Collagen alpha 1(I) gene (COL1A1) is repressed by RFX family.

28 Pages·2005·0.75 MB·English

JBC Papers in Press. Published on March 23, 2005 as Manuscript M413191200
Collagen alpha 1(I) gene (COL1A1) is repressed by RFX
family
.
Sengupta, P.K.1,2, Xu, Y. 1, Wang, L. 1, Widom, R. L. 1,3 and Smith, B.D. 1,2
From the 1Dept. of Biochemistry and 3Arthritis Center’ Boston University School of
Medicine, Boston, MA 02118 and 2VA Boston Healthcare System, Boston, MA 02118
USA
Running title: COL1A1 repressed by RFX family
Address correspondence to: Barbara D. Smith, Ph.D. 1 Department of Biochemistry,
Boston University School of Medicine, 715 Albany St., Boston, MA 02118. Tel:
[phone] FAX: [phone] Email: [email protected]
Key words: collagen, DNA binding proteins, RFX, DNA methylation, transcription,
interferon-gamma, cancer

Abstract collagen expression. The DNA methylation
Collagen type I is composed of three inhibitor, 5-aza-2'-deoxycytidine (aza-dC)
polypeptide chains transcribed from two increases collagen gene expression with time in
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separate genes (COL1A1 and COL1A2) with human cancer cells. On the other hand, RFX5 o
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different promoters requiring coordinate interacts with both collagen type I genes with a lo
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regulation. Our recent publications, similar binding affinity and represses both de
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centering on COL1A2 regulation, promoters equally in transient transfections. Two fro
m
demonstrate that methylation in the first exon dominant negative forms of RFX5 activate both h
of COL1A2 at a regulatory factor for X box collagen genes coordinately. Finally, CIITA RNAi ttp
(RFX) site (at -1 to +20) occurs in human experiments indicate that CIITA induction is ://ww
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cancer cells and correlates with increased required for IFN-γ mediated repression of both .jb
RFX1 binding and decreased collagen collagen type I genes. c.o
transcription (Sengupta et al, JBC 274: Introduction: brg/
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36649-55, 1999; Sengupta, et al. Biochim g
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Biophys Acta 1443:75089, 1998; Sengupta et Collagen, a major extracellular matrix es
t o
al. Cancer Res 63: 1789-97, 2003). In normal component, plays a significant role influencing cell n
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cheislltso,c omRFpXat5ib ilcitoym pclelaxs s aloIIn g trwanitsha ctmivaatjoorr bI ehcaovlliaogr eann, d tmhea inmtaoinsti nga btiusnsduaen stt rumcetumreb e(r1 ).o fT ytphee pril 4, 2
(CIITA) is induced by IFN-γ to occupy this collagen family, is composed of two identical α1(I) 01
9
site and repress collagen transcription (Xu et polypeptide chains and a similar, but distinct
al. JBC 278:49134-44, 2003; Sengupta et al. polypeptide chain designated α2(I). Collagen
JBC 277: 24926-37, 2002; Xu et al. JBC expression is controlled in a developmental and
41319-41332, 2004). In this paper, we tissue-specific manner. In adult tissue, type I gene
demonstrate that COL1A1 has an RFX expression is largely inactivated, but stimulated after
consensus binding site surrounding the injury. The two type I collagen genes (COL1A1 and
transcription start site (-11 to +10) that COL1A2) are within separate chromosomes with
contains three methylation sites rather than different regulatory sequences in their promoters, yet
one in the COL1A2 gene RFX binding site. they need to be regulated coordinately to ensure
RFX1 interacts weakly with the proper function.
unmethylated COL1A1 site, and binds with Synthesis of collagen by cells in culture is
higher affinity to the methylated site. RFX1 down-regulated upon oncogenic transformation with
represses the unmethylated COL1A1 less viruses or with chemical carcinogens (2-5). We have
efficiently than COL1A2. COL1A1 promoter previously demonstrated (5) that collagen α2(I) is
activity is sensitive to DNA methylation and not expressed in a tumorigenic cell line, W8, after
the COL1A1 gene is methylated in human treatment of the parental liver epithelial-like cell
cancer cells with coordinately decreased line, K16, with the carcinogen 2-N-(acetoxyacetyl)-
1
amino- fluorine. Analysis of genomic DNA IFN-γ (17,18). In fact, blocking CIITA expression
isolated from W8 cells revealed that the by RNAi also blocks IFN-γ mediated repression
promoter-5’ region of the α2(I) gene, COL1A2, (16).
was methylated (6). Furthermore, reporter Since our published studies to date center on the
constructs containing the COL1A2 promoter COL1A2 gene, it was important to determine
region (218-bp), as well as the first exon (54-bp) whether the COL1A1 gene is regulated in a similar
of the COL1A2 gene, were inactivated by DNA manner. In this publication, we demonstrate that
methylation in transient transfection experiments the α1(I) gene, COL1A1, is also methylated in
and in vitro transcription assays (6,7). The human cancer cells and this methylation occurs, in
inhibition of reporter gene expression was most cases, coordinately with COL1A2 gene. In
attributable to CpG methylation within the first addition, the COL1A1 gene contains a methylation
exon surrounding the transcription start site of specific binding site for the RFX family at the
the COL1A2 gene. Finally, the collagen transcription start site farther upstream than in the
transcription start site (-1 to +20) contains a low COL1A2 gene. The RFX proteins bind to both
affinity-binding site for the regulatory factor for genes and act as repressors, but do so with different
X box (RFX) family (8,9). The binding affinity binding affinities and transcriptional activities.
of RFX1 is increased if the CpG site at +7 is
methylated or mutated to thymidine on the Methods D
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coding strand. Cell culture, treatments, transfection and w
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RFX1 is a member of the RFX family of luciferase assay: loa
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closely related proteins, RFX1-4 (10-13), that Human lung fibroblasts, IMR-90, (IMR, ed
can bind methylated DNA sequences with NJ), fibrosarcoma cells (HT1080, American Type from
higher affinity within a sequence-specific 14-bp Culture Collection, MD), hepatocarcinoma cells http
consensus sequence. Methylation-dependent (SNU387, SNU398, SNU449, PLC/PRF/5) and ://w
binding sites have been located for RFX at the breast epithelial carcinoma cells (Hs578T) were w
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beginning of human genes such as hypoxanthine maintained in Dulbecco’s modified Eagle’s medium .jb
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phosphoribosyl transferase (HPRT), α- (DMEM) (Invitrogen, Carlsbad, CA) supplemented .org
galactosidase A, human leukocyte antigens, and with 10% fetal bovine serum (FBS, Hyclone). Media b/
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the apoferritin H gene (14) suggesting a role for for Hs578T contained additional insulin (1 µg/ml). gu
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this protein family in DNA methylation induced A second breast carcinoma cell line (MCF-7) was st o
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gene repression. Most importantly, the RFX maintained in high glucose Dulbecco's modified A
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proteins repress COL1A2 gene expression in Eagle's medium. The colon cancer cell line ril 4
transient transfection as well as in vitro (HCT116) was maintained in McCoys medium. , 2
0
transcription assays (8). The IMR-90 cell line was used for 19
Our data suggest that RFX5 complex and transfection studies. Prior to transfection, cells were
CIITA play an important role in repression of plated in 6-well culture plates at density of 3X105
COL1A2 gene when fibroblasts cells are treated per well and incubated at 37 oC with 5% CO for 16-
2
with IFN-γ (15,16). RFX5, a member of the 24 hours. Transfections were performed using
RFX family binds to DNA in a sequence- Lipofectamine reagent (Invitrogen, Carlsbad, CA)
specific and methylation-insensitive manner (8). according to manufacturer’s protocol. Three hours
Previously, we demonstrated that the RFX5 after transfection, cells were transferred to DMEM
complex of transcription factors can also bind to with 10% FBS and harvested 24 hours later. Cells
COL1A2 at the transcription start site (8,9). were then lysed in 1X reporter lysis buffer
Other components of the RFX5 complex, RFXB (Promega, Madison, WI) and luciferase activities
and RFXAP, contribute to repression of were assayed using a luciferase reporter assay
COL1A2 gene expression (15) as well as class II system (Promega, Madison, WI). Luciferase activity
transactivator (CIITA) in fibroblasts (8). RFX5 was normalized for total protein and transfection
and CIITA play an important role inducing efficiency. Transfection efficiency was monitored by
major histocompatability class II (MHC II) gene fluorescent readings for co-transfected GFP
expression and are induced in fibroblasts by construct (0.1 µg). Experiments were routinely
2
performed in duplicate plates. The ratio of constructs were co-transfected with collagen reporter
experimental to control (reporter plasmid with constructs as described previously (8,15,16).
empty vector) X 100 was calculated for each Electrophoretic mobility gel shift assay
experiment with the control values set at 100. Nuclear extracts were prepared essentially
Values from three or more experiments were according to Dignam et al. (20) with some
averaged and presented in this paper. modifications. Extractions of protein from isolated
IFN-γ treatment: In several studies nuclei were performed at higher salt conditions than
IMR-90 cells were treated with IFN-γ. IMR-90 normal using 500 mM NaCl or 420 mM NaCl rather
fibroblasts were plated in p35 tissue culture than 350 mM NaCl in buffer C. All buffers
dishes at 4X105 cells/dish for mRNA studies and contained the protease inhibitors leupeptin (40
maintained in DMEM with 10% FBS for 16-24 µg/ml), aprotinin (200 µg/ml), pepstatin A (40
hours. Cells were pretreated in DMEM with µg/ml) and phenyl methyl sulfonyl fluoride (0.5
0.4% FBS for 16 hours prior to IFN-γ treatment mM) as well as the phosphatase inhibitor
100U/ml in 0.4% DMEM for 24 hours. orthovanadate (1 mM). Protein concentration of the
Aza-dC treatment: HT1080Cells were extracts was determined by the Bradford reagent
plated at 250,000 cells per p35 mm well plate using bovine serum albumin as a standard. Collagen
and treated with 1 µM of 5-aza-2’-deoxycytidine sequences or MDBP/RFX consensus sequences
(aza-dC) daily for 0, 1, 2 or 3 days before (Table 1) with Hind III overhangs were synthesized D
o
harvesting. (Oligo Etc.; CT and Integrated DNA Technology, w
n
Plasmids USA) as complementary strands, annealed to make loa
d
The COL1A1 promoter plasmids were double stranded oligonucleotides and radiolabeled ed
all cloned into a pGL2-luciferase vector from the using the α32P- dATP and the Klenow fragment to from
-804hCOL1-luc plasmid described previously fill in the Hind III overhang. For DNA mobility http
(19). All constructs have the exact same 3' end shift assay, the binding reaction was performed for ://w
as the -804hCOL1-Luc (+114). The -311COL1- 30 mins at room temperature in 20 µl of binding ww
luc was constructed using exonuclease III buffer containing 90,000-100,000 CPM/200 fmol of .jb
c
digestion. First, -804COL1-Luc was digested labeled probe, 1 µg of poly(dI-dC).poly(dI-dC) and .org
with KpnI and XhoI (both cut upstream of -804) nuclear extract containing 4.5-5.0 µg of protein. by/
g
to linearize the plasmid. Then the plasmid was Double stranded annealed complementary u
e
s
treated with exonuclease III followed by mung oligonucleotides for mpBr322 (Oligo Etc., CT and t o
n
bean nuclease to remove single stranded ends. Integrated DNA Technology, USA) were used as A
p
The resulting DNA was self-ligated to competitors. Separation of free radiolabeled DNA ril 4
recircularize -311COL1-Luc. The -112COL1- from DNA-protein complexes was carried out on a , 2
0
Luc construct was amplified by PCR using a 20 4-5% non-denaturing polyacrylamide gel with a 19
nt primer at -112 and a luciferase primer (GL2 standard Tris-borate electrophoresis buffer (TBE) at
primer from Promega) downstream. The PCR 300 V in the cold (4oC). Autoradiography was
product was cloned into pCR2.1 by TA cloning. performed by overnight exposure to Kodak Biomax
The fragment was then excised with KpnI and film (Eastman Kodak Company, NY USA). The
HindIII and cloned into pGL2basic. These intensities of the bands were quantified using Instant
constructs were verified by DNA sequencing. Imager (Packard Instrument Company, CT, USA).
In some experiments, plasmids were Methylation status
methylated by Sss1 methylase using Bisulfite modification of genomic DNA:
manufactures recommendations (New England DNA, isolated from cells, was modified by
Biolabs, Inc, MA) as described previously (7). bisulfite treatment to analyze methylation of
Unmethylated plasmids were incubated in the collagen at the +7 CpG site as previously described
reaction mixture with S-adenosyl-methionine (8,21). The bisulfite modification causes
and purified in the same way as the methylated unmethylated cytosines (Cs) to be converted to
plasmids. In several experiments, full-length uracil. Methylated cytosines are resistant to
expression constructs for RFX1, Flag-RFX5, deamination. PCR was used to amplify the modified
Flag-RFXB/ANK, Flag-CIITA and His-RFXAP DNA replacing uracil residues with thymine.
3
Primers were designed using a converted pcDNA3 vectors(23) and cloned into pLenti6/V5-D-
sequence (Cs to Ts) in a region that did not TOPO vector using a directional TOPO cloning kit
contain any possible CpG methylation sites. (Invitrogen). Constructs were sequenced to be sure
Separate primers were designed to amplify the there were no mutations, deletions, or extra bases.
coding strand (-65 to +151) or the template PCR Shagging-based RNA interference (RNAi)
strand (-71 to +144). (see Table 2). The PCR Three different reverse primers were
product was separated on 2% low melting designed by RNAi oligo retriever program on Cold
agarose gel and purified using the Qiaquick Spring harbor web site
Qiagen gel extraction kit (Qiagen, Valencia, (http://katahdin.cshl.org:9331/RNAi/html/rnai.html)
CA). and cloned into pLenti6-V5-TOPO vectors as
Methylation Sensitive-Single Nucleotide previously described (16). A pGEM1-U6 vector was
Primer Extension (MS-SNuPE) assay used as the template to clone U6 promoter driving
For determining methylation at shRNA. PCR reactions were performed by 95 oC
individual sites, we used methylation sensitive 3min; 30 cycles of 95 oC for 30 sec, 55 oC for 30 sec,
single nucleotide primer extension (MS-SNuPE) and 72 oC for 1min followed by one cycle of 72 oC
(22). This method established whether the for 10 min. PCR products were separated on 1%
nucleotide at +7 remained a C (methylated) Agarose gel, sliced from gel, purified by a Qiagen
residue or has been converted to T gel extraction kit and ligated into pLenti6-V5-TOPO
D
o
(unmethylated) as described previously (8,21). vector using a Viralpower directional cloning kit. w
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The MS-SNuPE primers are in Table I and their Ligation products were transformed into TOP10 lo
a
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relationship to the collagen gene is diagrammed competent cells and positive clones were screened ed
in Fig. 1A. The reaction mixture contained gel by restriction digestion and direct sequencing. fro
m
purified PCR fragments, primers, 1X Taq Generation of lentiviral stocks and transduction h
ttp
polymerase buffer, and 1 µCi radiolabeled [32P] Viral packaging cells (293FT, Invitrogen, ://w
dCTP or dTTP to identify methylated or Carlsbad, CA) were maintained in DMEM w
w
unmethylated residues respectively for coding supplemented with 10% FBS, 1% penicillin- .jb
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strand, and [32P] dGTP or dATP to identify streptomycin and 500 µg/ml geneticin (Invitrogen, .o
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methylated or unmethylated residues Carlsbad, CA). The lentiviral stock was prepared by b/
y
respectively for template strand (Promega, transfecting our constructs (RFX5, RFX5∆1, gu
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Wisconsin). After denaturation at 95oC for 3 RFX5∆5) or three shRNA clones as well as a LacZ st o
minutes, the primers were annealed at 40oC for 2 construct expressing β-galactosidase plus an empty n A
m(cionduitnegs (sttermanpdl)a,t e asntdra nedx)t eonrd 5e0do Ca tf o7r2 2oC m ifnourt e1s TinOtoP O29 v3eFcTto cre alllso nugs iwngit hL iVpiorfaelcPtoawmeinr ep a2c0k0a0g irnega gmeinxt pril 4, 2
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minute with Taq polymerase (1 unit) (Promega, according to manufacturer’s protocol (Invitrogen, 19
Wisconsin). The reaction was stopped by Carlsbad, CA). After 48-72 hours, Viruses
addition of 10 µl of stop buffer. The reaction containing supernatants were harvested, centrifuged
products were heated at 95oC for 2 minutes and stored in 1 ml aliquots. Titers of lentiviral stocks
before loading on to a 15% acrylamide- 7M urea were performed according to manufacturer’s
gel. The radioactivity of the specific bands was protocol using HT1080 cells in order to determine
quantified by a Packard Instant Imager (Perkin- the number of virus particles. IMR-90 cells were
Elmer) and pictured by autoradiography. The infected with virus at the MOI of 1. Cells were
percent of methylation was calculated by treated with or without 100U/ml IFN-γ for additional
dividing the amount of radiolabeled dCTP or 24 hours before harvesting. Proteins were extracted
dGTP incorporated into the primer by the total using RIPA buffer (1xPBS, 0.1% SDS, 1% NP-40,
radioactivity multiplied by 100. 0.5% sodium deoxycholate) and analyzed by SDS-
Construction of Dominant negative clones in PAGE gel followed by Western blot. RNAs were
lentivirus vectors extracted and analyzed as described in the following
The wild type RFX5 and the two section.
dominant negative constructs, RFX5∆1 and RNA isolation and real-time PCR:
RFX5∆5, were amplified from the original
4
Cells were harvested and RNA was As stated in the introduction, most of our
extracted using an RNeasy RNA isolation kit previous studies indicate that the RFX family
(Qiagen, Valencia, CA) according to represses the collagen α2(I) gene, COL1A2. The
manufacturer’s protocol. Reverse transcriptase collagen α1(I) gene, COL1A1 is homologous to the
reactions were performed using a SuperScript α2(I) gene mainly in the first 20 bases and
First-strand Synthesis System (Invitrogen, surrounding the TATA sequence (Fig 1A).
Carlsbad, CA) according to manufacturer’s Therefore, we hypothesized that RFX1 would bind
protocol. Real-time PCR reactions were to the first 20 bases of the homologous gene. To our
performed on ABI Prism 7700 sequence surprise, RFX1 did not form a high affinity complex
detection PCR machine (Applied Biosystems,
on this α1(I) sequence with or without methylation
Foster City, CA) according to manufacturer’s
(Fig. 1B) and the first 20 bases of α1(I) did not
protocol. The oligonucleotide primers (forward
compete with the RFX1 complex on the α2(I)
and reverse) and Taqman probes are described in
sequence at 50 or 20 fold molar excess (data not
Table 3.
shown).
Westerns:
However, there is a site upstream in the α1(I)
For detection of expressed proteins,
gene, between -11 and +6 (Figure 1A, underlined),
fibroblasts were extracted using RIPA buffer
that is a closer match to the RFX consensus
{1x phosphate-buffered saline (PBS), 1% NP-
sequence. Therefore, proteins binding to oligos at D
o
40, 0.5% sodium deoxycholate, 0.1% SDS} with w
both α1(Ι) sites were compared to proteins binding n
freshly added PMSF (100 µg /ml RIPA) and lo
a
to the α2(I)-binding site (Fig 1B). There was no d
protease inhibitor tablet (Roche, Mannheim, ed
Germany). The protein concentration was complex formation on the α1(I) gene at the fro
homologous site (-1 to 20). There was complex m
determined by the Bradford protein assay (Bio- h
formation on the upstream site between -11 and +10 ttp
Rad, Hercules, CA). Proteins in extracts were that contains three methylation sites (stars). In both ://w
separated by 10% polyacrylamide gel w
cases, there are two closely spaced complexes w
electrophoresis with pre-stained markers (Bio- .jb
Rad, Hercules, CA) for estimating molecular migrating in these gels suggesting that other family c.o
weight and efficiency of transfer to blots. members of the RFX family or other proteins are brg/
Proteins were transferred to nitrocellulose binding to these sites. y g
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Further studies indicate that RFX5 also interacts e
membranes (Bio-Rad, Hercules, CA) in a Mini- with the alpha1(I) transcription start site when RFX1 st on
Trans-Blot Cell (Bio-Rad, Hercules, CA). The A
is removed by methylated RFX1 consensus p
membranes were blocked with 5% milk powder sequence (Fig 1C). It seems that RFX1 has more ril 4
in Tris buffered saline (TBST) (0.05% Tween affinity for alpha 2(I) than alpha1(I) whereas RFX5 , 20
20, 150 mM NaCl, 100 mM Tris-HCl, pH 7.4) 1
9
interacts slightly better with alpha1(I) than alpha 2(I)
buffer at 4oC overnight and incubated for three
(Fig 1C).
hours with monoclonal anti-V5 (1:1000) (Sigma,
Methylation of COL1A1 inhibits promoter
St. Louis, MO), monoclonal anti-β-actin
activity
(1:1,000) (Sigma), or monoclonal anti-CIITA
The RFX consensus sequence on the COL1A1
(1:100) (7-1H, SantaCruz) as indicated. After 3
promoter contains several potential methylation
washes with TBST, the membranes were
sites. Since we previously found that COL1A2
incubated with appropriate secondary antibodies
promoter activity is sensitive to methylation
anti-mouse IgG (1:1000) (Amersham)
(7,24,25), experiments were performed to determine
conjugated to hourseradish peroxidase, for
whether COL1A1 transcription is also methylation
another 1hour at room temperature. Then protein
sensitive. Three human COL1A1 promoter
blots were visualized using ECL reagent (NEN,
constructs of different lengths were methylated using
Boston, MA) on a Kodak image station (Pierce).
Sss 1 methylase as previously described (7,25) and

transfected into IMR-90 cells. Promoter activities of
Results
all three COL1A1 constructs were dramatically
RFX proteins interacts with COL1A1 site (-11
down-regulated when they were methylated (Figure
to +6) at the transcription start site with
2). Therefore, it is clear that the human COL1A1
different affinity than COL1A2.
5
promoter is methylation sensitive, similar to the different days with the demethylating agent, aza-dC.
COL1A2 (7,25). The steady state levels of COL1A1 increased in a
COL1A1 gene is methylated in human cancer time dependent manner with aza-dC treatment
cells. similar to COL1A2 (Fig. 3C) (21). The data shown
Since COL1A2 gene is methylated in human is a representative experiment performed three times
cancer cells, the methylation status of the in duplicate. Therefore, the expression of alpha 1(I)
COL1A1 gene was evaluated. Genomic DNA mRNA is dependent, in part, on the methylation of
extracted from six cancer cell lines and one the gene.
normal cell line was modified by bisulfite RFX1 represses the COL1A1 promoter activity
technique and analyzed using single nucleotide less than RFX5 in transient transfection.
primer extension (SNuPE) assays as we have Co-transfection studies have been performed
reported previously (8). Bisulfite treatment using alpha 1(I) promoter (-311 to +114) luciferase
converts cytosines to uracil in single stranded constructs. RFX1 is a weaker repressor of the alpha
DNA under conditions that do not alter 5- 1(I) promoter (Fig 4A) than the alpha2(I) (8). On
methyl-cytosine. After bisulfite modification, the other hand, RFX5 represses alpha1(I) (Fig 4B)
the collagen promoter and first exon regions better than alpha2(I) (8). This may be the result of
were amplified by PCR using primers for the different binding affinities seen in gel shift analysis
coding strand (Table I). In order to analyze (Fig. 1). Co-transfection with the other RFX5
D
o
methylation within the collagen RFX binding complex proteins (RFXB and RFXAP) repressed the w
n
site, specific primers (Fig. 1A and Table I) were alpha1(I) promoter (Fig 4C) similar to the alpha 2(I) lo
a
d
annealed to a sequence at the +6 CpG site promoter (8). Since these proteins are induced ed
followed by single nucleotide primer extension during IFN-γ treatment (15), we have confirmed fro
m
with radiolabeled nucleotides. The methylation results by others (27) that IFN-γ represses alpha1(I) h
ttp
status of the COL1A1 gene was compared in this promoter activity as well as the endogenous ://w
experiment to the methylation status on the COL1A1 steady state mRNA levels (Figure 4D). w
w
coding strand of +7 CpG site in the COL1A2 Dominant negative RFX5 mutants increase both .jb
c
gene. There was coordinate methylation in all collagen gene expression and decreases MHC II .org
but one cell line (PLC/PRF/5) where, like W8 expression. b/
y
cells, the COL1A1 gene was not methylated Since two mutated forms of RFX5 acted as gu
e
whereas the COL1A2 was methylated (Figure dominant negative proteins activating COL1A2 st o
3A). promoter activity and blocking IFN-γ repression in n A
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Next, steady state mRNA levels were gene expression. Since human lung fibroblasts have 19
measured by real time RT-PCR to determine if low transfection efficiency, an efficient gene transfer
collagen expression is inversely correlated to system was needed to transfect cells that were not
collagen gene methylation. All the samples rapidly dividing. We decided to use the lentiviral
were normalized to 18S rRNA and compared to expression system (Invitrogen) for several reasons.
the lung fibroblast cell mRNA as the standard in First, lentiviral vectors can transduce slow or even
each real-time experiment using the 2(-Delta non-dividing cells (28-31). Second, transgenes
Delta C(T)) method (26) (Fig. 3B). At least introduced with the lentiviral backbone are more
three samples in duplicate were analyzed for resistant to transcriptional silencing common to
each cell line (bar graph, Fig. 3B). In general, other retrovirus transduced cells (32). Third, this
COL1A1 expression decreased with increasing vector can be used in animal studies since it can be
methylation surrounding the collagen start site expressed during development (30,33). Finally,
and correlated with COL1A2. lentiviral-based gene therapy has begun using a third
Collagen alpha 1(I) expression is reactivated generation of vectors with elimination of certain
by demethylation using aza-dC genes (34-37) and the use of multiple constructs to
To investigate whether the expression of package a non-replication competent lentivirus (33).
mRNA for collagen is inactivated by To analyze transduction efficiency, whole cell
methylation, the HT1080 cells were treated for (IMR-90) extracts were prepared and Western
6
analyses were performed after transduction to and RFX5∆5 dramatically inhibited the IFN-γ
examine the expression of the viral clones using induced HLA-DRA expression (Fig. 5D, one-sample
an anti-V5 antibody. The anti-V5 antibody two tailed t-test, p<0.01 for both RFX5∆1 and
recognizes the epitope present on the expressed RFX5∆5) strongly arguing that RFX5 is an
proteins. All four constructs were expressed as important factor for both collagen and MHC II
proteins of correct sizes and expression levels of transcription.
all four constructs were comparable (Figure 5A). Since Lentiviral infection can induce an
Next, steady state levels of COL1A1 mRNA
IFN-γ response in some cells and since CIITA
were measured after viral infections in IMR-90
expression is markedly induced by IFN-γ, viral
cells. Infections of the cells with either the
expression alone could induce CIITA expression.
empty vector or LacZ construct did not have any
Therefore, CIITA expression in cells after
effect on COL1A1 mRNA levels with or without
transduction was analyzed as a negative control. As
IFN-γ treatment (Fig. 5B). Infection of the cells
expected, IFN-γ markedly up-regulated CIITA
with RFX5 construct inhibited COL1A1 mRNA
expression. Most importantly, none of the infections
expression by about 15% on average without
had any effect on expression of CIITA mRNA,
IFN-γ treatment (Fig. 5B) (one-sample two
confirming that dominant effects of RFX5 mutants
tailed t-test, p<0.01). In the presence of IFN-γ, were gene-specific rather than artifacts of lentiviral
RFX5 infection down-regulated COL1A1 infection (Fig. 5E). D
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mRNA level by 52% (one-sample two tailed t- CIITA is a repressor of COL1A1 gene expression. wn
lo
test, p<0.05) indicating that RFX5 was a CIITA is recruited to the COL1A2 gene, in ad
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nvievgoa.t iv eB ortehg uRlaFtXor5 ∆o1f acnodl laRgFenX 5e∆x5p reinsfseiocnti onins pIFaNrt -tγh rionudguhce Rd FrXep5r easnsdio ins oanf CimOpLo1rAta2n t trmanedsciaritpotri oonf d from
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activated COL1A1 mRNA expression with or (16). Therefore, several experiments were ttp
without IFN-γ treatment (Fig. 5B, without IFN-γ conducted to determine if COL1A1 was also ://w
w
one-sample two tailed T-test, p<0.1 for RFX5∆1 sensitive to CIITA mediated repression. First, co- w
and p<0.05 for RFX5∆5; with IFN-γ one-way transfection experiments indicated that CIITA .jbc
.o
ANOVA, p<0.05 for both RFX5∆1 and repressed COL1A1 promoter activity in a dose rg
RFX5∆5), confirming that these two mutants responsive manner (Fig 6A). To determine if CIITA by g/
had dominant negative effects on endogenous increases repression beyond RFX5, CIITA was co- ue
s
COL1A1 gene expression. However, in the transfected alone and with the RFX5 complex with t on
slightly additive effect (Fig.6B). Deletion mutations A
presence of IFN-γ, the repression of mRNA p
steady state levels is only partially reversed and in CIITA had the same effects on both collagen ril 4
promoters (data not shown) suggesting a role for , 2
do not reach control levels, possibly because 0
1
CIITA in repression of both collagen genes. 9
RFX5 complex protein levels increase with IFN-
The next question is whether CIITA is an
γ treatment (15). Steady state COL1A2 mRNA
important COL1A1 repressor induced and activated
levels were also activated by RFX5∆1 and
in fibroblasts during IFN-γ treatment. In our earlier
RFX5∆5 with or without IFN-γ treatment (Fig.
publication (16) we used RNAi to CIITA in
5C). These data suggested that both RFX5∆1
lentiviral vectors to knock-down the IFN-γ
and RFX5∆5 acted as dominant negative factors
stimulated expression of CIITA in human lung
coordinately on endogenous collagen type I
fibroblasts. Briefly, short hairpin RNA (shRNA)
transcription.
sequences against different regions of CIITA were
Since RFX5 has been reported to be an
cloned into a lentiviral vector. Three viral stocks
effective activator of MHC II expression (38),
were generated using lentiviral vectors containing
similar experiments were performed to examine
different CIITA shRNA sequences to infect human
the effect of RFX5 mutants on the endogenous
lung fibroblasts. Cells were transduced and treated
MHC II expression in human lung fibroblast
48 hours after infection with IFN-γ for an additional
cells. RFX5 tended to enhance the HLA-DRA
24 hours before harvesting. Two clones, named
mRNA expression by IFN-γ (Fig. 5D, one-
pLenti6-CSH4 and pLenti6-CSH6 respectively, were
sample two tailed t-test, p<0.1). Both RFX5∆1
able to inhibit IFN-γ-induced expression of CIITA at
7
both mRNA and protein levels (16). Expression gene is methylated at the collagen transcription start
of certain MHC-II molecule, HLA-DRA, was site within a RFX consensus binding site (-11 to
also examined and CIITA shRNA could +10). The RFX binding region in COL1A1 gene
specifically block the stimulation of HLA-DRA contains the transcription start site and 3 rather than
expression by IFN-γ. Moreover, the CIITA 1 methylation sites. COL1A1 construct was
RNAi were capable of alleviating the IFN-γ- extremely sensitive to methylation. Others have also
mediated down-regulation of COL1A2 mature reported that methylation of the COL1A1 promoter
mRNA suggesting that CIITA is required for constructs is accompanied by down-regulation of
IFN-γ-mediated COL1A2 transcription promoter activity (39-42). The mechanism for
repression. Here we demonstrate that CIITA is promoter repression was described as indirect (41),
possibly through chromatin (42). Indeed, recent
also required for IFN-γ-mediated COL1A1
evidence demonstrates that chromatin changes and
repression (Figure 7A). The relative mRNA
DNA methylation are linked and are important in
levels of collagen decreased when CIITA
cancer (43,44) (45). The linkage occurs in part since
protein levels increased as demonstrated by the
certain methylation binding factors (46) and/or
linear regression plot of four experiments
transcription factors such as Rb (47), recruit co-
performed in duplicate (Figure 7B). This data
repressor complexes that contain histone modifiying
confirms that CIITA is a key regulator of
enzymes that may induce a more compact chromatin
collagen type I expression. D
o
In summary, COL1A1 has an RFX configuration (48,49). Our preliminary data suggest w
n
that RFX1 interacts with histone deacetylases and lo
consensus binding site in a similar region to the a
d
COL1A2 gene that contains three methylation DNA methyltranserases that may alter the histone ed
sites rather than one. The COL1A1 gene is acetylation and methylate DNA surrounding the from
aberrantly methylated in human cancer cells and collagen transcription start site when RFX1 interacts http
the transcription of COL1A1 is down-regulated with DNA (Sengupta, unpublished data). ://w
in cancer cells, a process that can be reversed by COL1A1 methylation has been noted in w
w
Aza-dC. RFX1 represses COL1A1 less aging periodontal fibroblasts (40), in MOV virus .jb
c
efficiently than COL1A2. On the other hand, integration (50-52) and during development (53), but .org
RFX5 complex and CIITA repress both type I it has not been reported previously in human cancer b/
y
cells. Since COL1A2 RFX1 site is methylated in g
gene in response to IFN-γ. u
e
human cancer cells, the same cells were examined st o
Discussion: for methylation status at the +6 CpG site in n A
p
COL1A2P revtrioanussclyri,p twioen desmtaornt strasitteed thhaats thae cCeOllLs 1tAha1t. a lsToh ec oCntOaiLn1eAd1 C gOeLn1eA w2 ams emtheytlhaytiloante. d Ains ril 4, 2
0
expected, the COL1A1 expression was inversely 1
methylation responsive binding site for RFX 9
proportional to the DNA methylation status. In
family mostly within the first exon (-1 to +20)
addition, COL1A1 expression was increased with
(7,9). The RFX1 protein represses collagen
time in the presence of aza-dC, an agent that
transcription and binds with higher affinity when
decreases overall DNA methylation similar to other
the C at +7 is methylated on the coding strand or
studies with transfected constructs (42). Thus,
mutated from C to T (7-9). In addition, the +7
COL1A1 joins the multiple examples of genes that
site as well as several other CpG sites in the
are aberrantly methylated in aberrant cancers, such
surrounding area become methylated in cancer
as cell cycle-related (RB1, p16INK4A), DNA repair
cell lines and in tumors(21). The COL1A1 gene
(hMLH1, MGMT), adhesion molecule (E cadherin),
has limited homology to the COL1A2 gene with
and extracellular matrix (thrombospondin, TIMP3)
a crucial base missing within the RFX binding
genes {for review see (44)}. Recent investigation of
site placing the CpG site at +6. Therefore, since
the hypermethylated regions have focused on so
these two genes need to be coordinately
called "CpG islands" that are short regions (0.5-4
expressed for normal collagen production, it was
kb) with high numbers (>100) of methylated CpG
important to determine if the COL1A1 gene also
sites that are normally not methylated. These
contains a functional RFX binding site. In the
regions are usually within promoters of genes close
present study, we demonstrate that the COL1A1
to the start site. In the case of both collagen type I
8
genes the major methylation sites within RFX1 treatment and both RFX5 mutants repress this
site are located near the transcription start site. activation. This data suggests that the RFX5 DNA
The COL1A1 region is in a limited CpG like binding domain and the CIITA interaction domain is
island with more CpG sites than COL1A2. important for IFN-γ mediated coordinate repression
Since the COL1A1 promoter/first exon is highly of type I collagen.
sensitive to DNA methylation and RFX1, a CIITA is considered as a master regulator of
repressor for this gene, binds with higher affinity MHC-II gene expression (55,56). However, this
to the methylated sequence, RFX1 may be protein also enhances the transcription of the
responsible in part for repression of COL1A1 on invariant chains coordinately regulated with classical
methylated sequences. MHC-II, suggesting that it could be an important
Our previous data demonstrate that protein for antigen presentation (57,58). Recently,
RFX5 also interacts with the COL1A2 start site, it has been established using microarray analysis that
as judged by gel shift migration patterns and multiple genes are both activated and suppressed by
blocking of binding by RFX5 and RFXB CIITA (59). COL1A2 is one of the genes repressed
antibodies (8,9). This was the first by CIITA (8,60) along with IL-4 (61,62) cathepsin E
demonstration that RFX5 complex can form on (63) and Fas ligand (64). This paper demonstrates
DNA other than on the X-box site in the MHC-II that CIITA coordinately represses transcription of
or MHC-I promoters. RFX5 complex interacts both type I collagen genes. D
o
with the COL1A1 gene with similar affinity to We and others have demonstrated that w
n
COL1A2 on gel shift analysis. RFX5 complex CIITA is dramatically induced in human lung lo
a
d
assembly causes repression of COL1A2 and fibroblasts by IFN-γ as judged by mRNA and ed
activation of MHC-II (23,54). Using mutational protein levels (16,65-67). When CIITA is co- fro
m
studies to examine protein-protein interactions transfected with either collagen type I promoter, h
ttp
and DNA-protein interactions, it was promoter activity is greatly reduced when RFX5 ://w
demonstrated that all three components are complex is active (16). Most important, when w
w
critical for COL1A2 (15). Most likely, all three shRNA is used to block the CIITA expression, IFN-γ .jb
c
components are necessary for COL1A1 mediated repression of collagen type I is .org
repression as well. coordinately attenuated. b/
y
IFN-γ treatment of human lung In conclusion, both collagen type I genes are gu
e
fibroblasts increases the expression of RFX5 coordinately regulated by RFX family members st o
n
complex proteins, increases nuclear location, through a binding site close to the transcription start A
p
and enhances complex formation of RFX5 (15). site (model figure 8). Both collagen genes are ril 4
Furthermore, mutants of RFX5 containing either methylated in several human cancer cells and the , 2
0
deletions of the DNA binding domain (RFX5∆1) degree of methylation is inversely correlated with 19
or the proline rich CIITA interaction domain collagen type I expression. RFX1-RFX3 may be
(RFX5∆5) can compete with wild-type RFX5 involved in this process since they tend to bind to
and reverse the COL1A2 promoter repression methylated genes with higher affinities,
(15). When these same cDNAs are cloned into underscoring a potential role for RFX1 in collagen
lentivirus to increase the efficiency of gene down-regulation during carcinogenesis. On the
expression in human lung fibroblasts, the other hand, RFX5 and CIITA may be important
endogenous levels of both collagen type I genes proteins in IFN-γ mediated repression of both
increase by approximately 20 -30 percent with collagen type I genes. In the human lung fibroblasts,
or without IFN-γ. This is a small, but significant increased levels of RFX5 and CIITA also activate
increase in endogenous steady state mRNA MHC II. Therefore, RFX5/CIITA proteins may be
levels. Strikingly, the human lung fibroblasts essential for initiating inflammatory response and
express MHC II after 24 hours of IFN-γ delaying tissue repair.

9
References

1. Hirtenlehne, K., Pec, M., Kubista, E., and Singer, C. F. (2002) Eur Cytokine Netw 13, 234-240
2. Adams, S. L., Sobel, M. E., Howard, B. H., Olden, K., Yamada, K. M., de Crombrugghe, B., and
Pastan, I. (1977) Proc Natl Acad Sci U S A 74, 3399-3403
3. Rowe, D. W., Moen, R. C., Davidson, J. M., Byers, P. H., Bornstein, P., and Palmiter, R. D.
(1978) Biochemistry 17, 1581-1590
4. Sandmeyer, S., Smith, R., Kiehn, D., and Bornstein, P. (1981) Cancer Res 41, 830-838
5. Smith, B. D., and Niles, R. (1980) Biochemistry 19, 1820-1825
6. Smith, B. D., and Marsilio, E. (1988) Biochem J 253, 269-273
7. Sengupta, P. K., and Smith, B. D. (1998) Biochim Biophys Acta 1443, 75-89
8. Sengupta, P. K., Fargo, J., and Smith, B. D. (2002) J Biol Chem 277, 24926-24937.
9. Sengupta, P. K., Erhlich, M., and Smith, B. D. (1999) J Biol Chem 274, 36649-36655.
10. Emery, P., Durand, B., Mach, B., and Reith, W. (1996) Nucleic Acids Res 24, 803-807
11. Reith, W., Ucla, C., Barras, E., Gaud, A., Durand, B., Herrero-Sanchez, C., Kobr, M., and Mach,
B. (1994) Mol Cell Biol 14, 1230-1244
12. Huang, L. H., Wang, R., Gama-Sosa, M. A., Shenoy, S., and Ehrlich, M. (1984) Nature 308, 293-
295
D
o
13. Zhang, X. Y., Jabrane-Ferrat, N., Asiedu, C. K., Samac, S., Peterlin, B. M., and Ehrlich, M. w
n
(1993) Mol Cell Biol 13, 6810-6818 lo
a
d
14. Zhang, X. Y., Asiedu, C. K., Supakar, P. C., Khan, R., Ehrlich, K. C., and Ehrlich, M. (1990) ed
Nucleic Acids Res 18, 6253-6260 fro
m
15. Xu, Y., Wang, L., Buttice, G., Sengupta, P. K., and Smith, B. D. (2003) J Biol Chem 278, 49134- h
ttp
49144 ://w
16. Xu, Y., Wang, L., Buttice, G., Sengupta, P. K., and Smith, B. D. (2004) J Biol Chem 279, 41319- w
w
41332 .jb
c
17. Villard, J., Masternak, K., Lisowska-Grospierre, B., Fischer, A., and Reith, W. (2001) Medicine .o
rg
(Baltimore) 80, 405-418. b/
y
18. Reith, W., and Mach, B. (2001) Annu Rev Immunol 19, 331-373 g
u
e
19. Widom, R. L., Lee, J. Y., Joseph, C., Gordon-Froome, I., and Korn, J. H. (2001) Matrix Biol 20, st o
451-462 n A
p
20. D58ig2n-5a9m8, J. D., Martin, P. L., Shastry, B. S., and Roeder, R. G. (1983) Methods Enzymol 101, ril 4, 2
0
21. Sengupta, S., Smith, E. M., Kim, K., Murnane, M. J., and Smith, B. D. (2003) Cancer Res 63, 1
9
1789-1797
22. Gonzalgo, M. L., and Jones, P. A. (1997) Nucleic Acids Res 25, 2529-2531.
23. DeSandro, A. M., Nagarajan, U. M., and Boss, J. M. (2000) Mol Cell Biol 20, 6587-6599.
24. Smith, B. D. (1990) Annals N.Y. Acad. Sci 580, 459-461
25. Guenette, D. K., Ritzenthaler, J. D., Foley, J., Jackson, J. D., and Smith, B. D. (1992) Biochem J
283, 699-703
26. Livak, K. J., and Schmittgen, T. D. (2001) Methods 25, 402-408.
27. Yuan, W., Yufit, T., Li, L., Mori, Y., Chen, S. J., and Varga, J. (1999) J Cell Physiol 179, 97-108
28. Naldini, L., Blomer, U., Gallay, P., Ory, D., Mulligan, R., Gage, F. H., Verma, I. M., and Trono,
D. (1996) Science 272, 263-267
29. Chen, M., Li, W., Fan, J., Kasahara, N., and Woodley, D. (2003) Clin Exp Dermatol 28, 193-199
30. Ravot, E., Comolli, G., Lori, F., and Lisziewicz, J. (2002) J Gene Med 4, 161-169
31. Dishart, K. L., Denby, L., George, S. J., Nicklin, S. A., Yendluri, S., Tuerk, M. J., Kelley, M. P.,
Donahue, B. A., Newby, A. C., Harding, T., and Baker, A. H. (2003) J Mol Cell Cardiol 35, 739-
748
32. Gerolami, R., Uch, R., Jordier, F., Chapel, S., Bagnis, C., Brechot, C., and Mannoni, P. (2000)
Cancer Gene Ther 7, 1286-1292
10