Selected Transcription Factors Research Articles

DOF, MYB and TCP transcription factors: Their possible roles on barley germination and seedling establishment

Seed germination is a multi-staged complex process during seed plant life cycle, and it is tightly regulated through a coordinated expression of diverse genes in diverse tissues. As regulatory molecules of gene expression, determination of transcription factors is crucial to understanding molecular basis and regulatory network of germination process and seedling establishment. However, limited data on the contributions of these transcription factors to the germination of crop barley (Hordeum vulgare L.) are available. Here, we investigated the expression profiles of selected transcription factors from different families (DOF, MYB and TCP) with qRT-PCR analysis in various tissues including coleoptiles, leaves and roots following the germination. Analysis of MYB and DOF gene expression profiles indicated that there were differing expressions in different aged tissues, HvMYB5 and HvDOF2 being the most outstanding one in the oldest tissue, 15-day-old root. On the other hand, investigated TCP genes were lowly expressed compared to selected MYB and DOF genes, except HvTCP3, where the highest expression was observed in 15-day-old root tissue. The obtained expression profiles illustrate the importance of potential regulatory roles of transcription factors in early developmental stages of barley germination and seedling establishment.

Regulation and Dynamics of Gene Expression During the Life Cycle of Fusarium graminearum.

Fungal pathogens survive harsh environments and overcome physical, temporal, and chemical barriers to colonize their hosts and reproduce. Fusarium graminearum was one of the first fungal plant pathogens for which transcriptomic tools were developed, making analysis of gene expression a cornerstone approach in studying its biology. The analysis of gene expression in diverse in vitro conditions and during infection of different cereal crops has revealed subsets of both unique and shared transcriptionally regulated genes. Together with genetic studies, these approaches have enhanced our understanding of the development and infection cycle of this economically important pathogen. Here, we will outline recent advances in transcriptional profiling during sporogenesis, spore germination, vegetative growth, and host infection. Several transcriptional regulators have been identified as essential components in these responses and the role of select transcription factors will be highlighted. Finally, we describe some of the gaps in our understanding of F. graminearum biology and how expression analysis could help to address these gaps.

P0511ENHANCER AND SUPER-ENHANCER DYNAMICS IN REPAIR AFTER ISCHEMIC ACUTE KIDNEY INJURY

Abstract Background and Aims The endogenous repair process of the mammalian kidney allows rapid recovery after acute kidney injury (AKI) through robust proliferation of tubular epithelial cells. There is currently limited understanding of which transcriptional regulators activate these repair programs and how transcriptional dysregulation leads to maladaptive repair. Here we investigate the existence of enhancer dynamics in the regenerating mouse kidney. Method RNA-seq and ChIP-seq (H3K27ac, H3K4m3, BRD4, POL2 and selected transcription factors) were performed on samples from repairing kidney cortex 2 days after ischemia/reperfusion injury (IRI) to identify activated genes, transcription factors, enhancer and super-enhancers associated with kidney repair. Further we investigated the role of super-enhancer activation in kidney repair through pharmacological BET inhibition using the small molecule JQ1 in vitro and in acute kidney injury models in vivo. Results Response to kidney injury leads to genome-wide alteration in enhancer repertoire in-vivo. We identified 16,781 enhancer sites (H3K27ac and BRD4 positive, H3K4me3 negative binding) active in SHAM and IRI samples; 6,512 lost and 9,774 gained after IRI. The lost and gained enhancer sites can be annotated to 62% and 63% of down- and up-regulated transcripts at day 2 after kidney injury, respectively. Super-enhancer analysis revealed 164 lost and 216 gained super-enhancer sites at IRI day 2. 385 super-enhancers maintain activity before and after injury. ChIP-seq profiles of selected transcription factors based on motif analysis show specific binding at corresponding enhancer sites. We observed lost enhancer binding of HNF4A and GR mainly at kidney related enhancer elements. In contrast, STAT3 showed increased binding at injury induces enhancer elements. No dynamic was observed for STAT5. Both transcription factor groups show corresponding mRNA changes after injury. Pharmacological inhibition of enhancer and super-enhancer activity by BRD4 inhibition (JQ1: 50mg/kg/day) before IRI leads to suppression of 40% of injury-induced transcripts associated with cell cycle regulation and significantly increased mortality between days 2 and 3 after AKI. Conclusion This is the first demonstration of enhancer and super-enhancer function in the repairing kidney. In addition, our data call attention to potential caveats for use of small molecule inhibitors of BET proteins that are currently being tested in clinical trials in cancer patients who are at risk for AKI. Our analyses of enhancer dynamics after kidney injury in vivo have the potential to identify new targets for therapeutic intervention.

Transcriptome Analysis Reveals Important Transcription Factor Families and Reproductive Biological Processes of Flower Development in Celery (Apium graveolens L.)

There are few reports on the reproductive biology of celery, which produces small flowers in a long flowering period. Anther development was analyzed by paraffin sectioning and related genes were examined by transcriptome sequencing and qPCR. The development process was divided into nine stages based on the significant changes in the cell and tissue morphologies. These stages included: archesporial stage, sporogenous cell stage, microspore mother cell stage, dyad and tetrad stage, mononuclear microspore stage, late uninucleate microspore stage, binuclear cell stage, mature pollen stage, and dehiscence stage. A total of 1074 differentially expressed genes were identified by transcriptome sequencing in the early flower bud, middle flower bud, and early flowering period. Functional annotation indicated that these genes were involved in physiological and biochemical processes such as ribosomes metabolism, sugar metabolism, and amino acid metabolism. Transcription factors such as C2H2, AP2/ERF, bZIP, WRKY, and MYB played key regulatory roles in anther development and had different regulatory capabilities at various stages. The expression patterns based on qPCR and transcriptome data of the selected transcription factor genes showed consistency, suggesting that these genes played an important role in different flower development stages. These results provide a theoretical basis for molecular breeding of new celery varieties with pollen abortion. Furthermore, they have enriched research on the reproductive biology of celery and the Apiaceae family.

Nkx2.1 downregulation is involved in brain abnormality induced by excess retinoic acid.

Abnormal development of central nervous system (CNS) caused by neural tube defects is not only a major contributor in the prevalence of stillbirths and neonatal deaths but also causes lifelong physical disability in surviving infants. Due to insufficient known investigated causes, CNS developmental abnormality has brought sever burden on health around the world. From previous results of high throughput transcriptome sequencing, we selected transcription factor Nkx2.1 as a candidate to investigate its role on brain abnormalities induced by excessive retinoic acid. The result of in situ hybridization showed that Nkx2.1 was mainly expressed in mouse brain. After the Nkx2.1 gene was silenced, retarded proliferation and accelerated apoptosis were found in mouse Neuro-2a (N2a) cells. Furthermore, our results indicated that the main components of sonic hedgehog (Shh) signaling pathway were affected in Nkx2.1-silenced cells, implying that Nkx2.1 plays an important role in the development of mouse brain by regulating Shh signaling pathway.

Tumor progression and chromatin landscape of lung cancer are regulated by the lineage factor GATA6

Lineage selective transcription factors (TFs) are important regulators of tumorigenesis, but their biological functions are often context dependent with undefined epigenetic mechanisms of action. In this study, we uncover a conditional role for the endodermal and pulmonary specifying TF GATA6 in lung adenocarcinoma (LUAD) progression. Impairing Gata6 in genetically engineered mouse models reduces the proliferation and increases the differentiation of Kras mutant LUAD tumors. These effects are influenced by the epithelial cell type that is targeted for transformation and genetic context of Kras-mediated tumor initiation. In LUAD cells derived from surfactant protein C expressing progenitors, we identify multiple genomic loci that are bound by GATA6. Moreover, suppression of Gata6 in these cells significantly alters chromatin accessibility, particularly at distal enhancer elements. Analogous to its paradoxical activity in lung development, GATA6 expression fluctuates during different stages of LUAD progression and can epigenetically control diverse transcriptional programs associated with bone morphogenetic protein signaling, alveolar specification, and tumor suppression. These findings reveal how GATA6 can modulate the chromatin landscape of lung cancer cells to control their proliferation and divergent lineage dependencies during tumor progression.

Conditional Immortalization of Lymphoid Progenitors via Tetracycline-Regulated LMO2 Expression.

Conditional immortalization of hematopoietic progenitors through lentiviral expression of selected transcription factors in hematopoietic stem and progenitor cells provides a promising tool to study stem cell and leukemia biology. In this study, to generate conditionally immortalized lymphoid progenitor (ciLP) cell lines, murine hematopoietic progenitor cells were transduced with an inducible lentiviral "all-in-one" vector expressing LMO2 under doxycycline (DOX) stimulation and the reverse tetracycline-regulated transactivator (rtTA3). For selection of LMO2-expressing ciLPs (LMO2-ciLPs) and longitudinal manipulation in T cell differentiation lymphoid conditions, we developed a robust approach based on coculture with OP9-DL1 stromal cells and improved cytokine conditions allowing a controlled balance between cell proliferation and differentiation in vitro. LMO2-ciLP cell lines with the highest proliferation, vector copy number, and similar insertion pattern were selected for LMO2 "on/off" in vitro study. LMO2 expression under DOX induction resulted in a double negative (DN) 2 differentiation arrest and a propagation of CD44+CD25- myeloid cell population characterized by lymphoid and myeloid phenotypes, respectively. Both DN2 and CD44+CD25- myeloid cell subpopulations expressed c-KIT, suggesting that LMO2-ciLPs were similar to uncommitted progenitors under DOX supplementation. DOX removal resulted in cessation of ectopic LMO2 expression and LMO2-ciLPs continued T cell lymphoid differentiation accompanied by c-KIT downregulation and interleukin 7 receptor expression. Switching off LMO2 expression was accompanied by increased Notch signaling and significant reduction of the CD44+CD25- myeloid cell population under T cell differentiation lymphoid conditions. Although vector insertions in cooperation with LMO2 expression could influence the fate of LMO2-ciLPs and additional experiments are required to evaluate it, our approach provides a promising tool to investigate mechanisms underlying stem cell, leukemia, and lymphocyte biology, leading to novel approaches for disease modeling and therapy evaluation.

TCF7L2 regulates postmitotic differentiation programmes and excitability patterns in the thalamus.

ABSTRACTNeuronal phenotypes are controlled by terminal selector transcription factors in invertebrates, but only a few examples of such regulators have been provided in vertebrates. We hypothesised that TCF7L2 regulates different stages of postmitotic differentiation in the thalamus, and functions as a thalamic terminal selector. To investigate this hypothesis, we used complete and conditional knockouts of Tcf7l2 in mice. The connectivity and clustering of neurons were disrupted in the thalamo-habenular region in Tcf7l2−/− embryos. The expression of subregional thalamic and habenular transcription factors was lost and region-specific cell migration and axon guidance genes were downregulated. In mice with a postnatal Tcf7l2 knockout, the induction of genes that confer thalamic terminal electrophysiological features was impaired. Many of these genes proved to be direct targets of TCF7L2. The role of TCF7L2 in terminal selection was functionally confirmed by impaired firing modes in thalamic neurons in the mutant mice. These data corroborate the existence of master regulators in the vertebrate brain that control stage-specific genetic programmes and regional subroutines, maintain regional transcriptional network during embryonic development, and induce terminal selection postnatally.

MA17.01 Cell Lineage and Chromatin Landscape of Lung Cancer Are Controlled by GATA6

Thoracic malignancies are histologically and biologically heterogeneous. The underlying causes of this heterogeneity are believed to be linked to the complex cellular ontogenies of lung cancers and their relationship to pulmonary development. Lineage selective transcription factors (TFs) are critical determinants of airway cell differentiation and homeostasis, but their biological requirements are often conditional. Analogously, several developmental TFs can paradoxically enhance or inhibit lung cancer progression, depending on cellular and epigenetic contexts which remain largely undefined. In this study, we report a novel function for the endodermal and pulmonary specifying TF GATA6 in lung cancer. To understand the role of GATA6 in lung tumorigenesis we used a genetically engineer mouse model (GEMMs) harboring Kras p53 mutations using progenitor cell specific gene targeting. We combined GEMMs biology with integrated analysis of the transcriptome and the chromatin landscape of lung cells derived from GATA6 deficient tumors. In this study, we uncover a conditional role for the endodermal and pulmonary specifying TF GATA6 during the initiation and progression of Kras mutant lung adenocarcinoma (LUAD). Inhibition of Gata6 in genetically engineered mouse models dampens the proliferation and increases the differentiation of LUAD tumors. These effects are influenced by the epithelial cell type that is targeted for transformation, demonstrating that GATA6 expression is an important molecular determinant of the cell of origin in Kras mutant lung cancer. In LUAD cells derived from surfactant protein C expressing progenitors, we identify multiple genomic loci that are bound by GATA6. Moreover, suppression of Gata6 in these cells significantly alters chromatin accessibility, particularly at distal enhancer elements. Analogous to its paradoxical activity in the developing lungs, GATA6 expression fluctuates during different stages of LUAD progression and can epigenetically control diverse lineage programs associated with cell proliferation, alveolar specification, BMP signaling, and epithelial plasticity. In summary: 1) GATA6 expression varies during different stages of disease progression in the lung adenocarcinoma (LUAD) subtype 2) Suppression of Gata6 can diminish the proliferation and progression of LUAD in a manner that is influenced by the transforming progenitor of origin 3) GATA6 differentially modulates chromatin accessibility across the genome of LUAD cells 4) This epigenomic mechanism results in the activation of different lineage specific programs, including the BMP signaling pathway. These findings reveal how GATA6 can modulate the chromatin landscape of lung cancer cells to control their divergent lineage dependencies during tumor progression. D.X.N. has received research funding from AstraZeneca, Inc.

Coordinate regulation of ELF5 and EHF at the chr11p13 CF modifier region.

E74‐like factor 5 (ELF5) and ETS‐homologous factor (EHF) are epithelial selective ETS family transcription factors (TFs) encoded by genes at chr11p13, a region associated with cystic fibrosis (CF) lung disease severity. EHF controls many key processes in lung epithelial function so its regulatory mechanisms are important. Using CRISPR/Cas9 technology, we removed three key cis‐regulatory elements (CREs) from the chr11p13 region and also activated multiple open chromatin sites with CRISPRa in airway epithelial cells. Deletion of the CREs caused subtle changes in chromatin architecture and site‐specific increases in EHF and ELF5. CRISPRa had most effect on ELF5 transcription. ELF5 levels are low in airway cells but higher in LNCaP (prostate) and T47D (breast) cancer cells. ATAC‐seq in these lines revealed novel peaks of open chromatin at the 5’ end of chr11p13 associated with an expressed ELF5 gene. Furthermore, 4C‐seq assays identified direct interactions between the active ELF5 promoter and sites within the EHF locus, suggesting coordinate regulation between these TFs. ChIP‐seq for ELF5 in T47D cells revealed ELF5 occupancy within EHF introns 1 and 6, and siRNA‐mediated depletion of ELF5 enhanced EHF expression. These results define a new role for ELF5 in lung epithelial biology.

Abstract 5204: ICAM-1 and IL-17E upregulate FOXO1 to suppress the expression of oncogenic PKC-ι in human melanoma

Abstract Throughout the span of the last 15 years, the number of cases of melanoma has been on a steady increase. Approximately, 92,000 new melanoma cases will be diagnosed in the United States in 2018. Our previous studies demonstrated that protein kinase C-iota (PKC-ι) is crusial for melanoma progression, which promotes the activation of nuclear factor (NF)-κB through a PI3K/AKT manner, thereby supporting survival and progression. In addition, PKC-ι induced the metastasis of melanoma cells by stimulating ephithilial-mesenchmal transition. Results also showed that PKC- specific inhibitors diminished the expression of both PKC-ι and phospo-PKC-ι, suggesting that PKC-ι plays a role in regulating its own expression in melanoma cells in-vitro {Ratnayake, W.S., et al. Cell Adhes. Migr. (2018) https://doi.org/10.1080/19336918.2018.1471323}. In this study, we report the transcriptional regulation mechanisms of PRKCI (PKC-ι gene) expression in melanoma. PROMO and Genomatix Matinspector methods were used to identify c-Jun, interferon-stimulated gene factor 3 (ISGF3), paired box gene 3 (PAX3), early growth response protein 1 (EGR1) and forkhead box protein O1 (FOXO1), which bind on or near the promoter sequence of the PRKCI and they were analyzed for their roles in PKC-ι expression. siRNAs were used to silence selected transcription factors, and the results revealed that, silencing of FOXO1 and c-Jun significantly altered the expression of PRKCI. In addtion, we knocked down NF-κB in order to demonstrate changes on activities of c-Jun and FOXO1. Both phosphorylated and total PKC-ι levels increased upon FOXO1 silencing and decreased upon c-Jun silencing, suggesting that c-Jun turns is an upregulator, while FOXO1 acts as a downregulator of PRKCI expression. Multiplex ELISA assays were used to analyze multiple pathways and the expression of cytokines with respect to crosstalk between signaling pathways affected by specific inhibiton PKC-ι. Western blot data for all siRNA treatments of PKC-ι, NF-κB, FOXO1 and c-Jun were supported by real-time qPCR. Taken together, results suggested that the regulation of PKC-ι expression was strongly associated with FOXO1 and c-Jun through the trascriptional deactivation/activation. In addition, we observed a significant decrease in the mRNA levels of both interleukin (IL)-6 and IL-8, with a significant increase in the levels of IL-17E and intercellular adhesion molecule 1 (ICAM-1) upon the knockdown of expression of PKC-ι in both cell lines. This suggested that PKC-ι expression was affected by said cytokines in an autocrine modus. Overall, the findings suggest that PKC-ι inhibition suppresses its own expression, diminishing oncogenic signaling such as NF-κB, PI3K/AKT and JNK/c-Jun while upregulating anti-tumor signaling such as ICAM-1/FOXO1, thus rendering it an effective novel biomarker for use in the design of novel targeted therapeutics for melanoma metastasis. Citation Format: Wishrawana Sarathi Ratnayake, Christopher Apostolatos, Sloan Breedy, André Apostolatos, Mildred Acevedo-Duncan. ICAM-1 and IL-17E upregulate FOXO1 to suppress the expression of oncogenic PKC-ι in human melanoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 5204.

Association of metabolism-related genes polymorphisms with adenocarcinoma of the oesophagogastric junction: Evidence from 2261 subjects.

The etiology of adenocarcinoma of the esophagogastric junction (AEG) remains unclear. It is believed that the increasing of AEG may be correlated with the elevated ratio of obesity and overweight. Thus, metabolism‐related genes and variants may play important roles in the occurrence and progress of AEG. The current investigation involved 720 patients with AEG and 1541 healthy controls. We selected transcription factor 7‐like 2 (TCF7L2) rs7903146 and rs290481, INS rs689 and INSR rs1799817 single‐nucleotide polymorphisms (SNPs), and explored the association of these SNPs with lymph node status and risk of AEG. The polymerase chain reaction was harnessed to identify the genotyping of four polymorphisms. We found that TCF7L2 rs290481 (T > C) and INSR rs1799817 (G > A) polymorphisms were associated with the increased susceptibility of AEG (P = .007 and 0.004 for TCF7L2 rs290481 in TC vs TT and TC/CC vs TT models, and P = .040 for INSR rs1799817 in GA/AA vs GG model). We also conducted a subgroup analysis by different cancer stage. We identified that TCF7L2 rs290481, INS rs689, and INSR rs1799817 SNPs increased the susceptibility of AEG in different cancer stage subgroups. In addition, we found that rs290481 SNP in TCF7L2 gene increased the risk of lymph node metastasis in drinking patients with AEG. However, the association of INSR rs1799817 SNP with a decreased risk of lymph node metastasis in smoking patients with AEG was found. Our findings highlight that TCF7L2 rs290481, INS rs689, and INSR rs1799817 polymorphisms may increase the risk of AEG. In addition, TCF7L2 rs290481 and INSR rs1799817 SNPs may influence the lymph node metastasis in patients with AEG.

Direct Neuronal Reprogramming Reveals Unknown Functions for Known Transcription Factors.

In recent years, the need to derive sources of specialized cell types to be employed for cell replacement therapies and modeling studies has triggered a fast acceleration of novel cell reprogramming methods. In particular, in neuroscience, a number of protocols for the efficient differentiation of somatic or pluripotent stem cells have been established to obtain a renewable source of different neuronal cell types. Alternatively, several neuronal populations have been generated through direct reprogramming/transdifferentiation, which concerns the conversion of fully differentiated somatic cells into induced neurons. This is achieved through the forced expression of selected transcription factors (TFs) in the donor cell population. The reprogramming cocktail is chosen after an accurate screening process involving lists of TFs enriched into desired cell lineages. In some instances, this type of studies has revealed the crucial role of TFs whose function in the differentiation of a given specific cell type had been neglected or underestimated. Herein, we will speculate on how the in vitro studies have served to better understand physiological mechanisms of neuronal development in vivo.

Brachypodium histone deacetylase BdHD1 positively regulates ABA and drought stress responses

Despite recent evidence that HDACs are involved in the environmental stress responses of plants, their roles in the abiotic stress responses of monocot plants remain largely unexplored. We investigated a HDAC gene, Bradi3g08060 (BdHD1), in Brachypodium distachyon. The Brachypodium BdHD1-overexpression plants displayed a hypersensitive phenotype to ABA and exhibited better survival under drought conditions. On the other hand, the RNA-interference plants were insensitive to ABA and showed low survival under drought stress. At the genome-wide level, overexpression of BdHD1 led to lower H3K9 acetylation at the transcriptional start sites of 230 genes than in the wild type plants under the drought treatment. We validated our ChIP-Seq data on 10 selected transcription factor genes from the 230 drought-specific genes. These genes exhibited much lower expression in the BdHD1-overexpression plants compared to the wild type plants under drought stress. We further identified an ABA-inducible transcription factor gene BdWRKY24 that was repressed in BdHD1-OE plants, but highly expressed in RNA-interference plants under drought stress. These results indicate that BdHD1 plays a positive role in ABA sensitivity and drought stress tolerance and they provide a link between the role of BdHD1 and the drought stress response at a genome-wide level in Brachypodium.

Oligonucleotide transcription factor decoys as tools to control bacterial transcription

Transcription Factor Decoys (TFDs) are short synthetic oligonucleotides that contain the binding site for specific bacterial transcription factors. When translocated into bacterial cytoplasm they can rapidly kill cells when targeted against essential bacterial pathways. Translocation is currently achieved by combination of the TFD with a proprietary lipidic delivery agent, CM2, to form nanoparticles. These interact with highly conserved anionic phospholipids, such as Cardiolipin, to effect delivery to both Gram-positive and Gram-negative bacteria. Simplifying translocation would be an advance that would allow serial screening of large libraries of TFDs to delineate genetic regulatory networks in numerous types of bacteria, including emerging strains. To achieve this we have combined key chemical moieties of the CM2 delivery molecule to the oligonucleotide conjugate by Click chemistry and show that these discrete conjugates are capable of translocation. Confocal laser scanning microscopy was used to monitor the uptake of the TFD-conjugates to E. coli and in parallel their effect on the targeted genetic pathways was confirmed with reporter strains and plating under selective conditions. Hence, it was confirmed that these conjugates can be used as tools to efficiently and specifically modify gene expression by inhibition of selected transcription factors.

Timing mechanism of sexually dimorphic nervous system differentiation.

The molecular mechanisms that control the timing of sexual differentiation in the brain are poorly understood. We found that the timing of sexually dimorphic differentiation of postmitotic, sex-shared neurons in the nervous system of the Caenorhabditis elegans male is controlled by the temporally regulated miRNA let-7 and its target lin-41, a translational regulator. lin-41 acts through lin-29a, an isoform of a conserved Zn finger transcription factor, expressed in a subset of sex-shared neurons only in the male. Ectopic lin-29a is sufficient to impose male-specific features at earlier stages of development and in the opposite sex. The temporal, sexual and spatial specificity of lin-29a expression is controlled intersectionally through the lin-28/let-7/lin-41 heterochronic pathway, sex chromosome configuration and neuron-type-specific terminal selector transcription factors. Two Doublesex-like transcription factors represent additional sex- and neuron-type specific targets of LIN-41 and are regulated in a similar intersectional manner.

Derivation of Hematopoietic Stem and Progenitor Cells from Human Pluripotent Stem Cells.

Hematopoietic stem cell (HSC) transplantation is a curative treatment for hematologic malignancies and innate immunodeficiency, but its applications are limited to matched donors, and the availability of umbilical cord blood of immune types. Therefore, derivation of HSCs from patient-specific human induced pluripotent stem cells (hiPSCs) is a holy grail in regenerative medicine. However, derivation of HSCs from iPSCs has proven elusive. Here, the authors developed a method to derive hematopoietic stem and progenitor cells (HSPCs) by combining two established methods. The first method mimics embryonic development by directed differentiation of iPSCs into a cellular state termed hemogenic endothelium (HE) by stepwise exposure to different combinations of morphogens and cytokines. In the second method, transcription factors are induced in HE cells for specification into HSCs. By combining these approaches, the authors identified a set of select transcription factors that programmed HE cells into HSPCs with long-term and multilineage capacity in vivo. In this chapter, I provide an overview of this technology, technical tips, and future applications.

FOXO1 regulates oncogenic PKC-ι expression in melanoma inversely to c-Jun in an autocrine manner via IL-17E and ICAM-1 activation

Regardless of abundant efforts to enhance primary prevention and early detection, the number of melanoma cases in the United States has increased steadily over the past 15 years, thus greatly affecting public health and the economy. In previous studies, we demonstrated protein kinase C‑ι (PKC‑ι) to be an oncogene in melanoma, which promotes the activation of nuclear factor (NF)‑κB, thereby supporting survival and progression. In addition, we demonstrated that PKC‑ι induced the metastasis of melanoma cells by activating Vimentin, and PKC‑ι inhibition downregulated epithilial‑mesencymal transition (EMT), while inducing apoptosis. Of note, PKC‑ι specific inhibitors downregulated the expression of both PKC‑ι and phosphorylated PKC‑ι, suggesting that PKC‑ι plays a role in regulating its own expression in melanoma. In this study, we report the underlaying mechanisms of the transcriptional regulation of PKC‑ι (PRKCI gene) expression in melanoma. c‑Jun, interferon‑stimulated gene factor 3 (ISGF3), paired box gene 3 (PAX3), early growth response protein 1 (EGR1) and Forkhead box protein O1 (FOXO1), which bind on or near the promoter sequence of the PRKCI gene, were analyzed for their role in PKC‑ι regulation in SK‑MEL‑2 and MeWo cell lines. We silenced selected transcription factors using siRNA, and the results revealed that the silencing of c‑Jun and FOXO1 significantly altered the expression of PRKCI. The levels of both phosphorylated and total PKC‑ι increased upon FOXO1 silencing and decreased upon c‑Jun silencing, suggesting that c‑Jun acts as an upregulator, while FOXO1 acts as a downregulator of PRKCI expression. We also used a multiplex ELISA to analyze multiple pathways other than NF‑κB that were affected by treatment with PKC‑ι inhibitor. The silencing of NF‑κB p65 and PKC‑ι by siRNA suggested that the regulation of PKC‑ι expression was strongly associated with FOXO1. In addition, we observed a significant decrease in the mRNA levels of both interleukin (IL)‑6 and IL‑8, with a significant increase in the levels of IL‑17E and intercellular adhesion molecule 1 (ICAM‑1) upon the knockdown of expression of PKC‑ι in both cell lines. This suggested that PKC‑ι expression was affected by these cytokines in an autocrine manner. Overall, the findings of this study suggest that PKC‑ι inhibition suppresses its own expression, diminishing oncogenic signaling, while upregulating anti‑tumor signaling, thus rendering it an effective novel biomarker for use in the design of novel targeted therapeutics for melanoma.

FOXO1 regulates oncogenic PKC-ι expression in melanoma inversely to c-Jun in an autocrine manner via IL-17E and ICAM-1 activation

Regardless of abundant efforts to enhance primary prevention and early detection, the number of melanoma cases in the United States has increased steadily over the past 15 years, thus greatly affecting public health and the economy. In previous studies, we demonstrated protein kinase C‑ι (PKC‑ι) to be an oncogene in melanoma, which promotes the activation of nuclear factor (NF)‑κB, thereby supporting survival and progression. In addition, we demonstrated that PKC‑ι induced the metastasis of melanoma cells by activating Vimentin, and PKC‑ι inhibition downregulated epithilial‑mesencymal transition (EMT), while inducing apoptosis. Of note, PKC‑ι specific inhibitors downregulated the expression of both PKC‑ι and phosphorylated PKC‑ι, suggesting that PKC‑ι plays a role in regulating its own expression in melanoma. In this study, we report the underlaying mechanisms of the transcriptional regulation of PKC‑ι (PRKCI gene) expression in melanoma. c‑Jun, interferon‑stimulated gene factor 3 (ISGF3), paired box gene 3 (PAX3), early growth response protein 1 (EGR1) and Forkhead box protein O1 (FOXO1), which bind on or near the promoter sequence of the PRKCI gene, were analyzed for their role in PKC‑ι regulation in SK‑MEL‑2 and MeWo cell lines. We silenced selected transcription factors using siRNA, and the results revealed that the silencing of c‑Jun and FOXO1 significantly altered the expression of PRKCI. The levels of both phosphorylated and total PKC‑ι increased upon FOXO1 silencing and decreased upon c‑Jun silencing, suggesting that c‑Jun acts as an upregulator, while FOXO1 acts as a downregulator of PRKCI expression. We also used a multiplex ELISA to analyze multiple pathways other than NF‑κB that were affected by treatment with PKC‑ι inhibitor. The silencing of NF‑κB p65 and PKC‑ι by siRNA suggested that the regulation of PKC‑ι expression was strongly associated with FOXO1. In addition, we observed a significant decrease in the mRNA levels of both interleukin (IL)‑6 and IL‑8, with a significant increase in the levels of IL‑17E and intercellular adhesion molecule 1 (ICAM‑1) upon the knockdown of expression of PKC‑ι in both cell lines. This suggested that PKC‑ι expression was affected by these cytokines in an autocrine manner. Overall, the findings of this study suggest that PKC‑ι inhibition suppresses its own expression, diminishing oncogenic signaling, while upregulating anti‑tumor signaling, thus rendering it an effective novel biomarker for use in the design of novel targeted therapeutics for melanoma.

Abstract 2013: microRNAs targeting EMT transcription factors in breast cancer and their relation to lymph node metastasis

Abstract Metastases derived from primary tumors are responsible for the high cancer-associated death rates. Lymph nodes near to the primary breast tumor have a high chance to develop a secondary tumor, representing one of the first signs of metastasis. For this reason, inhibition of metastasis should be a major goal for breast cancer treatment. Metastasis is a non-random process that initiates with a phenotypic change of the tumor cells, named Epithelial-Mesenchymal Transition (EMT), and is leaded by the transcription factors SNAIL, SLUG, ZEB and TWIST. Because these changes in tumor cells must be plastic and reversible, epigenetic alterations are necessary for EMT. In this sense, microRNAs have emerged as candidate molecular biomarkers and novel therapeutic targets associated to metastasis. The aim of this study is to identify microRNAs that regulate the expression of EMT-transcription factors in breast cancer tumors, and that are involved in lymph node metastasis. For this purpose, we used microRNA microarray data from 50 fresh frozen breast tumors, 28 from patients with lymph node metastasis. Transcription factor expression was assayed by immunohistochemistry. Microarray data analysis using RankProd (R package) revealed approximately 40 microRNAs down-regulated in breast tumors expressing EMT-transcription factors (p<0.05). Using in silico analysis, we predicted 28 microRNAs to be regulators of SNAIL, SLUG and/or TWIST. Four not previously validated microRNAs were selected: miR-202, miR-210, miR-331 and miR-34b. We used luciferase reporter assays to assess the regulation of the 3´UTR of selected transcription factors by each microRNA, at two different concentrations: 10nM and 50nM. We found that miR-210 caused a 50% decrease in luciferase activity through SNAIL and SLUG 3'UTR when tested at 50nM. miR-331, predicted as regulator of SLUG 3'UTR, decreased luciferase activity at both evaluated concentrations in a dose-dependent manner, in a 20% and a 37%. In the case of TWIST, miR-34b at 50nM, decreased luciferase activity in a 65%. No inhibition of luciferase activity was found with mir202. In addition to transcription factors, two of these microRNAs have been predicted as regulators of chemokine receptors CXCR4 and CCR7. Both receptors direct lymphocyte migration to lymphoid organs, and their role has been also implicated in cancer. Our results suggest that down regulation of specific microRNAs, and up regulation of their targets, may induce a metastatic behavior of primary breast tumor cells, promoting invasion and colonization of lymph nodes. In this sense, changes in the expression of microRNAs may serve as biomarkers of breast cancer prognosis in patients. Citation Format: Elisa Pérez-Moreno, Valentina Zavala, Gabriela Valarezo, Wanda Fernández, Pilar Carvallo. microRNAs targeting EMT transcription factors in breast cancer and their relation to lymph node metastasis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2013.