Promoter Regions Of Target Genes Research Articles

MiR-17-5p enhances pancreatic cancer proliferation by altering cell cycle profiles via disruption of RBL2/E2F4-repressing complexes

The members of the miR-17-92 cluster are upregulated in various cancers and function as a cluster of oncogenic miRNA. Our study characterized a new function of miR-17-5p, a member of the miR-17-92 cluster, in regulating cell proliferation in pancreatic cancer. Our results indicate that miR-17-5p was up-regulated in pancreatic adenocarcinoma and directly targeted the retinoblastoma-like protein 2 (RBL2), a tumor suppressor belonging to the Rb family. High levels of miR-17-5p and low levels of RBL2 were associated with poor prognosis. RBL2 interacted with the transcription factor E2F4 and bound to the promoter regions of the E2F target genes. Disruption of the RBL2/E2F4 complex by miR-17-5p overexpression shifted the activity of E2F from gene repressing to gene activating, which induced cell cycle entry and proliferation. These results suggest that miR-17-5p promoted proliferation in pancreatic ductal adenocarcinoma cells (PDAC), and altered cell cycle profiles in vivo and in vitro, by disrupting the RBL2/E2F4-associated gene repressing complexes via direct targeting of RBL2. The new regulatory network, involving miR-17-5p and RBL2, emerges as a new target of PDAC treatment.

Characterization of redox-active cysteine residues of persulfide-responsive transcriptional repressor SqrR

ABSTRACTWe recently identified the novel persulfide sensor SqrR that functions as a master regulator of sulfide-dependent gene expression in the purple photosynthetic bacterium Rhodobacter capsulatus. SqrR binds to the promoter regions of target genes to repress their expression in the absence of sulfide, and the repressor activity is negated by sulfide treatment. SqrR has 3 cysteine residues, 2 of which are conserved in SqrR homologs from other bacteria: Cys41 and Cys107. SqrR forms an intramolecular tetrasulfide bond between Cys41 and Cys107 when exposed to persulfide, which results in loss of the DNA-binding activity in vitro. Here, we address the mechanism through which these cysteine residues are modified by persulfides. We show that the predicted pKa value of Cys107, as revealed by a putative SqrR structural model, is lower than that of Cys41. Furthermore, C41S SqrR in which Cys41 was changed to serine forms an intermolecular disulfide-bond between Cys107 of 2 SqrRs, suggesting high nucleophilic reactivity of Cy107. These data suggest that Cys107 and Cys41 function as attacking Cys and resolving Cys, respectively; this occurs during tetrasulfide-bond formation of WT SqrR, when it is exposed to persulfide.

Identification of HDA15-PIF1 as a key repression module directing the transcriptional network of seed germination in the dark.

Light is a major external factor in regulating seed germination. Photoreceptor phytochrome B (PHYB) plays a predominant role in promoting seed germination in the initial phase after imbibition, partially by repressing phytochrome-interacting factor1 (PIF1). However, the mechanism underlying the PHYB-PIF1-mediated transcription regulation remains largely unclear. Here, we identified that histone deacetylase15 (HDA15) is a negative component of PHYB-dependent seed germination. Overexpression of HDA15 in Arabidopsis inhibits PHYB-dependent seed germination, whereas loss of function of HDA15 increases PHYB-dependent seed germination. Genetic evidence indicated that HDA15 acts downstream of PHYB and represses seed germination dependent on PIF1. Furthermore, HDA15 interacts with PIF1 both in vitro and in vivo. Genome-wide transcriptome analysis revealed that HDA15 and PIF1 co-regulate the transcription of the light-responsive genes involved in multiple hormonal signaling pathways and cellular processes in germinating seeds in the dark. In addition, PIF1 recruits HDA15 to the promoter regions of target genes and represses their expression by decreasing the histone H3 acetylation levels in the dark. Taken together, our analysis uncovered the role of histone deacetylation in the light-regulated seed germination process and identified that HDA15-PIF1 acts as a key repression module directing the transcription network of seed germination.

Elucidation of the calcineurin-Crz1 stress response transcriptional network in the human fungal pathogen Cryptococcus neoformans.

Calcineurin is a highly conserved Ca2+/calmodulin-dependent serine/threonine-specific protein phosphatase that orchestrates cellular Ca2+ signaling responses. In Cryptococcus neoformans, calcineurin is activated by multiple stresses including high temperature, and is essential for stress adaptation and virulence. The transcription factor Crz1 is a major calcineurin effector in Saccharomyces cerevisiae and other fungi. Calcineurin dephosphorylates Crz1, thereby enabling Crz1 nuclear translocation and transcription of target genes. Here we show that loss of Crz1 confers phenotypes intermediate between wild-type and calcineurin mutants, and demonstrate that deletion of the calcineurin docking domain results in the inability of Crz1 to translocate into the nucleus under thermal stress. RNA-sequencing revealed 102 genes that are regulated in a calcineurin-Crz1-dependent manner at 37°C. The majority of genes were down-regulated in cna1Δ and crz1Δ mutants, indicating these genes are normally activated by the calcineurin-Crz1 pathway at high temperature. About 58% of calcineurin-Crz1 target genes have unknown functions, while genes with known or predicted functions are involved in cell wall remodeling, calcium transport, and pheromone production. We identified three calcineurin-dependent response element motifs within the promoter regions of calcineurin-Crz1 target genes, and show that Crz1 binding to target gene promoters is increased upon thermal stress in a calcineurin-dependent fashion. Additionally, we found a large set of genes independently regulated by calcineurin, and Crz1 regulates 59 genes independently of calcineurin. Given the intermediate crz1Δ mutant phenotype, and our recent evidence for a calcineurin regulatory network impacting mRNA in P-bodies and stress granules independently of Crz1, calcineurin likely acts on factors beyond Crz1 that govern mRNA expression/stability to operate a branched transcriptional/post-transcriptional stress response network necessary for fungal virulence. Taken together, our findings reveal the core calcineurin-Crz1 stress response cascade is maintained from ascomycetes to a pathogenic basidiomycete fungus, but its output in C. neoformans appears to be adapted to promote fungal virulence.

DNA-binding mechanism of the Hippo pathway transcription factor TEAD4.

TEA domain (TEAD) family transcription factors are key regulators in development, tissue homeostasis and cancer progression. TEAD4 acts as a critical downstream effector of the evolutionarily conserved Hippo signaling pathway. The well-studied oncogenic protein YAP forms a complex with TEAD4 to regulate gene transcription; so does the tumor suppressor VGLL4. Although it is known that TEAD proteins can bind promoter regions of target genes through the TEA domain, the specific and detailed mechanism of DNA recognition by the TEA domain remains partially understood. Here, we report the crystal structure of TEAD4 TEA domain in complex with a muscle-CAT DNA element. The structure revealed extensive interactions between the TEA domain and the DNA duplex involving both the major and minor grooves of DNA helix. The DNA recognition helix, α3 helix, determines the specificity of the TEA domain binding to DNA sequence. Structure-guided biochemical analysis identified two major binding sites on the interface of the TEA domain-DNA complex. Mutation of TEAD4 at either site substantially decreases its occupancy on the promoter region of target genes, and largely impaired YAP-induced TEAD4 transactivation and target gene transcription, leading to inhibition of growth and colony formation of gastric cancer cell HGC-27. Collectively, our work provides a structural basis for understanding the regulatory mechanism of TEAD-mediated gene transcription.

Coumarin-chalcone hybrid instigates DNA damage by minor groove binding and stabilizes p53 through post translational modifications

S009-131, a coumarin-chalcone hybrid, had been shown to possess anti-proliferative and anti-tumour effect by triggering apoptosis. In this report, we investigated role of DNA damage signalling pathway in S009-131 induced cancer cell death. Here we show that S009-131 causes DNA damage by potential binding to the minor groove which led to the phosphorylation and activation of ATM and DNA-PK, but not ATR, at earlier time points in order to initiate repair process. S009-131 induced DNA damage response triggered activation of p53 through phosphorylation at its key residues. Pharmacological inhibition of PIKKs abrogated S009-131 induced phosphorylation of p53 at Ser 15. DNA damage induced phosphorylation resulted in reduced proteasomal degradation of p53 by disrupting p53-MDM2 interaction. Additionally, our docking studies revealed that S009-131 might also contribute to increased cellular p53 level by occupying p53 binding pocket of MDM2. Posttranslational modifications of p53 upon S009-131 treatment led to enhanced affinity of p53 towards responsive elements (p53-RE) in the promoter regions of target genes and increased transcriptional efficiency. Together, the results suggest that S009-131 cleaves DNA through minor groove binding and eventually activates PIKKs associated DNA damage response signalling to promote stabilization and enhanced transcriptional activity of p53 through posttranslational modifications at key residues.

MKL1 defines the H3K4Me3 landscape for NF-\u03baB dependent inflammatory response

Macrophage-dependent inflammatory response is considered a pivotal biological process that contributes to a host of diseases when aberrantly activated. The underlying epigenetic mechanism is not completely understood. We report here that MKL1 was both sufficient and necessary for p65-dependent pro-inflammatory transcriptional program in immortalized macrophages, in primary human and mouse macrophages, and in an animal model of systemic inflammation (endotoxic shock). Extensive chromatin immunoprecipitation (ChIP) profiling and ChIP-seq analyses revealed that MKL1 deficiency erased key histone modifications synonymous with transactivation on p65 target promoters. Specifically, MKL1 defined histone H3K4 trimethylation landscape for NF-κB dependent transcription. MKL1 recruited an H3K4 trimethyltransferase SET1 to the promoter regions of p65 target genes. There, our work has identified a novel modifier of p65-dependent pro-inflammatory transcription, which may serve as potential therapeutic targets in treating inflammation related diseases.

The regulation of Jmjd3 upon the expression of NF-κB downstream inflammatory genes in LPS activated vascular endothelial cells

Inflammatory mediators and adhesion molecules have been implicated in a variety of diseases including atherosclerosis. As both the mediator-releasing and targeted cells, vascular endothelial cells play key role in pathological processes. NF-κB signaling regulates a cluster of inflammatory factors in LPS-activated vascular endothelial cells but the underlying mechanisms remain largely unknown. Here, we investigated the epigenetic regulation of LPS upon the expression of inflammatory mediators and adhesion molecules. We found that LPS treatment promoted jmjd3 expression, enhanced Jmjd3 nuclear accumulation in human vascular endothelial cells. In addition, LPS enhanced the demethylation of H3K27me3, a specific substrate of Jmjd3. LPS treatment recruited Jmjd3 and NF-κB to the promoter region of target genes, suggesting Jmjd3 synergizes with NF-κB to activate the expression of target genes. We further found that Jmjd3 attenuated the methylation status in promoter region of target genes, culminating in target gene expression. Our findings unveil epigenetic regulations of LPS upon NF-κB pathway and identify Jmjd3 as a critical modulator of NF-κB pathway and potential therapeutic target for NF-κB related diseases including atherosclerosis.

P53 and RAD9, the DNA Damage Response, and Regulation of Transcription Networks.

The way cells respond to DNA damage is important since inefficient repair or misrepair of lesions can have deleterious consequences, including mutation, genomic instability, neurodegenerative disorders, premature aging, cancer or death. Whether damage occurs spontaneously as a byproduct of normal metabolic processes, or after exposure to exogenous agents, cells muster a coordinated, complex DNA damage response (DDR) to mitigate potential harmful effects. A variety of activities are involved to promote cell survival, and include DNA repair, DNA damage tolerance, as well as transient cell cycle arrest to provide time for repair before entry into critical cell cycle phases, an event that could be lethal if traversal occurs while damage is present. When such damage is prolonged or not repairable, senescence, apoptosis or autophagy is induced. One major level of DDR regulation occurs via the orchestrated transcriptional control of select sets of genes encoding proteins that mediate the response. p53 is a transcription factor that transactivates specific DDR downstream genes through binding DNA consensus sequences usually in or near target gene promoter regions. The profile of p53-regulated genes activated at any given time varies, and is dependent upon type of DNA damage or stress experienced, exact composition of the consensus DNA binding sequence, presence of other DNA binding proteins, as well as cell context. RAD9 is another protein critical for the response of cells to DNA damage, and can also selectively regulate gene transcription. The limited studies addressing the role of RAD9 in transcription regulation indicate that the protein transactivates at least one of its target genes, p21/waf1/cip1, by binding to DNA sequences demonstrated to be a p53 response element. NEIL1 is also regulated by RAD9 through a similar DNA sequence, though not yet directly verified as a bonafide p53 response element. These findings suggest a novel pathway whereby p53 and RAD9 control the DDR through a shared mechanism involving an overlapping network of downstream target genes. Details and unresolved questions about how these proteins coordinate or compete to execute the DDR through transcriptional reprogramming, as well as biological implications, are discussed.

Identification of downstream metastasis-associated target genes regulated by LSD1 in colon cancer cells.

PurposeThis study aims to identify downstream target genes regulated by lysine-specific demethylase 1 (LSD1) in colon cancer cells and investigate the molecular mechanisms of LSD1 influencing invasion and metastasis of colon cancer.MethodWe obtained the expression changes of downstream target genes regulated by small-interfering RNA-LSD1 and LSD1-overexpression via gene expression profiling in two human colon cancer cell lines. An Affymetrix Human Transcriptome Array 2.0 was used to identify differentially expressed genes (DEGs). We screened out LSD1-target gene associated with proliferation, metastasis, and invasion from DEGs via Gene Ontology and Pathway Studio. Subsequently, four key genes (CABYR, FOXF2, TLE4, and CDH1) were computationally predicted as metastasis-related LSD1-target genes. ChIp-PCR was applied after RT-PCR and Western blot validations to detect the occupancy of LSD1-target gene promoter-bound LSD1.ResultA total of 3633 DEGs were significantly upregulated, and 4642 DEGs were downregulated in LSD1-silenced SW620 cells. A total of 4047 DEGs and 4240 DEGs were upregulated and downregulated in LSD1-overexpressed HT-29 cells, respectively. RT-PCR and Western blot validated the microarray analysis results. ChIP assay results demonstrated that LSD1 might be negative regulators for target genes CABYR and CDH1. The expression level of LSD1 is negatively correlated with mono- and dimethylation of histone H3 lysine4(H3K4) at LSD1- target gene promoter region. No significant mono-methylation and dimethylation of H3 lysine9 methylation was detected at the promoter region of CABYR and CDH1.ConclusionLSD1- depletion contributed to the upregulation of CABYR and CDH1 through enhancing the dimethylation of H3K4 at the LSD1-target genes promoter. LSD1- overexpression mediated the downregulation of CABYR and CDH1expression through decreasing the mono- and dimethylation of H3K4 at LSD1-target gene promoter in colon cancer cells. CABYR and CDH1 might be potential LSD1-target genes in colon carcinogenesis.

TRBP maintains mammalian embryonic neural stem cell properties by acting as a novel transcriptional coactivator of the Notch signaling pathway.

Transactivation response element RNA-binding protein (TRBP; TARBP2) is known to play important roles in human immunodeficiency virus (HIV) replication and microRNA biogenesis. However, recent studies implicate TRBP in a variety of biological processes as a mediator of cross-talk between signal transduction pathways. Here, we provide the first evidence that TRBP is required for efficient neurosphere formation and for the expression of neural stem cell markers and Notch target genes in primary neural progenitor cells in vitro Consistent with this, introduction of TRBP into the mouse embryonic brain in utero increased the fraction of cells expressing Sox2 in the ventricular zone. We also show that TRBP physically interacts with the Notch transcriptional coactivation complex through C promoter-binding factor 1 (CBF1; RBPJ) and strengthens the association between the Notch intracellular domain (NICD) and CBF1, resulting in increased NICD recruitment to the promoter region of a Notch target gene. Our data indicate that TRBP is a novel transcriptional coactivator of the Notch signaling pathway, playing an important role in neural stem cell regulation during mammalian brain development.

The effect of the epigenome on the Vitamin D Receptor target genes

Background. Humans produce vitamin D3 by exposure to sunlight that includes ultraviolet B (UVB) radiation, but also via the intake of nutrients. In the body, the Vitamin D3 is converted to its active form, which is called 1, 25(OH)2D3. This can bind to the Vitamin D Receptor (VDR), which is located on several genes which are called the VDR target genes. Objective. The present study investigated which genes are regulated by the 1, 25(OH)2D3 and checked whether the epigenetic modifications are measured in two cell lines within the ENCODE project. The data of the epigenetic modifications will be linked to the promoter regions of the VDR target genes. In this way the epigenetic control of the VDR target genes could be investigated in more detail. Methods. The human VDR target genes were identified with an extensive literature search. The promoter regions of the VDR target genes were found with Genomatix. Two types of epigenetic modifications, DNA methylation and histone modification, which were determined in two cell lines, IMR90 and H1ESC, in the ENCODE project were linked to the promoter regions of the VDR target genes. In the network analysis program Cytoscape the VDR target genes and the measured epigenetic modifications were integrated and visualized in an epigenetics-VDR target genes network. Results. In total 185 VDR target genes were found in 25 literature papers. There were 528 promoter regions in the 185 VDR target genes identified. In the H1ESC were 445 histone modifications linked to the VDR target gene promoters and in the IMR90 were 259 histone modifications linked to the VDR target gene promoters. In the IMR90 were 158 promoter regions with a DNA methylation region and in the H1ESC were this 177 promoter regions with a DNA methylation region. In the combined network of DNA methylation and histone modifications were in the IMR90 99 genes and in the H1ESC 111 genes selected. Conclusion. The epigenetics-VDR target genes network can be used in further research to understand the role of the epigenetic control of the VDR target genes.

Role of the Polycomb Methyltransferase Ezh1 in Myelodysplastic Syndrome Induced By Ezh2 Insufficiency

Ezh1 and Ezh2, the catalytic components of polycomb-repressive complex 2 (PRC2), negatively control gene expression by catalyzing mono, di, and tri-methylation of histone H3 at lysine 27 (H3K27me1/me2/me3). Loss-of-function mutations of EZH2, but not those of EZH1, have been found in patients with hematologic malignancies such as myelodysplastic syndrome (MDS), myeloproliferative neoplasms (MPNs), and MDS/MPN overlap disorders.We previously demonstrated that hematopoietic cell-specific Ezh2 knockout mice (Ezh2Δ/Δ) developed hematologic malignancies including MDS and MDS/MPN. Although deletion of Ezh1, another enzymatic component of PRC2, (Ezh1-/-) did not significantly affect global H3K27me3 levels or hematopoiesis, deletion of both Ezh1 and Ezh2 in mice (Ezh1-/-Ezh2Δ/Δ) caused rapid exhaustion of hematopoietic stem cells (HSCs). Given that only Ezh1 and Ezh2 are known as enzymatic components of PRC2, we concluded that residual PRC2 enzymatic activity is required for HSC maintenance and development of hematologic malignancies in the setting of EZH2 insufficiency frequently observed in MDS. However, the role of Ezh1 in Ezh2-insufficient hematologic malignancies is still not fully understood since hematopoiesis could not be maintained in Ezh1-/-Ezh2Δ/Δ mice. Here we analyzed the impact of Ezh1 heterozygosity on Ezh2-null hematopoiesis (Ezh1+/-Ezh2Δ/Δ), in which PRC2 activity is mediated by a single allele of Ezh1, for better understanding of Ezh2-deficient hematologic malignancies.We first transplanted BM cells from Ezh1+/-Ezh2flox/flox CD45.2 mice with CD45.1 wild-type competitor cells into lethally irradiated CD45.1 recipient mice and deleted Ezh2 by intraperitoneal injection of tamoxifen. Ezh1+/-Ezh2Δ/Δ cells exhibited a lower repopulation capacity than Ezh2Δ/Δ but established persistent repopulation for at least 6 months after the deletion of Ezh2 while double knockout cells (Ezh1-/-Ezh2Δ/Δ) were outcompeted by competitor cells immediately. We next transplanted BM cells from Ezh1+/-Ezh2flox/flox CD45.2 mice without CD45.1 wild-type competitor cells into lethally irradiated CD45.1 recipient mice and deleted Ezh2 by intraperitoneal injection of tamoxifen. Importantly, recipient mice reconstituted with Ezh1+/-Ezh2Δ/Δ cells exhibited MDS-like phenotypes including anemia and morphological myelodysplasia, which were more pronounced than those of Ezh2Δ/Δ mice. Ezh1+/-Ezh2Δ/Δ mice also showed more advanced hematological abnormalities such as erythroid differentiation block, increased apoptosis of erythroid cells, and extramedullary hematopoiesis in the spleen than Ezh2Δ/Δ mice did. These results suggest that Ezh1 heterozygosity promotes the development of myelodysplasia in the setting of Ezh2insufficiency.Next we examined the molecular mechanism by which the loss of Ezh1 promotes myelodysplasia. Western blot and ChIP-sequence analyses revealed that global levels of H3K27me3 were not significantly changed but H3K27me3 levels at promoter regions of the PRC2 target genes were obviously reduced by Ezh1 heterozygosity in Ezh2Δ/Δ HSPCs. As a consequence, PRC2 target genes were highly de-repressed in Ezh1+/-Ezh2Δ/Δ LSK HSPCs compared with Ezh2Δ/Δ HSPCs. Among these, several genes appeared to be associated with MDS such as S100A9, encoding an inflammatory protein implicated in dyserythropoiesis in MDS. Furthermore, gene set enrichment analysis showed that the genes highly expressed in myeloid cells were positively enriched by Ezh1 heterozygosity in Ezh2Δ/ΔHSPCs.These findings indicate that dosage of Ezh1 is critical in the maintenance of Ezh2-insufficient hematopoiesis as well as the progression of MDS with Ezh2 insufficiency. DisclosuresNo relevant conflicts of interest to declare.

Identification and characterization of promoters and cis-regulatory elements of genes involved in secondary metabolites production in hop (Humulus lupulus. L)

Molecular and biochemical studies have shown that gene contains single or combination of different cis-acting regulatory elements are actively controlling the transcriptional regulation of associated genes, downstream effects of these result in the modulation of various biological pathways such as biotic/abiotic stress responses, hormonal responses to growth and development processes and secondary metabolite production. Therefore, the identification of promoters and their cis-regulatory elements is one of intriguing area to study the dynamic complex regulatory network of genes activities by integrating computational, comparative, structural and functional genomics. Several bioinformatics servers or database have been established to predict the cis-acting elements present in the promoter region of target gene and their association with the expression profiles in the TFs. The aim of this study is to predict possible cis-acting regulatory elements that have putative role in the transcriptional regulation of a dynamic network of metabolite gene activities controlling prenylflavonoid and bitter acids biosynthesis in hop (Humulus lupulus). Recent release of hop draft genome enabled us to predict the possible cis-acting regulatory elements by extracting 2kbp of 5′ regulatory regions of genes important for lupulin metabolome biosynthesis, using Plant CARE, PLACE and Genomatix Matinspector professional databases. The result reveals the plausible role of cis-acting regulatory elements in the regulation of gene expression primarily involved in lupulin metabolome biosynthesis including under various stress conditions.

ITAR: a web server for identifying target genes of transcription factors using ChIP-seq or ChIP-chip data.

BackgroundChromatin immunoprecipitation followed by massively parallel DNA sequencing (ChIP-seq) or microarray hybridization (ChIP-chip) has been widely used to determine the genomic occupation of transcription factors (TFs). We have previously developed a probabilistic method, called TIP (Target Identification from Profiles), to identify TF target genes using ChIP-seq/ChIP-chip data. To achieve high specificity, TIP applies a conservative method to estimate significance of target genes, with the trade-off being a relatively low sensitivity of target gene identification compared to other methods. Additionally, TIP’s output does not render binding-peak locations or intensity, information highly useful for visualization and general experimental biological use, while the variability of ChIP-seq/ChIP-chip file formats has made input into TIP more difficult than desired.DescriptionTo improve upon these facets, here we present are fined TIP with key extensions. First, it implements a Gaussian mixture model for p-value estimation, increasing target gene identification sensitivity and more accurately capturing the shape of TF binding profile distributions. Second, it enables the incorporation of TF binding-peak data by identifying their locations in significant target gene promoter regions and quantifies their strengths. Finally, for full ease of implementation we have incorporated it into a web server (http://syslab3.nchu.edu.tw/iTAR/) that enables flexibility of input file format, can be used across multiple species and genome assembly versions, and is freely available for public use. The web server additionally performs GO enrichment analysis for the identified target genes to reveal the potential function of the corresponding TF.ConclusionsThe iTAR web server provides a user-friendly interface and supports target gene identification in seven species, ranging from yeast to human. To facilitate investigating the quality of ChIP-seq/ChIP-chip data, the web server generates the chart of the characteristic binding profiles and the density plot of normalized regulatory scores. The iTAR web server is a useful tool in identifying TF target genes from ChIP-seq/ChIP-chip data and discovering biological insights.Electronic supplementary materialThe online version of this article (doi:10.1186/s12864-016-2963-0) contains supplementary material, which is available to authorized users.

Abstract 4228: Imaging and targeting of hypoxic microenvironments in prostate cancer

Abstract Cancer cells display an adaptive response to hypoxia through the activation of several genes mediated by the binding of hypoxia inducible factors (HIFs) to hypoxia response elements (HRE) in the promoter region of target gene that results in their increased transcription [1]. HIFs promote key steps in tumorigenesis, including angiogenesis, metabolism, proliferation, metastasis, and differentiation [1]. Bacterial or yeast cytosine deaminase (yCD) converts the nontoxic prodrug 5-fluorocytosine (5-FC) to the anti-cancer drug 5-fluorouracil (5-FU) that is widely used in cancer treatment [2]. Using a lentivirus approach, we established controlled expression of yCD by HRE in prostate cancer cells (PC-3). These cells also report on HIF-1α expression with regulated luciferase (Luc) expression, allowing detection of hypoxia, and the generation of 5-FU from 5-FC by yCD in the presence of hypoxia. Transduction efficiency and reporter activity in response to hypoxia was evaluated by performing luciferase assays, and bioluminescence imaging (BLI) of cells in vitro or in vivo using a Xenogen IVIS Spectrum system. Cell viability in vitro in response to hypoxia in the presence of 5-FC was assessed by MTS assay. In vivo studies were performed by inoculating 2×10⁁6 PC-3-HRE-Luc cells and PC-3-HRE-yCD+Luc cells on either flank of 5-week-old male severely combined immune deficient (SCID) mice. BLI was performed once tumors reached ∼200mm3 followed by 5-FC injection through the tail vein (200mg/kg) and intraperitoneally (250mg/kg). BLI was performed 3 days after the first 5-FC injection and continued through the treatment protocol. At the end of the treatment protocol, tumors were excised, and a part of the tumor was processed for immunohistochemistry. Bioluminescence was detected in both PC3-HRE-Luc and PC-3-HRE-yCD+Luc cells only in response to the hypoxia mimetic cobalt chloride or hypoxia (1% O2) confirming the regulation of luciferase by hypoxia and activation of CD. Expression of yCD and its ability to convert the prodrug 5-FC to 5-FU, with increased cell kill was evident under hypoxia. In vivo, engineered PC-3-HRE-yCD+Luc cells reported hypoxia, and showed significant reduction of hypoxic regions and tumor volume. Morphologically, PC-3-HRE-yCD+Luc tumors exhibited extensive necrosis. We are currently evaluating the effects of eliminating hypoxic cancer cells on distant metastasis as well as on aggressive subpopulations such as cancer stem cells in the primary tumor.

Abstract 2658: Genome-wide mistargeting of oncogenic SWI/SNF(BAF) complexes in SMARCB1(BAF47)-deficient sarcomas

Abstract SMARCB1/BAF47/INI1 is a core subunit of the mammalian SWI/SNF (BAF) family of ATP-dependent chromatin remodeling complexes, which remodel nucleosome architecture to achieve coordinated regulation of gene expression. Genetic loss of SMARCB1 has been identified in several cancer types, including malignant rhabdoid tumor (MRT, 98%) and epithelioid sarcoma (EpS, 90%), strongly implicating this event as the oncogenic driver in these malignancies. However, the precise mechanism underpinning the tumor suppressive function of BAF47 to date remains unclear. In order to elucidate the underlying mechanism and to identify direct genetic targets of aberrant BAF complexes in this context, we comprehensively evaluated the effects of BAF47 reintroduction in BAF47-deficient sarcomas with respect to complex subunit and associated protein factor composition and stability, global chromatin structure, and gene regulation. Reintroduced BAF47 stably integrated into BAF complexes, and remarkably, stabilized a highly specific set of BAF subunits, resulting in an increased complex molecular weight and stoichiometric nuclear abundance. These biochemical changes inducing the formation of wild-type complexes in MRT and EpS cell settings were directly linked to reproducible changes in BAF complex localization genome-wide, particularly, in the targeting to H3K4me3-marked promoter regions of direct target genes to establish DNA accessibility. Importantly, changes in BAF47-dependent BAF complex targeting between oncogenic and induced wild-type conditions were reproducibly associated with differential chromatin architecture and gene expression signatures hallmark to both MRT and EpS, and uniformly resulted in proliferative senescence of MRT and EpS cell lines in culture. These studies highlight, for the first time, the full spectrum of structural and functional contributions of the BAF47 subunit, implicating its role as a keystone in heteromorphic BAF complex assembly; BAF47 is required for the stable integration of several BAF subunits and novel interacting factors, which we determine govern specific genome-wide targeting mechanisms and chromatin-templated activities. These results reveal the mechanisms underlying the oncogenesis of BAF47-deficient sarcomas and point toward novel therapeutic strategies for this group of human sarcomas. Citation Format: Robert Nakayama, Robert T. Williams, Seth H. Cassel, Mingxiang Teng, Rafael A. Irizarry, George D. Demetri, Cigall Kadoch. Genome-wide mistargeting of oncogenic SWI/SNF(BAF) complexes in SMARCB1(BAF47)-deficient sarcomas. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2658.

Rex in Clostridium kluyveri is a global redox-sensing transcriptional regulator

Clostridium kluyveri is unique in fermenting ethanol and acetate to butyrate, caproate, and H2. The genes encoding butyrate-producing enzymes, including electron-bifurcating butyryl-CoA dehydrogenase/electron transfer flavoprotein complex and NADH-dependent reduced ferredoxin:NADP+ oxidoreductase, form a cluster, which is preceded by a gene annotated as the transcriptional regulator Rex. Northern blotting and RT-PCR experiments indicated that the gene cluster forms a large transcriptional unit that possibly includes several small transcriptional units. The deduced Rex protein contains a winged helix DNA-binding domain and a Rossmann fold potentially interacting with NAD(H). Bioinformatics analysis revealed that Rex can bind the promoter regions of numerous genes, which are involved in carbon and energy metabolism, including NADH oxidation, hydrogen production, ATP synthesis, butyrate formation, and succinate metabolism. Rex may regulate the transcription of genes encoding certain transcriptional regulators and transporters. Electrophoretic mobility shift and isothermal titration calorimetry assays revealed that Rex specifically formed protein-DNA complexes with the promoter regions of target genes, which could be inhibited by NADH but restored by an excess amount of NAD+. These results suggest that Rex plays a key role in the carbon and energy metabolism of C. kluyveri as a global transcriptional regulator in response to the cellular NADH/NAD+ ratio.

BRG1 in the Nucleus Accumbens Regulates Cocaine-Seeking Behavior

BackgroundDrug addiction is defined as a chronic disease characterized by compulsive drug seeking and episodes of relapse despite prolonged periods of drug abstinence. Neurobiological adaptations, including transcriptional and epigenetic alterations in the nucleus accumbens, are thought to contribute to this life-long disease state. We previously demonstrated that the transcription factor SMAD3 is increased after 7 days of withdrawal from cocaine self-administration. However, it is still unknown which additional factors participate in the process of chromatin remodeling and facilitate the binding of SMAD3 to promoter regions of target genes. Here, we examined the possible interaction of BRG1—also known as SMARCA4, an adenosine triphosphatase–containing chromatin remodeler—and SMAD3 in response to cocaine exposure. MethodsThe expression of BRG1, as well as its binding to SMAD3 and target gene promoter regions, was evaluated in the nucleus accumbens and dorsal striatum of rats using western blotting, co-immunoprecipitation, and chromatin immunoprecipitation following abstinence from cocaine self-administration. Rats were assessed for cocaine-seeking behaviors after either intra-accumbal injections of the BRG1 inhibitor PFI3 or viral-mediated overexpression of BRG1. ResultsAfter withdrawal from cocaine self-administration, BRG1 expression and complex formation with SMAD3 are increased in the nucleus accumbens, resulting in increased binding of BRG1 to the promoter regions of Ctnnb1, Mef2d, and Dbn1. Intra-accumbal infusion of PFI3 attenuated, whereas viral overexpression of Brg1 enhanced, cocaine-reinstatement behavior. ConclusionsBRG1 is a key mediator of the SMAD3-dependent regulation of cellular and behavioral plasticity that mediates cocaine seeking after a period of withdrawal.

Recruitment of Polycomb Repressive Complex 2 is Essential to Suppress the Target Chromatin in Arabidopsis

ABSTRACTThe polycomb group (PcG) proteins play important roles in repressing gene expression by modifying chromatin. PcG proteins usually form multiple protein complexes, such as Polycomb Repressive Complex 1 (PRC1) and PRC2 complexes, to repress gene expression. At least three PRC2 complexes have been identified in Arabidopsis that are required for normal plant development. PRC2 complexes repress gene expression through trimethylating lysine 27 on histone 3. Sequentially, PRC1 complexes recognize and bind to H3K27me3 mark generated by PRC2 complexes and monoubiquitylate lysine 119. However, neither the recruitment of PRC nor the regulation mechanisms of PRC functions are well understood. Recent findings suggest that the recruitment of PRC2 complexes to the target chromatin is not simple. Polycomb Response Elements (PREs) in the promoter regions of several PRC2 target genes, transcriptional factors, long noncoding RNAs (lncRNAs), and PRC2 associated-proteins have been documented to be involved in PRC2 targeting. In this review, we focus on the recent findings on PRC2 targeting and their regulatory roles in plant development by repressing gene expression.