single batch for comparative analyses. Cells are plated at 3.0 × 105 cells per 20-mm well in six-well cell culture dishes, incubated overnight in DMEM-10% (v/v) FCS, washed twice with PBS, and incubated overnight (14-16 hr) with DMEM without FCS. The medium is then replaced with FCS-free DMEM containing insulin (I), dexamethasone (DEX), and/or dibutyrylcAMP (dBcAMP); the cells are then incubated for 24 hr and harvested for determination of luciferase activity. Analyses using a singly cloned cell line containing pGLE2-7.0, H4S1, are presented in the figures, but analyses using other independently cloned cells lines are performed to ensure the results reported for H4S1.

Reporter Minigene Assays Secreted alkaline phosphatase (SEAP) activity is measured from culture medium (100/zl) collected from the transfected cells according to the directions of the supplier of the assay kit (Tropix). This medium is mixed with 1 × dilution buffer (300/A) and heated to 65° for 30 min, and then 100/.d of heat-treated medium is mixed with 100/zl of assay buffer containing a mixture of different alkaline phosphatase inhibitors. These inhibitors allow the detection of the secreted placental alkaline phosphatase while minimizing the interference from nonheat inactivated endogenous phosphatase activities. The reaction buffer containing CSPD ® (Tropix) chemiluminescent substrate (100 ~1) is added to the reaction tube and after 20 min of incuba14F. M. Ausebel, "Current Protocols in Molecular Biology," pp. 9.13-9.14. John Wiley & Sons, New York, 1995.

484

REGULATION AND EXPRESSION

[431

tion at room temperature, the luminescence is measured with a model 20e luminometer (Turner Designs, Sunnyvale, CA). 13 Luciferase (LUC) activity is measured according to the directions of the assay kit supplier (Promega). Transfected cells are washed twice with PBS and covered with 200/zl (per 20-mm well) of 1 × reporter lysis buffer, incubated for 15 min at room temperature, and scraped into a 1.5-ml microcentrifuge tube. After vortex mixing for 10 sec and microcentrifuge centrifugation for 10 sec, 20 /zl of cell extract is mixed with 100 ~1 of luciferase assay reagent (470 /zM luciferin, 270 /xM coenzyme A, and 530 tzM ATP) and luminescence determined with the Turner Designs model 20e luminometer. All assay values are measured in duplicate or triplicate to ensure proper values. All transient transfections are performed in triplicate in the same multiwell dish and, similarly, all stably transfected cells are assayed in triplicate. Luciferase activity from each sample is corrected for SEAP activity in medium collected prior to serum starvation and exposure of the cells to hormonal effectors. The corrected luciferase activities from the triplicate samples are averaged and the Student t test performed on the basis of the mean and the standard deviation (Sn-1).

Gel Retardation (Gel Shift) Assay Nuclear Extract Preparation. H4IIEC3 cells are grown to confluency (5 × 150 mm dishes), washed and scraped into 20 ml of cold PBS, centrifuged for 10 min at 1000 rpm, and rewashed in PBS. They are then resuspended and incubated on ice for 10 min in 5 ml of 10 mM HEPES (pH 7.9), 1.5 mM MgCIE, 10 mM KC1, 0.5 mM dithiothreitol (DT-F), and 0.5 mM phenylmethylsulfonyl fluoride (PMSF) and then centrifuged, and the pellet is resuspended in fresh buffer to which 0.05% (v/v) Nonidet P-40 has been added. The pellet is homogenized (20 strokes) in a tight Dounce homogenizer and the pellet recentrifuged. The pellet is resuspended in 2 ml of 5 mM HEPES (pH 7.9), 1.5 mM MgCIa, 0.2 mM EDTA, 26% (v/v) glycerol, 0.5 mM DTr, and 0.5 mM PMSF, and to this suspension concentrated NaC1 is added to 420 mM and incubated on ice for 30 min. The lysate is centrifuged at 12,000 rpm for 20 min in an Sw41 Beckman (Fullerton, CA) rotor, dialyzed through 0.0025-tzm pore size VS membrane (Millipore, Bedford, MA) against 5 mM HEPES (pH 7.9), 100 mM KC1, 0.2 mM EDTA, 26% (v/v) glycerol, 0.5 mM DTT, and 0.5 mM PMSF at 4 °, and frozen in 50-/zl aliquots in liquid nitrogen. 15 Gel Retardation Assay. Template D N A from the upstream E2 promoter 15p. E. Berg, D. W. Williams, R. B. Cohen, R.-L. Qian, M. Mittelman, and A. N. Schechter Nucleic Acids Res. 17, 8833 (1989).

[43]

BCKAD GENEEXPRESSIONIN HEPATOMACELLLINES

485

region ( - 4 7 to - 1 4 0 bp upstream from the start site) is amplified by polymerase chain reaction (PCR), purified, and eluted into Tris-EDTA (TE). The gel-purified D N A (3 pmol) is end labeled with [T-s2p]ATP by T4 polynucleotide kinase in a 10-/xl reaction volume at 37 ° for 30 min. The reaction is terminated with 1/zl of 0.5 M E D T A and diluted in TE to 200 /zl. One microliter of the end-labeled template is mixed with nuclear extract (approximately 3 txg), incubated at room temperature for 20 min, and loaded in 1 x gel shift binding buffer [5 x buffer: 20% (v/v) glycerol, 5 mM MgCI:,, 2.5 mM EDTA, 2.5 mM DTI?, 250 mM NaC1, 50 mM Tris (pH 7.0), poly(dI-dC) (0.25 mg/ml)] onto a 5% (w/v) nondenaturing polyacrylamide gel containing 2.5% (v/v) glycerol. After electrophoresis at 100 V for 3 hr in 0.5 x TBE, the gel is dried under vacuum and exposed to X-ray film at --80°. 15'16

Results

Glucocorticoids Affecting BCKAD E2 RNA Accumulation in H4IIEC3 After exposure of H4IIEC3 cells to titrated amounts of dexamethasone (DEX), steady state levels of BCKAD E2, but not Elot, subunit R N A increased (Fig. 1). Studies were also performed with dibutyryl-cAMP (dBcAMP), an agent known to interact synergistically with dexamethasone under certain conditions. In contrast to dexamethasone, which consistently produced increased accumulation of E2 RNA, exposure of cells to dBcAMP alone had no consistent effect on the accumulation of the E2 transcript. However, low levels of dBcAMP in combination with dexamethasone caused an enhanced increase in the observed level of E2 RNA, usually threefold, by 24 hr after treatment.

Glucocorticoids and Insulin Affecting E2 Minigene Expression in H4IIEC3 Using an E2 minigene plasmid (pGLE2-7.0) containing 7.0 kb of 5' murine genomic sequence with promoter activity directing expression of the reporter gene luciferase, stably transfected H4IIEC3 cells were isolated and tested for their responsiveness to dexamethasone (DEX), dBcAMP, and insulin (Fig. 2). Similar to the results observed with R N A levels, exposure to D E X produced twofold increases in luciferase, dBcAMP alone produced no increases, and in combination, dBcAMP appeared to augment 16F. M. Ausebel, "Current Protocolsin MolecularBiology,"pp. 12.03-12.2.10.John Wiley& Sons, New York, 1995.

486

1431

REGULATION AND EXPRESSION

1

2

3 4

5

6

7

8

9 101112

E2 EIo

Actin rPS2

Dex

d Bc

- 01 0 5 . 1

.5 1.0

.1 1.0 .Ol .1 .5 1.o .1

.1

uM

mM

FIc. 1. Effect of glucocorticoids on BCKAD subunit RNA in H4IIEC3 cells. Northern blot hybridization analysis of 20 tzg of total cellular RNA prepared from pooled samples from three individual plates of cells grown in media containing the indicated concentrations of dexamethasone (Dex) and dibutyryl-cAMP (dBc) is shown. Rehybridization with actin and rPS2 probes as control for loading is indicated. Densitometric analysis comparing lane 1 and 11 suggested a 3.8-fold increase in BCKAD E2 RNA in the combined presence of 0.1/~M Dex/0.1 mM dBc in this experiment.

the DEX-induced increase to about threefold within 24 hr. Further increases were observed at 48 hr, up to six- to sevenfold (data not shown). Titration analysis of D E X exposure of H4S1 cells demonstrated increases in relative promoter activity from 0.001 to 1.0/zM in the presence of a constant amount of dBcAMP known to augment the D E X response (data not shown). To confirm the involvement of a glucocorticoid-receptor mediated mechanism in the activation of the E2 gene, we treated H4S1 cells with a combination of dexamethasone and the glucocorticoid receptor antagonist RU486 (Roussel Uclaf, Romainville, France), which prevented the DEX-associated increase in E2 promoter activity (data not shown). This finding suggested that the glucocorticoid receptor participated in the regulation of E2 by glucocorticolds. In contrast to glucocorticoids, exposure of transfected H4IIEC3 cells to insulin (I) resulted in decreases in luciferase activity (about twofold) at 24 hr. The presence of insulin along with D E X or D E X plus dBcAMP

[431

BCKAD

GENE EXPRESSION IN HEPATOMA CELL LINES

487

300' >

• ca

200 0_1

E_

22 _~ o~

1oo

0 Ctr

Ins

Dex

D/I

dBc

Did

D/d/I

Treatment FIG. 2. Hormonal effects on H4IIEC3 cells stably transfected with BCKAD 7.0-kb E2 promoter-luciferase minigene DNA. Cloned isolate H4S1 cells derived from stably transfected H4IIEC3 cells were plated in triplicate, grown overnight in DMEM-10% FCS, washed with PBS, incubated for 16 hr in FCS-free medium, and then exposed to dexamethasone (DEX or D, 1.(1/~M), insulin (INS or I, 0.1/xM), and/or dBcAMP (dBc or d, 0.5 mM), or not exposed (Ctr), for 24 hr in FCS-free medium. Cells were then harvested for luciferase assay as described in General Methodology. The relative promoter activity represents the ratio of the mean (-+ SD) luciferase activity per treatment group (n = 3) to the activity of control (Ctr) untreated cells, and is expressed as a percentage [control cells (Ctr) by definition had 100% relative promoter activity].

did not result in the othewise consistent increase in E2 promoter activity observed in association with glucocorticoid exposure alone (Fig. 2). This was further investigated by examination of H4IIEC3 cells transiently transfected with E2-7.0 minigenes and subsequently exposed to these hormones. The results of a typical experiment, showing the ratio of luciferase to secreted alkaline phosphatase activities, are shown in Table I. Using the presence of SEAP activity measured after transfection but prior to the addition of hormone, one could standardize for the relative efficiencies of individually transfected dishes. A number of experiments have confirmed that the presence of glucocorticoids increases the BCKAD E2-7.0 promoter activity by two- to fourfold in H4IIEC3 cells. In contrast, the presence of insulin appears to consistently suppress this increase, but to a variable extent. The range of insulin concentration required to produce this effect appears to be from 10 to 100 nM in these cells, based on titration experiments.

488

REGULATION AND EXPRESSION

[431

TABLE I EFFECT OF DEXAMETHASONE, dBcAMP, AND INSULINON B C K A D PROMOTER ACTIVITY IN TRANSIENTLY TRANSFECTED H4IIEC3 CELLS

Luciferase activity Additive~

LUC/SEAP b

Fold differencec

None DEX dBcAMP DEX/dB Insulin DEX/Ins dB/Ins DEX/dB/Ins

2.429 _ 0.069 6.601 _+ 0.155 3.041 _ 0.047 8.830 _ 0.147 2.484 ± 0.120 3.717 ± 0.327 2.792 ± 0.196 5.067 _+ 0.149

1.00 2.71 1.22 3.64 1.02 1.55 1.15 2.09

Dexamethasone (DEX), 1.0/~M; dBcAMP (dB), 0.1 mM; insulin (Ins), 0.1/xM. b Values represent mean ratios __+ SD of luciferase (LUC) to secreted alkaline phosphatase (SEAP) activities determined for each transfected dish (n = 3). c Fold increase in promoter activity (LUC/SEAP) compared with control. a

Regional Localization of Sequences Involved in Regulation of BCKAD E2 Promoter Activity To identify the region that contained cis-acting elements responsible for the regulation of B C K A D E2 p r o m o t e r activity, a series of luciferaseexpressing minigenes containing decreasing amounts of E2 genomic D N A directly upstream from the transcription start site was assayed. The 0.3-kb E2-containing minigene demonstrated notable increases in response to D E X or D E X / d B c A M P , which could be affected by the presence of insulin (data not shown). This finding suggested that this proximal p r o m o t e r region contained sequences sufficient for glucocorticoid-mediated activation. To narrow further the critical p r o m o t e r region-containing sequences needed for this glucocorticoid effect, a series of minigenes with progressively shorter regions of the B C K A D p r o m o t e r D N A (from 315 to 44 bp upstream from the transcription initiation site) was assayed by both transfection studies and linker scanning analysis. The minimum B C K A D p r o m o t e r sequence that could be demonstrated contained 44 bases upstream from the transcription start site. 13 The utility of using H 4 I I E C 3 cells for identification of potential D N A protein-binding sites in the proximal E2 p r o m o t e r region was tested by preparation of nuclear extracts and D N A gel shift analysis

[43]

BCKAD

PROBE CELL EXTRACT

I

GENE EXPRESSION

IN HEPATOMA

CELL LINES

489

E2 GL53C I

H411EC3 =

=

" =

tt) ..J iv"

El ..I tY

r~ -.I n¢

t,9 (.9 0

vr,.. ~

¢q

tY

u~

(.9 O

f..

A'-'~

B----D~

c ---I~

Fx6. 3. Gel retardation analysis of the BCKAD E2 upstream promoter region. Oligonucleotide DNA prepared from the murine BCKAD E2 promoter region ( - 4 7 to -140) by PCR amplification was gel purified, end labeled with [7-32p]ATP, and incubated with 3 /xg of H4IIEC3 nuclear extract and the indicated competitor DNA (100x) at room temperature for 20 min. The DNA-protein reaction mix was then separated by nondenaturing PAGE and the gel dried and exposed for autoradiography. (-) No competitor; WT, unaltered wildtype murine sequence; GR1-GR7, GR71, GR72, sequences with linker scanning mutations; GRL1-GRL7, series of contiguously deleted sequences; hE2, amplified DNA derived from the corresponding region of the human BCKAD E2 promoter. 16a

of E2 D N A containing sequences from - 4 7 to -140 bases from the start site. Using these nuclear extracts, several shifted bands were identified, indicating binding of nuclear protein to the labeled promoter sequence (see Fig. 3, bands A and B). All the shifted bands were efficiently competed with excess unlabeled probe but not with oligonucleotides containing SP1 or AP2 consensus sequences. The top, slower migrating band (band 16a K. S. Lau, W. J. Herring, J. L. Chuang, M. McKean, D. J. Danner, R. P. Cox, and D. T. Chuang, J. Biol. Chem. 267, 24090 (1992).

490

REGULATION AND EXPRESSION

[431

A) was easy to compete with as little as a 100x excess of unlabeled probe, but the lower, faster migrating band (band B) required at least a 200× excess. This same promoter DNA region ( - 4 7 to -140) was PCR amplified from a series of minigenes containing contiguous linker scanning mutations or contiguously deleted sequences along this region. 13,17 Each of these prepared DNAs was used at 100x excess to determine competition for binding with the prepared H4IIEC3 nuclear proteins. As shown in Fig. 3, the upper, slowly migrating band was completely competed with all the mutant competitors as efficiently as the wild-type (WT) sequence, with the exception of competitor D N A containing linker scanning mutations in or near the reverse CAAT box site (pGR6, pGR7, pGR71, pGR72) at - 7 8 to - 8 3 bp upstream from the transcription start site. 13Similarly, the competitor DNAs containing sequences whose deletions extended down to but did not include the CAAT box ( p G R L I - p G R L 5 ) competed the shifted band efficiently. The D N A with deletions termininating near or within the region of bp - 7 8 to -83, GRL6: to - 8 5 and GRL7: to -76, failed to compete out the shifted band. More detailed binding kinetic studies are required to establish the dynamics of DNA-protein interaction in this region. However, these studies indicate that this cell line will provide a useful model with which to identify potentially important sites of DNA protein binding that may be involved in the regulation of E2 promoter activity in hepatic cells.

Summary The rat hepatoma cell line H4IIEC3 has demonstrated a response to both insulin and glucocorticoids in its accumulation of BCKAD subunit RNAs. It is amenable to BCKAD promoter minigene transfection analyses, demonstrating positive (glucocorticoids) and negative (insulin) regulatory effects. These cells can therefore be used as a model to identify cis-acting sites responsible for regulation of BCKAD subunit promoter activity.

Acknowledgments These studies were supported in part by a Life and Health Insurance Medical Research Fund grant to J. M. Chinsky and presented as a portion of the doctoral thesis submitted by P. A. Costeas.

17F. M. Ausebel, "Current Protocols in Molecular Biology," pp. 8.5.1-8.5.9. John Wiley & Sons, New York, 1995.