Pharmacologic inhibition of bromodomain and extra-terminal (BET) proteins is currently being explored as a new therapeutic approach in cancer. Some studies have also implicated BET proteins as ...regulators of cell identity and differentiation through their interactions with lineage-specific factors. However, the role of BET proteins has not yet been investigated in melanocyte differentiation. Melanocyte inducing transcription factor (MITF) is the master regulator of melanocyte differentiation, essential for pigmentation and melanocyte survival. In this study, we tested the hypothesis that BET proteins regulate melanocyte differentiation through interactions with MITF.
Here we show that chemical inhibition of BET proteins prevents differentiation of unpigmented melanoblasts into pigmented melanocytes and results in de-pigmentation of differentiated melanocytes. BET inhibition also slowed cell growth, without causing cell death, increasing the number of cells in G1. Transcriptional profiling revealed that BET inhibition resulted in decreased expression of pigment-specific genes, including many MITF targets. The expression of pigment-specific genes was also down-regulated in melanoma cells, but to a lesser extent. We found that RNAi depletion of the BET family members, bromodomain-containing protein 4 (BRD4) and bromodomain-containing protein 2 (BRD2) inhibited expression of two melanin synthesis enzymes, TYR and TYRP1. Both BRD4 and BRD2 were detected on melanocyte promoters surrounding MITF-binding sites, were associated with open chromatin structure, and promoted MITF binding to these sites. Furthermore, BRD4 and BRD2 physically interacted with MITF.
These findings indicate a requirement for BET proteins in the regulation of pigmentation and melanocyte differentiation. We identified changes in pigmentation specific gene expression that occur upon BET inhibition in melanoblasts, melanocytes, and melanoma cells.
Summary
Microphthalmia‐associated transcription factor (MITF) is a survival factor in melanocytes and melanoma cells. MITF regulates expression of antiapoptotic genes and promotes lineage‐specific ...survival in response to ultraviolet (UV) radiation and to chemotherapeutics. SWI/SNF chromatin‐remodeling enzymes interact with MITF to regulate MITF target gene expression. We determined that the catalytic subunit, BRG1, of the SWI/SNF complex protects melanoma cells against UV‐induced death. BRG1 prevents apoptosis in UV‐irradiated melanoma cells by activating expression of the melanoma inhibitor of apoptosis (ML‐IAP). Down‐regulation of ML‐IAP compromises BRG1‐mediated survival of melanoma cells in response to UV radiation. BRG1 regulates ML‐IAP expression by cooperating with MITF to promote transcriptionally permissive chromatin structure on the ML‐IAP promoter. The alternative catalytic subunit, BRM, and the BRG1‐associated factor, BAF180, were found to be dispensable for elevated expression of ML‐IAP in melanoma cells. Thus, we illuminate a lineage‐specific mechanism by which a specific SWI/SNF subunit, BRG1, modulates the cellular response to DNA damage by regulating an antiapoptotic gene and implicate this subunit of the SWI/SNF complex in mediating the prosurvival function of MITF.
•Expression of BRM and BRG1 is modulated by the ERK1/2 pathway in melanoma.•Suppression of ERK1/2 activity by MEK or BRAF inhibitors enhances BRM expression.•Activation of BRM by ERK1/2 inhibition is ...associated with chromatin modifications.•BRM has an anti-proliferative effect in melanoma cells that harbor oncogenic BRAF.•BRM becomes a pro-survival factor when ERK1/2 activity is suppressed.
Brahma (BRM) and Brahma-related gene 1(BRG1) are catalytic subunits of SWItch/sucrose non-fermentable (SWI/SNF) chromatin remodeling complexes. BRM is epigenetically silenced in a wide-range of tumors. Mutations in the v-raf murine sarcoma viral oncogene homolog B1 (BRAF) gene occur frequently in melanoma and lead to constitutive activation of the mitogen-activated protein kinase (MAPK)/extracellular signal regulated kinase (ERK1/2) pathway. We tested the hypothesis that BRM expression is modulated by oncogenic BRAF and phosphorylation of ERK1/2 in melanocytes and melanoma cells. Expression of oncogenic BRAF in melanocytes and melanoma cells that are wild-type for BRAF decreased BRM expression and increased BRG1 expression. Inhibition of mitogen-activated protein/extracellular signal-regulated kinase kinase (MEK) or selective inhibition of BRAF in melanoma cells that harbor oncogenic BRAF increased BRM expression and decreased BRG1 expression. Increased BRM expression was associated with increased histone acetylation on the BRM promoter. Over-expression of BRM in melanoma cells that harbor oncogenic BRAF promoted changes in cell cycle progression and apoptosis consistent with a tumor suppressive role. Upon inhibition of BRAF(V600E) with PLX4032, BRM promoted survival. PLX4032 induced changes in BRM function were correlated with increased acetylation of the BRM protein. This study provides insights into the epigenetic consequences of inhibiting oncogenic BRAF in melanoma through modulation of SWI/SNF subunit expression and function.
Mutations in SOX10 cause neurocristopathies which display varying degrees of hypopigmentation. Using a sensitized mutagenesis screen, we identified Smarca4 as a modifier gene that exacerbates the ...phenotypic severity of Sox10 haplo-insufficient mice. Conditional deletion of Smarca4 in SOX10 expressing cells resulted in reduced numbers of cranial and ventral trunk melanoblasts. To define the requirement for the Smarca4 -encoded BRG1 subunit of the SWI/SNF chromatin remodeling complex, we employed in vitro models of melanocyte differentiation in which induction of melanocyte-specific gene expression is closely linked to chromatin alterations. We found that BRG1 was required for expression of Dct, Tyrp1 and Tyr, genes that are regulated by SOX10 and MITF and for chromatin remodeling at distal and proximal regulatory sites. SOX10 was found to physically interact with BRG1 in differentiating melanocytes and binding of SOX10 to the Tyrp1 distal enhancer temporally coincided with recruitment of BRG1. Our data show that SOX10 cooperates with MITF to facilitate BRG1 binding to distal enhancers of melanocyte-specific genes. Thus, BRG1 is a SOX10 co-activator, required to establish the melanocyte lineage and promote expression of genes important for melanocyte function.
Summary
Microphthalmia‐associated transcription factor (
MITF
) is a survival factor in melanocytes and melanoma cells.
MITF
regulates expression of antiapoptotic genes and promotes lineage‐specific ...survival in response to ultraviolet (
UV
) radiation and to chemotherapeutics.
SWI
/
SNF
chromatin‐remodeling enzymes interact with
MITF
to regulate
MITF
target gene expression. We determined that the catalytic subunit,
BRG
1, of the
SWI
/
SNF
complex protects melanoma cells against
UV
‐induced death.
BRG
1 prevents apoptosis in
UV
‐irradiated melanoma cells by activating expression of the melanoma inhibitor of apoptosis (
ML
‐
IAP
). Down‐regulation of
ML
‐
IAP
compromises
BRG
1‐mediated survival of melanoma cells in response to
UV
radiation.
BRG
1 regulates
ML
‐
IAP
expression by cooperating with
MITF
to promote transcriptionally permissive chromatin structure on the
ML
‐
IAP
promoter. The alternative catalytic subunit,
BRM
, and the
BRG
1‐associated factor,
BAF
180, were found to be dispensable for elevated expression of
ML
‐
IAP
in melanoma cells. Thus, we illuminate a lineage‐specific mechanism by which a specific
SWI
/
SNF
subunit,
BRG
1, modulates the cellular response to
DNA
damage by regulating an antiapoptotic gene and implicate this subunit of the
SWI
/
SNF
complex in mediating the prosurvival function of
MITF
.
Bromodomains belong to a family of evolutionarily conserved protein modules originally found in proteins associated with chromatin and in nearly all nuclear histone acetyltransferases. Bromodomains ...modulate enzyme activities, protein assembly, and protein-protein interactions via lysine acetylation with broad implications in a wide variety of cellular events, such as transcriptional activation and chromatin remodeling. They are the sole protein domains which function as acetyl-lysine binding residues and are epigenetic readers which bind to the acetylated residues regulating chromatin structure and gene expression. We assessed two groups of bromodomain containing proteins with their relevance to melanocyte differentiation and melanoma. In the first study, the function of bromodomain (BRD) and extra-C terminal domain (BET) protein family in modulating pigmentation and melanocyte differentiation was deduced. In this study, we utilized an in vitro murine model of immortalized murine melanoblasts (melb-a cells) which can be differentiated by treatment with alpha-MSH. Alpha-MSH upon binding to melanocortin 1 receptor activates the microphthalmia-associated transcription factor (MITF) which is the master regulator of melanocyte differentiation and melanoma oncogene. MITF in turn activates genes required for melanin production, proliferation, and survival. JQ1 is a cell permeable small molecule inhibitor of BET proteins which binds to BRD4 with the highest affinity and specificity, inhibiting its binding to the acetylated lysine residues. JQ1 has an excellent shape complementarity with the acetyl-lysine binding cavity of bromodomains of BET family. Treating melb-a cells with JQ1 compromised MITF target gene expression, melanin synthesis, and reduced proliferation of melb-a cells as well as neonatal human epidermal melanocytes (NHEM) and melanoma cells. Furthermore, we determined that BRD4 occupied the promoters of MITF target genes that regulate pigmentation. Treatment with JQ1 inhibited the binding of both BRD4 and MITF to these promoters. Moreover, treating the melb-a cells as well as NHEMs disrupts histone H3 lysine 4 trimethylation (H3K4me3) which is an active mark of transcription. The effects on gene expression were recapitulated by siRNA mediated silencing of BRD4. We showed a physical interaction between the two proteins. Thus, we hypothesize that BRD4 plays a crucial role in melanocyte differentiation by interacting with MITF and can be a potential novel target for treatment of hyperpigmentation disorders and in melanoma. In the second project, the role of bromodomain containing proteins of an ATP-dependent chromatin remodeling complex SWI/SNF were assessed. Ultraviolet (UV)-radiation causes DNA damage and is implicated in the etiology of cutaneous melanoma, an aggressive malignancy that is notoriously chemoresistant. SWI/SNF chromatin remodeling enzymes have critical functions in the regulation of gene expression. Importantly, PBAF specific SWI/SNF subunits are disrupted in a number of human cancers including melanoma. We were interested in assessing a potential tumor suppressive role of PBAF complexes in the context of melanocytes and melanoma cells. We hypothesized that the PBAF specific SWI/SNF complex mediates tumor suppression by activating p53 target genes in response to UV-radiation. ARID2 has been identified as a gene that is frequently mutated in melanoma. We surveyed a panel of melanoma cell lines and found that a subset of melanoma cells is deficient in the expression of the other PBAF specific subunits, BAF180 and BRD7. Our data indicate that BRG1, BAF180, BRD7, and ARID2 promote expression of multiple p53 target genes as well as cell cycle arrest in UV-irradiated melanocytes and melanoma cells. BRG1, the central ATPase in PBAF SWI/SNF complex promoted cell cycle arrest in melanoma cells. Thus, our work suggests that a specific configuration of the SWI/SNF complex may have a tumor suppressive role in melanocytes and that aberrant expression or mutations of SWI/SNF components may contribute to melanoma tumorigenicity. (Abstract shortened by ProQuest.)
Globalization has added new dimensions to consumers’ decision-making. Consumer Ethnocentrism is among the critical variables affecting consumers’ decisions to purchase foreign products. Consumer ...Ethnocentrism research has gained momentum in the last few decades, and a copious amount of literature is available on this subject. This article presents an overview of Consumer Ethnocentrism literature and a compressive review of research work on the relationship between “Consumer Ethnocentrism” and “Product Evaluation” with critical insights from previous literature findings. There is a fractured opinion on the subject of whether Consumer Ethnocentrism leads to a lower quality evaluation of foreign products or whether consumer preference for the domestic product is purely out of a feeling that it is unfair to buy a foreign product. We also provided future research directions using TMC (Theory, Methodology, and Context) framework in line with earlier studies.
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