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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. Chromatin immunoprecipitations (ChIPs) were performed in BRG1 deficient SK-MEL5+empty vector (EV) or SK-MEL5+BRG1 cells using a FLAG antibody or control IgG, and run on an agarose gel stained with ethidium bromide. 1% input is shown. MITF target promoters were amplified with primers that span the MITF binding site. CD25 is a negative control. B. ChIPs from A were quantified by real-time PCR. The results presented are an average of two or more independent experiments done in triplicate. C. Change in BRG1 recruitment to MITF target promoters in SK-MEL5+BRG1 cells expressing siRNAs that target MITF compared to SK-MEL5+BRG1 cells expressing a control siRNA. BRG1 recruitment was analyzed by ChIP analysis as in Fig. 4B. The data is the average of two independent experiments performed in triplicate. All values are statistically significant at p<.01. D. Fold increase in trimethylation at histone H3 (K4) and RNA polymerase II (phsophoS2) recruitment at MITF target promoters in SK-MEL5 expressing BRG1 compared to that in control SK-MEL5 cells. The data is the average of two independent experiments performed in triplicate. E. MITF target gene expression in SK-MEL5+empty vector (EV) and SK-MEL5+BRG1 cells. mRNA levels were quantified by real-time RT-PCR. These results are an average of greater than three independent experiments performed in triplicate.
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Figure 4: A. Chromatin immunoprecipitations (ChIPs) were performed in BRG1 deficient SK-MEL5+empty vector (EV) or SK-MEL5+BRG1 cells using a FLAG antibody or control IgG, and run on an agarose gel stained with ethidium bromide. 1% input is shown. MITF target promoters were amplified with primers that span the MITF binding site. CD25 is a negative control. B. ChIPs from A were quantified by real-time PCR. The results presented are an average of two or more independent experiments done in triplicate. C. Change in BRG1 recruitment to MITF target promoters in SK-MEL5+BRG1 cells expressing siRNAs that target MITF compared to SK-MEL5+BRG1 cells expressing a control siRNA. BRG1 recruitment was analyzed by ChIP analysis as in Fig. 4B. The data is the average of two independent experiments performed in triplicate. All values are statistically significant at p<.01. D. Fold increase in trimethylation at histone H3 (K4) and RNA polymerase II (phsophoS2) recruitment at MITF target promoters in SK-MEL5 expressing BRG1 compared to that in control SK-MEL5 cells. The data is the average of two independent experiments performed in triplicate. E. MITF target gene expression in SK-MEL5+empty vector (EV) and SK-MEL5+BRG1 cells. mRNA levels were quantified by real-time RT-PCR. These results are an average of greater than three independent experiments performed in triplicate.

Mentions: We performed chromatin immunoprecipitations (ChIPs) to determine whether ectopically expressed BRG1 in SK-MEL5 cells is recruited to different classes of MITF target genes that regulate differentiation, proliferation, and survival. We detected BRG1 on the promoters of genes that encode enzymes needed for melanin synthesis, tyrosinase, tyrosinase related protein 1 (TRP1), and dopachrome tautomerase (DCT) (Fig.4A and B). We also detected BRG1 on the RAB27A promoter. RAB27A is a recently identified MITF target gene that regulates peripheral transport of mature melanosomes (Chiaverini et al., 2008). We also detected BRG1 on the p21CIP1 promoter, an anti-proliferative MITF target gene, associated with increased melanocyte differentiation (Carreira et al., 2005). Thus, BRG1 was recruited to multiple promoters that regulate genes important for pigmentation and overall differentiation status.


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. Chromatin immunoprecipitations (ChIPs) were performed in BRG1 deficient SK-MEL5+empty vector (EV) or SK-MEL5+BRG1 cells using a FLAG antibody or control IgG, and run on an agarose gel stained with ethidium bromide. 1% input is shown. MITF target promoters were amplified with primers that span the MITF binding site. CD25 is a negative control. B. ChIPs from A were quantified by real-time PCR. The results presented are an average of two or more independent experiments done in triplicate. C. Change in BRG1 recruitment to MITF target promoters in SK-MEL5+BRG1 cells expressing siRNAs that target MITF compared to SK-MEL5+BRG1 cells expressing a control siRNA. BRG1 recruitment was analyzed by ChIP analysis as in Fig. 4B. The data is the average of two independent experiments performed in triplicate. All values are statistically significant at p<.01. D. Fold increase in trimethylation at histone H3 (K4) and RNA polymerase II (phsophoS2) recruitment at MITF target promoters in SK-MEL5 expressing BRG1 compared to that in control SK-MEL5 cells. The data is the average of two independent experiments performed in triplicate. E. MITF target gene expression in SK-MEL5+empty vector (EV) and SK-MEL5+BRG1 cells. mRNA levels were quantified by real-time RT-PCR. These results are an average of greater than three independent experiments performed in triplicate.
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Figure 4: A. Chromatin immunoprecipitations (ChIPs) were performed in BRG1 deficient SK-MEL5+empty vector (EV) or SK-MEL5+BRG1 cells using a FLAG antibody or control IgG, and run on an agarose gel stained with ethidium bromide. 1% input is shown. MITF target promoters were amplified with primers that span the MITF binding site. CD25 is a negative control. B. ChIPs from A were quantified by real-time PCR. The results presented are an average of two or more independent experiments done in triplicate. C. Change in BRG1 recruitment to MITF target promoters in SK-MEL5+BRG1 cells expressing siRNAs that target MITF compared to SK-MEL5+BRG1 cells expressing a control siRNA. BRG1 recruitment was analyzed by ChIP analysis as in Fig. 4B. The data is the average of two independent experiments performed in triplicate. All values are statistically significant at p<.01. D. Fold increase in trimethylation at histone H3 (K4) and RNA polymerase II (phsophoS2) recruitment at MITF target promoters in SK-MEL5 expressing BRG1 compared to that in control SK-MEL5 cells. The data is the average of two independent experiments performed in triplicate. E. MITF target gene expression in SK-MEL5+empty vector (EV) and SK-MEL5+BRG1 cells. mRNA levels were quantified by real-time RT-PCR. These results are an average of greater than three independent experiments performed in triplicate.
Mentions: We performed chromatin immunoprecipitations (ChIPs) to determine whether ectopically expressed BRG1 in SK-MEL5 cells is recruited to different classes of MITF target genes that regulate differentiation, proliferation, and survival. We detected BRG1 on the promoters of genes that encode enzymes needed for melanin synthesis, tyrosinase, tyrosinase related protein 1 (TRP1), and dopachrome tautomerase (DCT) (Fig.4A and B). We also detected BRG1 on the RAB27A promoter. RAB27A is a recently identified MITF target gene that regulates peripheral transport of mature melanosomes (Chiaverini et al., 2008). We also detected BRG1 on the p21CIP1 promoter, an anti-proliferative MITF target gene, associated with increased melanocyte differentiation (Carreira et al., 2005). Thus, BRG1 was recruited to multiple promoters that regulate genes important for pigmentation and overall differentiation status.

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