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Human Molecular Genetics Advance Access originally published online on February 24, 2005
Human Molecular Genetics 2005 14(7):973-982; doi:10.1093/hmg/ddi091
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© The Author 2005. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions{at}oupjournals.org

Transcriptional targets of the chromatin-remodelling factor SMARCA4/BRG1 in lung cancer cells

Pedro P. Medina1, Julian Carretero1, Esteban Ballestar2, Barbara Angulo1, Fernando Lopez-Rios3, Manel Esteller2 and Montserrat Sanchez-Cespedes1,*

1Lung Cancer Group and 2Cancer Epigenetics Group, Molecular Pathology Programme, Spanish National Cancer Centre (CNIO), Madrid, Spain and 3Pathology Department, Hospital Universitario 12 de Octubre, Madrid, Spain

* To whom correspondence should be addressed at: Molecular Pathology Programme, Spanish National Cancer Centre (CNIO), C/Melchor Fernandez Almagro 3, 28029 Madrid, Spain. Tel: +34 912246954; Fax: +34 912246923; Email: msanchez{at}cnio.es

Received January 13, 2005; Accepted February 17, 2005


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
BRG1, also called SMARCA4, is the catalytic subunit of the SWI/SNF chromatin-remodelling complex and influences transcriptional regulation by disrupting histone–DNA contacts in an ATP-dependent manner. BRG1 and other members of the SWI/SNF complex become inactivated in tumours, implying a role in cancer development. To understand the contribution of BRG1 to lung tumourigenesis, we restored BRG1 in H1299 lung cancer cells and used cDNA microarray analysis to identify changes in gene expression. Forty-three transcripts became activated, whereas two were repressed. Chromatin immunoprecipitation of resulting candidate genes revealed that the CYP3A4 and ZNF185 promoters recruited BRG1 and that recruitment to the CYP3A4 promoter was specific to this gene and did not involve the CYP3A5 or CYP3A7 family members. Moreover, specifically BRG1 but not its homologue BRM was recruited to the CYP3A4 and ZNF185 promoters. To explore their potential relevance in lung tumours, levels of CYP3A4 and ZNF185 transcripts were evaluated in seven additional lung cancer cell lines. CYP3A4 was undetectable in any of the lung cancer cells tested, and only the CYP3A5 family member was present in the A549 and Calu-3 cells. In contrast, the amount of ZNF185 transcript clearly varied among lung cancer cell lines and severely reduced levels were observed in BRG1-deficient cells, except those of A427. We extended these observations to 27 lung primary tumours using real-time RT–PCR (TaqMan) and observed that four (15%) and 14 (52%) of them had BRG1 and ZNF185 downregulation, respectively, when compared with normal lung. No significant correlation between reduced levels of BRG1 and ZNF185 was observed, indicating that additional mechanisms to BRG1 inactivation may contribute to the loss of ZNF185 expression in lung tumours. In conclusion, our results provide evidence that transcriptional activation of ZNF185 and CYP3A4 is mediated by direct association of BRG1 with their promoters and also indicate that a decreased level of ZNF185 is a common feature of lung tumours and may be of biological relevance in lung carcinogenesis.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
The regulation of gene expression in eukaryotic organisms depends on a combination of multiple factors, including the availability of transcriptional factors, the packaging of DNA into chromatin and the architectural organization of the chromatin within the nucleus (1Go,2Go). Two major types of multiprotein complex activities modulate the structure of the chromatin and its capability to coordinate the binding of transcriptional factors to promoters or enhancers: histone-modifying activities (e.g. histone deacetylases, histone methyltransferases) and chromatin-remodelling complexes, which alter chromatin structure in an ATP-dependent fashion. There is increasing evidence of a role for some components of chromatin-remodelling complexes in cancer development (3Go–5Go).

SWI/SNF is an evolutionarily conserved complex composed of several subunits that remodel the chromatin structure in an ATPase-dependent manner (2Go). Several of these components are altered in human tumours, such as the gene that codes for the hSNF5/INI1 subunit, mutated in tumours from the central nervous system, kidney and in soft-tissue rhabdoid tumours (6Go). Moreover, the hSWI/SNF brahma-related gene 1 (BRG1), also known as SMARCA4, which codes for one of the two alternative ATPases of the hSWI/SNF complex, is homozygously inactivated in several cancer cell lines derived from lung, prostate, breast and pancreatic cancer (7Go,8Go). In addition to cancer cell lines, BRG1 gene point mutations and loss of BRG1 protein expression have been observed in lung primary tumours (9Go–11Go). Furthermore, mice heterozygous for BRG1 mutations have an increased predisposition to developing epithelial tumours (12Go), implying an active role of BRG1 gene inactivation during lung cancer development. Finally, several lines of evidence demonstrate that the SWI/SNF chromatin-remodelling system is involved in cell-growth control (13Go) and differentiation partly because of its interaction with cancer-related proteins such as RB, B-catenin, FANCA and LKB1 (14Go–16Go).

Whereas the yeast Swi/Snf is estimated to regulate the transcription of 5–6% of the genome (1Go,17Go), in mammalian cells, the total number of genes regulated by the SWI/SNF is unknown and, specifically, few direct transcriptional targets have been identified in human cells (18Go–24Go). Genes that appear to be regulated by BRG1 include colony-stimulating factor 1 (CSF1) (20Go), heat shock factor 1 (HSF1) (21Go), E2F1 (23Go), several IFN-alpha inducible genes (24Go), p21 (25Go) and fos (26Go). This paucity of information limits our understanding of how hSWI/SNF inactivation contributes to tumour development.

In the present study, we search for novel genes regulated through the hSWI/SNF chromatin-remodelling complex and evaluate their relevance in lung carcinogenesis by analyzing global changes in gene expression after restoring wild-type BRG1 activity in the lung cancer cell line H1299, followed by chromatin immunoprecipitation (ChIP) in candidate gene promoters.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Massive transcriptional activation after restoring wild-type BRG1 expression in lung cancer deficient cells
To identify in vivo transcriptional targets of the BRG1 gene, we used the H1299 lung cancer cell line, which carries a deletion that results in a truncated protein (7Go). The homozygous deletion of the cDNA of BRG1 is depicted in Figure 1A. BRG1 wild-type activity in these cells was restored by transient transfection with a mammalian expression vector (pCMV) containing the full-length wild-type cDNA of BRG1 (pCMV-BRG1). As a control, we transfected with pCMV-K798R, a mutant form of the BRG1 gene, which has been demonstrated to lack ATPase activity (19Go), and with empty vector (pCMV). In our system, the efficiency of gene transfer at 24 h was ~33%, as indicated by immunofluorescence (data not shown). The ectopic expression of wild-type BRG1 is shown by western blotting in Figure 1B and C.



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  		Figure 1. Expression of ectopic BRG1 in H1299 lung cancer cells and endogenous BRG1 in cancer cell lines. (A) A 70 bp frameshift deletion at the RNA level of the BRG1 in the H1299 cells (7Go); (B) western blot analysis of ectopic wild-type BRG1 using an anti-HA antibody in the H1299 cells at various times following transfection with pCMV-BRG1 or pCMV; (C) western blot analysis of endogenous BRG1 in different cancer cell lines and ectopic BRG1 in the H1299 cells transfected with pCMV-BRG1 or pCMV. BRG1 is shown at ~180 kDa.

 
For the cDNA microarray analysis, only genes with>2-fold induction or repression in both duplicates, 24 h after BRG1 ectopic expression were counted. To eliminate background noise in the analysis, we considered only genes that were induced or repressed at two or more time-points and discarded those transcripts that were deregulated after treatment with empty vector or with pCMV-K798R. Adopting these criteria, 43 and two transcripts were found to be activated and repressed, respectively. Thirty-four of the 43 upregulated transcripts corresponded to known genes, whereas the rest were ESTs (Table 1). The table also shows the changes in gene expression at different times after BRG1 overexpression and in the controls (transfection with pCMV-K798R and with empty vector at 24 h). As expected, BRG1 was the transcript with the greatest upregulation in cells overexpressing wild-type (16-fold upregulation at 24 and 48 h) and mutant BRG1 (8-fold upregulation at 24 h). Among the transcripts with the highest levels of up- or downregulation were cytochrome P450 (CYP3A4), transcriptional regulators (CEBPB, HDAC4, LDB1, MXD4, NFKB2, TBX2), transcripts involved in cell adhesion (ECM2, PCDH1, CDH11, CDH16, CNTN1) and transcripts involved in cell-cycle regulation (CDKN2D and CCNA2). The BRM transcript appeared upregulated in cells overexpressing wild-type and mutant BRG1. However, this is probably due to cross-hybridization of the BRG1 transcript with the BRM clone AI23475, with which it shares a strong sequence homology. No cross-hybridization was observed with the BRM clone AA481026 [GenBank] , which has a very different sequence (Table 1). Semiquantitative RT–PCR analysis of some strongly activated genes confirmed the cDNA microarray observations (Fig. 2A).


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  		Table 1. Genes modified by wild-type BRG1 (BRG1), mutant BRG1 (K798R) or empty vector (pCMV) transient transfection in H1299 cells at various times following transfection, as indicated
 


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  		Figure 2. Identification of genes transcriptionally regulated by the hSWI/SNF complex. (A) Semi-quantitative RT–PCR analysis of mRNA of several transcripts in H1299 cells, in H1299 cells transfected with empty vector (pCMV), with wild-type BRG1 (pCMV-BRG1) (24 h after transfection) or with wild-type BRM (pCMV-BRM) (24 h after transfection). The amount of each RNA template was controlled through amplification of GAPDH. (B) BRG1 and BRM occupancy in several gene promoters analyzed by ChIP assay. Input and ‘unbound’ fractions of the no-antibody (No Ab) control are shown for cells transfected with empty vector (pCMV) and with wild-type BRG1 (pCMV-BRG1) or with empty vector and wild-type BRM (pCG-BRM). (C) Partial sequence of the promoter for the distinct CYP3A family members is compared with the sequence obtained from the ChIP analysis in BRG1 immunoprecipitates and input DNA in pCMV-BRG1 cells. Nucleotide differences are indicated in blue or black and confirm the specificity of BRG1 for CYP3A4 but not for the CYP3A5 or CYP3A7 gene.

 
We also tested the ability of BRG1 to induce growth arrest in the H1299 cells. To do this, we used FACS assay to monitor the effects on cell proliferation of the H1299 cells transfected with BRG1. Our data showed a lack of significant cell growth arrest at G1/S in the H1299 cells overexpressing wild-type BRG1 when compared with cells transfected with mutant BRG1 (data not shown).

Transcriptional activation of the CYP3A4 and ZNF185 genes by the hSWI/SNF chromatin-remodelling complex in the H1299 lung cancer cells
To elucidate whether transcriptional upregulation was a direct consequence of the activity in the gene promoter of the BRG1-containing hSWI/SNF complex, we performed ChIP assays using anti-HA and anti-BRG1 (kindly provided by Dr Robert Kingston) antibodies to precipitate chromatin of H1299 cells expressing ectopic wild-type BRG1. In parallel, chromatin isolated from cells transfected with the empty vector was also immunoprecipitated. Several of the genes found to be upregulated in the global expression study were evaluated. Interestingly, two of them, CYP3A4 and ZNF185, exhibited binding of BRG1 to their promoters, as indicated by the exclusive specific appearance of a band in the bound fraction of the anti-BRG1 or anti-HA samples in cells transfected with BRG1 (Fig. 2B). No bands were observed in the ‘no antibody’ controls or in samples obtained with the earlier antibodies in chromatin from cells transfected with the empty vector, providing further evidence for the specificity of our results. Such observations indicate that the CYP3A4 and ZNF185 genes are targets for the hSWI/SNF complex in H1299 cells. In contrast, other upregulated genes did not exhibit BRG1 bound to their promoters (Fig. 2B), indicating that their transcriptional activation was independent of chromatin remodelling throughout the hSWI/SNF complex. Although not upregulated by ectopic expression of wild-type BRG1, we subjected the CSF1 and p21 promoters to ChIP analysis because they were known to be targeted by the hSWI/SNF complex in the SW-13 cells (20Go) and in the SW-13 and ALAB cells (25Go), respectively. Neither the CSF1 nor the p21 gene promoters were immunoprecipitated with anti-HA or anti-BRG1 antibodies in the H1299 lung cancer cells, suggesting a target specificity of the SWI/SNF complex in the distinct tumours.

The H1299 cells express low levels of BRM, the catalytic subunit for a subset of SWI/SNF complexes, highly homologous to BRG1. It is unclear whether BRG1 and BRM regulate a common set of promoters. To ascertain whether BRM could also associate to CYP3A4 and ZNF185 promoters and activate gene transcription in H1299 cells, where BRG1 is inactive, we overexpressed wild-type BRM protein in the H1299 cells. No CYP3A4 or ZNF185 gene overexpression was observed (Fig. 2A). Consistently, ChIP analysis against endogenous BRM protein did not detect binding of BRM to the CYP3A4 or ZNF185 gene promoter (Fig. 2B). Taken together, these observations demonstrate that binding to the CYP3A4 and ZNF185 promoters to allow transcriptional activation is specifically mediated by BRG1.

The CYP3A4 gene is located at the CYP3A locus, which contains at least three different genes (CYP3A4, CYP3A5, CYP3A7); all of which, including their promoters, share a high degree of sequence homology. To identify the specific gene promoter from the CYP3A locus that recruited the BRG1 protein, we sequenced the PCR product obtained from the ChIP analysis. The primers used for the PCR reaction completely matched the CYP3A4 and CYP3A7 gene promoters and were very similar to the CYP3A5 gene promoter. Figure 2C shows how BRG1 specifically bound to CYP3A4 promoter but not CYP3A5 or CYP3A7 promoters, indicating that the hSWI/SNF complex specifically regulates CYP3A4 gene transcription in H1299 lung cancer cells. Moreover, direct sequencing of the RT–PCR product obtained from H1299 cells transfected with wild-type BRG1 (Fig. 2A) confirmed that the transcriptional activation observed was that of the CYP3A4 gene and not of the CYP3A7 or CYP3A5 gene (data not shown).

BRG1, ZNF185 and CYP3A4 gene expression in lung cancer cell lines and lung primary tumours
To explore their relevance further and to characterize the expression patterns of BRG1, ZNF185 and CYP3A4 in lung tumours, we first tested seven additional non-small cell lung cancer cell lines (H23, A427, H441, H522, A549, H2126, Calu-3). Levels of BRG1 protein were evaluated by western blotting and compared to those of ZNF185 and CYP3A4 transcripts. In agreement with previously published observations, four of the cell lines had no detectable, or extremely low, levels of BRG1 protein (H23, A427, H522, A549) (Fig. 3A). BRG1 protein expression matched the levels of BRG1 mRNA assessed by real-time RT–PCR (Fig. 3B). To test the expression of the three members of the CYP3A family, CYP3A4, CYP3A5 and CYP3A7, which have highly homologous sequences, we used an approach involving PCR followed by restriction fragment length polymorphism (RFLP) analysis. None of the cell lines tested had a detectable endogenous CYP3A4 transcript (Fig. 3A). The Calu-3 and A549 lung cancer cells expressed a member of the CYP3A family that was identified as CYP3A5 (data not shown). Real-time (TaqMan) RT–PCR was used to analyze the levels of the ZNF185 transcript (Fig. 3C). Four out of five lung cancer cell lines with no detectable BRG1 protein also had an absence, or severely reduced levels, of ZNF185 transcript, implying a role for BRG1 in the transcriptional activation of the ZNF185 gene. The only exception was the A427 lung adenocarcinoma cells, which had no BRG1 protein but exhibited average levels of the ZNF185.



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  		Figure 3. Analysis of BRG1, CYP3A4 and ZNF185 gene expression in lung cancer cell lines and lung primary tumours. (A) Western blot of BRG1 protein and semi-quantitative RT–PCR for the ZNF185 and CYP3A transcripts in eight lung cancer cell lines. The amount of each RNA template was controlled through amplification of GAPDH. (B and C) Real-time (TaqMan) quantitative RT–PCR for the analysis of BRG1 and ZNF185 gene expression, respectively, in lung cancer cell lines and primary tumours. All data were standardized by TBP ({Delta}Ct, cycle difference). The bar indicates the median {Delta}Ct value from three different normal lung tissues.

 
To extend these observations to primary tumours, we measured the expression of the BRG1 and ZNF185 transcripts in 27 non-small cell lung tumours, using real-time (TaqMan) RT–PCR. Normal lung tissue from three patients was used to calculate basal levels. Expression of the BRG1 transcript varied widely among tumours. We arbitrarily chose the values of {Delta}Ct greater than –2 to describe complete absence or downregulation and of {Delta}Ct less than –4 to describe upregulation of BRG1 transcript when compared with normal lung tissue (median value of {Delta}Ct=–2.9). Downregulation of BRG1 was observed in four (15%) and five (62%) lung primary tumours and cancer cell lines, respectively, whereas upregulation was detected in six (22%) and two (25%) lung primary tumours and cancer cell lines, respectively (Fig. 3B). BRG1 downregulation was detected only in adenocarcinomas when compared with squamous cell carcinomas, although these differences were not statistically significant. Regarding the ZNF185 transcript, we chose values of {Delta}Ct greater than 0 to signify downregulation and of {Delta}Ct less than –2.5 to signify upregulation when compared with values obtained for normal lung (median value of {Delta}Ct=–1.6). Downregulation of ZNF185 was observed in 14 (52%) and five (62.5%) of lung primary tumours and cancer cell lines, respectively, whereas upregulation was detected in only three (11%) and one (12.5%) lung primary tumours and cancer cell lines, respectively (Fig. 3C). No association was observed between BRG1 and ZNF185 downregulation.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
MATERIALS AND METHODS
 REFERENCES
 
The presence of somatic point mutations and deletions in lung primary tumours and lung cancer cell lines is the evidence of a critical role for the BRG1 gene in lung carcinogenesis (7Go,10Go). Furthermore, severely reduced levels of BRG1 protein were previously reported in lung cancer (8Go,9Go,11Go), and loss of BRG1 protein has been proposed as a predictor of recurrence for patients diagnosed with this type of cancer. Thus, investigation of the genes targeted by BRG1 is an essential issue in cancer research.

To our knowledge, this is the first report of the use of a global gene expression approach to identify targets of the BRG1 protein in mammalian lung cancer cells. We have demonstrated that overexpression of wild-type BRG1 in a lung cancer deficient cell line triggers generalized transcriptional activation rather than repression. Similarly, Liu et al. (20Go) reported massive activation of gene expression following re-introduction of BRG1 in SW-13 phaeochromocytoma-derived cells, which are also BRG1-deficient. These observations suggest that BRG1 is mainly involved in the activation of gene expression. However, Hendricks et al. (25Go) reported that 70 and 65 transcripts became activated and repressed, respectively, after restoring BRG1 wild-type activity in the BRG1-deficient breast cancer cell line ALAB. Others have also concluded a role for BRG1 in transcriptional repression (reviewed in 27).

Several gene promoters have so far been identified in mammalian cells as targets of the hSWI/SNF complex, including the human CSF1 and the cell-cycle regulator P21 (20Go,22Go,25Go). However, in our system, the p21 and CSF1 promoters were not recruited by ectopic BRG1 protein. A BRG1-mediated induction of p21 transcript was previously reported after re-introduction of wild-type BRG1 in BRG1-deficient cancer cells from distinct tumour locations such as the breast cancer-(ALAB), the phaeochromocytoma (SW-13) and the cervical cancer (HeLa) derived cells (22Go,25Go). However, some distinct characteristics were detected between cell lines including the 50-fold and 5-fold induction of p21 observed in the ALAB and SW13 cells, respectively (25Go), or the BRG1-mediated activation of p21 transcription dependent of p53 status in the SW13 (22Go) but not in the ALAB cells (25Go). Interestingly, the H1299 cells are p53 mutant, suggesting that this may be the reason for the lack of p21 induction and for the lack of significant cell growth arrest in these cells following expression of ectopic wild-type BRG1. Similarly, differences in CSF1 induction after re-introduction of wild-type BRG1 in the SW-13 versus ALAB, were also reported (25Go). Taken together, these observations are potentially interesting because they suggest that BRG1-mediated gene expression may obey tissue-specific or genetic background-specific responses of cancer cells to the chromatin-remodelling activity of the hSWI/SNF complex.

We have identified the CYP3A4 and ZNF185 genes as novel transcriptional targets of the hSWI/SNF complex. The CYP3A4 protein belongs to the cytochrome P450 (CYP) family, which are haeme-containing enzymes, which metabolize a wide variety of endogenous substrates and xenobiotics including carcinogens (28Go). CYP3A4 gene transcription is induced by several nuclear receptors such as the pregnane X receptor (PXR), the orphan receptor CAR, the vitamin D3 receptor (VDR) and the glucocorticoid receptor (GR) (29Go). Interestingly, some of these receptors, such as the VDR and the GR, regulate gene transcription through their interaction with components of the SWI/SNF complex (30Go,31Go). Thus, our observations provide further evidence of an interaction between these nuclear receptors and the hSWI/SNF complex. CYP3A4 is located within the CYP3A locus that consists of at least three different genes and several pseudogenes, which are highly homologous in sequence and probably originated from a common gene ancestor (32Go). Notably, our results show that BRG1 is specifically recruited to the CYP3A4 but not to the CYP3A5 or CYP3A7 gene promoter. This observation agrees with previous findings that some of the nuclear receptors that bind to the SWI/SNF complex, such as the PXR, specifically regulate CYP3A4 but not CYP3A5 gene transcription (33Go). In normal lung epithelia, the predominant CYP3A transcript is CYP3A5, whereas that of CYP3A4 is present in 20% of the individuals and that of CYP3A7 is rarely detected (34Go). Endogenous CYP3A4 transcript was not detected in any of the eight lung cancer cell lines evaluated, indicating that the cells from which the tumours originated either did not carry CYP3A4 or were lost during tumour development. The latter explanation could account for the H1299 cells that expressed CYP3A4 after restoring BRG1 wild-type but not mutant activity. Still, how CYP3A4 loss contributes to tumour development remains to be fully understood, but one possibility is that CYP3A4 loss permits carcinogens to remain inside the cell for longer, thereby facilitating tumourigenesis.

The ZNF185 protein encodes a LIM-domain zinc finger protein that may be involved in protein–protein interactions. To date, little is known about the biological function of ZNF185 or its possible involvement in cancer development. Expression of ZNF185 has been reported to be specific to some adult tissues such as prostate, testis, ovary, peripheral blood or placenta (35Go). Our data show that ZNF185 is moderately expressed in normal lung and that severely decreased levels of the ZNF185 transcript are common in lung primary tumours and cancer cell lines. Supporting our observations, downregulation of ZNF185 is known to occur in head and neck squamous cell carcinoma, a tumour that is very similar to the lung cancer squamous cell carcinoma cell type (36Go). In lung cancer cell lines, absence of BRG1 protein occurred in parallel with the downregulation of ZNF185, although ZNF185 downregulation was far more common than BRG1 downregulation in primary tumours. Although the presence of inactivating missense point mutations at the coding region can inactivate BRG1 without altering the levels of the transcript (37Go), the presence of these alterations represents less than one-tenth of BRG1 mutations in lung cancer (7Go,10Go), suggesting additional mechanisms to BRG1 inactivation must exist, which lead to decreased expression of ZNF185 in lung tumours. Among these potential mechanisms are inactivation of other hSWI/SNF components, point mutations and deletions at the ZNF185 gene and gene promoter hypermethylation. As evidence for the latter, recent data demonstrated transcriptional silencing by gene promoter hypermethylation in prostate tumours (38Go).

BRM is another ATPase, highly homologous to BRG1, which can be alternatively used by the SWI/SNF complex as a catalyitic subunit. Previous data have shown that in cancer cells carrying simultaneous absence of BRG1 and BRM proteins, either BRM or BRG1 can induce RB-mediated cell growth arrest (8Go) or restore CD44 expression (39Go), indicating that BRM can functionally substitute for BRG1 and vice versa. However, it has also been reported certain specificity of both ATPases in transcriptional regulation (8Go,18Go). In the present work, we demonstrated that transcriptional activation of CYP3A4 and ZNF185 is exclusively mediated by BRG1, further supporting that both ATPases are not completely interchangeable.

In conclusion, our results demonstrate that CYP3A4 and ZNF185 genes are transcriptionally regulated through the hSWI/SNF complex and give insight into how inactivation of chromatin-remodelling complexes may contribute to lung carcinogenesis. Our observations that decreased ZNF185 expression is a frequent feature of lung primary tumours and lung cancer cell lines, further highlighting the relevance of this protein in cancer development. Additional research, both functional and genetic/epigenetic, will definitively establish the precise role of ZNF185 and CYP3A4 in lung cancer development


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
REFERENCES
 
Cell lines and primary tumours
Cell lines were obtained from the American Type Culture Collection (ATCC, Rockville, MD, USA) and grown under recommended conditions. All cells tested negative for mycoplasma infection. Tumour tissues from 27 lung cancer patients (13 squamous cell carcinomas and 14 adenocarcinomas) were provided by the CNIO Tumour Bank Network, CNIO (Madrid, Spain), in collaboration with the Hospital Universitario 12 de Octubre. Cases were selected, on the basis of the availability of frozen tissue.

Expression vectors and transfection assays
H1299 cells were plated in culture flasks in RPMI (Sigma Chemical, Spain) medium containing 10% (v/v) fetal bovine serum, 2 mM L-glutamine, 50 mg/ml penicillin/streptomycin and 2.5 µg/ml fungizone. The cultures were maintained at 37°C in a humidified atmosphere of 5% CO2/95%O2. Cells were cultured up to 70% confluence in 75 cm2 flasks and transfected with pCMV-BRG1-HA vector (which carries wild-type BRG1) (26Go), pCMV-BRG1(K798R)-HA vector (which carries the mutant form K798R) (26Go) and the empty vector, following the manufacturer's protocol (Transfast Transfection Reagent, Promega, WI, USA). Protein and total RNA were extracted from the cells at different times (6, 12, 24 and 48 h), following transfection with wild-type BRG1, or 24 h following transfection with BRG1-K798R, or with empty vector.

For BRM transfection, we used the pCG-HA-BRM vector (40Go) using the same protocols. Proteins and RNA were extracted 24 h following transfection.

Immunocytochemical identification of BRG1 protein
For subcellular localization of BRG1 protein, we performed an immunocytochemical assay. Cells growing on coverslips were transfected with pCMV-BRG1-HA. After 24 h, the coverslips were fixed in cold methanol (2 min at –20°C). The preparation was washed in PBS and blocked with 3% BSA/0.1% Triton X-100 in PBS. Primary mouse anti-HA antibody (Santa Cruz Biotechnology, Santa Cruz, CA, USA) was diluted 1 : 200 and incubated for 1 h. Excess antibody was removed by washing three times in blocking medium. Labelling was revealed with anti-mouse IgG-Alexa488 (Molecular Probes, Eugene, OR, USA) at 1 : 200 and incubated for 1 h. Cell nuclei were revealed by incubation with 1 µM DAPI (48,6-diamidino-2-phenylindole) for 5 min. All preparations were mounted with DePeX mounting medium (BDH Laboratory Supplies, Poole, UK). Fluorescence was analyzed under the microscope.

Flow cytometry
The H1299 lung cancer cells transfected with either pCMV-BRG1-HA or pCMV-BRG1(K798R)-HA and used for FACS analysis. Twenty-four and 48 h after transfection, the cells were trypsinized, fixated with cold ethanol 70% and permeabilizated with Triton 0.25%, incubated with Monoclonal Mouse HA antibody (Covance Research Products, Denver, PA, USA) and after with the Anti-mouse FITC conjugated. Then, the cells were stained with propidium iodide and data acquisition was performed using a flow cytometer FACS Calibur (Becton Dickinson, Mountain View, CA, USA). Only the BRG1 positive cells were selected for measures of the cell cycle.

Semi-quantitative RT–PCR, real-time quantitative RT–PCR (TaqMan) and western blotting
Total RNA was collected from cell lines using the RNeasy kit (Qiagen Inc., Valencia, CA, USA) and from primary tumours as previously described (41Go) using the Trizol reagent (Life Technologies, Inc., Grand Island, NY, USA), followed by the RNeasy kit and digestion with RNase-free DNase I, following the manufacturers' instructions. A small aliquot of RNA was separated for quantification and for quality control. Reverse transcription was performed with 1 µg RNA following the manufacturer's protocol (Reverse Transcription System, Promega, WI, USA) using a random hexamer primer (primary tumours) or oligo-dTs (cell lines).

For RT–PCR, 30 ng of cDNA was amplified using the following cycling conditions: 2 min at 94°C for initial denaturation and 28–35 cycles of amplification (for 30 s at 95°C, 30 s at 55°C and 30 s at 72°C). After the final cycle, a final extension (5 min at 72°C) was added. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used to normalize variations in the input cDNA. The primer sequences were as follows: for GAPDH, 5'-TCTTCTTTTGCGTCGCCAG-3' (forward) and 5'-AGCCCCAGCCTTCTCCA-3' (reverse); for BRG1, 5'-GCTCATGGCTGAAGATGAGG-3' (forward) and 5'-CAGGCGTCTGTCCTTCTGC-3' (reverse); for ZNF185, 5'-GCGGATCTGAGCAACTTGTC-3' (forward) and 5'-TTCTGGGGTACTGGGATCTG-3' (reverse) for CYP3A4, 5'-GGTGATGATTCCAAGCTATG-3' (forward) and 5'-GGAAATAGTCCCGTGAGAAG-3' (reverse).

To quantify BRG1 RNA levels in lung cancer cell lines and primary tumours, we performed real-time quantitative PCR analysis (TaqMan Technology) using commercially available assay-on demand probe–primer sets (Applied Biosystem, Foster City, CA, USA). For TaqMan PCR assay, cDNA from 25 ng of total RNA were used to measure gene expression with an ABI Prism 7700 sequence detection system and TaqMan Universal PCR Master Mix reagent (Applied Biosystems). Target cDNA was amplified in duplicate using the following cycling conditions: 2 min at 50°C, 10 min at 95°C and then 40 cycles of amplification (95°C for 15 s and 60°C for 30 s). The TATA binding protein (TBP) (Human TBP Pre-Developed TaqMan Assay Reagents, Applied Biosystems) was used to normalize variations in the input cDNA. The threshold cycle (Ct) was determined and relative gene expression was calculated as: {Delta}Ct=Cttarget–CtTBP (cycle difference).

For western blotting, cells were scraped from the dishes into lysis buffer and 50 µg of total protein was separated by SDS–PAGE, as previously described, and blotted with rabbit anti-BRG1 H88 (1 : 500; Santa Cruz Biotechnology, Santa Cruz, CA, USA) or mouse anti-HA antibody (Santa Cruz Biotechnology). The secondary antibody anti-rabbit-IgG : HRP or anti-mouse-IgG : HRP (Santa Cruz Biotechnology) was added to give a final dilution of 1 : 3000.

Preparations of labelled cDNA and hybridization of microarray
Thirty-five micrograms of total RNA were used for cDNA microarray analysis. Fluorescent-labelled cDNA was synthesized and hybridized to the CNIO OncoChipTM, as previously described (39Go). The CNIO OncoChipTM is a cDNA microarray, which has been especially designed for looking at genes involved in cancer and includes a core of 2489 cancer-relevant genes in addition to genes involved in drug response, tissue-specific genes and control genes. There are a total of 6386 genes represented by 7237 clones. RNA from H1299 cells (labelled with Cy3) was hybridized against RNA from the H1299-treated cells (labelled with Cy5), as described previously (39Go). Slides were scanned for Cy3 and Cy5 fluorescence using Scanarray 5000 XL (GSI Lumonics Kanata, Ontario, Canada) and quantified using the Quantarray (GSI Lumonics) and/or GenePix Pro 5.0 programs (Axon instruments Inc., Union City, CA, USA).

Data analysis and normalization
Fluorescence intensity from each array element was subtracted from the local background. To normalize the data, Cy5/Cy3 ratios were global-median normalized. Substandard spots were manually flagged and excluded from further analysis. The Cy5/Cy3 ratios of the duplicated spots in the array were averaged. All ratio values were semi-log transformed (base 2). Inconsistent duplicates were excluded, whereas uniform duplicated spots were averaged. Gene profiles with <70% of available data were excluded from further analysis. This process yielded 6854 transcripts that were suitable for analysis.

Chromatin immunoprecipitation assay
ChIP assays were performed as previously described (42Go). Antibody against BRG1 was kindly provided by Dr Robert Kingston. The antibody against BRM, BRM N-19, was obtained from Santa Cruz Biotechnology. Preliminary fixation experiments were performed over a predetermined time-course. Cells were then fixed in 1% formaldehyde for between 5 min and 1 h, and final conditions were chosen, which yielded the best combination of in vivo fixed chromatin, high DNA recovery and small average size of chromatin fragments. In our analyses, chromatin was sheared to an average length of 0.25–1 kb. PCR amplification was performed in 25 µl with specific primers for each of the analyzed promoters. For each promoter, the sensitivity of PCR amplification was evaluated in serial dilutions of total DNA collected after sonication (input fraction). Two independent ChIP experiments were performed. In PCR amplifications, the input DNA, antibody control and ‘bound’ fractions for each antibody were run in 2% agarose gels.

Primer sequences were as follows: for ZNF185, 5'-CGGAAACAGGAAGCAAAGAC-3' (forward) and 5'-CTGTTCTCACCAGACTGGCA-3' (reverse); for TNFSF13, 5'-AGGAGGCAGACTTCCTCCTG-3' (forward) and 5'-CTTACATAAGGCTCTGGACG-3' (reverse); for CYPA34, 5'-ATAAGAACCCAGAACCCTTGG-3' (forward) and 5'-CCTGCACAGCAGTGATTCAG-3' (reverse); for TNFSF10, 5'-CACTGAAGCCCTTCCTTCTC-3' (forward) and 5'-AAATGGGCTTGAGGTGAGTG-3' (reverse); for CSF1, 5'-GTCCCTTGGGACGATCATAG-3' (forward) and 5'-GTGGTTTATGGGAAATCACCC-3' (reverse).

RT–PCR and RFLP analysis
To identify which CYP3A transcript was present in particular lung cancer cell lines, we performed RT–PCR (primer sequences available upon request), thereby obtaining a PCR fragment of 272 bp. Afterwards, RFLP analysis was carried out with HinfI and AluI restriction enzymes. Digestion with HinfI (Promega, Madison, WI, USA) yields two distinct fragments (120 and 152 bp) if either the CYP3A5 or the CYP3A7 transcripts are present, or three distinct fragments (68, 88 and 120 bp) if the CYP3A4 transcript is present. On the other hand, restriction analysis with AluI yields two distinct fragments (144 and 128 bp) if either the CYP3A4 or the CYP3A7 transcripts are present, or an uncut fragment of 272 bp if the CYP3A5 transcript is present. Digested products were resolved on 2% agarose gels.


   ACKNOWLEDGEMENTS
 
We thank all our collaborators in the Tumour Bank Network for providing normal and lung tumour tissues. The authors acknowledge the provision of the BRG1 expression vectors by Dr Daniel M. Murphy, University of Virginia and of the BRM expression vector by Drs Muchardt and Yaniv from the Institute Pasteur, Paris. We also thank Dr Robert Kingston for his generous gift of the anti-BRG1 antibody. P.P.M. is a fellow of the Comunidad Autonoma de Madrid, J.C. is a postdoctoral fellow of the Excmo. Ayuntamiento de Madrid and M.S.-C. and E.B. are supported by the Ramon y Cajal Programme of the Ministerio de Ciencia y Tecnología, Spain. The research was partially supported by a grant from the Ministerio de Ciencia y Tecnología (SAF2002-01595) and the Comunidad Autonoma de Madrid (CAM 08.1/0032/2003 1).


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 

  1. Sudarsanam, P. and Winston, F. (2000) The Swi/Snf family nucleosome-remodeling complexes and transcriptional control. Trends Genet., 16, 345–351.[CrossRef][ISI][Medline]

  2. Peterson, C.L. (2002) Chromatin remodeling: nucleosomes bulging at the seams. Curr. Biol., 12, R245–R247.[CrossRef][ISI][Medline]

  3. Klochendler-Yeivin, A., Muchardt, C. and Yaniv, M. (2002) SWI/SNF chromatin remodelling and cancer. Curr. Opin. Genet. Dev., 12, 73–79.[CrossRef][ISI][Medline]

  4. Neely, K.E. and Workman, J.L. (2002) The complexity of chromatin remodelling and its links to cancer. Biochim. Biophys. Acta, 1603, 19–29.[Medline]

  5. Roberts, C.W. and Orkin, S.H. (2004) The SWI/SNF complex-chromatin and cancer. Nat. Rev. Cancer, 4, 133–142.[ISI][Medline]

  6. Versteege, I., Sevenet, N., Lange, J., Rousseau-Merck, M.F., Ambros, P., Handgretinger, R., Aurias, A. and Delattre, O. (1998) Truncating mutations of hSNF5/INI1 in aggressive paediatric cancer. Nature, 394, 203–206.[CrossRef][Medline]

  7. Wong, A.K., Shanahan, F., Chen, Y., Lian, L., Ha, P., Hendricks, K., Ghaffari, S., Iliev, D., Penn, B., Woodland, A.M. et al. (2000) BRG1, a component of the SWI-SNF complex, is mutated in multiple human tumor cell lines. Cancer Res., 60, 6171–6177.[Abstract/Free Full Text]

  8. Reisman, D.N., Strobeck, M.W., Betz, B.L., Sciariotta, J., Funkhouser, W., Jr, Murchardt, C., Yaniv, M., Sherman, L.S., Knudsen, E.S., and Weissman, B.E. (2002) Concomitant down-regulation of BRM and BRG1 in human tumor cell lines: differential effects on RB-mediated growth arrest vs. CD44 expression. Oncogene, 21, 1196–1207.[CrossRef][ISI][Medline]

  9. Reisman, D.N., Sciarrotta, J., Wang, W., Funkhouser, W.K. and Weissman, B.E. (2003) Loss of BRG1/BRM in human lung cancer cell lines and primary lung cancers: correlation with poor prognosis. Cancer Res., 63, 560–566.[Abstract/Free Full Text]

  10. Medina, P.P., Carretero, J., Fraga, M.F., Esteller, M., Sidransky, D. and Sanchez-Cespedes, M. (2004) Genetic and epigenetic screening for gene alterations of the chromatin-remodeling factor, SMARCA4/BRG1 in lung tumors. Genes Chromosomes Cancer, 41, 170–177.[CrossRef][ISI][Medline]

  11. Fukuoka, J., Fujii, T., Shih, J.H., Dracheva, T., Meerzaman, D., Player, A., Hong, K., Settnek, S., Gupta, A., Buetow, K. et al. (2004) Chromatin remodeling factors and BRM/BRG1 expression as prognostic indicators in non-small cell lung cancer. Clin. Cancer Res., 10, 4314–4324.[Abstract/Free Full Text]

  12. Bultman, S., Gebuhr, T., Yee, D., La Mantia, C., Nicholson, J., Gilliam, A., Randazzo, F., Metzger, D., Chambon, P., Crabtree, G., Magnuson, T. (2000) A Brg1 null mutation in the mouse reveals functional differences among mammalian SWI/SNF complexes. Mol. Cell, 6, 1287–1295.[CrossRef][ISI][Medline]

  13. Krebs, J.E., Fry, C.J., Samuels, M.L. and Peterson, C.L. (2000) Global role for chromatin remodeling enzymes in mitotic gene expression. Cell, 102, 587–598.[CrossRef][ISI][Medline]

  14. Barker, N., Hurlstone, A., Musisi, H., Miles, A., Bienz, M. and Clevers, H. (2001) The chromatin remodelling factor Brg-1 interacts with beta-catenin to promote target gene activation. EMBO J., 20, 4935–4943.[CrossRef][ISI][Medline]

  15. Marignani, P., Kanai, F. and Carpenter, C.L.(2001) LKB1 associates with Brg1 and is necessary for Brg1-induced growth arrest. J. Biol. Chem., 276, 32415–32418.[Abstract/Free Full Text]

  16. Otsuki, T., Furukawa, Y., Ikeda, K., Endo, H., Yamashita, T., Shinohara, A., Iwamatsu, A., Ozawa, K. and Liu, J.M. (2001) Fanconi anemia protein, FANCA, associates with BRG1, a component of the human SWI/SNF complex. Hum. Mol. Genet., 10, 2651–2660.[Abstract/Free Full Text]

  17. Sudarsanam, P., Iyer, V.R., Brown, P.O. and Winston, F. (2000) Whole-genome expression analysis of snf/swi mutants of Saccharomyces cerevisiae. Proc. Natl Acad, Sci. USA, 97, 3364–3369.[Abstract/Free Full Text]

  18. Kadam, S. and Emerson, B.M. (2003) Transcriptional specificity of human SWI/SNF BRG1 and BRM chromatin remodeling complexes. Mol. Cell, 11, 377–389.[CrossRef][ISI][Medline]

  19. Pattenden, S.G., Klose, R., Karaskov, E. and Bremner, R. (2002) Interferon-gamma-induced chromatin remodeling at the CIITA locus is BRG1 dependent. EMBO J., 21, 1978–1986.[CrossRef][ISI][Medline]

  20. Liu, R., Liu, H., Chen, X., Kirby, M., Brown, P.O. and Zhao, K. (2001) Regulation of CSF1 promoter by the SWI/SNF-like BAF complex. Cell, 106, 309–318.[CrossRef][ISI][Medline]

  21. Sullivan, E.K., Weirich, C.S., Guyon, J.R., Sif, S. and Kingston, R.E. (2001) Transcriptional activation domains of human heat shock factor 1 recruit human SWI/SNF. Mol. Cell. Biol., 21, 5826–5837.[Abstract/Free Full Text]

  22. Kang, H., Cui, K. and Zhao, K. (2004) BRG1 controls the activity of the retinoblastoma protein via regulation of p21(CIP1/WAF1/SDI). Mol. Cell. Biol., 24, 1188–1199.[Abstract/Free Full Text]

  23. Liu, K., Luo, Y., Lin, F.T., and Lin, W.C. (2004) TopBP1 recruits Brg1/Brm to repress E2F1-induced apoptosis, a novel pRb-independent and E2F1-specific control for cell survival. Genes Dev., 18, 673–686.[Abstract/Free Full Text]

  24. Huang, M., Qian, F., Hu, Y., Ang, C., Li, Z., and Wen, Z. (2002) Chromatin-remodelling factor BRG1 selectively activates a subset of interferon-alpha-inducible genes. Nat. Cell. Biol., 4, 774–781.[CrossRef][ISI][Medline]

  25. Hendricks, K.B., Shanahan, F. and Lees, E. (2004) Role for BRG1 in cell cycle control and tumor suppression. Mol. Cell. Biol., 24, 362–376.[Abstract/Free Full Text]

  26. Murphy, D.J., Hardy, S. and Engel, D.A. (1999) Human SWI–SNF component BRG1 represses transcription of the c-fos gene. Mol. Cell. Biol., 19, 2724–2733.[Abstract/Free Full Text]

  27. Martens, J.A. and Winston, F. (2003) Recent advances in understanding chromatin remodeling by Swi/Snf complexes. Curr. Opin. Genet. Dev., 13, 136–142.[CrossRef][ISI][Medline]

  28. Nelson, D.R. (1999) Cytochrome P450 and the individuality of species. Arch. Biochem. Biophys., 369, 1–10.

  29. Pascussi, J.M., Gerbal-Chaloin, S., Drocourt, L., Maurel, P. and Vilarem, M.J. (2003) The expression of CYP2B6, CYP2C9 and CYP3A4 genes: a tangle of networks of nuclear and steroid receptors. Biochim. Biophys. Acta, 1619, 243–253.[Medline]

  30. Hsiao, P.W., Fryer, C.J., Trotter, K.W., Wang, W. and Archer, T.K. (2003) BAF60a mediates critical interactions between nuclear receptors and the BRG1 chromatin-remodeling complex for transactivation. Mol. Cell. Biol., 23, 6210–6220.[Abstract/Free Full Text]

  31. Fryer, C.J. and Archer, T.K. (1998) Chromatin remodelling by the glucocorticoid receptor requires the BRG1 complex. Nature, 393, 88–91.[CrossRef][Medline]

  32. Meyer, U.A. and Wojnowski, L. (2001) Genomic organization of the human CYP3A locus: identification of a new, inducible CYP3A gene. Pharmacogenetics, 11, 111–121.[CrossRef][ISI][Medline]

  33. Blumberg, B., Sabbagh, W.Jr, Juguilon, H., Bolado, J. Jr, van Meter, C.M., Ong, E.S. and Evans, R.M. (1998) SXR, a novel steroid and xenobiotic-sensing nuclear receptor. Genes Dev., 12, 3195–3205.[Abstract/Free Full Text]

  34. Anttila, S., Hukkanen, J., Hakkola, J., Stjernvall, T., Beaune, P., Edwards, R.J., Boobis, A.R., Pelkonen, O. and Raunio, H. (1997) Expression and localization of CYP3A4 and CYP3A5 in human lung. Am. J. Respir. Cell Mol. Biol., 16, 242–249.[Abstract]

  35. Heiss, N.S., Gloeckner, G., Bachner, D., Kioschis, P., Klauck, S.M., Hinzmann, B., Rosenthal, A., Herman, G.E. and Poustka, A. (1997) Genomic structure of a novel LIM domain gene (ZNF185) in Xq28 and comparisons with the orthologous murine transcript. Genomics, 43, 329–338.[CrossRef][ISI][Medline]

  36. Gonzalez, H.E., Gujrati, M., Frederick, M., Henderson, Y., Arumugam, J., Spring, P.W., Mitsudo, K., Kim, H.W. and Clayman, G.L. (2003) Identification of 9 genes differentially expressed in head and neck squamous cell carcinoma. Arch. Otolaryngol. Head Neck Surg., 129, 754–759.[Abstract/Free Full Text]

  37. Richmond, E. and Peterson, C.L. (1996) Functional analysis of the DNA-stimulated ATPase domain of yeast SWI2/SNF2. Nucleic Acids Res., 24, 3685–3692.[Abstract/Free Full Text]

  38. Vanaja, D.K., Cheville, J.C., Iturria, S.J. and Young, C.Y. (2003) Transcriptional silencing of zinc finger protein 185 identified by expression profiling is associated with prostate cancer progression. Cancer Res., 63, 3877–3882.[Abstract/Free Full Text]

  39. Strobeck, M.W., Reisman, D.N., Gunawardena, R.W., Betz, B.L., Angus, S.P., Knudsen, K.E., Kowalik, T.F., Weissman, B.E. and Knudsen, E.S. (2002) Compensation of BRG-1 function by Brm: insight into the role of the core SWI–SNF subunits in retinoblastoma tumor suppressor signaling. J. Biol. Chem., 277, 4782–4789.[Abstract/Free Full Text]

  40. Bourachot, B., Yaniv, M., and Muchardt, C. (2003) Growth inhibition by the mammalian SWI±SNF subunit Brm is regulated by acetylation. EMBO J., 22, 6505–6515.[CrossRef][ISI][Medline]

  41. Fernandez, P., Carretero, J., Medina, P.P., Jimenez, A.I., Rodriguez-Perales, S., Paz, M.F., Cigudosa, J.C., Esteller, M., Lombardia, L., Morente, M, et al. (2004) Distinctive gene expression of human lung adenocarcinomas carrying LKB1 mutations. Oncogene, 23, 5084–5091.[CrossRef][ISI][Medline]

  42. Fournier, C., Goto, Y., Ballestar, E., Delaval, K., Hever, A., Esteller, M. and Feil, R. (2002) Allele-specific histone lysine methylation marks regulatory regions at imprinted mouse genes. EMBO J., 21, 6560–6570.[CrossRef][ISI][Medline]


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