Modulation of extracellular matrix/adhesion molecule expression by BRG1 is associated with increased melanoma invasiveness

<p>Abstract</p> <p>Background</p> <p>Metastatic melanoma is an aggressive malignancy that is resistant to therapy and has a poor prognosis. The progression of primary melanoma to metastatic disease is a multi-step process that requires dynamic regulation of gene expression through currently uncharacterized epigenetic mechanisms. Epigenetic regulation of gene expression often involves changes in chromatin structure that are catalyzed by chromatin remodeling enzymes. Understanding the mechanisms involved in the regulation of gene expression during metastasis is important for developing an effective strategy to treat metastatic melanoma. SWI/SNF enzymes are multisubunit complexes that contain either BRG1 or BRM as the catalytic subunit. We previously demonstrated that heterogeneous SWI/SNF complexes containing either BRG1 or BRM are epigenetic modulators that regulate important aspects of the melanoma phenotype and are required for melanoma tumorigenicity in vitro.</p> <p>Results</p> <p>To characterize BRG1 expression during melanoma progression, we assayed expression of BRG1 in patient derived normal skin and in melanoma specimen. BRG1 mRNA levels were significantly higher in stage IV melanomas compared to stage III tumors and to normal skin. To determine the role of BRG1 in regulating the expression of genes involved in melanoma metastasis, we expressed BRG1 in a melanoma cell line that lacks BRG1 expression and examined changes in extracellular matrix and adhesion molecule expression. We found that BRG1 modulated the expression of a subset of extracellular matrix remodeling enzymes and adhesion proteins. Furthermore, BRG1 altered melanoma adhesion to different extracellular matrix components. Expression of BRG1 in melanoma cells that lack BRG1 increased invasive ability while down-regulation of BRG1 inhibited invasive ability in vitro. Activation of metalloproteinase (MMP) 2 expression greatly contributed to the BRG1 induced increase in melanoma invasiveness. We found that BRG1 is recruited to the MMP2 promoter and directly activates expression of this metastasis associated gene.</p> <p>Conclusions</p> <p>We provide evidence that BRG1 expression increases during melanoma progression. Our study has identified BRG1 target genes that play an important role in melanoma metastasis and we show that BRG1 promotes melanoma invasive ability in vitro. These results suggest that increased BRG1 levels promote the epigenetic changes in gene expression required for melanoma metastasis to proceed.</p

Functional screen identifies regulators of murine hematopoietic stem cell repopulation.

Understanding the molecular regulation of hematopoietic stem and progenitor cell (HSPC) engraftment is paramount to improving transplant outcomes. To discover novel regulators of HSPC repopulation, we transplanted >1,300 mice with shRNA-transduced HSPCs within 24 h of isolation and transduction to focus on detecting genes regulating repopulation. We identified 17 regulators of HSPC repopulation: Arhgef5, Armcx1, Cadps2, Crispld1, Emcn, Foxa3, Fstl1, Glis2, Gprasp2, Gpr56, Myct1, Nbea, P2ry14, Smarca2, Sox4, Stat4, and Zfp251. Knockdown of each of these genes yielded a loss of function, except in the cases of Armcx1 and Gprasp2, whose loss enhanced hematopoietic stem cell (HSC) repopulation. The discovery of multiple genes regulating vesicular trafficking, cell surface receptor turnover, and secretion of extracellular matrix components suggests active cross talk between HSCs and the niche and that HSCs may actively condition the niche to promote engraftment. We validated that Foxa3 is required for HSC repopulating activity, as Foxa3(-/-) HSC fails to repopulate ablated hosts efficiently, implicating for the first time Foxa genes as regulators of HSPCs. We further show that Foxa3 likely regulates the HSC response to hematologic stress. Each gene discovered here offers a window into the novel processes that regulate stable HSPC engraftment into an ablated host

The Cancer Moonshot Immuno-Oncology Translational Network at 5: accelerating cancer immunotherapies.

The Immuno-Oncology Translational Network (IOTN) was established in 2018 as part of the Cancer Moonshot. In 2022, President Joe Biden set new goals to reduce the cancer death rate by half within 25 years and improve the lives of people with cancer and cancer survivors. The IOTN is focused on accelerating translation of cancer immunology research, from bench to bedside, and improving immunotherapy outcomes across a wide array of cancers in the adult population. The unique structure and team science approach of the IOTN is designed to accelerate discovery and evaluation of novel immune-based therapeutic and prevention strategies. In this article, we describe IOTN progress to date, including new initiatives and the development of a robust set of resources to advance cancer immunology research. We summarize new insights by IOTN researchers, some of which are ripe for translation for several types of cancers. Looking to the future, we identify barriers to the translation of immuno-oncology concepts into clinical trials and key areas for action and improvements that are suitable for high-yield investments. Based on these experiences, we recommend novel National Institutes of Health funding mechanisms and development of new resources to address these barriers

Dominant negative BRM and BRG1 inhibit SOX10-mediated activation of myelin genes.

<p>Cell lines that express dominant negative BRM or BRG1 were either infected with a pBabe control vector or pBabe-SOX10 in the presence or absence of tetracycline and then cultured in low serum media to promote differentiation. (A) Western Blot showing expression of SOX10 in cells that were cultured in the presence and absence of tetracycline and the expression of FLAG-tagged dominant negative BRM (left) in the H17 cell line and BRG1 (right) in the B22 cell line, when cells were cultured in the absence of tetracycline. Protein expression was detected from cell extracts and ERK1/2 was used as a loading control (B) Quantitative RT-PCR (qRT-PCR) of SOX10 target genes from pBabe or pBabe-SOX10 infected H17 (left) and B22 (right) cells. KROX20, MPZ, and MBP expression was normalized to expression of RPL7. The data are representative of at least four experiments and are the average of two independent experiments performed in triplicate. Standard error bars and statistical significance are shown (**p<0.01).</p

Dominant negative BRG1 inhibits synergistic activation of myelin genes by SOX10 and KROX20.

<p>Cell lines that express dominant negative BRG1 were infected with an empty vector, pBabe-SOX10, pBabe-KROX20, or pBabe-SOX10 together with pBabe-KROX20 in the presence or absence of tetracycline and then cultured in low serum media to promote differentiation. (A) Western Blot showing expression of SOX10 and KROX20 in the presence and absence of tetracycline and the expression of FLAG-tagged dominant negative BRG1 in the B22 cell line when cells were cultured in the absence of tetracycline. Protein expression was detected from nuclear extracts and a non-specific band was used as a loading control. (B) Quantitative RT-PCR (qRT-PCR) of SOX10 target genes from pBabe or pBabe-SOX10, pBabe-SOX10, pBabe-KROX20, or pBabe-SOX10 together with pBabe-Krox20 infected cells. Expression of MPZ and MBP was normalized to expression of RPL7. The data are representative of greater than three experiments and are the average of two independent experiments performed in triplicate. Standard error bars and statistical significance are shown (**p<0.01).</p

Dominant negative BRG1 inhibits expression of SOX10 target genes in S16 Schwann cells.

<p>S16 cells were transfected with GFP (not shown), empty vector (EV), or pBabe-dominant negative BRG1 (dnBRG1). Transfection efficiency was monitored by GFP and was determined to be approximately 30%. (A) Cell extracts were prepared and subjected to Western analysis with antibodies to the Flag epitope, SOX10, or KROX20. Tubulin is shown as a loading control. Quantitative RT-PCR (qRT-PCR) of SOX10 (B), KROX20 (C), MPZ (D), and MBP (E) from empty vector (EV) or pBabe-dnBRG1 transfected cells. Expression of each gene was normalized to that of 18S rRNA. The data are the average of three independent experiments performed in triplicate. Standard error bars are shown (**p<0.01).</p

Eutherian-Specific Functions of BetaM Acquired through <i>Atp1b4</i> Gene Co-Option in the Regulation of MyoD Expression

Vertebrate ATP1B4 genes represent a rare instance of orthologous gene co-option, resulting in radically different functions of the encoded BetaM proteins. In lower vertebrates, BetaM is a Na, K-ATPase β-subunit that is a component of ion pumps in the plasma membrane. In placental mammals, BetaM lost its ancestral role and, through structural alterations of the N-terminal domain, became a skeletal and cardiac muscle-specific protein of the inner nuclear membrane, highly expressed during late fetal and early postnatal development. We previously determined that BetaM directly interacts with the transcriptional co-regulator SKI-interacting protein (SKIP) and is implicated in the regulation of gene expression. This prompted us to investigate a potential role for BetaM in the regulation of muscle-specific gene expression in neonatal skeletal muscle and cultured C2C12 myoblasts. We found that BetaM can stimulate expression of the muscle regulatory factor (MRF), MyoD, independently of SKIP. BetaM binds to the distal regulatory region (DRR) of MyoD, promotes epigenetic changes associated with activation of transcription, and recruits the SWI/SNF chromatin remodeling subunit, BRG1. These results indicate that eutherian BetaM regulates muscle gene expression by promoting changes in chromatin structure. These evolutionarily acquired new functions of BetaM might be very essential and provide evolutionary advantages to placental mammals

The requirement for BRG1 during Schwann cell differentiation and myelination.

<p>A model illustrating the stepwise requirement for BRG1 during Schwann cell differentiation and myelination based on the current study as well as two recent studies [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0069037#B11" target="_blank">11</a>,<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0069037#B30" target="_blank">30</a>]..</p

SOX10 is required to recruit BRG1 to the MPZ promoter in S16 Schwann cells.

<p>A. Cell extracts were prepared from S16 cells that were transfected with siControl or siSOX10 and subjected to Western analysis with antibodies to SOX10, KROX20, and antisera to BRG1 (The starred top band (*) in the KROX20 Western may be a non-specific band. It was not used in the quantitation of KROX20 expression shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0069037#pone-0069037-g005" target="_blank">Figure 5B</a>. ERK1/2 is shown as a loading control. B. Quantitation of KROX20 protein levels relative to ERK1/2 protein levels by ImageJ software. The data are the average of three independent experiments. Standard error bars and statistical significance are shown (**p<0.01). C–E. Quantitative RT-PCR (qRT-PCR) of KROX20, MPZ, and MBP mRNA levels. F–H. ChIPs were performed with control IgG, antibodies to SOX10 or KROX20, or antisera to BRG1 on chromatin from S16 cells that were transfected with siControl or siSOX10. Enrichment on the MPZ promoter and intron is relative to control IgG and normalized to a control region, Ig2a enhancer. There was minimal variation in the ChIP signal at the Ig2A enhancer. (F) Detection of SOX10 interactions with the MPZ promoter. (G) Detection of KROX20 interactions with the MPZ promoter. (H) Detection of BRG1 interactions with the MPZ promoter. The data are representative of three independent experiments that were assayed three times. Standard errors bars and statistical significance are shown (**p<0.01, *p<0.05).</p

Eutherian-Specific Functions of BetaM Acquired through Atp1b4 Gene Co-Option in the Regulation of MyoD Expression

Vertebrate ATP1B4 genes represent a rare instance of orthologous gene co-option, resulting in radically different functions of the encoded BetaM proteins. In lower vertebrates, BetaM is a Na, K-ATPase &beta;-subunit that is a component of ion pumps in the plasma membrane. In placental mammals, BetaM lost its ancestral role and, through structural alterations of the N-terminal domain, became a skeletal and cardiac muscle-specific protein of the inner nuclear membrane, highly expressed during late fetal and early postnatal development. We previously determined that BetaM directly interacts with the transcriptional co-regulator SKI-interacting protein (SKIP) and is implicated in the regulation of gene expression. This prompted us to investigate a potential role for BetaM in the regulation of muscle-specific gene expression in neonatal skeletal muscle and cultured C2C12 myoblasts. We found that BetaM can stimulate expression of the muscle regulatory factor (MRF), MyoD, independently of SKIP. BetaM binds to the distal regulatory region (DRR) of MyoD, promotes epigenetic changes associated with activation of transcription, and recruits the SWI/SNF chromatin remodeling subunit, BRG1. These results indicate that eutherian BetaM regulates muscle gene expression by promoting changes in chromatin structure. These evolutionarily acquired new functions of BetaM might be very essential and provide evolutionary advantages to placental mammals