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Scientists have used pharmacological inhibitors of polycomb proteins to restore the expression of tumor suppressor genes and stop cancer proliferation and invasion. A major limitation of this approach is that key transcriptional activators, such as TP53 and BAF SWI/SNF, are often mutated in cancer. Poor clinical results for polycomb-targeting therapies in solid cancers, including triple-negative breast cancer (TNBC), could discourage the further development of epigenetic monotherapies. Here, we performed epigenome actuation with a synthetic reader-actuator (SRA) that binds trimethylated histone H3 lysine 27 in polycomb chromatin and modulates core transcriptional activators. In SRA-expressing TNBC BT-549 cells, 122 genes become upregulated ≥2-fold, including the genes involved in cell death, cell cycle arrest, and migration inhibition. The SRA-expressing spheroids showed reduced size in Matrigel and loss of invasion. Therefore, targeting Mediator-recruiting regulators to silenced chromatin can activate tumor suppressors and stimulate anti-cancer phenotypes, and further development of robust gene regulators might benefit TNBC patients.
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Scientists have used small molecule inhibitors and genetic knockdown of gene-silencing polycomb repressive complexes (PRC1/2) to determine if restoring the expression of tumor suppressor genes can block proliferation and invasion of cancer cells. A major limitation of this approach is that inhibitors can not restore key transcriptional activators that are mutated in many cancers, such as p53 and members of the BRAF SWI/SNF complex. Furthermore, small molecule inhibitors can alter the activity of, rather than inhibit, the polycomb enzyme EZH2. While chromatin has been shown to play a major role in gene regulation in cancer, poor clinical results for polycomb chromatin-targeting therapies for diseases like triple-negative breast cancer (TNBC) could discourage further development of this emerging avenue for treatment. To overcome the limitations of inhibiting polycomb to study epigenetic regulation, we developed an engineered chromatin protein to manipulate transcription. The synthetic reader-actuator (SRA) is a fusion protein that directly binds a target chromatin modification and regulates gene expression. Here, we report the activity of an SRA built from polycomb chromodomain and VP64 modules that bind H3K27me3 and subunits of the Mediator complex, respectively. In SRA-expressing BT-549 cells, we identified 122 upregulated differentially expressed genes (UpDEGs, ≥ 2-fold activation, adjusted p < 0.05). On-target epigenetic regulation was determined by identifying UpDEGs at H3K27me3-enriched, closed chromatin. SRA activity induced activation of genes involved in cell death, cell cycle arrest, and the inhibition of migration and invasion. SRA-expressing BT-549 cells showed reduced spheroid size in Matrigel over time, loss of invasion, and activation of apoptosis. These results show that Mediator-recruiting regulators broadly targeted to silenced chromatin activate silenced tumor suppressor genes and stimulate anti-cancer phenotypes. Therefore further development of gene-activating epigenetic therapies might benefit TNBC patients.
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To elucidate principles operating in native biological systems and to develop novel biotechnologies, synthetic biology aims to build and integrate synthetic gene circuits within native transcriptional networks. The utility of synthetic gene circuits for cell engineering relies on the ability to control the expression of all constituent transgene components. Transgene silencing, defined as the loss of expression over time, persists as an obstacle for engineering primary cells and stem cells with transgenic cargos. In this review, we highlight the challenge that transgene silencing poses to the robust engineering of mammalian cells, outline potential molecular mechanisms of silencing, and present approaches for preventing transgene silencing. We conclude with a perspective identifying future research directions for improving the performance of synthetic gene circuits.
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Redes Reguladoras de Genes , Ingeniería Genética , Animales , Transgenes/genética , Comunicación Celular , Mamíferos/genéticaRESUMEN
Chromatin is a system of proteins and DNA that regulates chromosome organization and gene expression in eukaryotes. Essential features that support these processes include biochemical marks on histones and DNA, 'writer' enzymes that generate or remove these marks and proteins that translate the marks into transcriptional regulation: reader-effectors. Here, we review recent studies that reveal how reader-effectors drive chromatin-mediated processes. Advances in proteomics and epigenomics have accelerated the discovery of chromatin marks and their correlation with gene states, outpacing our understanding of the corresponding reader-effectors. Therefore, we summarize the current state of knowledge and open questions about how reader-effectors impact cellular function and human disease and discuss how synthetic biology can deepen our knowledge of reader-effector activity.
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Cromatina , Epigenómica , Cromatina/genética , ADN/metabolismo , Epigénesis Genética , Histonas/metabolismo , HumanosRESUMEN
Rhabdomyosarcoma (RMS) is an aggressive pediatric soft tissue sarcoma. There are two main subtypes of RMS, alveolar rhabdomyosarcoma (ARMS) and embryonal rhabdomyosarcoma. ARMS typically encompasses fusion-positive rhabdomyosarcoma, which expresses either PAX3-FOXO1 or PAX7-FOXO1 fusion proteins. There are no targeted therapies for ARMS; however, recent studies have begun to illustrate the cooperation between epigenetic proteins and the PAX3-FOXO1 fusion, indicating that epigenetic proteins may serve as targets in ARMS. Here, we investigate the contribution of BMI1, given the established role of this epigenetic regulator in sustaining aggression in cancer. We determined that BMI1 is expressed across ARMS tumors, patient-derived xenografts, and cell lines. We depleted BMI1 using RNAi and inhibitors (PTC-209 and PTC-028) and found that this leads to a decrease in cell growth/increase in apoptosis in vitro, and delays tumor growth in vivo. Our data suggest that BMI1 inhibition activates the Hippo pathway via phosphorylation of LATS1/2 and subsequent reduction in YAP levels and YAP/TAZ target genes. These results identify BMI1 as a potential therapeutic vulnerability in ARMS and warrant further investigation of BMI1 in ARMS and other sarcomas.
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Proliferación Celular/fisiología , Epigénesis Genética/fisiología , Complejo Represivo Polycomb 1/fisiología , Rabdomiosarcoma/patología , Apoptosis/fisiología , Línea Celular Tumoral , Xenoinjertos , Vía de Señalización Hippo , Humanos , Fosforilación , Complejo Represivo Polycomb 1/genética , Interferencia de ARN , Rabdomiosarcoma/metabolismoRESUMEN
Neuroblastoma is an aggressive pediatric malignancy of the neural crest with suboptimal cure rates and a striking predilection for widespread metastases, underscoring the need to identify novel therapeutic vulnerabilities. We recently identified the RNA binding protein LIN28B as a driver in high-risk neuroblastoma and demonstrated it promotes oncogenic cell proliferation by coordinating a RAN-Aurora kinase A network. Here, we demonstrate that LIN28B influences another key hallmark of cancer, metastatic dissemination. Using a murine xenograft model of neuroblastoma dissemination, we show that LIN28B promotes metastasis. We demonstrate that this is in part due to the effects of LIN28B on self-renewal and migration, providing an understanding of how LIN28B shapes the metastatic phenotype. Our studies reveal that the let-7 family, which LIN28B inhibits, decreases self-renewal and migration. Next, we identify PDZ Binding Kinase (PBK) as a novel LIN28B target. PBK is a serine/threonine kinase that promotes the proliferation and self-renewal of neural stem cells and serves as an oncogenic driver in multiple aggressive malignancies. We demonstrate that PBK is both a novel direct target of let-7i and that MYCN regulates PBK expression, thus elucidating two oncogenic drivers that converge on PBK. Functionally, PBK promotes self-renewal and migration, phenocopying LIN28B. Taken together, our findings define a role for LIN28B in neuroblastoma metastasis and define the targetable kinase PBK as a potential novel vulnerability in metastatic neuroblastoma.
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Genetic testing for BRCA1, a DNA repair protein, can identify carriers of pathogenic variants associated with a substantially increased risk for breast and ovarian cancers. However, an association with increased risk is unclear for a large fraction of BRCA1 variants present in the human population. Most of these variants of uncertain clinical significance lead to amino acid changes in the BRCA1 protein. Functional assays are valuable tools to assess the potential pathogenicity of these variants. Here, we systematically probed the effects of substitutions in the C terminus of BRCA1: the N- and C-terminal borders of its tandem BRCT domain, the BRCT-[N-C] linker region, and the α1 and α'1 helices in BRCT-[N] and -[C]. Using a validated transcriptional assay based on a fusion of the GAL4 DNA-binding domain to the BRCA1 C terminus (amino acids 1396-1863), we assessed the functional impact of 99 missense variants of BRCA1. We include the data obtained for these 99 missense variants in a joint analysis to generate the likelihood of pathogenicity for 347 missense variants in BRCA1 using VarCall, a Bayesian integrative statistical model. The results from this analysis increase our understanding of BRCA1 regions less tolerant to changes, identify functional borders of structural domains, and predict the likelihood of pathogenicity for 98% of all BRCA1 missense variants in this region recorded in the population. This knowledge will be critical for improving risk assessment and clinical treatment of carriers of BRCA1 variants.