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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. SK-MEL5, SK-MEL5+empty vector (EV), SK-MEL5+BRG1 cell pellets. Cells were grown to 90% confluency, pelleted, and photographed. B. Detection by Western blotting of tyrosinase and TRP1 in human melanocytes, SK-MEL5, SKMEL5+empty vector (EV), and SK-MEL5+BRG1. Tubulin is a loading control. C. Sensitivity of BRG1 deficient and BRG1 expressing melanoma cells to cisplatin. SK-MEL5, SK-MEL5+empty vector (EV), SK-MEL5+BRG1 cells were treated with 20 μM cisplatin for 72 h and assayed. These results were analyzed by Student’s t test and are representative of two independent experiments performed in triplicate.
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Figure 3: A. SK-MEL5, SK-MEL5+empty vector (EV), SK-MEL5+BRG1 cell pellets. Cells were grown to 90% confluency, pelleted, and photographed. B. Detection by Western blotting of tyrosinase and TRP1 in human melanocytes, SK-MEL5, SKMEL5+empty vector (EV), and SK-MEL5+BRG1. Tubulin is a loading control. C. Sensitivity of BRG1 deficient and BRG1 expressing melanoma cells to cisplatin. SK-MEL5, SK-MEL5+empty vector (EV), SK-MEL5+BRG1 cells were treated with 20 μM cisplatin for 72 h and assayed. These results were analyzed by Student’s t test and are representative of two independent experiments performed in triplicate.

Mentions: Melanoma cells are often less differentiated than their normal counterparts (Eberle et al., 1995). SK-MEL5 cells retain MITF expression but are amelanotic, suggesting that differentiation specific gene expression is suppressed downstream of MITF (note MITF expression, Fig. 2D). We found that ectopic expression of BRG1 in these cells led to a noticeable increase in pigmentation (Fig. 3A). Consistently, we detected increased expression of tyrosinase and tyrosinase related protein 1 (TRP1), two enzymes important for melanin synthesis (Fig. 3B). These results suggest that BRG1 can promote melanoma differentiation by activating MITF target gene expression. Over-expression of BRM to approximately four times the endogenous levels (supple. Fig. 1A, B, and C) did not increase visible pigmentation nor did it activate expression of most MITF target genes (supple. Fig. 1D and 1E), suggesting that when expressed at these levels, BRM cannot compensate for BRG1 in promoting differentiation of SK-MEL5 melanoma cells.


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. SK-MEL5, SK-MEL5+empty vector (EV), SK-MEL5+BRG1 cell pellets. Cells were grown to 90% confluency, pelleted, and photographed. B. Detection by Western blotting of tyrosinase and TRP1 in human melanocytes, SK-MEL5, SKMEL5+empty vector (EV), and SK-MEL5+BRG1. Tubulin is a loading control. C. Sensitivity of BRG1 deficient and BRG1 expressing melanoma cells to cisplatin. SK-MEL5, SK-MEL5+empty vector (EV), SK-MEL5+BRG1 cells were treated with 20 μM cisplatin for 72 h and assayed. These results were analyzed by Student’s t test and are representative of two independent experiments performed in triplicate.
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Related In: Results  -  Collection

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Figure 3: A. SK-MEL5, SK-MEL5+empty vector (EV), SK-MEL5+BRG1 cell pellets. Cells were grown to 90% confluency, pelleted, and photographed. B. Detection by Western blotting of tyrosinase and TRP1 in human melanocytes, SK-MEL5, SKMEL5+empty vector (EV), and SK-MEL5+BRG1. Tubulin is a loading control. C. Sensitivity of BRG1 deficient and BRG1 expressing melanoma cells to cisplatin. SK-MEL5, SK-MEL5+empty vector (EV), SK-MEL5+BRG1 cells were treated with 20 μM cisplatin for 72 h and assayed. These results were analyzed by Student’s t test and are representative of two independent experiments performed in triplicate.
Mentions: Melanoma cells are often less differentiated than their normal counterparts (Eberle et al., 1995). SK-MEL5 cells retain MITF expression but are amelanotic, suggesting that differentiation specific gene expression is suppressed downstream of MITF (note MITF expression, Fig. 2D). We found that ectopic expression of BRG1 in these cells led to a noticeable increase in pigmentation (Fig. 3A). Consistently, we detected increased expression of tyrosinase and tyrosinase related protein 1 (TRP1), two enzymes important for melanin synthesis (Fig. 3B). These results suggest that BRG1 can promote melanoma differentiation by activating MITF target gene expression. Over-expression of BRM to approximately four times the endogenous levels (supple. Fig. 1A, B, and C) did not increase visible pigmentation nor did it activate expression of most MITF target genes (supple. Fig. 1D and 1E), suggesting that when expressed at these levels, BRM cannot compensate for BRG1 in promoting differentiation of SK-MEL5 melanoma cells.

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