- Email: [email protected]
Down-regulation of vimentin gene expression during myogenesis is controlled by a 5′-flanking sequence
235
Gene, 78 (1989) 235-242
Elsevier GEN 02962
Down-regulation sequence (Recombinant
of vimentin gene expression
DNA; muscle
Christina M. Sax
differentiation;
during myogenesis
intermediate
a,*,Francis X. Farrell b and Zendra E.
is controlled
by a 5’4lanking
filaments; GC box; Spl factor; gene repression)
Zehner b
a Department of Human Genetics and b Department of Biochemistry and Molecular Biophysics and The Massey Cancer Center, Medical College of Virginia/Virginia Commonwealth University,Richmond, VA 23298 (U.S.A.) Received by J. Piatigorsky: 6 July 1988 Revised: 14 November 1988 Accepted: 6 December 1988/2 February 1989
SUMMARY
During myogenesis, the intermediate filament proteins vimentin and desmin are differentially expressed. While desmin levels increase dramatically, vimentin mRNA levels decrease substantially. Here, we show that transfected whole- and mini-vimentin-coding genes (Vim) are expressed in libroblasts (mouse L cells) and down-regulated during muscle cell differentiation in culture. Functional assays with 5’-end Vim: :cat constructs demonstrate that this repression is controlled by a 5’-element (nt -321 to -160). This region is distinct from Vim promoter elements (nt -160 to + 71) which do not contribute to vimentin’s down-regulation during myogenesis.
Muscle development is characterized by a 5-6fold reduction in overall gene expression (Paterson and Bishop, 1977). Against this backdrop of repression a smaller group of muscle-specific genes is selectively activated. Within the IFP family there is a
switch both in the type of IFP synthesized and in its filament distribution during myogenesis (Bennett et al., 1979; Gard and Lazarides, 1980; Holtzer et al., 1982; Tokuyasu et al., 1984; 1985). Whether or not vimentin is still actively synthesized in the adult myotube is still a matter of debate, but it is certain that its levels are significantly reduced, if
Correspondenceto: Dr. Z.E. Zehner,
Abbreviations:
dynamic
INTRODUCTION
Biophysics,
Box 614 MCV
University,
Richmond,
Fax (804)225-FAXX
Station,
Dept. of Biochem. Virginia
VA 23298 (U.S.A.)
Molec.
Commonwealth
Tel. (804)786 8753
(3299).
transferase;
Laboratory
Biology,
National
Eye Institute,
of Molecular
(U.S.A.)
Tel. (301)4963234.
N.I.H.
and Developmental
Bethesda,
MD 20892
CAT, chloramphenicol
tary to DNA; Des, gene coding for desmin; modified
* Present address:
bp, base pair(s);
Eagle’s medium;
htk, herpes
kinase gene; IFP, intermediate nucleotide(s);
pfh, promoter
factor described tk, gene coding
for vimentin;
by Dynan
filament protein;
0 1989 Elsevier
Science Publishers
B.V. (Biomedical
Division)
Dulbecco’s
kb, 1000 bp; nt,
and Tjian (Cell 32: 669-680,
for TK; TK, thymidine ‘, designates
DMEM,
simplex virus thymidine
of the htk gene; Spl, transcriptional
a truncated
(5’ or 3’); ::, novel joint (fusion).
0378-l 119/89/$03.50
acetyl-
car, gene coding for CAT; cRNA, RNA complemen-
kinase;
1983);
vim, gene coding
gene at the indicated
side
236
not absent, in terminally differentiated tissue (Granger and Lazarides, 1979; Tokuyasu et al., 1985). On the other hand, the synthesis of desmin increases dramatically during myogenesis both in vivo and in muscle cultures (Bennett et al., 1979; Capetanaki et al., 1984; Holtzer et al., 1982). We are investigating the mech~ism(s) by which these two IFP-coding genes, vimentin and desmin, are noncoordinately regulated during myogenesis. Sequences internal and adjacent to the polyadenylation site have been found important for the down-re~lation of two non-muscle genes, chicken tk and p-actin genes, respectively (Merrill et al., 1984; DePonti-Zilli et al., 1988). Moreover, /J-actin gene repression appears to occur by an unknown post-transcriptional initiation event. Using vimentin 5’-end:cat assays it was recently reported that 5’ sequences were important for f/im expression during myogenesis (Pieper et al., 1987). However, Vfm expression down-regulated in only one (C2C12) of the two (C2C12, T983) muscle cell lines studied and the responsible control regions were quite different, i.e., upstream from nt position -599 for expression in T983 cells, or nt positions 176 to + 101 for C2Cl2 muscle cell lines. In addition, the C2C 12 muscle cell line has been shown to anomalously express several muscle-sp~i~c genes at the myoblast stage (Seiler-Tuyns et al., 1984; Minty et al., 1986) and behaves as a predetermined muscle cell already expressing muscle-specific rruns-modulating factors at an early stage (Minty et al., 1986). Here, we have shown that sequences from within the Vim gene are not required for gene repression. Down-regulation is attributable to 5’-end sequences. Furthermore, we have separated the 5’-end region into an enhancer element (nt -321 to -160), responsible for vimentin down-regulation, and a promoter element (nt -160 to + 71) which contributes to overall Vim gene activity but not gene repression.
EXPERIMENTAL
AND DISCUSSION
(a) Vimentin mRNA synthesis duringmyogenesis in culture
It is well documented that both vimentin protein and mRNA levels decline during in vivo muscle
development (Gard and Lazarides, 1980; Granger and Lazarides, 1979; Tokuyasu et al., 1984; 1985); however, this has not been fully established for all muscle cell lines in culture. In fact, Vim expression increases in the mouse T983 myogenic line during A.3
2
6. L6 EQ ABCDE
C.
20 O-
_I iWyoblast Aligned
I-2 days
5 days
14 days
Days Post-Fusion
Fig. 1. Northern analysis of vimentin mRNA synthesis during myogenesis in ovo and in culture (Panel A) Total RNA was isolated from 14-day embryonic (lane 1) and l-day post-hatch (lane 2) chicken breast muscle (Paterson and Roberts, 1981), analyzed (10 pg) on 6.6% formaldehyde/l% agarose gels (Maniatis et al., 1982), transferred to nitrocellulose (Thomas, 1980), and hybridized with a chicken Vim cDNA probe, E8 (Zehner and Paterson, 198317).(Panel B) Total RNA was isolated (Chirgwin et al., 1979) from cultures of rat L6E9 cells (Nadal-Ginard, 1978) at the folIowing stages of ~fferentiation: myoblasts (lane A), alignment (lane B), and post-fusion day 1 (lane C), day 5 (lane D), and day 14 (lane E). Vimentin mRNA was quantitated by cRNA mapping (Melton et al., 1984). 5 pg of total RNA from each timepoint was annealed to 2.5 x lo5 dpm of an SP6-generated probe specific for exon 1 of vimentin (probe is referred to as cRNA; see Fig. 2A) at 65”Cfor 16 h. UnanneaIed regions were digested with 0.4 fig/ml RNaseA and 0.02 pgjml RNaseTl at 30°C for 30 min. Samples were then treated with proteinase K, phenol-chloroform extracted, and sized on 6% polyacrylamide/8 M urea gels. rv denotes a segment of radiolabelled cRNA (430 nt) protected from RNase digestion by vimentin mRNA isolated from L6E9 cells. (Panel C) The autoradiogram in panel B was scanned by densitometry and the resultant values expressed as Y0of myoblast level (panel B, lane A) for each stage of myogenesis.
231
d~erentiation (Pieper et al., 1987). Therefore, we have compared vimentin mRNA levels during myogenesis in ovo to a rat skeletal myogenic cell line, L6E9 (Nadai-Ginard, 1978). Total RNA was isolated from 14-day embryonic and l-day post-hatch chicken breast muscle (Paterson and Roberts, 1981) and analyzed (see Fig. 1A) by Northern blot (Thomas, 1980) using a chicken vimentin cDNA probe (Zehner and Paterson, 1983a). The level of vimentin mRNA is maximal by day 14 in ovo (lane 1) and decreases substantially (46%) by 1 day post-hatch (lane 2). Previously, we have shown (Zehner and Paterson, 1983b) that multiple mRNA species are generated from the chicken
gene by dif%rential utilization of several polyadenylation sites (see Fig. 2A). The L6E9 myogenic cell line was grown as described (Nadal-Ginard, 1978) in DMEM plus 20% fetal calf serum for proliferative myoblasts and switched to differentiation media (5 y0 horse serum) to promote myogenesis. Vimentin mRNA was quantitated in total RNA isolated from myoblasts, aligned l-, 5, and 16day post-fusion L6E9 cultures (Chirgwin et al., 1979) by RNA mapping (Melton et al., 1984) using a uniformly labelled cRNA probe to exon 1 (see Fig. 2A). Due to species heterogeneity in the 5’-end of exon 1, this probe protects a 430~nt species in rat under mild RNase digestion conditions vim
Fig. 2. Expression of transfected vimentin-coding (fim) gene constructs in L-cells. (Panel A) A schematic presentation of the Vim whole and mini-gene constructs. The chicken vimentin gene is encoded by nine exons (l-9) and is flanked by four poly(A) sites as shown (A = functional, a = nonfunctional) (Zehner and Paterson, 1983b). The position of the Vim-specific cRNA probe used in transcriptional mapping (Figs. lB, 2B, 3A) is denoted. Phage 1A contains the entire chicken Vim gene as well as 3.0 and 4.5 kb of 5’- and 3’-flanking sequence, respectively, between the arms of phage I Charon4A (Zehner and Paterson, 1983a). The mini-gene-carrying plasmid, pVim-mini, was constructed in pUC18 from sub-clones of the chicken Vim gene and contains exons 1, 2, and 9 (fused in intron 2 to 8) and is flanked by 800 and 700 bp on its 5’- and 3’-end, respectively (Zehner et al., 1987). Shown here are the two genomic fragments which have been fused together in pVim-mini. (Panel B) Expression of the transfected Vim whole and mini-gene constructs in mouse L cells. Equimolar mounts (4 fmoles, adjusted to 10 pg with pBR322) of phage IA (IA) and pVim-rn~i (M) were co-transfected with pSV2neo (10 pg) (Southern and Berg, 1982) into mouse L cells (Earle et al., 1943) via CaCl, co-precipitation (Graham and Van der Eb, 1973). Stable transformants were selected (48 h post-transfection) for two weeks in DMEM + 10% fetal calf serum + 300 lg/ml of the aminoglycoside G418 (Colbtre-Garapin et al., 1981). Total RNA was extracted and vimentin mRNA levels (in 20 pg) quantitated by cRNA mapping as described in Fig. 1B (using 8 x lo5 dpm of cRNA probe). cv denotes the cRNA species (605 nt) protected from RNase digestion by mRNA arising from exon 1 of the transfected gene, while mv represents a cRNA species (430 nt) protected by mRNA arising from the endogenous mouse Vim gene. Due to species heterogeneity in the S-end of exon 1, chicken and mouse vimentin mRNA protect a different sized fragment of the cRNA from RNase digestion.
238
(such that not every mismatch is digested). While the level of vimentin mRNA decreases during differentiation in ovo and in L6E9 cultures, the level of 28s and 18s rRNA remains constant (data not shown). Only the pertinent region of the gel is shown in Fig. IB. Scanning densitometry (Fig. 1C) shows that the L6E9 cell line exhibits a progressive decrease in vimentin mRNA abundance during myogenesis. Moreover, this decrease corresponds to changes in vimentin mRNA levels during in vivo development (Fig. lA), and establishes the usefulness of this myogenic cell line for further studies.
(b) Expression of Vim whole and mini-genes
The chicken Vim gene (Zehner et al., 1987) spans 8.5 kb of DNA and contains nine exons (Fig. 2A). To localize the DNA sequences controlling Vim gene expression during myogenesis, we initiated transfection studies with the whole vim gene and compared its expression pattern to a selected mini-gene. Phage 1A contained the entire Vim gene plus considerable 5’- (3 kb) and 3’-flanking (4.5 kb) DNA (Fig. 2A). A mini-gene-carrying plasmid (pVim-mini)
prim-mini
Transformed Cultures
100
illCItY
,
n ’ s
80
I Ij
60
f
40
n q
Myoblasts
pVim-mlnl endogenous
Myotubes
Fig. 3. Expression of transfected Vim whole and mini-genes during L6E9 myogenesis. (Panel A) Rat L6E9 myoblasts were co-transfected with pSVZneo (20 pg) and phage 1A (I A) or pVim-mini (Mini) as described in Fig. 2. Stable transformants were selected for two weeks in DMEM + 20% fetal calf serum + 300 &ml G418, and cultures maint~ned as myoblasts. Transformed cultures were split into two and grown in either proliferation media (DMEM + 20% fetal calf serum) for 24 h (myoblasts), or differentiation media (DMEM + 5% horse serum) for 7 days (myotubes). Total RNA was extracted from myoblasts (B) and myotubes (T), and vimentin mRNA levels quantitated by cRNA mapping using 20 ng of total RNA and 8 x lo5 dpm of cRNA probe, as described in Fig. 1B. cv, rv, Denote the cRNA species protected from RNase digestion by mRNA arising from the transfected and endogenous vimentin gene, respectively. The top of panel A was exposed to film 1 more day than the bottom half. (Panel B and C) The autoradiograms in A were scanned by densitometry and the amount of vimentin mRNA arising from the transfected Vim whole and mini-gene, or the endogenous gene expressed as the % of myoblast levels.
239
containing exons 1, 2, and 9 (fused in intron 2 to 8) plus 800 bp and 700 bp of 5’- and 3’-flanking DNA, respectively, was constructed in the plasmid pUC18. Approximately 80% of the gene’s internal region is deleted in pVim-mini. Equimolar amounts of phage 1A or pVim-mini were co-precipitated with the selectable vector pSV2neo (Southern and Berg, 1982) and co-transfected via calcium phosphate (Graham and Van der Eb, 1973) into mouse L cells (Earle et al., 1943). Mouse L cells were chosen because 3% of the total cell protein is vimentin (Lilienbaum et al., 1986). Both pVim-mini and phage 1A (a 1 Chat-on 4A derivative described in Fig. 2) are expressed in L-cells as judged by the appearance of a 630-nt protected cRNA species in Fig. 2B. The endogenous vimentin mRNA band is apparent in equal amounts in both transfections. A first glance it would appear that the mini-gene expresses live-fold more RNA than phage 1A. However, this is probably accentuated by differences in transfection efficiency between a small supercoiled plasmid and a large linear piece of DNA (Weintraub et al., 1986). More importantly, pVim-mini is capable of producing mRNA in amounts comparable to those produced by the endogenous gene. We next asked whether or not the transfected whole and pVim-mini genes could be appropriately down-regulated during L6E9 myogenesis in culture. Equimolar amounts of both constructs and pSV2neo were co-transfected into the L6E9 muscle cell line and RNA analyzed as above (Fig. 3A). As in L-cells, pVim-mini expression is greater than phage 1A expression. The changing levels of mRNA during myogenesis were quantitated by densitometric scanning (Fig. 3B and 3C). Both phage 1A and pVim-mini were expressed in myoblasts and appropriately down-regulated to 13 % and 20x, respectively, of myoblast levels by day 7 post-fusion, whereas pSV2neo mRNA levels remained constant in L6E9 myoblasts and myotubes stably transformed with plasmids pSV2neo and pVim-mini (data not shown). In this experiment, endogenous vimentin gene activity dropped 50-60% (compare to Fig. 1C). The 2.5fold higher activity of the endogenous gene, relative to the transfected gene, might reflect (i) preferential mRNA stabilization of native vimentin mRNA, or (ii), expression differences between the native gene located in its natural position in chromatin and
the pool of transfected genes randomly integrated into a variety of chromosomal locations. Such minor differences have been observed by others (Feinstein et al., 1982). Nevertheless, it is clear that pVim-mini is appropriately expressed in both mouse L cells and rat myoblasts. Moreover, it is down-regulated, as is the native gene during myogenesis, while pSV2neo expression remains constant. Therefore, deletion of 80% of the Vim gene internal sequences does not interfere with its normal expression pattern. of vimentin 5’-end Vim::cat fusions
(c) Expression in vivo
Our results indicate that sequences which repress Vim gene expression during myogenesis must be conlined to the remaining 20% of pVim-mini or the 5’and 3’-flanking regions. Functional studies with various vimentin 5’-end Vim::cat constructs (pcV-321, pcV-160, and pcV-496/-162) suggest that chicken Vim expression is controlled by at least two regions: (i) containing a tissue-specific enhancer (nt -321 to -160) which increases gene activity in some celltypes (fibroblasts), and (ii) promoter elements consisting of GC boxes, the transcription factor Spl, and a CCAAT box (Sax et al., 1988). Here, we have analyzed the expression of these same 5’-end Vim::cat constructs in L6E9 cells during myogenesis (Fig. 4 and Table I). The htk promoter (nt -110 to -40) closely resembles the 5’-region (nt -90 to + 1) of the Vim gene in that both contain multiple GC boxes separated by a reversely GCl
B
18 bp -360
- 505 -460
CAT GC5 GC3 GC4 + 1 exon
GC2
-280
-225
-115
1
-75 -90-55 pcV-321
-321
+1 pcW496/-162
-496
-162 P -160
Fig. 4. Schematic chicken (GCl-5), similar
diagram
of the
Vim gene. The 5’-end a CCAAT (75%,
SV40 enhancer
loo%, (Herr
pcV-160 +1
5’-flanking
region
of Vim contains
box (CAT), three
elements
and 88%, respectively) and Clarke,
(A,B, and C)
to regions
(Quax et al., 1983) Vim genes.
fragments
(Sax et al., 1988) fused to cat in the expression et al.,
pcV-4961-162
1983),
giving
and pcV-160.
rise
1987) and
+ 1 refers to the major
cap site. Shown here are the three promoter (Dente
in the
1986), and an 1%bp region
with 72% identity to the human (Rittling and Baserga, hamster
of the
five GC boxes
to the
previously
vector
plasmids
p8CAT pcV-321,
240
TABLE
I of the 5 ’ end Vim : cat and pTK-CAT
Expression
plasmids
during
myogenesis car-gene
fusion”
CAT enzyme
activity b
% CAT activity remaining
Myoblasts
Myotubes
pcV-321
7.3 f 2.1
2.3
pcV-4961-162
3.1 k 0.6
0.62 f 0.07
pcV-160
0.6
0.64
20% 106%
pTKCAT
0.5
0.44
88%
a pcV-321, pcV-496/-162, moter
flanking
p8CAT
fragments
and pcV-160 contain
chicken
described
contains
using pSV2neo
and myotubes
plasmids
transformations
(one week post-fusion),
the number of pmoles of [ r4C]chloramphenicol of total cellular promoterless
protein,
plasmid,
(20 pg) were
minus the background
(Gorman
et al.,
in both myoand expressed acetylated
as
per fig
(< 0.03) of the
p8CAT.
’ Relative activity is represented remaining
the hrk
(20 pg) into L6E9 cells as
in Fig. 3A. CAT activity was assayed
1982) twice from three separate
plasmid
et al., 1986).
Vim : :cut or pTK-CAT
stably co-transformed
Vim pro-
fused to cat in the parental
fused to cat (Miksicek
b The 5’-end
activity
31%
(Fig. 4; Sax et al., 1988). pTK-CAT
promoter
blasts
+ 0.6
c
in myotubes,
as the percent with respect
by 5’-flanking Vim fragments, the decrease in expression of pcV-321 and pcV-4961-162 during myogenesis suggests that down-regulation of CAT activity is due to a decrease in transcription initiation directed by these Vim fragments. We conclude that sequences which control the down-regulation of vimentin expression during myogenesis lie in the common region -321 to -160. Our result is in agreement with Pieper et al. (1987) who found the region -176 to + 101 important for hamster Vim downregulation during C2C12 myogenesis. Here, we have further separated this control region into two functional elements, i.e. a repressible enhancer and a constitutive promoter region. Although our studies suggest that Vim down-regulation during myogenesis is, at least in part, due to a decrease in transcription initiation, additional effects occurring post-initiation cannot be ruled out at this time. Such events might include a difference in elongation rate in myoblasts and myotubes.
of CAT enzyme to that present
(d) Significance
in
myoblasts.
oriented CCAAT box (Jones et al., 1985; Zehner et al., 1987). In addition, htk and pTK-CAT (htk nt-105 to + 56 fused to cat) have been shown to be equally active throughout differentiation of the myogenie line MM14 (Merrill et al., 1984; Jaynes et al., 1986). Therefore, as a control for GC box activity, we have compared the expression of our various constructs to pTK-CAT (Miksicek et al., 1986). Both pcV-160 (chicken Vim nt -160 to + 1 fused to cat) and pTK-CAT are expressed in myoblasts at low levels which may reflect the level of GC box promoter activity at this stage in differentiation. Moreover, this level does not change significantly upon differentiation to myotubes. In contrast, both pcV-321 (chicken vimentin nt -321 to + 1 fused to cut) and pcV-4961-162 (chicken Vim nt -496 to -162 fused to cat) are amply expressed in myoblasts and their expression declines 69% and 80 %, respectively, during differentiation to myotubes. That activity differences between plasmids are not due to transfection variability is suggested by constant pSV2neo RNA levels in all myotube samples (slot-blot not shown). Since transcription of the cut gene is directed
Previously, we have shown that a tissue-specific enhancer is located in the region -321 to -160 (Sax et al., 1988). Here we show that this region specititally represses activity of a heterologous gene during myogenesis in culture. We suggest that the downregulation of the Vim gene during myogenesis may be due to the decreased synthesis of a positive-acting transcription factor(s) which binds in this region. This implies a different mechanism than that recently proposed for the j?-actin gene (DePonti-Zilli et al., 1988). In the case of a tissue-specific expressed gene like vimentin, the decreased synthesis of a positive transcription factor would be a simple alternative for repressing gene activity
ACKNOWLEDGEMENTS
The authors wish to thank Dr. Bernard0 Nadal-Ginard for the L6E9 myogenic cell line. This research was supported by Public Health Service Grant AM33310 from the National Institutes of Health and by grants from the Muscular Dystrophy Association and the Jeffress Trust.
241 REFERENCES Bennett, G.S., Fellini, S.A., Toyama, Y. and Holtzer, H.: Redistribution of intermediate filament subunits during skeletal myogenesis and maturation in vitro. J. Cell Biol. 82 (1979) 577-584. Capetanaki, Y.G., Ngai, J. and Lazarides, E.: Characterization and regulation in the expression of a gene coding for the intermediate filament protein desmin. Proc. Natl. Acad. Sci. USA 81 (1984) 6909-6913. Chirgwin, J.M., Przybyla, A.E., MacDonald, R.J. and Rutter, W.J.: Isolation of biologically active ribonucleic acid from sources enriched in ribonuclease. Biochemistry 18 (1979) 5292-5299. Colbere-Garapin, F., Horodniceanu, F., Kourilsky, P. and Garapin, A.-C.: A new dominant hybrid selective marker for higher eukaryotic cells. J. Mol. Biol. 150 (1981) 1-14. Dente, L., Cesareni, G. and Cortese, R.: pEMBL: A new family of single stranded plasmids. Nucleic Acids Res. 11 (1983) 1645-1655. DePonti-Zilli, L., Seiler-Tuyns, A. and Paterson, B.M.: A 40-base-pair sequence in the 3’ end of the beta-actin gene regulates mRNA transcription during myogenesis. Proc. Natl. Acad. Sci. USA 85 (1988) 1389-1393. Dynan, W.S. and Tjian, R.: Isolation oftranscription factors that discriminate between different promoters recognized by RNA polymerase II. Cell 32 (1983) 669-680. Earle, W.R., Schilling, E.L., Stark,T.H., Straus, N.P., Brown, F. and Shelton, E.: Production of malignancy in vitro, IV. The mouse Bbroblast cultures and changes seen in the living cells. J. Natl. Cancer Inst. 4 (1943) 165-212. Feinstein, S.C., Ross, S.R. and Yamamoto, K.R.: Chromosomal position effects determine transcriptional potential of integrated mammary tumor virus DNA. J. Mol. Biol. 156 (1982) 549-566. Gard, D.L. and Lazarides, E.: The synthesis and distribution of desmin and vimentin during myogenesis in vitro. Cell 19 (1980) 263-275. Gorman, C.M., Moffat, L.F. and Howard, B.H.: Recombinant genomes which express chloramphenicol acetyltransferase in mammalian cells. Mol. Cell. Biol. 2 (1982) 1044-1051. Graham, F.L. and Van der Eb, A.J.: A new technique for the assay of infectivity of human adenovirus 5 DNA. Virology 52 (1973) 456-467. Granger, B.L. and Lazarides, E.: Desmin and vimentin co-exist at the periphery of the myofibril Z disc. Cell 18 (1979) 1053-1063. Herr, W. and Clarke, J.: The SV40 enhancer is composed of multiple functional elements that can compensate for one another. Cell 45 (1986) 461-470. Holtzer, H., Bennett, G.S., Tapscott, S.J., Croop, J.M. and Toyama, Y.: Intermediate-size filaments: changes in synthesis and distribution in cells of the myogenic and neurogenic lineages. Cold Spring Harbor Symp. Quant. Biol. 46 (1982) 3 17-329. Jaynes, J.B., Chamberlain, J.S., Buskin, J.N., Johnson, J.E. and Hauschka, S.D.: Transcriptional regulation of the muscle creatine kinase gene and regulated expression in transfected mouse myoblasts. Mol. Cell. Biol. 6 (1986) 2855-2864.
Jones, K.A., Yamamoto, K.R. and Tjian, R.: Two distinct transcription factors bind the HSV thymidine kinase promoter in vitro. Cell 42 (1985) 559-572. Lilienbaum, A., Legagneux, V., Portier, M.-M., Dellagi, K. and Paulin, D.: Vimentin gene: expression in human lymphocytes and in Burkitt’s lymphoma cells. EMBO J. 5 (1986) 2809-2814. Maniatis, T., Fritsch, E.F. and Sambrook, J.: Molecular Cloning. A Laboratory Manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, 1982, pp. 202-203. Melton, D.A., Krien, P.A., Rebagliati, M.R., Maniatis, T., Zinn, K. and Green, M.R.: Efficient in vitro synthesis of biologically active RNA and RNA hybridization probes from plasmids containing a bacteriophage Sp6 promoter. Nucleic Acids Res. 12 (1984) 7035-7056. Merrill, G.F., Hauschka, S.D. and McKnight, S.L.: Tk enzyme expression in differentiating muscle cells is regulated through an internal segment of the cellular rk gene. Mol. Cell. Biol. 4 (1984) 1777-1784. Minty, A., Blau, H. and Kedes, L.: Two-level regulation of cardiac actin gene transcription: muscle-specific modulating factors can accumulate before gene activation. Mol. Cell. Biol. 6 (1986) 2137-2148. Miksicek, R., Heber, A., S&mid, W., Danesch, U., Posseckert, G., Beato, M. and Shutz, G.: Glucocorticoid responsiveness of the transcriptional enhancer of Moloney murine sarcoma virus. Cell 46 (1986) 283-290. Nadal-Ginard, B.: Commitment, fusion, and biochemical differentiation of a myogenic cell line in the absence of DNA synthesis. Cell 15 (1978) 855-864. Paterson, B.M. and Bishop, J.O.: Changes in the mRNA population of chick myoblasts during myogenesis in vitro. Cell 12 (1977) 751-765. Paterson, B.M. and Roberts, B.: Structural gene identification utilizing eukaryotic cell-free translational systems. In Chirikjian, J.G. and Papas, T.S. (Eds.), Structural Analysis of Nucleic Acids, Vol. 2. Elsevier, North Holland, 1981, pp. 417-437. Pieper, F.R., Slobbe, R.L., Ramaekers, F.C.S., Cuypers, H.T. and Bloemendal, H.: Upstream regions ofthe hamster desmin and vimentin genes regulate expression during in vitro myogenesis. EMBO J. 6 (1987) 3611-3618. Quax, W., Egberts, W.V., Hendricks, W., Quax-Jeuken, Y. and Bloemendal, H.: The structure of the vimentin gene. Cell 35 (1983) 215-223. Rittling, S.R. and Baserga, R.: Functional analysis and growth factor regulation of the human vimentin promoter. Mol. Cell. Biol. 7 (1987) 3908-3915. Sax, CM., Farrell, F.X., Tobian, J.A. and Zehner, Z.E.: Multiple elements are required for expression of an intermediate filament gene. Nucleic Acids Res. 16 (1988) 8057-8076. Seiler-Tuyns, A., Eldridge, J. and Paterson, B.M.: Expression and regulation of chicken actin genes introduced into mouse myogenic and nonmyogenic cells. Proc. Natl. Acad. Sci. USA 8 1 (1984) 2980-2984. Southern, P.J. and Berg, P.: Transformation of mammalian cells to antibiotic resistance with a bacterial gene under control of the SV40 early region promoter. J. Mol. Appl. Genet. 1(1982) 327-341.
242
Thomas,
P.S.: Hybridization
fragments
transferred
ofdenatured
to nitrocellulose.
RNA and small DNA
fected
Proc. Natl. Acad. Sci.
115-122.
USA 77 (1980) 5201-5205. Tokuyasu,
K.T., Maher,
of vimentin vivo.
Zehner,
P.A. and
and desmin
Singer,
in developing
I. Immunofluorescence
study.
S.J.: Distributions chick
myotubes
in
J. Cell. Biol. (1980)
vimentin
P.A. and Singer, S.J.: Distributions
and desmin in developing
II. Immunoelectron
microscopic
chick myotubes study.
of
in vivo.
J. Cell Biol.
100
(1985) 1157-1166. Weintraub.
H., Cheng, P.F. and Conrad,
of trans-
topology.
gene. Nucleotide
and comparison
to the hamster
Cell 46 (1986)
B.M. and Sax, C.M.: sequence,
regulatory
gene. J. Biol. Chem.
262 (1987) 8112-8120. Z.E.
chicken mRNAs. Zehner,
and
Paterson,
vimentin
B.M.:
Characterization
gene: single copy gene producing
Proc. Natl. Acad.
Z.E. and Paterson,
during myogenesis: K.: Expression
on DNA
Z.E., Li, Y., Roe, B.A., Paterson,
elements, Zehner,
K.T., Maher,
depends
The chicken vimentin
1961-1972. Tokuyasu,
DNA
Sci. USA 80 (1983a) B.M.: Vimentin
two functional
of the multiple 911-915.
gene expression
transcripts
from a single
copy gene. Nucleic Acids Res. 11 (1983b) 8317-8332.
Gene, 78 (1989) 235-242
Elsevier GEN 02962
Down-regulation sequence (Recombinant
of vimentin gene expression
DNA; muscle
Christina M. Sax
differentiation;
during myogenesis
intermediate
a,*,Francis X. Farrell b and Zendra E.
is controlled
by a 5’4lanking
filaments; GC box; Spl factor; gene repression)
Zehner b
a Department of Human Genetics and b Department of Biochemistry and Molecular Biophysics and The Massey Cancer Center, Medical College of Virginia/Virginia Commonwealth University,Richmond, VA 23298 (U.S.A.) Received by J. Piatigorsky: 6 July 1988 Revised: 14 November 1988 Accepted: 6 December 1988/2 February 1989
SUMMARY
During myogenesis, the intermediate filament proteins vimentin and desmin are differentially expressed. While desmin levels increase dramatically, vimentin mRNA levels decrease substantially. Here, we show that transfected whole- and mini-vimentin-coding genes (Vim) are expressed in libroblasts (mouse L cells) and down-regulated during muscle cell differentiation in culture. Functional assays with 5’-end Vim: :cat constructs demonstrate that this repression is controlled by a 5’-element (nt -321 to -160). This region is distinct from Vim promoter elements (nt -160 to + 71) which do not contribute to vimentin’s down-regulation during myogenesis.
Muscle development is characterized by a 5-6fold reduction in overall gene expression (Paterson and Bishop, 1977). Against this backdrop of repression a smaller group of muscle-specific genes is selectively activated. Within the IFP family there is a
switch both in the type of IFP synthesized and in its filament distribution during myogenesis (Bennett et al., 1979; Gard and Lazarides, 1980; Holtzer et al., 1982; Tokuyasu et al., 1984; 1985). Whether or not vimentin is still actively synthesized in the adult myotube is still a matter of debate, but it is certain that its levels are significantly reduced, if
Correspondenceto: Dr. Z.E. Zehner,
Abbreviations:
dynamic
INTRODUCTION
Biophysics,
Box 614 MCV
University,
Richmond,
Fax (804)225-FAXX
Station,
Dept. of Biochem. Virginia
VA 23298 (U.S.A.)
Molec.
Commonwealth
Tel. (804)786 8753
(3299).
transferase;
Laboratory
Biology,
National
Eye Institute,
of Molecular
(U.S.A.)
Tel. (301)4963234.
N.I.H.
and Developmental
Bethesda,
MD 20892
CAT, chloramphenicol
tary to DNA; Des, gene coding for desmin; modified
* Present address:
bp, base pair(s);
Eagle’s medium;
htk, herpes
kinase gene; IFP, intermediate nucleotide(s);
pfh, promoter
factor described tk, gene coding
for vimentin;
by Dynan
filament protein;
0 1989 Elsevier
Science Publishers
B.V. (Biomedical
Division)
Dulbecco’s
kb, 1000 bp; nt,
and Tjian (Cell 32: 669-680,
for TK; TK, thymidine ‘, designates
DMEM,
simplex virus thymidine
of the htk gene; Spl, transcriptional
a truncated
(5’ or 3’); ::, novel joint (fusion).
0378-l 119/89/$03.50
acetyl-
car, gene coding for CAT; cRNA, RNA complemen-
kinase;
1983);
vim, gene coding
gene at the indicated
side
236
not absent, in terminally differentiated tissue (Granger and Lazarides, 1979; Tokuyasu et al., 1985). On the other hand, the synthesis of desmin increases dramatically during myogenesis both in vivo and in muscle cultures (Bennett et al., 1979; Capetanaki et al., 1984; Holtzer et al., 1982). We are investigating the mech~ism(s) by which these two IFP-coding genes, vimentin and desmin, are noncoordinately regulated during myogenesis. Sequences internal and adjacent to the polyadenylation site have been found important for the down-re~lation of two non-muscle genes, chicken tk and p-actin genes, respectively (Merrill et al., 1984; DePonti-Zilli et al., 1988). Moreover, /J-actin gene repression appears to occur by an unknown post-transcriptional initiation event. Using vimentin 5’-end:cat assays it was recently reported that 5’ sequences were important for f/im expression during myogenesis (Pieper et al., 1987). However, Vfm expression down-regulated in only one (C2C12) of the two (C2C12, T983) muscle cell lines studied and the responsible control regions were quite different, i.e., upstream from nt position -599 for expression in T983 cells, or nt positions 176 to + 101 for C2Cl2 muscle cell lines. In addition, the C2C 12 muscle cell line has been shown to anomalously express several muscle-sp~i~c genes at the myoblast stage (Seiler-Tuyns et al., 1984; Minty et al., 1986) and behaves as a predetermined muscle cell already expressing muscle-specific rruns-modulating factors at an early stage (Minty et al., 1986). Here, we have shown that sequences from within the Vim gene are not required for gene repression. Down-regulation is attributable to 5’-end sequences. Furthermore, we have separated the 5’-end region into an enhancer element (nt -321 to -160), responsible for vimentin down-regulation, and a promoter element (nt -160 to + 71) which contributes to overall Vim gene activity but not gene repression.
EXPERIMENTAL
AND DISCUSSION
(a) Vimentin mRNA synthesis duringmyogenesis in culture
It is well documented that both vimentin protein and mRNA levels decline during in vivo muscle
development (Gard and Lazarides, 1980; Granger and Lazarides, 1979; Tokuyasu et al., 1984; 1985); however, this has not been fully established for all muscle cell lines in culture. In fact, Vim expression increases in the mouse T983 myogenic line during A.3
2
6. L6 EQ ABCDE
C.
20 O-
_I iWyoblast Aligned
I-2 days
5 days
14 days
Days Post-Fusion
Fig. 1. Northern analysis of vimentin mRNA synthesis during myogenesis in ovo and in culture (Panel A) Total RNA was isolated from 14-day embryonic (lane 1) and l-day post-hatch (lane 2) chicken breast muscle (Paterson and Roberts, 1981), analyzed (10 pg) on 6.6% formaldehyde/l% agarose gels (Maniatis et al., 1982), transferred to nitrocellulose (Thomas, 1980), and hybridized with a chicken Vim cDNA probe, E8 (Zehner and Paterson, 198317).(Panel B) Total RNA was isolated (Chirgwin et al., 1979) from cultures of rat L6E9 cells (Nadal-Ginard, 1978) at the folIowing stages of ~fferentiation: myoblasts (lane A), alignment (lane B), and post-fusion day 1 (lane C), day 5 (lane D), and day 14 (lane E). Vimentin mRNA was quantitated by cRNA mapping (Melton et al., 1984). 5 pg of total RNA from each timepoint was annealed to 2.5 x lo5 dpm of an SP6-generated probe specific for exon 1 of vimentin (probe is referred to as cRNA; see Fig. 2A) at 65”Cfor 16 h. UnanneaIed regions were digested with 0.4 fig/ml RNaseA and 0.02 pgjml RNaseTl at 30°C for 30 min. Samples were then treated with proteinase K, phenol-chloroform extracted, and sized on 6% polyacrylamide/8 M urea gels. rv denotes a segment of radiolabelled cRNA (430 nt) protected from RNase digestion by vimentin mRNA isolated from L6E9 cells. (Panel C) The autoradiogram in panel B was scanned by densitometry and the resultant values expressed as Y0of myoblast level (panel B, lane A) for each stage of myogenesis.
231
d~erentiation (Pieper et al., 1987). Therefore, we have compared vimentin mRNA levels during myogenesis in ovo to a rat skeletal myogenic cell line, L6E9 (Nadai-Ginard, 1978). Total RNA was isolated from 14-day embryonic and l-day post-hatch chicken breast muscle (Paterson and Roberts, 1981) and analyzed (see Fig. 1A) by Northern blot (Thomas, 1980) using a chicken vimentin cDNA probe (Zehner and Paterson, 1983a). The level of vimentin mRNA is maximal by day 14 in ovo (lane 1) and decreases substantially (46%) by 1 day post-hatch (lane 2). Previously, we have shown (Zehner and Paterson, 1983b) that multiple mRNA species are generated from the chicken
gene by dif%rential utilization of several polyadenylation sites (see Fig. 2A). The L6E9 myogenic cell line was grown as described (Nadal-Ginard, 1978) in DMEM plus 20% fetal calf serum for proliferative myoblasts and switched to differentiation media (5 y0 horse serum) to promote myogenesis. Vimentin mRNA was quantitated in total RNA isolated from myoblasts, aligned l-, 5, and 16day post-fusion L6E9 cultures (Chirgwin et al., 1979) by RNA mapping (Melton et al., 1984) using a uniformly labelled cRNA probe to exon 1 (see Fig. 2A). Due to species heterogeneity in the 5’-end of exon 1, this probe protects a 430~nt species in rat under mild RNase digestion conditions vim
Fig. 2. Expression of transfected vimentin-coding (fim) gene constructs in L-cells. (Panel A) A schematic presentation of the Vim whole and mini-gene constructs. The chicken vimentin gene is encoded by nine exons (l-9) and is flanked by four poly(A) sites as shown (A = functional, a = nonfunctional) (Zehner and Paterson, 1983b). The position of the Vim-specific cRNA probe used in transcriptional mapping (Figs. lB, 2B, 3A) is denoted. Phage 1A contains the entire chicken Vim gene as well as 3.0 and 4.5 kb of 5’- and 3’-flanking sequence, respectively, between the arms of phage I Charon4A (Zehner and Paterson, 1983a). The mini-gene-carrying plasmid, pVim-mini, was constructed in pUC18 from sub-clones of the chicken Vim gene and contains exons 1, 2, and 9 (fused in intron 2 to 8) and is flanked by 800 and 700 bp on its 5’- and 3’-end, respectively (Zehner et al., 1987). Shown here are the two genomic fragments which have been fused together in pVim-mini. (Panel B) Expression of the transfected Vim whole and mini-gene constructs in mouse L cells. Equimolar mounts (4 fmoles, adjusted to 10 pg with pBR322) of phage IA (IA) and pVim-rn~i (M) were co-transfected with pSV2neo (10 pg) (Southern and Berg, 1982) into mouse L cells (Earle et al., 1943) via CaCl, co-precipitation (Graham and Van der Eb, 1973). Stable transformants were selected (48 h post-transfection) for two weeks in DMEM + 10% fetal calf serum + 300 lg/ml of the aminoglycoside G418 (Colbtre-Garapin et al., 1981). Total RNA was extracted and vimentin mRNA levels (in 20 pg) quantitated by cRNA mapping as described in Fig. 1B (using 8 x lo5 dpm of cRNA probe). cv denotes the cRNA species (605 nt) protected from RNase digestion by mRNA arising from exon 1 of the transfected gene, while mv represents a cRNA species (430 nt) protected by mRNA arising from the endogenous mouse Vim gene. Due to species heterogeneity in the S-end of exon 1, chicken and mouse vimentin mRNA protect a different sized fragment of the cRNA from RNase digestion.
238
(such that not every mismatch is digested). While the level of vimentin mRNA decreases during differentiation in ovo and in L6E9 cultures, the level of 28s and 18s rRNA remains constant (data not shown). Only the pertinent region of the gel is shown in Fig. IB. Scanning densitometry (Fig. 1C) shows that the L6E9 cell line exhibits a progressive decrease in vimentin mRNA abundance during myogenesis. Moreover, this decrease corresponds to changes in vimentin mRNA levels during in vivo development (Fig. lA), and establishes the usefulness of this myogenic cell line for further studies.
(b) Expression of Vim whole and mini-genes
The chicken Vim gene (Zehner et al., 1987) spans 8.5 kb of DNA and contains nine exons (Fig. 2A). To localize the DNA sequences controlling Vim gene expression during myogenesis, we initiated transfection studies with the whole vim gene and compared its expression pattern to a selected mini-gene. Phage 1A contained the entire Vim gene plus considerable 5’- (3 kb) and 3’-flanking (4.5 kb) DNA (Fig. 2A). A mini-gene-carrying plasmid (pVim-mini)
prim-mini
Transformed Cultures
100
illCItY
,
n ’ s
80
I Ij
60
f
40
n q
Myoblasts
pVim-mlnl endogenous
Myotubes
Fig. 3. Expression of transfected Vim whole and mini-genes during L6E9 myogenesis. (Panel A) Rat L6E9 myoblasts were co-transfected with pSVZneo (20 pg) and phage 1A (I A) or pVim-mini (Mini) as described in Fig. 2. Stable transformants were selected for two weeks in DMEM + 20% fetal calf serum + 300 &ml G418, and cultures maint~ned as myoblasts. Transformed cultures were split into two and grown in either proliferation media (DMEM + 20% fetal calf serum) for 24 h (myoblasts), or differentiation media (DMEM + 5% horse serum) for 7 days (myotubes). Total RNA was extracted from myoblasts (B) and myotubes (T), and vimentin mRNA levels quantitated by cRNA mapping using 20 ng of total RNA and 8 x lo5 dpm of cRNA probe, as described in Fig. 1B. cv, rv, Denote the cRNA species protected from RNase digestion by mRNA arising from the transfected and endogenous vimentin gene, respectively. The top of panel A was exposed to film 1 more day than the bottom half. (Panel B and C) The autoradiograms in A were scanned by densitometry and the amount of vimentin mRNA arising from the transfected Vim whole and mini-gene, or the endogenous gene expressed as the % of myoblast levels.
239
containing exons 1, 2, and 9 (fused in intron 2 to 8) plus 800 bp and 700 bp of 5’- and 3’-flanking DNA, respectively, was constructed in the plasmid pUC18. Approximately 80% of the gene’s internal region is deleted in pVim-mini. Equimolar amounts of phage 1A or pVim-mini were co-precipitated with the selectable vector pSV2neo (Southern and Berg, 1982) and co-transfected via calcium phosphate (Graham and Van der Eb, 1973) into mouse L cells (Earle et al., 1943). Mouse L cells were chosen because 3% of the total cell protein is vimentin (Lilienbaum et al., 1986). Both pVim-mini and phage 1A (a 1 Chat-on 4A derivative described in Fig. 2) are expressed in L-cells as judged by the appearance of a 630-nt protected cRNA species in Fig. 2B. The endogenous vimentin mRNA band is apparent in equal amounts in both transfections. A first glance it would appear that the mini-gene expresses live-fold more RNA than phage 1A. However, this is probably accentuated by differences in transfection efficiency between a small supercoiled plasmid and a large linear piece of DNA (Weintraub et al., 1986). More importantly, pVim-mini is capable of producing mRNA in amounts comparable to those produced by the endogenous gene. We next asked whether or not the transfected whole and pVim-mini genes could be appropriately down-regulated during L6E9 myogenesis in culture. Equimolar amounts of both constructs and pSV2neo were co-transfected into the L6E9 muscle cell line and RNA analyzed as above (Fig. 3A). As in L-cells, pVim-mini expression is greater than phage 1A expression. The changing levels of mRNA during myogenesis were quantitated by densitometric scanning (Fig. 3B and 3C). Both phage 1A and pVim-mini were expressed in myoblasts and appropriately down-regulated to 13 % and 20x, respectively, of myoblast levels by day 7 post-fusion, whereas pSV2neo mRNA levels remained constant in L6E9 myoblasts and myotubes stably transformed with plasmids pSV2neo and pVim-mini (data not shown). In this experiment, endogenous vimentin gene activity dropped 50-60% (compare to Fig. 1C). The 2.5fold higher activity of the endogenous gene, relative to the transfected gene, might reflect (i) preferential mRNA stabilization of native vimentin mRNA, or (ii), expression differences between the native gene located in its natural position in chromatin and
the pool of transfected genes randomly integrated into a variety of chromosomal locations. Such minor differences have been observed by others (Feinstein et al., 1982). Nevertheless, it is clear that pVim-mini is appropriately expressed in both mouse L cells and rat myoblasts. Moreover, it is down-regulated, as is the native gene during myogenesis, while pSV2neo expression remains constant. Therefore, deletion of 80% of the Vim gene internal sequences does not interfere with its normal expression pattern. of vimentin 5’-end Vim::cat fusions
(c) Expression in vivo
Our results indicate that sequences which repress Vim gene expression during myogenesis must be conlined to the remaining 20% of pVim-mini or the 5’and 3’-flanking regions. Functional studies with various vimentin 5’-end Vim::cat constructs (pcV-321, pcV-160, and pcV-496/-162) suggest that chicken Vim expression is controlled by at least two regions: (i) containing a tissue-specific enhancer (nt -321 to -160) which increases gene activity in some celltypes (fibroblasts), and (ii) promoter elements consisting of GC boxes, the transcription factor Spl, and a CCAAT box (Sax et al., 1988). Here, we have analyzed the expression of these same 5’-end Vim::cat constructs in L6E9 cells during myogenesis (Fig. 4 and Table I). The htk promoter (nt -110 to -40) closely resembles the 5’-region (nt -90 to + 1) of the Vim gene in that both contain multiple GC boxes separated by a reversely GCl
B
18 bp -360
- 505 -460
CAT GC5 GC3 GC4 + 1 exon
GC2
-280
-225
-115
1
-75 -90-55 pcV-321
-321
+1 pcW496/-162
-496
-162 P -160
Fig. 4. Schematic chicken (GCl-5), similar
diagram
of the
Vim gene. The 5’-end a CCAAT (75%,
SV40 enhancer
loo%, (Herr
pcV-160 +1
5’-flanking
region
of Vim contains
box (CAT), three
elements
and 88%, respectively) and Clarke,
(A,B, and C)
to regions
(Quax et al., 1983) Vim genes.
fragments
(Sax et al., 1988) fused to cat in the expression et al.,
pcV-4961-162
1983),
giving
and pcV-160.
rise
1987) and
+ 1 refers to the major
cap site. Shown here are the three promoter (Dente
in the
1986), and an 1%bp region
with 72% identity to the human (Rittling and Baserga, hamster
of the
five GC boxes
to the
previously
vector
plasmids
p8CAT pcV-321,
240
TABLE
I of the 5 ’ end Vim : cat and pTK-CAT
Expression
plasmids
during
myogenesis car-gene
fusion”
CAT enzyme
activity b
% CAT activity remaining
Myoblasts
Myotubes
pcV-321
7.3 f 2.1
2.3
pcV-4961-162
3.1 k 0.6
0.62 f 0.07
pcV-160
0.6
0.64
20% 106%
pTKCAT
0.5
0.44
88%
a pcV-321, pcV-496/-162, moter
flanking
p8CAT
fragments
and pcV-160 contain
chicken
described
contains
using pSV2neo
and myotubes
plasmids
transformations
(one week post-fusion),
the number of pmoles of [ r4C]chloramphenicol of total cellular promoterless
protein,
plasmid,
(20 pg) were
minus the background
(Gorman
et al.,
in both myoand expressed acetylated
as
per fig
(< 0.03) of the
p8CAT.
’ Relative activity is represented remaining
the hrk
(20 pg) into L6E9 cells as
in Fig. 3A. CAT activity was assayed
1982) twice from three separate
plasmid
et al., 1986).
Vim : :cut or pTK-CAT
stably co-transformed
Vim pro-
fused to cat in the parental
fused to cat (Miksicek
b The 5’-end
activity
31%
(Fig. 4; Sax et al., 1988). pTK-CAT
promoter
blasts
+ 0.6
c
in myotubes,
as the percent with respect
by 5’-flanking Vim fragments, the decrease in expression of pcV-321 and pcV-4961-162 during myogenesis suggests that down-regulation of CAT activity is due to a decrease in transcription initiation directed by these Vim fragments. We conclude that sequences which control the down-regulation of vimentin expression during myogenesis lie in the common region -321 to -160. Our result is in agreement with Pieper et al. (1987) who found the region -176 to + 101 important for hamster Vim downregulation during C2C12 myogenesis. Here, we have further separated this control region into two functional elements, i.e. a repressible enhancer and a constitutive promoter region. Although our studies suggest that Vim down-regulation during myogenesis is, at least in part, due to a decrease in transcription initiation, additional effects occurring post-initiation cannot be ruled out at this time. Such events might include a difference in elongation rate in myoblasts and myotubes.
of CAT enzyme to that present
(d) Significance
in
myoblasts.
oriented CCAAT box (Jones et al., 1985; Zehner et al., 1987). In addition, htk and pTK-CAT (htk nt-105 to + 56 fused to cat) have been shown to be equally active throughout differentiation of the myogenie line MM14 (Merrill et al., 1984; Jaynes et al., 1986). Therefore, as a control for GC box activity, we have compared the expression of our various constructs to pTK-CAT (Miksicek et al., 1986). Both pcV-160 (chicken Vim nt -160 to + 1 fused to cat) and pTK-CAT are expressed in myoblasts at low levels which may reflect the level of GC box promoter activity at this stage in differentiation. Moreover, this level does not change significantly upon differentiation to myotubes. In contrast, both pcV-321 (chicken vimentin nt -321 to + 1 fused to cut) and pcV-4961-162 (chicken Vim nt -496 to -162 fused to cat) are amply expressed in myoblasts and their expression declines 69% and 80 %, respectively, during differentiation to myotubes. That activity differences between plasmids are not due to transfection variability is suggested by constant pSV2neo RNA levels in all myotube samples (slot-blot not shown). Since transcription of the cut gene is directed
Previously, we have shown that a tissue-specific enhancer is located in the region -321 to -160 (Sax et al., 1988). Here we show that this region specititally represses activity of a heterologous gene during myogenesis in culture. We suggest that the downregulation of the Vim gene during myogenesis may be due to the decreased synthesis of a positive-acting transcription factor(s) which binds in this region. This implies a different mechanism than that recently proposed for the j?-actin gene (DePonti-Zilli et al., 1988). In the case of a tissue-specific expressed gene like vimentin, the decreased synthesis of a positive transcription factor would be a simple alternative for repressing gene activity
ACKNOWLEDGEMENTS
The authors wish to thank Dr. Bernard0 Nadal-Ginard for the L6E9 myogenic cell line. This research was supported by Public Health Service Grant AM33310 from the National Institutes of Health and by grants from the Muscular Dystrophy Association and the Jeffress Trust.
241 REFERENCES Bennett, G.S., Fellini, S.A., Toyama, Y. and Holtzer, H.: Redistribution of intermediate filament subunits during skeletal myogenesis and maturation in vitro. J. Cell Biol. 82 (1979) 577-584. Capetanaki, Y.G., Ngai, J. and Lazarides, E.: Characterization and regulation in the expression of a gene coding for the intermediate filament protein desmin. Proc. Natl. Acad. Sci. USA 81 (1984) 6909-6913. Chirgwin, J.M., Przybyla, A.E., MacDonald, R.J. and Rutter, W.J.: Isolation of biologically active ribonucleic acid from sources enriched in ribonuclease. Biochemistry 18 (1979) 5292-5299. Colbere-Garapin, F., Horodniceanu, F., Kourilsky, P. and Garapin, A.-C.: A new dominant hybrid selective marker for higher eukaryotic cells. J. Mol. Biol. 150 (1981) 1-14. Dente, L., Cesareni, G. and Cortese, R.: pEMBL: A new family of single stranded plasmids. Nucleic Acids Res. 11 (1983) 1645-1655. DePonti-Zilli, L., Seiler-Tuyns, A. and Paterson, B.M.: A 40-base-pair sequence in the 3’ end of the beta-actin gene regulates mRNA transcription during myogenesis. Proc. Natl. Acad. Sci. USA 85 (1988) 1389-1393. Dynan, W.S. and Tjian, R.: Isolation oftranscription factors that discriminate between different promoters recognized by RNA polymerase II. Cell 32 (1983) 669-680. Earle, W.R., Schilling, E.L., Stark,T.H., Straus, N.P., Brown, F. and Shelton, E.: Production of malignancy in vitro, IV. The mouse Bbroblast cultures and changes seen in the living cells. J. Natl. Cancer Inst. 4 (1943) 165-212. Feinstein, S.C., Ross, S.R. and Yamamoto, K.R.: Chromosomal position effects determine transcriptional potential of integrated mammary tumor virus DNA. J. Mol. Biol. 156 (1982) 549-566. Gard, D.L. and Lazarides, E.: The synthesis and distribution of desmin and vimentin during myogenesis in vitro. Cell 19 (1980) 263-275. Gorman, C.M., Moffat, L.F. and Howard, B.H.: Recombinant genomes which express chloramphenicol acetyltransferase in mammalian cells. Mol. Cell. Biol. 2 (1982) 1044-1051. Graham, F.L. and Van der Eb, A.J.: A new technique for the assay of infectivity of human adenovirus 5 DNA. Virology 52 (1973) 456-467. Granger, B.L. and Lazarides, E.: Desmin and vimentin co-exist at the periphery of the myofibril Z disc. Cell 18 (1979) 1053-1063. Herr, W. and Clarke, J.: The SV40 enhancer is composed of multiple functional elements that can compensate for one another. Cell 45 (1986) 461-470. Holtzer, H., Bennett, G.S., Tapscott, S.J., Croop, J.M. and Toyama, Y.: Intermediate-size filaments: changes in synthesis and distribution in cells of the myogenic and neurogenic lineages. Cold Spring Harbor Symp. Quant. Biol. 46 (1982) 3 17-329. Jaynes, J.B., Chamberlain, J.S., Buskin, J.N., Johnson, J.E. and Hauschka, S.D.: Transcriptional regulation of the muscle creatine kinase gene and regulated expression in transfected mouse myoblasts. Mol. Cell. Biol. 6 (1986) 2855-2864.
Jones, K.A., Yamamoto, K.R. and Tjian, R.: Two distinct transcription factors bind the HSV thymidine kinase promoter in vitro. Cell 42 (1985) 559-572. Lilienbaum, A., Legagneux, V., Portier, M.-M., Dellagi, K. and Paulin, D.: Vimentin gene: expression in human lymphocytes and in Burkitt’s lymphoma cells. EMBO J. 5 (1986) 2809-2814. Maniatis, T., Fritsch, E.F. and Sambrook, J.: Molecular Cloning. A Laboratory Manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, 1982, pp. 202-203. Melton, D.A., Krien, P.A., Rebagliati, M.R., Maniatis, T., Zinn, K. and Green, M.R.: Efficient in vitro synthesis of biologically active RNA and RNA hybridization probes from plasmids containing a bacteriophage Sp6 promoter. Nucleic Acids Res. 12 (1984) 7035-7056. Merrill, G.F., Hauschka, S.D. and McKnight, S.L.: Tk enzyme expression in differentiating muscle cells is regulated through an internal segment of the cellular rk gene. Mol. Cell. Biol. 4 (1984) 1777-1784. Minty, A., Blau, H. and Kedes, L.: Two-level regulation of cardiac actin gene transcription: muscle-specific modulating factors can accumulate before gene activation. Mol. Cell. Biol. 6 (1986) 2137-2148. Miksicek, R., Heber, A., S&mid, W., Danesch, U., Posseckert, G., Beato, M. and Shutz, G.: Glucocorticoid responsiveness of the transcriptional enhancer of Moloney murine sarcoma virus. Cell 46 (1986) 283-290. Nadal-Ginard, B.: Commitment, fusion, and biochemical differentiation of a myogenic cell line in the absence of DNA synthesis. Cell 15 (1978) 855-864. Paterson, B.M. and Bishop, J.O.: Changes in the mRNA population of chick myoblasts during myogenesis in vitro. Cell 12 (1977) 751-765. Paterson, B.M. and Roberts, B.: Structural gene identification utilizing eukaryotic cell-free translational systems. In Chirikjian, J.G. and Papas, T.S. (Eds.), Structural Analysis of Nucleic Acids, Vol. 2. Elsevier, North Holland, 1981, pp. 417-437. Pieper, F.R., Slobbe, R.L., Ramaekers, F.C.S., Cuypers, H.T. and Bloemendal, H.: Upstream regions ofthe hamster desmin and vimentin genes regulate expression during in vitro myogenesis. EMBO J. 6 (1987) 3611-3618. Quax, W., Egberts, W.V., Hendricks, W., Quax-Jeuken, Y. and Bloemendal, H.: The structure of the vimentin gene. Cell 35 (1983) 215-223. Rittling, S.R. and Baserga, R.: Functional analysis and growth factor regulation of the human vimentin promoter. Mol. Cell. Biol. 7 (1987) 3908-3915. Sax, CM., Farrell, F.X., Tobian, J.A. and Zehner, Z.E.: Multiple elements are required for expression of an intermediate filament gene. Nucleic Acids Res. 16 (1988) 8057-8076. Seiler-Tuyns, A., Eldridge, J. and Paterson, B.M.: Expression and regulation of chicken actin genes introduced into mouse myogenic and nonmyogenic cells. Proc. Natl. Acad. Sci. USA 8 1 (1984) 2980-2984. Southern, P.J. and Berg, P.: Transformation of mammalian cells to antibiotic resistance with a bacterial gene under control of the SV40 early region promoter. J. Mol. Appl. Genet. 1(1982) 327-341.
242
Thomas,
P.S.: Hybridization
fragments
transferred
ofdenatured
to nitrocellulose.
RNA and small DNA
fected
Proc. Natl. Acad. Sci.
115-122.
USA 77 (1980) 5201-5205. Tokuyasu,
K.T., Maher,
of vimentin vivo.
Zehner,
P.A. and
and desmin
Singer,
in developing
I. Immunofluorescence
study.
S.J.: Distributions chick
myotubes
in
J. Cell. Biol. (1980)
vimentin
P.A. and Singer, S.J.: Distributions
and desmin in developing
II. Immunoelectron
microscopic
chick myotubes study.
of
in vivo.
J. Cell Biol.
100
(1985) 1157-1166. Weintraub.
H., Cheng, P.F. and Conrad,
of trans-
topology.
gene. Nucleotide
and comparison
to the hamster
Cell 46 (1986)
B.M. and Sax, C.M.: sequence,
regulatory
gene. J. Biol. Chem.
262 (1987) 8112-8120. Z.E.
chicken mRNAs. Zehner,
and
Paterson,
vimentin
B.M.:
Characterization
gene: single copy gene producing
Proc. Natl. Acad.
Z.E. and Paterson,
during myogenesis: K.: Expression
on DNA
Z.E., Li, Y., Roe, B.A., Paterson,
elements, Zehner,
K.T., Maher,
depends
The chicken vimentin
1961-1972. Tokuyasu,
DNA
Sci. USA 80 (1983a) B.M.: Vimentin
two functional
of the multiple 911-915.
gene expression
transcripts
from a single
copy gene. Nucleic Acids Res. 11 (1983b) 8317-8332.