Regulation of mouse Scgb3a1 gene expression by NF-Y and association of CpG methylation with its tissue-specific expression
© Tomita and Kimura; licensee BioMed Central Ltd. 2008
Received: 23 July 2007
Accepted: 14 January 2008
Published: 14 January 2008
Secretoglobin (SCGB) 3A1 is a secretory protein of small molecular weight with tumor suppressor function. It is highly expressed in lung and trachea in both human and mouse, with additional tissues expressing the protein that differ depending on the species. However, little is known about the function and transcriptional regulation of this gene in normal mouse tissues.
By reporter gene transfection and gel mobility shift analyses, we demonstrated that expression of the mouse Scgb3a1 gene is regulated by a PU-box binding protein and a ubiquitous transcription factor NF-Y that respectively binds to the PU-boxes located at -99 to -105 bp and -158 to -164 bp, and the "CCAAT" binding sites located at -425 to -429 bp and -498 to -502 bp from the transcription start site of the gene. However, the effect of PU-box binding protein on transcriptional activation is minimal as compared to NF-Y, suggesting that NF-Y is a more critical transcription factor for mouse Scgb3a1 gene transcription. Despite that NF-Y is a ubiquitous factor, Scgb3a1 is highly expressed only in mouse lung and mtCC cells that are derived from SV40 transformed mouse Clara cells, but not in ten other mouse tissues/cells examined. Gene methylation analysis revealed that within 600 bp of the Scgb3a1 gene promoter region, there are nine CpG methylation sites present, of which two CpGs closest to the transcription start site of the gene are unmethylated in the tissues/cells expressing SCGB3A1.
A ubiquitous transcription factor NF-Y binds to and activates expression of the mouse Scgb3a1 gene and tissue-specific expression of the gene is associated with CpG methylation of the promoter.
Secretoglobin (SCGB) 3A1, also called high in normal-1 (HIN-1)  or uteroglobin-related protein 2 (UGRP2) , is a member of the SCGB gene superfamily that consists of small secretory proteins . HIN-1 was originally identified as a tumor suppressor gene because its expression was silenced by methylation in the majority of human breast carcinomas . UGRP2 was independently identified as a homologous gene to UGRP1 (also called SCGB3A2) that is a downstream target for the homeodomain transcription factor NKX2-1, also called TITF1, TTF1, NKX2.1, or T/EBP . Mouse SCGB3A1 and SCGB3A2 share 33% amino acid sequence identity .
In humans, SCGB3A1 is highly expressed in the trachea, lung, salivary gland, prostate, esophagus, duodenum and mammary gland [1, 2, 4], whereas in mouse, it is primarily expressed in the trachea and lung, and weakly expressed in the heart, stomach, and small intestine [2, 4, 5]. Methylation patterns of the human SCGB3A1 gene promoter have been extensively studied and a correlation of methylation and loss of SCGB3A1 expression and malignant phenotypes is well established in many human cancers including breast, prostate, lung, and pancreatic carcinomas [6–8]. The AKT signaling pathway is responsible for the SCGB3A1's tumor suppressor function as characterized by inhibition of cell growth, cell migration and invasion . In this connection, it was shown that EGF and TGFγ increase SCGB3A1 expression through activation of the ERK-MAPK and phosphoinositide-3 kinase-AKT pathways . Further, the expression of SCGB3A1 is restricted to terminally differentiated airway epithelial cells and is up-regulated during retinoic acid induced differentiation of bronchial epithelial cells, suggesting that SCGB3A1 may be involved in the acquisition or maintenance of the terminally differentiated epithelial phenotype .
Under interleukin (IL)-4 and IL-13 stimulation, SCGB3A1 expression is up-regulated through binding of STAT6 to the STAT binding element located in -201 to -209 bp of the mouse Scgb3a1 gene promoter , suggesting that SCGB3A1 may also play a role in inflammation. In fact, a recent report demonstrated that SCGB3A2 (UGRP1), a homologous gene to SCGB3A1, suppresses allergic airway inflammation when a mouse model for allergic airway inflammation is subjected to intranasal administration of recombinant adenovirus expressing SCGB3A2 . STAT6 is the only transcription factor downstream of IL-4 and IL-13 signaling thus far demonstrated that directly binds to the Scgb3a1 promoter and regulates expression of the gene . It is not known what transcription factors are involved in constitutive expression of the mouse Scgb3a1.
In this study, we demonstrate that a ubiquitous transcription factor NF-Y is an important transcription factor for controlling expression of the mouse Scgb3a1 gene through its binding to the responsive "CCAAT" element located at -425 to -429, and -498 to -502 bp upstream of the Scgb3a1 gene. The methylation pattern of the mouse Scgb3a1 gene is examined and the association of the two closest CpGs to the transcription start site in tissue-specific expression of the mouse Scgb3a1 gene is discussed.
Analysis of the mouse Scgb3a1 gene promoter
NF-Y is involved in the mouse Scgb3a1 gene transcription
In order to determine which NF-Y binding element is responsible for Scgb3a1 gene transactivation, mutant reporter plasmids were constructed by introducing mutations singly or doubly into two of the "CCAAT" binding sites in the -598 construct (Fig. 3B). Mutations introduced were the same as those used for EMSA (see Fig. 3A). When the proximal "CCAAT" binding site was mutated (Mut 1), luciferase reporter activity with co-transfection of NF-Y expression plasmid was reduced to approximately one-third as compared to wild-type, whereas Mut 2 having the distal "CCAAT" site mutated, displayed about one-half the luciferase activity of wild-type. With both mutations together, luciferase activity was further reduced to approximately one-fourth of that of wild type. These results suggest that the proximal NF-Y binding site may be slightly more responsible, however, both NF-Y binding sites are required for Scgb3a1 gene activation.
NF-Y is a dominant transcription factor over PU-box binding protein in the mouse Scgb3a1 gene promoter activity
To determine which transcription factor between NF-Y and PU-box binding protein is more critical for the regulation of mouse Scgb3a1 gene, co-transfection experiments were carried out in COS-1 cells using the construct -598 and -598 PU-box1&2 double mutant with and without NF-Y. While both constructs had very little reporter activity without NF-Y (also see Fig. 4A), the promoter activity was markedly increased by NF-Y over-expression regardless of the presence of PU-box double mutation (Fig. 4C). The importance of NF-Y in mouse Scgb3a1 gene transcription was further demonstrated by co-transfection into COS-1 cells of the -598 construct and an expression plasmid for PU.1 (purine rich box-1)/Spi-1 (SFFV (spleen focusforming virus) proviral integration site-1), one of the PU box binding ETS family transcription factors [25, 26], although we do not know what ETS family member of transcription factor is involved in mouse Scgb3a1 gene transcription. In this case, Scgb3a1 promoter activity was slightly increased by the addition of PU.1/Spi-1 expression plasmid both in the absence and presence of NF-Y, however, again the effect of NF-Y on the promoter activity was far more robust than the PU-box binding transcription factor (Fig. 4D). These results suggest that NF-Y may be a dominant transcription factor over PU-box binding protein for mouse Scgb3a1 gene transcription. We therefore focused on NF-Y in further studies.
Analysis of NF-Y binding to its specific binding sites in the mouse Scgb3a1 gene promoter
Correlation between Scgb3a1 promoter methylation and gene expression in various mouse tissues/cells
Proximal 2 CpGs Methylation
Methylated + Unmethylated
CpG methylation plays a role in the regulation of mouse Scgb3a1 gene
We report here for the first time that a ubiquitous transcription factor NF-Y is important for regulation of the mouse Scgb3a1 gene, while DNA methylation appears to be associated with tissue-specific expression of the gene. NF-Y recognizes a "CCAAT" penta-nucleotide that can be found at every 500 bp in random genomic DNAs based on mathematical calculation. With additional high stringency, in which the "CCAAT" box is expanded to a 9-nucleotide element as (G/A)(G/A)CCAAT(C/G)(A/G)(G/C) , this sequence appears every 16 kbp in frequency. A "CCAAT" box is found in the vicinity of promoter region (between -60 and -100 bp of the major start site in both orientations) with significant high frequency (30%), suggesting that NF-Y may play a pivotal role in controlling expression of many genes . In the current study, CCAAT boxes were located further upstream (-425 to -429 bp and -498 to -502 bp), away from the vicinity of Scgb3a1 gene transcription start site.
Transfection analysis in COS-1 cells revealed that the -59 and -273 constructs increased reporter activity two-fold in an NF-Y dependent manner (see Fig. 2A). However, no "CCAAT" binding sequence was found between -59 bp and the transcription start site, nor did EMSA reveal any NF-Y binding in this region (data not shown). While an exact "CCAAT" sequence is required for NF-Y binding, "CCACT" sequence was reported to similarly function as the NF-Y binding site in regulation of Oncoprotein18 gene . A low stringency survey revealed the presence of two "CCACT" sequences in the Scgb3a1 gene promoter at -44 to -48 bp and -113 to -117 bp, however, neither bound NF-Y as determined by EMSA (data not shown). Further, when NFYAm29, a dominant negative form of NF-YA was co-transfected with the constructs -598, an approximately two-fold higher luciferase activity as compared to control remained even though a heterotrimer with NF-Yam29 cannot bind DNA (Fig. 2A) . Moreover, mutation of the two NF-Y binding sites did not completely abolish NF-Y activation of the -598 construct luciferase activity (Fig. 3B). The exact reason for these phenomena is not known. However, it has been established that NF-Y interacts with TATA-binding protein (TBP) , TFIID , and coactivators such as p300  and P/CAF  that modify gene expression. The interaction of NF-Y with these factors could stabilize basic transcription machinery and/or its association with DNA, which in turn results in a slight increase in Scgb3a1 reporter activity with co-transfection of constructs -598 and NF-YAm29, and/or the constructs -59/-273 and NF-Y expression plasmid. Note that in these reporter assays, three expression constructs, NF-YA, NF-YB, and NF-YC were simultaneously co-transfected along with a reporter construct. The fact that only cells that have taken up all four plasmids can produce a complete form of NF-Y that can activate the promoter suggests that the 20-fold increase of Scgb3a1 promoter activity obtained in this co-transfection assay system may represent only a fraction of naturally occurring NF-Y regulated Scgb3a1 expression.
The importance of NF-Y, in particular the NF-YA subunit in mouse Scgb3a1 gene expression was demonstrated by reporter activities using a various combination of NF-Y subunits and reduced SCGB3A1 mRNA expression by NF-YA shRNA. In the latter, both NF-YA protein and SCGB3A1 mRNA levels were suppressed at similar levels of approximately 50–60% of the control. This incomplete suppression may be due to the fact that NF-Y is a ubiquitous transcription factor and regulates many genes including housekeeping genes and those involved in cell cycle control, that account for approximately 20% of all genes [20, 22, 34]. Thus, complete suppression of NF-Y expression may likely result in suppression of cell proliferation and cell death. Indeed, a knockout mouse for NF-YA is embryonic lethal, and embryonic fibroblasts prepared from this mouse demonstrate halted cell proliferation and DNA synthesis .
An important factor controlling tissue-specific expression of genes is CpG methylation . This may particularly play a role in suppressing expression of NF-Y-responsive genes in many tissues since NF-Y is ubiquitously expressed [20–22] and is responsible for recruitment of RNA polymerase II . However, the possibility exists that NF-Y may cooperate with another transcription factor(s), which could render expression of genes tissue and/or stage-specific. For instance, NF-Y and C/EBPα interact with each other and synergistically activate the mouse amelogenin gene, which contributes to physiological regulation during amelogenesis . Whether this could be the case in the regulation of mouse Scgb3a1 gene expression remains to be determined.
DNA methylation is known to suppress gene expression through two basic mechanisms [38, 39]; first, methylation of cytosine bases inhibit association between DNA-binding factors and their cognate DNA recognition sequences, resulting in direct inhibition of transcriptional activation. Second, proteins that recognize methyl-CpG such as methyl-CpG-binding proteins elicit repressive potential of methylated DNA by recruiting co-repressor molecules to silence gene transcription and to modify surrounding chromatin, including histone modification. In the case of the mouse Scgb3a1 gene, the second mechanism most likely plays a role in silencing expression of the gene since two CpGs are located closest to the transcription start site, which is 3–400 bp downstream of the NF-Y binding sites. These two CpGs in the mouse Scgb3a1 gene promoter are partially or completely unmethylated in mouse lung and mtCC cells, respectively. The partial methylation observed in the lung is the result of a mixture of methylated and unmethylated CpGs at approximately a 1:2 ratio. Although we do not have a proof, the methylated CpGs might be at least partially derived from non-epithelial cells such as mesenchymal cells that are contained in whole lung from which genomic DNA for methylation analysis was prepared. We found that the two CpGs were totally methylated when embryonic lung mesenchymal cells that do not express SCGB3A1 were separately prepared from epithelial cells and subjected to sequencing (Tomita and Kimura, unpublished results). SCGB3A1 is known to be expressed only in the airway epithelium . However, very low SCGB3A1 expression was observed in the heart even though the two CpGs closest to the transcription start site were methylated. The reason for this difference is not known. A small percentage of these CpGs might be unmethylated in a specific part of the heart and not abundant enough to be detected particularly using mRNA prepared from whole heart. In this regard, the expression site in the heart is not known. Alternatively, another transcription factor(s) enriched in the heart might be responsible for the low SCGB3A1 expression in this tissue. EMSA using methylated CpG oligonucleotides revealed the presence of additional shifted bands as compared to unmethylated oligonucleotides. This could be due to binding of one of the methyl-CpG-binding proteins [38, 40] or other DNA-binding proteins (transcription factors or co-factors) to these sites. We do not know which methyl-CpG-binding protein or DNA-binding protein is responsible for the new band, however the results at least suggest that CpG methylation affects binding of DNA-binding proteins within the region around proximal and distal CpGs in the mouse Scgb3a1 gene promoter. Previously we reported that a STAT binding element located at -201- to -209 bp of the mouse Scgb3a1 gene promoter is responsible for the IL-3/14-induced increase of Scgb3a1 gene expression in mtCC and mouse embryo lung primary cells . This STAT element is located to close to the 3' most methylated CpG. Whether any changes in methylation status in this CpG under IL-3/14 induction remains to be determined.
Lastly, in the human SCGB3A1 gene promoter, 138 potential methylation CpG sites are found within 1,500 bp of the promoter region . High frequency of methylation of these CpGs is associated with loss of SCGB3A1 expression in many human cancers [6–8]. In contrast, there are only 9 CpGs within 600 bp of the mouse Scgb3a1 gene promoter. This remarkable species difference in CpG islands can be explained by little significant homology in DNA sequences between the human and mouse gene promoters, suggesting that mouse Scgb3a1 and human SCGB3A1 may be under different regulation. This could explain the different patterns of SCGB3A1 expression between these two species. Whether the difference in gene regulation has any implication in functional role of SCGB3A1 between mouse and human requires additional studies.
A ubiquitous transcription factor, NF-Y appears to be a potential potent regulator of mouse Scgb3a1 gene expression through binding to two "CCAAT" boxes located at -425 to -429, and -498 to -502 bp in the Scgb3a1 gene promoter. Tissue-specific expression of SCGB3A1 may be due in part to methylation of the two CpG sites closest to the transcription start site of the gene.
Normal rabbit IgG (sc-2027), anti-NF-YA (sc-10779X) and anti-NF-YB (sc-13045) antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). All restriction endonucleases were obtained from New England Biolabs (Ipswich, MA).
Serial Scgb3a1 gene promoter deletion mutants were constructed with PCR using mouse genomic DNA and the following primers: forward primer for -59 construct 5'-CAAACTAGTTCCCAGGAGGGTGAGGTTCCAC-3', -91 construct 5'-TAAGCTAGCCCTTTCTGCCTGTTAGCTGAGCAAAC-3', -184 construct 5'-CACACTAGTTCAGGAGACCCTGCCAAGAG-3', -273 construct 5'-AGGACTAGTCAGACCACATTATCAAGGTGTAGG-3', and -598 construct 5'-CCCACTAGTCATTAGCAGCTACCTGTGGCCCAAC-3', and a common reverse primer 5'-AGAGGATCCCAGGACCTATAAGACAATCTTCC-3'. PCR products were double-digested with Spe I (Nhe I for -91 construct) and Bam HI, and cloned into the Nhe I – Bgl II site of the pGL4.11 vector (Promega, Madison, WI). These reporter constructs contained +24 bp of the 5' UTR of the SCGB3A1 mRNA sequence.
For construction of expression plasmids, total RNAs isolated from mouse embryonic lungs (embryonic day 16.5) by using TRIZOL (Invitrogen, Carlsbad, CA) were first treated with DNase I (Ambion, Austin, TX) and subjected to cDNA synthesis using Superscript II reverse transcriptase (Invitrogen). PCR reactions were carried out using the following primers: 5'-TTAGCGGCCGCATGGAGCAGTATACGACAAACAGCAATAG-3' and 5'-TAATCTAGATTAGGAAACTCGGATGATCTGTGTCATGG-3' for NF-YA, 5'-TAAGGTACCTTACATGACAATGGACGGCGACAGCTC-3' and 5'-TAATCTAGATCATGAAAACTGAATTTGCTGGACACCAG-3' for NF-YB, 5'-TAAGCGGCCGCACCATGTCCACAGAAGGAGGGTTTGG-3' and 5'-TTATCTAGAGCCCTCAGTCTCCAGTCACCTGG-3' for NF-YC, and 5'-TTAGCGGCCGCATGTTACAGGCGTGCAAAATGGAAGG-3' and 5'-TTATCTAGACGATCAGTGGGGCGGGAGG-3' for PU.1/Spi-1. PCR products were subcloned into the Not I – Xba I (NF-YA, NF-YC and Spi-1) or Kpn I – Xba I sites (NF-YB) of pcDNA3.1 vector (Invitrogen). The NF-Y binding site and PU-box mutations were generated by site-directed mutagenesis kit (Stratagene, La Jolla, CA). All plasmid sequences were confirmed by DNA sequence analyses (Beckman Coulter, model CEQ-200XL, Fullerton, CA).
Reporter gene assays
COS-1 and mtCC cells were seeded in 24-well tissue culture plates in DMEM with high glucose and L-glutamine (Invitrogen), supplemented with 10% FBS. A transfection cocktail contained 20 μl serum-free DMEM, 1 μl Fugene 6 (Roche Applied Science, Indianapolis, IN), 250 ng reporter construct, and 5 ng pGL4.74 Renilla luciferase vector (Promega) as an internal control, and whenever necessary, a various combination of three NF-Y expression plasmids (16.6 ng each, a total amount adjusted to 50 ng with a control vector). In order to have all plasmids transfected into a cell, the amount of each expression plasmid used were kept small and transfection was carried out using cells at 50% confluency. Cells were washed with PBS 48 h after transfection, and lysed in passive lysis buffer (Promega). Luciferase activity was determined using a luminometer (Pharmingen, model monolight 3010) with Dual-Luciferase Reporter Assay System (Promega). All reporter assays were carried out at least three times, each in duplicate, and the results were expressed as the mean ± SD.
For shRNA experiments, synthetic oligonucleotide designed to generate RNAi for mouse NF-YA (Invitrogen, Mmi515428) was inserted into a linearized vector pcDNA6.2-GW/miR (Invitrogen). mtCC cells transfected with Mmi515428/pcDNA6.2-GW/miR or control pcDNA6.2-GW/miR were maintained in media containing 2 μg/ml blasticidin (Invitrogen).
Nuclear extracts were prepared from COS-1 cells transfected with NF-Y expression plasmids, mouse lungs and mtCC cells . In the case of COS-1 cells, 1 ml of serum-free DMEM containing expression constructs (NF-YA, NF-YB, and NF-YC, 7 μg each) was mixed with 50 μl of Fugene HD (Roche Applied Science, Indianapolis, IN), which after 15 min incubation, added to COS-1 cells that were grown to 50–80% confluency in 150-mm dish. The media was changed 8 h after transfection, and 48 h after the media change, cells were washed and harvested in cold PBS. Cell suspension was centrifuged for 5 min at 1,000 rpm and the pellet was resuspended in 2 ml Buffer A (10 mM HEPES, pH 7.6, 15 mM KCl, 2 mM MgCl2, 0.1 mM EDTA, 1 mM DTT, 0.5 mM PMSF). After centrifugation at 2,500 rpm for 3 min, cell pellet was suspended in Buffer B (Buffer A + 0.2% IGEPAL), followed by re-centrifugation for 3 min at 2,500 rpm. Mouse lungs were homogenized using a dounce homogenizer in Sucrose buffer (250 mM sucrose, 10 mM HEPES, pH 7.6, 15 mM KCl, 2 mM MgCl2, 0.5 M EDTA, 1 mM DTT, 0.5 mM PMSF), and centrifuged for 3 min at 2,500 rpm. The nuclear pellets thus obtained were washed with Sucrose buffer mixed with Extraction buffer (50 mM HEPES, pH 7.9, 400 mM KCl, 0.1 mM EDTA, 10% Glycerol, 0.5 mM PMSF), and rotated in cold room for 30 min. Finally, the nuclear extract was obtained by centrifugation, and protein concentration was adjusted at 2 mg/ml by using Bradford protein assay (Bio-Rad Laboratories, Hercules, CA) with BSA as a standard.
Electrophoretic mobility shift assays (EMSA)
Oligonucleotides were radio-labeled with [γ-32P]ATP (PerkinElmer, Wellesley, MA) and T4 polynucleotide kinase (New England Biolabs). Nuclear extract (1 μl) was diluted in Binding buffer (0.1 μg/μl polydI-dC, 10 mM Tris, pH 8.0, 1 mM DTT, 80 mM KCl, 20% Glycerol, 0.04 μg/μl BSA) and incubated for 15 min at room temperature with radio labeled probe in the presence or absence of cold oligonucleotide as a competitor. For supershift analysis, the mixture was incubated with 1 μl of antibody solution for additional 15 min at room temperature. DNA-protein complexes were electrophoresed on a 4% polyacrylamide gel using 0.5× TBE buffer, and were visualized by exposing to phosphoimager screen (Storm 840, Amersham Biosciences, Piscataway, NJ).
Chromatin immunoprecipitation (ChIP) assays
DNA was isolated from mtCC, NIH3T3, and MLE15 cells that were fixed with 1% formaldehyde, followed by sonication. MLE15 was grown in HITES (RPMI-1640 supplemented with insulin, transferrin, sodium selenite [Sigma, St. Louis, MO], 10 mM hydrocortisone [Sigma], 10 mM γ-estradiol [Sigma], 10 mM HEPES [Invitrogen], and 2%FCS). Endogenous NF-Y together with fragmented DNAs was pulled down by anti-NF-YA or rabbit IgG as control. After reverse cross-linking, the Scgb3a1 promoter region containing the NF-Y binding site (200 bp) and a region derived from Scgb3a2 intron 1 + exon 2 as a reference (219 bp) were PCR amplified. PCR primers used were as follows: 5'-GCTATTAGGCATTCCTTTCTGGCTC-3' and 5'-CCACGGGCGATTATGTAGGTTC-3' for amplification of the NF-Y binding site, and 5'-TCTTCAGTCCTGTCACCAGATGTTCTAC-3' and 5'-CGAGAGGGATGGGATGGAGTCTTAG-3' for the reference. Sample solution was mixed with each primer set and Taq polymerase, followed by PCR reaction of 25 cycles with 94°C denaturation, 15 sec, 56.5°C annealing, 15 sec, and 72°C extension, 30 sec.
RNAs isolated from various organs of adult mouse and various mouse cell lines were subjected to RT-PCR analysis. PCR was carried out after cDNA synthesis using the following primers: 5'-CTCTACAGATCCCAGGCAGC-3' and 5'-CTGGAGCCTCTGATTGGGT-3' for NF-YA, 5'-TAGCTGGGAGGCATCTGTG-3' and 5'-AGGATCCACCACCTTTTTGA-3' for NF-YB, 5'-TTTCTTCCATGACTCTGGGC-3' and 5'-GCTGCTTTCTTCGCTGGA-3' for NF-YC, and 5'-GATGGCCAAGTGGCTTAATG-3' and 5'-TCTGTGTGGCTCTGCTCAGT-3' for SCGB3A1. PCR condition used was 94°C, 2 min, followed by 30 cycles of 94°C, 15 sec, 55°C, 15 sec, and 68°C, 30 sec.
Total RNA (3 μg) isolated from mtCC cells was electrophoresed on 1% agarose gel containing 0.22 M formaldehyde and transferred onto nitrocellulose membrane (Immobilon-Ny+, Millipore, Billerica, MA). Filters were hybridized with mouse SCGB3A1 and ribosomal protein B36 (loading control) as a probe. Hybridization was performed in Perfect Hybridization solution (Amersham Biosciences) at 68°C overnight. The membrane was washed twice with 2 × SSC containing 0.1% SDS at 68°C for 30 min, followed by exposure to phosphoimager screen. Data processing was carried out using ImageQuant TL 2005 software (Amersham Biosciences).
Nuclear extracts (3 μg) obtained from mtCC cells were run on 10% SDS-polyacrylamide gels, and proteins transferred onto PVDF membrane (Hydond-P, Amersham Biosciences). The membrane was gently shaken with PBS containing 5% skim milk at 4°C overnight, followed by incubation with anti-NF-YA or NF-YB antibody diluted in PBS containing 1% Tween 20 (PBST). After washing three times with PBST, the membrane was incubated with horseradish peroxidase conjugated anti-rabbit IgG (NA9340V, Amersham Biosciences) followed by further three times wash with PBST. Signals were directly detected and quantified using chemiluminescence reaction (Immobilon Western, Millipore) with CCD camera system and equipped software (Alpha Innotech Fluor Chem HD2, San Leandro, CA).
DNA methylation analysis
Genomic DNAs were isolated based on simplified procedure  from various tissues and cultured cells. Isolated DNAs were overnight digested with Bam HI at 37°C, ethanol precipitated after phenol-chloroform extraction and pellets were dissolved in TE buffer. EZ DNA methylation kit (Zymo Research, CA) was used to generate bisulfite treated DNA. Five hundred ng of DNA was incubated with bisulfite at 50°C for 16 h, and the product was purified and desulphonated through a spin-column. This procedure converts all non-methylated cytosine to uracil but does not affect 5-methyl cytosine. Two μl of final elution from the spin-column was used as template for two different PCRs with a primer set, 5'-GTGGTTTTTGGAAGAAAGGTTTAGTATTTGAGTTTAGG-3' and TACTAAACCCCCAAAAAAACTCACCAAAAATCAC-3' for proximal Scgb3a1 promoter region (343 bp), and 5'-GAGAGATTTAGAGTTTTTGGGATTTGTTGATTTAT-3' and 5'-CCCACCTCAATCCTAAAAATTTCCTCTTAAC-3' for distal Scgb3a1 promoter region (279 bp). Both PCR reactions were carried out at the same conditions; 94°C, 2 min, followed by 40 cycles of 94°C, 15 sec, 52.8°C, 15 sec, and 68°C, 30 sec. All PCR products were subjected to 2% agarose gel electrophoresis, and each band was excised and purified (gel extraction kit, Qiagen, Valencia, CA) for subsequent direct sequencing analysis to identify cytosine residues, but not thymine (uracil), that was the result of resistance to bisulfite reaction due to methylation. In some cases, PCR products were subcloned into pGEM-T easy vector (Promega) and individual clones were subjected to sequencing analysis. For confirmation of non-bisulfite treated genomic DNA sequence, PCR was carried out using the following primer sets: 5'-CAGAAAAATGTCACAGCCCCTC-3' and 5'-CCAACTTCCTCTTATGGTCAGTGGAC-3' to obtain 543-bp fragment of the distal Scgb3a1 promoter region containing the two CCAAT sites and 5'-CTTCCAGACCCAGAACCTACATAATC-3' and 5'-AGAGTCACTGAGCAGAGCCACAC-3' to obtain 471-bp fragment of the proximal Scgb3a1 promoter region. PCR condition used was 94°C, 2 min, followed by 30 cycles of 94°C, 15 sec, 58°C, 15 sec, and 68°C, 60 sec.
We would like to thank Drs. Francesco DeMayo (Baylor College of Medicine, Houston, TX) and Jeffrey Whitsett (Cincinnati Children's Hospital Medical Center, Cincinnati, OH) for providing mtCC cells and MLE15 cells, respectively, Frank Gonzalez (NCI, Bethesda, MD) for his critical review of the manuscript, and Eyal Rand (NCI, Bethesda, MD) for discussion on CpG methylation. This research was supported by the Intramural Research Program of the National Cancer Institute, Center for Cancer Research.
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