May 10, 2016 - 100-150 pg of DNA were incubated per reaction (150 pl) at 26 âC. ...... CEF ["'PIRNA (Fig. l), confirming the findings of Sandmeyer et al. (19) that ...
THEJOURNALOF BIOLOGICAL CHEMISTRY Vol. 258. No 9,Issue of May 10,pp. 5787-5793, 1983 Prinled in C S.A.
Transcriptional Control of the FibronectinGene in Chick Embryo Fibroblasts Transformed by Rous Sarcoma Virus* (Received for publication, December 6, 1982)
Jaya Sivaswami Tyagi, Hideyasu Hirano, Glenn T. MerlinoS, and Ira Pastan From the Laboratory of Molecular Biology, National Cancer Institute, National Institutes of Health, Bethesda, Maryland 20205
bridization of a fibronectin-specific cDNA probe to total cellular RNA (9). Fagan et al. (9) demonstrated that in CEF cultures infected with temperature-sensitive mutants of RSV, fibronectin mRNA levels did not increase until 10 h after shifting cells from the permissive to the nonpermissive temperature. Becausethe net levelof fibronectin mRNA could be reduced by a decrease in the rate of synthesis or an increase in the rate of breakdown, we used an in vitro transcription assay to measure these parameters in nuclei isolated from CEF andRSV-CEF.FiveoverlappingrecombinantDNA phages containing the entire chicken fibronectin gene have et al. (10). The geneis beenisolatedrecentlybyHirano approximately 48 kilobase pairs long and its mRNA-coding information is split intoat least 48 exons (10). In the present [a-:”P]GTPand hybridstudy, nuclear RNA was labeled with ized with various DNA fragments of the fibronectin gene located in three recombinant phage clones. Our data strongly suggest that the decreased levelof fibronectin mRNA in RSVCEF is due to transcriptional regulation of this gene. MATERIALSANDMETHODS
Isolation of Nuclei, Transcription in Nuclei, and f3‘P]RNA Extraction-Nuclei were isolated from CEF and RSV-CEF by the procedure of Groudine et al. (11). They were used immediately or stored a t -70 “C. With either fresh or stored nuclei (up to 5 months) similar results were obtained. Nuclear transcription and RNA extraction were performed essentially as described ( l l ) , except that the transcription mixture contained 20 m~ Tris-HC1 (pH 8.0), 30% glycerol, 2.5 m~ dithiothreitol, 5 mMMgC12, 110 mM KCI, and 500 p~ each of ATP, CTP, and UTP. [a-”PIGTP, 200 pCi (-1000-1200 Ci/ Fibronectin is a large glycoprotein (-450,000 daltons) pres- mmol, ICN), was usedto label elongating RNA chains. Approximately 100-150 pg of DNA were incubated per reaction (150 pl) at 26 “C. e n t o ncell surfaces, in connective tissues, in the blood, and in DNA Digestion, Electrophoresis, and Hybridization-Genomic other body fluids (1, 2). In addition to its interaction with DNA (15pg) or purified phage and plasmid DNAs (3 Kg) were digested cells, it interacts with many macromolecules including colla- by HindIII, EcoRI, or H i n d I I plus EcoRI (Boehringer Mannheim, gen, fibrinogen, fibrin, actin, and glycosaminoglycans ( 3 ) .To- New England Biolabs). DNA fragments were fractionated on 0.8% agarose gels of 5-mm thickness at 20 V for 16 h, transferred to gether with collagen, fibronectin plays an important role in cell adhesion, cell migration, and cell morphology (1-3). Fi- nitrocellulose fdters (BA 85, S & S) by the procedure of Southern (12), and the fdters were then baked at 75 “C for 2-3 h in uacuo. For bronectin is abundant in the matrix of normal cells but is the preparation of dot blots, DNA was spotted on nitrocellulose by reduced onthe surface of many transformed cells. This obser-the procedure of Kafatos et al. (13) using the Bethesda Research vation led to the inference that fibronectin may be involved Laboratories HybriDot apparatus. The prehybridization, hybridiza2). in the control of cell adhesion and mobility (1, tion, and washing of the filters were performed as described (11) Following transformation of CEF’ by RSV, fibronectin and except that 5-10 times concentrated Denhardt’s solution and 0.1% 5- t o 10- sodium dodecyl sulfate were included in the hybridlzation mixture. collagen protein levels are concomitantly diminished fold (4-8). T h e s t e a d y state level of fibronectin mRNA is RNA:DNA hybridizations were under conditions of DNA excess and were performed for 2-4 days at 42 & 1 “C. RNase A treatment of reduced approximately 8- to 10-fold when measured by hyfilters at 20 p g / d in 2 X SSC (37 “C, 30 min) reduced the background .~ and removed overhanging RNA tails. The filters were exposed to * The costs of publication of this article were defrayed in part by Kodak XAR-5 f i i with a DuPont Cronex Lightning Plus intensifying the payment of page charges. This article must therefore be hereby screen for various time periods. marked “aduertisement” in accordance with 18 U.S.C. Section 1734 DNA Probes Used-The pro-a1 and pro-a2 (Type I) collagen solely to indicate this fact. cDNAs located in pCol3 and pCol1, respectively, have been described $ Supported by a postdoctoral fellowship from the Arthritis Foun- earlier (14, 15). Plasmid pSrcEcoB containing -3 kb DNA coding for dation. the src gene of RSV in pBR322waskindly provided by Harold ’ The abbreviations used are: CEF, chick embryo fibroblast; HSV, Varmus (University of California, San Francisco). The EcoRI restricROUSsarcoma virus; kb, kilobase pair; bp, base pair. tion patterns of XFC40,XFC32, and XFC3B DNA (HindII pattern ~
5787
Downloaded from www.jbc.org by guest, on July 22, 2011
Rous sarcomavirus transformed-chickembryo fibroblasts contain decreased amounts of the adhesive protein fibronectin that results from a lowering of fibronectin mRNA levels (Fagan, J. B., Sobel, M. E., Yamada, K. M., de Crombrugghe,B., and Pastan, I. (1981) J.Biol. Chem. 256, 520-525). In the present study, nuclei isolated from chick embryo fibroblasts (CEF) and Rous sarcoma virus transformed-chick embryo fibroblasts (RSV-CEF) were used to measure relative rates of fibronectin RNA synthesis. E3’P]RNA was synthesized by preinitiated endogenous RNA polymerases in nuclei, and the radioactive RNA was subsequently hybridized to DNA fragments spanning approximately 40 kilobase pairs (-85%) of the fibronectin gene. Transformation of CEF by RSV resulted in a 7- to %foldreduction in the amount of 32P-labelednuclear RNA hybridizing with fibronectin DNA sequences when the nuclei were incubated for either 3.5 or 45 min. These results indicate that decreased transcription and not reduced RNA stability plays a majorrole in lowering fibronectin mRNA levels inRSV-CEF. Whennuclear RNA was transcribed in the presence of Sarkosyl, diminished RNA synthesis was still observed suggesting that reduced fibronectin transcription in RSV-CEF is not due to a block in RNA chain elongation. Our data indicate that transcriptional regulation of the fibronectin gene is animportant mechanism of lowering steady state levels of fibronectin mRNA in RSV-CEF.
5788
Gene
of Fibronectin Chick
Transcriptional Regulation
also) are shown in Fig. 2B. A recombinant plasmid containing -80% of the chick /?-actin genomic sequences in a 9.5-kb insert in pBR325 was the generous gift of Bruce Paterson (National Cancer Institute, National Institutes of Health). For hybridization to genomic DNA, the following purified fragments were used 5.6 kb (from hFC40), 6.2 kb (from XFCBB), and 2.0 kb (from XFC3B). These fragments were labeled with 32Pby nick translation.
optimal KC1 concentration in the reaction mixture was -110 m ~ Nuclei . were incubated with [~u-~*P]GTP for up to45 min to maximize 32Pincorporation into RNA. Net incorporation rates were approximately equal with CEFand RSV-CEF nuclei. Labeled RNAsynthesized in CEF nuclei gavehybridization signals with pro-a1 and pro-a2 (Type I) collagen cDNA fragments immobilized ona nitrocellulose filter. No hybridization was detected with RSV-CEF ["PIRNA(Fig. 1). RSV-CEF [32P]RNA hybridizedstrongly with pSrcEcoB, a plasmid containing RSV src gene sequences (Fig. 1).These results indicated that nuclei isolated from CEF and RSV-CEF retained their transcriptional fidelity and were suitable for studying the transcriptional regulation of the fibronectin gene.
RESULTS
Preliminary Characterization of Nuclei CEF and RSV-CEF nuclei were tested for their transcriptional activity in twoways: first, by measuringthe incorporation of [a-32P]GTPinto RNA, and second, by hybridization of the labeled RNA to specific DNAfragments. The incorporation of radioactivity into trichloroacetic acid-precipitable material was linear for a t least 25 min (data not shown). The
A IA
Kb
W
Y
""
""
w v ) O
""
" "
23-
l A > K
9.5-
6.6-
1.81.W
4 5-
3.12.8.b.'."
2 1-
1.7-
1.9-
6,
Pro a 2 ( T y p e I) Collagen
0.8-
(I)
Pro a l(Type I) Collagen
FIG. 3. Hybridization ofCEF
FIG. 1. Hybridization of ["PIRNA labeled in CEF and RSVCEF nuclei with collagen-specificDNA fragments. DNAs were spotted on the nitrocellulose as indicated. pCol 3 and pCol 1 DNAs (14,15)wereused to detect pro-a1 and pro-a2 collagen (Type I)specific RNAs, respectively. Src indicates pSrcEcoB plasmid. CEF lane, hybridization to CEF[:"P]RNA; RSV-CEF lane, hybridization to RSV-CEF [:"P]RNA. Input countswere 75 X IO'; cpm/filter (2 ml). Hybridizations were performed for 36 h and filters were exposed to xray film for 24 h.
in the phages as indicated in Fig. 1B.M indicates molecular weight markers in kb.
A FIG. 2. Restriction maps of X clones spanning the chick fibronectin gene. The location of AFC40, hFC32,
and RSV-CEF nuclear ['*PI
RNA with DNA fragments. A, ethidium bromide-staining pattern of DNA fragments subjected to blotting on nitrocellulose filters. B, hybridization pattern of CEF V2P]RNA. C, hybridization pattern of RSV-CEF [.'"I']RNA. Lanes a , b, c, d, and e are the same in all panels. Lane a, AFC3B cut by EcoRI and HindIII; lane b, XFC32 cleaved by EcoRI; lane c, AFC4O digested by EcoRI; lane d, pSrcEcoB cleaved by EcoRI; lane e, genomic /?-actin plasmid cleaved by EcoRI. Hybridization was for 90 h. Input counts were -8.5 X 10'' cpm (3 ml). Filters were exposed to x-ray film for 5 days. The broken lines point toward linear and partially digested /?-actin plasmid. Numbers preceding the solid lines indicate fragment sizes in kb or their location
pBR 3 2 2
and XFC3Bin the fibronectin gene is shown in A. The horizontal arrow indicates the direction of transcription of the fibronectin gene. The restriction maps of the X clones are shown in B. The vertical lines on the X clones indicate restriction endonuclease recognition sites. The numbers between these lines represent fragment sizes in kb. The location of fragments "a" and "b" is marked on hFC3B/RI + H 111. The long and short wavy lines represent the long and short arms of X, respectively. The thick dark lines indicate the location of repetitive sequences in the fibronectin gene. s.a. = short arm of X; La. = long arm of X; kbp = kilobase pairs.
0.6-
AFC32
-
AFC40
La.
AFC3B
a .I-s .a ..
t
5.8.
3'end of mRNA
s.a. P La.
t
5 'end of mRNA I
50
B
I
I
I
I
40
30
I
I
AFC40/EcoR 'I 0.6 1.1 12811 3.4 I 1
I
0
AFC3BIHind 111 !j.6
I MI
AFC32/EcoR I 5.7
I
10
0.9
t
0.6 0.9 111.81 I
I
I
20 KbP
12.31
AFC3B/RI+HIII I6
I
Kb
0.4 II
5)
1
0.6 1.0 0.6 ll2012011.71 3.5 I I1
'a"b'
I
I
-10
Downloaded from www.jbc.org by guest, on July 22, 2011
9.8-
9.1-
-, ,,
5789
Transcriptional Regulation of Chick Fibronectin Gene
RSV-CEF
