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Heterogeneous SWI/SNF chromatin remodeling complexes promote expression of microphthalmia-associated transcription factor target genes in melanoma.

Keenen B, Qi H, Saladi SV, Yeung M, de la Serna IL - Oncogene (2009)

Bottom Line: The microphthalmia-associated transcription factor (MITF) promotes melanocyte differentiation and cell-cycle arrest.SWI/SNF chromatin remodeling enzymes are multiprotein complexes composed of one of two related ATPases, BRG1 or BRM, and 9-12-associated factors (BAFs).Downregulation of the single ATPase, BRM, in SK-MEL5 cells inhibited expression of both differentiation-specific and pro-proliferative MITF target genes and inhibited tumorigenicity in vitro.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry and Cancer Biology, University of Toledo College of Medicine, Toledo, OH 43614, USA.

ABSTRACT
The microphthalmia-associated transcription factor (MITF) promotes melanocyte differentiation and cell-cycle arrest. Paradoxically, MITF also promotes melanoma survival and proliferation, acting like a lineage survival oncogene. Thus, it is critically important to understand the mechanisms that regulate MITF activity in melanoma cells. SWI/SNF chromatin remodeling enzymes are multiprotein complexes composed of one of two related ATPases, BRG1 or BRM, and 9-12-associated factors (BAFs). We previously determined that BRG1 interacts with MITF to promote melanocyte differentiation. However, it was unclear whether SWI/SNF enzymes regulate the expression of different classes of MITF target genes in melanoma. In this study, we characterized SWI/SNF subunit expression in melanoma cells and observed downregulation of BRG1 or BRM, but not concomitant loss of both ATPases. Re-introduction of BRG1 in BRG1-deficient SK-MEL5 cells enhanced expression of differentiation-specific MITF target genes and resistance to cisplatin. Downregulation of the single ATPase, BRM, in SK-MEL5 cells inhibited expression of both differentiation-specific and pro-proliferative MITF target genes and inhibited tumorigenicity in vitro. Our data suggest that heterogeneous SWI/SNF complexes composed of either the BRG1 or BRM subunit promote expression of distinct and overlapping MITF target genes and that at least one ATPase is required for melanoma tumorigenicity.

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A. Detection by Western blotting of ectopically expressed FLAG-BRG1 protein in BRG1 deficient SK-MEL5, SK-MEL5+empty vector (EV), or SK-MEL5+BRG1. FLAG-tagged BRG1 was detected with an antibody to the FLAG epitope and an antibody to BRG1. BRM levels in BRG1 deficient and BRG1 expressing cells were also probed. Tubulin is a loading control. B. Detection of ectopically expressed FLAG-BRG1 and BRM mRNA levels by real-time RT-PCR. BRG1 and BRM mRNA levels were standardized to 18S rRNA and are shown relative to those in melanocytes. C. Detection of FLAG-BRG1 by immunocytochemistry using a FLAG antibody. D. Coimmunoprecipitation of SWI/SNF components and MITF in BRG1 deficient and BRG1 expressing SK-MEL5 cells. Top panel: Cell lysates were immunoprecipitated with M2-FLAG agarose or control mouse IgG and blotted with the indicated antibodies. Bottom panel: Cell lysates were immunoprecipitated with MITF hybridoma supernatant or control mouse IgG and blotted with the indicated rabbit antibodies.
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Figure 2: A. Detection by Western blotting of ectopically expressed FLAG-BRG1 protein in BRG1 deficient SK-MEL5, SK-MEL5+empty vector (EV), or SK-MEL5+BRG1. FLAG-tagged BRG1 was detected with an antibody to the FLAG epitope and an antibody to BRG1. BRM levels in BRG1 deficient and BRG1 expressing cells were also probed. Tubulin is a loading control. B. Detection of ectopically expressed FLAG-BRG1 and BRM mRNA levels by real-time RT-PCR. BRG1 and BRM mRNA levels were standardized to 18S rRNA and are shown relative to those in melanocytes. C. Detection of FLAG-BRG1 by immunocytochemistry using a FLAG antibody. D. Coimmunoprecipitation of SWI/SNF components and MITF in BRG1 deficient and BRG1 expressing SK-MEL5 cells. Top panel: Cell lysates were immunoprecipitated with M2-FLAG agarose or control mouse IgG and blotted with the indicated antibodies. Bottom panel: Cell lysates were immunoprecipitated with MITF hybridoma supernatant or control mouse IgG and blotted with the indicated rabbit antibodies.

Mentions: Re-introduction of SWI/SNF components into cancer cells that lack expression has been demonstrated to result in cell cycle arrest, apoptosis, or differentiation, depending on the cancer cell type and the identity of the missing SWI/SNF subunit (Caramel et al., 2008; Dunaief et al., 1994; Wang et al., 2005; Yamamichi et al., 2007). To determine how BRG1 regulates the melanoma phenotype, we expressed epitope tagged BRG1 into BRG1 deficient SK-MEL5 cells by retroviral infection and selected a pool of cells. We obtained expression levels of BRG1 that were approximately twice that present in normal melanocytes (Fig. 2A and B). SK-Mel28 melanoma cells express BRG1 at approximately twice the levels present in melanocytes (Fig. 1A and B), thus the level of BRG1 expression that we obtained in SK-MEL5 cells is within a physiologically relevant range for melanocytic cells. We found that the selected cells grew slower (50% over a 6 day period) (data not shown) but could be propagated without loss of BRG1 expression (data not shown).


Heterogeneous SWI/SNF chromatin remodeling complexes promote expression of microphthalmia-associated transcription factor target genes in melanoma.

Keenen B, Qi H, Saladi SV, Yeung M, de la Serna IL - Oncogene (2009)

A. Detection by Western blotting of ectopically expressed FLAG-BRG1 protein in BRG1 deficient SK-MEL5, SK-MEL5+empty vector (EV), or SK-MEL5+BRG1. FLAG-tagged BRG1 was detected with an antibody to the FLAG epitope and an antibody to BRG1. BRM levels in BRG1 deficient and BRG1 expressing cells were also probed. Tubulin is a loading control. B. Detection of ectopically expressed FLAG-BRG1 and BRM mRNA levels by real-time RT-PCR. BRG1 and BRM mRNA levels were standardized to 18S rRNA and are shown relative to those in melanocytes. C. Detection of FLAG-BRG1 by immunocytochemistry using a FLAG antibody. D. Coimmunoprecipitation of SWI/SNF components and MITF in BRG1 deficient and BRG1 expressing SK-MEL5 cells. Top panel: Cell lysates were immunoprecipitated with M2-FLAG agarose or control mouse IgG and blotted with the indicated antibodies. Bottom panel: Cell lysates were immunoprecipitated with MITF hybridoma supernatant or control mouse IgG and blotted with the indicated rabbit antibodies.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2803337&req=5

Figure 2: A. Detection by Western blotting of ectopically expressed FLAG-BRG1 protein in BRG1 deficient SK-MEL5, SK-MEL5+empty vector (EV), or SK-MEL5+BRG1. FLAG-tagged BRG1 was detected with an antibody to the FLAG epitope and an antibody to BRG1. BRM levels in BRG1 deficient and BRG1 expressing cells were also probed. Tubulin is a loading control. B. Detection of ectopically expressed FLAG-BRG1 and BRM mRNA levels by real-time RT-PCR. BRG1 and BRM mRNA levels were standardized to 18S rRNA and are shown relative to those in melanocytes. C. Detection of FLAG-BRG1 by immunocytochemistry using a FLAG antibody. D. Coimmunoprecipitation of SWI/SNF components and MITF in BRG1 deficient and BRG1 expressing SK-MEL5 cells. Top panel: Cell lysates were immunoprecipitated with M2-FLAG agarose or control mouse IgG and blotted with the indicated antibodies. Bottom panel: Cell lysates were immunoprecipitated with MITF hybridoma supernatant or control mouse IgG and blotted with the indicated rabbit antibodies.
Mentions: Re-introduction of SWI/SNF components into cancer cells that lack expression has been demonstrated to result in cell cycle arrest, apoptosis, or differentiation, depending on the cancer cell type and the identity of the missing SWI/SNF subunit (Caramel et al., 2008; Dunaief et al., 1994; Wang et al., 2005; Yamamichi et al., 2007). To determine how BRG1 regulates the melanoma phenotype, we expressed epitope tagged BRG1 into BRG1 deficient SK-MEL5 cells by retroviral infection and selected a pool of cells. We obtained expression levels of BRG1 that were approximately twice that present in normal melanocytes (Fig. 2A and B). SK-Mel28 melanoma cells express BRG1 at approximately twice the levels present in melanocytes (Fig. 1A and B), thus the level of BRG1 expression that we obtained in SK-MEL5 cells is within a physiologically relevant range for melanocytic cells. We found that the selected cells grew slower (50% over a 6 day period) (data not shown) but could be propagated without loss of BRG1 expression (data not shown).

Bottom Line: The microphthalmia-associated transcription factor (MITF) promotes melanocyte differentiation and cell-cycle arrest.SWI/SNF chromatin remodeling enzymes are multiprotein complexes composed of one of two related ATPases, BRG1 or BRM, and 9-12-associated factors (BAFs).Downregulation of the single ATPase, BRM, in SK-MEL5 cells inhibited expression of both differentiation-specific and pro-proliferative MITF target genes and inhibited tumorigenicity in vitro.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry and Cancer Biology, University of Toledo College of Medicine, Toledo, OH 43614, USA.

ABSTRACT
The microphthalmia-associated transcription factor (MITF) promotes melanocyte differentiation and cell-cycle arrest. Paradoxically, MITF also promotes melanoma survival and proliferation, acting like a lineage survival oncogene. Thus, it is critically important to understand the mechanisms that regulate MITF activity in melanoma cells. SWI/SNF chromatin remodeling enzymes are multiprotein complexes composed of one of two related ATPases, BRG1 or BRM, and 9-12-associated factors (BAFs). We previously determined that BRG1 interacts with MITF to promote melanocyte differentiation. However, it was unclear whether SWI/SNF enzymes regulate the expression of different classes of MITF target genes in melanoma. In this study, we characterized SWI/SNF subunit expression in melanoma cells and observed downregulation of BRG1 or BRM, but not concomitant loss of both ATPases. Re-introduction of BRG1 in BRG1-deficient SK-MEL5 cells enhanced expression of differentiation-specific MITF target genes and resistance to cisplatin. Downregulation of the single ATPase, BRM, in SK-MEL5 cells inhibited expression of both differentiation-specific and pro-proliferative MITF target genes and inhibited tumorigenicity in vitro. Our data suggest that heterogeneous SWI/SNF complexes composed of either the BRG1 or BRM subunit promote expression of distinct and overlapping MITF target genes and that at least one ATPase is required for melanoma tumorigenicity.

Show MeSH
Related in: MedlinePlus