Direct Transcriptional Regulation Research Articles

Roles of Hoxb5 in the development of vagal and trunk neural crest cells.

Neural crest cells (NC) are a group of multipotent stem cells uniquely present in vertebrates. They are destined to form various organs according to their anterior-posterior (A-P) levels of origin in the neural tube (NT). They develop into a wide spectrum of cell lineages under the influence of signaling cascades, neural plate border genes and NC specifier genes. Although this complex gene regulatory network (GRN) specifies the fate of NC and the combinatory action of Hox genes executed at the time of NC induction governs the patterning of NC for the formation of specific structures along the A-P axis, not much information on how GRN and Hox genes directly interact and orchestrate is available. This review summarizes recent findings on the multiple roles of Hoxb5 on the survival and cell lineage differentiation of vagal and trunk NC cells during early development, by direct transcriptional regulation of NC specifier genes (Sox9 and Foxd3) of the GRN. We will also review findings on the transcriptional regulation of Ret by Hoxb5 in the population of the vagal NC that are committed to the enteric neuron and glia lineages. Functional redundancy between Hox proteins (Hoxa5 and Hoxc5) from the same paralogue group as Hoxb5, and the cooperative effects of Hox cofactors, collaborators and transcription factors in the Hoxb5 transcriptional regulation of target genes will also be discussed.

MiR-17 deregulates a core RUNX1-miRNA mechanism of CBF acute myeloid leukemia.

BackgroundCore Binding Factor acute myeloid leukemia (CBF-AML) with t(8;21) RUNX1-MTG8 or inv(16) CBFB-MYH11 fusion proteins often show upregulation of wild type or mutated KIT receptor. However, also non-CBF-AML frequently displays upregulated KIT expression. In the first part of this study we show that KIT expression can be also upregulated by miR-17, a regulator of RUNX1, the gene encoding a CBF subunit. Interestingly, both CBF leukemia fusion proteins and miR-17, which targets RUNX1-3′UTR, negatively affect a common core RUNX1-miRNA mechanism that forces myeloid cells into an undifferentiated, KIT-induced, proliferating state. In the second part of this study we took advantage of the conservation of the core RUNX1-miRNA mechanism in mouse and human, to mechanistically demonstrate in a mouse myeloid cell model that increased KIT-induced proliferation is per se a mechanism sufficient to delay myeloid differentiation.MethodsHuman (U937) or mouse (32D) myeloid clonal lines were used, respectively, to test: 1) the effect of RUNX1-MTG8 and CBFB-MYH11 fusion proteins, or upregulation of miR-17, on KIT-induced proliferation and myeloid differentiation, and 2) the effect of upregulation of KIT-induced proliferation per se on myeloid cell differentiation.ResultsIn the first part of this study we found that stable miR-17 upregulation affects, like the CBF-AML fusion proteins (RUNX1-MTG8 or CBFB-MYH11), a core RUNX1-miRNA mechanism leading to KIT-induced proliferation of differentiation-arrested U937 myeloid cells. In the second part of the study we harnessed the conservation of this core mechanism in human and mouse to demonstrate that the extent of KIT upregulation in 32D mouse myeloid cells with wild type RUNX1 can per se delay G-CSF-induced differentiation. The integrated information gathered from the two myeloid cell models shows that RUNX1 regulates myeloid differentiation not only by direct transcriptional regulation of coding and non-coding myeloid differentiation functions (e.g. miR-223), but also by modulating KIT-induced proliferation via non-coding miRNAs (e.g. miR-221).ConclusionsThe novelty of this study is dual. On the one hand, miRNAs (e.g. miR-17) can mimic the effects of CBF-AML fusion proteins by affecting a core RUNX1-miRNA mechanism of KIT-induced proliferation of undifferentiated myeloid cells. On the other hand, the extent of KIT-induced proliferation itself can modulate myeloid differentiation of cells with wild type RUNX1 function.Electronic supplementary materialThe online version of this article (doi:10.1186/s12943-014-0283-z) contains supplementary material, which is available to authorized users.

The DNA-binding network of Mycobacterium tuberculosis.

Mycobacterium tuberculosis (MTB) infects 30% of all humans and kills someone every 20–30 s. Here we report genome-wide binding for ~80% of all predicted MTB transcription factors (TFs), and assayed global expression following induction of each TF. The MTB DNA-binding network consists of ~16,000 binding events from 154 TFs. We identify >50 TF-DNA consensus motifs and >1,150 promoter-binding events directly associated with proximal gene regulation. An additional ~4,200 binding events are in promoter windows and represent strong candidates for direct transcriptional regulation under appropriate environmental conditions. However, we also identify >10,000 ‘dormant’ DNA-binding events that cannot be linked directly with proximal transcriptional control, suggesting that widespread DNA binding may be a common feature that should be considered when developing global models of coordinated gene expression.

MondoA-Mlx Transcriptional Activity Is Limited by mTOR-MondoA Interaction

Mammalian target of rapamycin (mTOR) integrates multiple signals, including nutrient status, growth factor availability, and stress, to regulate cellular and organismal growth. How mTOR regulates transcriptional programs in response to these diverse stimuli is poorly understood. MondoA and its obligate transcription partner Mlx are basic helix-loop-helix leucine zipper (bHLHZip) transcription factors that sense and execute a glucose-responsive transcriptional program. MondoA-Mlx complexes activate expression of thioredoxin-interacting protein (TXNIP), which is a potent inhibitor of cellular glucose uptake and aerobic glycolysis. Both mTOR and MondoA are central regulators of glucose metabolism, yet whether they interact physically or functionally is unknown. We show that inhibition of mTOR induces MondoA-dependent expression of TXNIP, coinciding with reduced glucose uptake. Mechanistically, mTOR binds to MondoA in the cytoplasm and prevents MondoA-Mlx complex formation, restricting MondoA's nuclear entry and reducing TXNIP expression. Further, we show that mTOR inhibitors and reactive oxygen species (ROS) regulate interaction between MondoA and mTOR in an opposing manner. Like mTOR's suppression of the MondoA-TXNIP axis, MondoA can also suppress mTOR complex 1 (mTORC1) activity via its direct transcriptional regulation of TXNIP. Collectively, these studies reveal a regulatory relationship between mTOR and the MondoA-TXNIP axis that we propose contributes to glucose homeostasis.

Differential binding of Lef1 and Msx1/2 transcription factors to Dkk1 CNEs correlates with reporter gene expression in vivo.

Besides the active Wnt signalling itself, the extracellular inhibition by Dkk1 is important for various embryonic developmental processes, such as optic vesicle differentiation and facial outgrowth. Although a feedback crosstalk of the active Wnt/β-catenin signaling and Dkk1 regulation has been suggested, the control of Dkk1 transcription by the Tcf/Lef1 mediated Wnt signalling and its connection to additional signalling factors has not been elucidated in vivo. Here, we used a combination of transgenic mouse approaches and biochemical analyses to unravel the direct Dkk1 transcriptional regulation via Tcf/Lefs. By using site directed mutagenesis, we tested several conserved Tcf/Lef1 binding sites within Dkk1 conserved non-coding elements (CNEs) and found that these are required for tissue specific reporter expression. In addition a conserved Msx1/2 binding site is required for retinal reporter expression and Msx2 but not Msx1 binds its conserved binding site within CNE195 in the optic cups. Within craniofacial expression domains, Lef1 interferes with Dkk1 directly via two conserved Tcf/Lef1 binding sites in the craniofacial enhancer CNE114, both of which are required for the general craniofacial Dkk1 reporter activation. Furthermore, these Tcf/Lef1 sites are commonly bound in the whisker hair bud mesenchyme but specifically Tcf/Lef1 (no. 2) is required for mandibular activation and repression of maxillar Dkk1 activation. Lastly, we tested the Tcf/Lef1 binding capacities of the Dkk1 promoter and found that although Lef1 binds the Dkk1 promoter, these sites are not sufficient for tissue specific Dkk1 activation. Together, we here present the importance of conserved Tcf/Lef1 and Msx1/2 sites that are required for differential Dkk1 transcriptional reporter activation in vivo. This requirement directly correlates with Lef1 and Msx1/2 interaction with these genomic loci.

Loss of ATF3 promotes Akt activation and prostate cancer development in a Pten knockout mouse model.

Activating transcription factor 3 (ATF3) responds to diverse cellular stresses, and regulates oncogenic activities (e.g., proliferation, survival and migration) through direct transcriptional regulation or protein-protein interactions. Although aberrant ATF3 expression is frequently found in human cancers, the role of ATF3 in tumorigenesis is poorly understood. Here we demonstrate that ATF3 suppresses the development of prostate cancer induced by knockout of the tumor suppressor Pten in mouse prostates. Where as the oncogenic stress elicited by Pten loss induced ATF3 expression in prostate epithelium, we found that ATF3 deficiency increased cell proliferation and promoted cell survival, leading to early onset of mouse prostatic intraepithelial neoplasia and the progression of prostate lesions to invasive adenocarcinoma. Importantly, loss of ATF3 promoted activation of the oncogenic AKT signaling evidenced by high levels of phosphorylated AKT and S6 proteins in ATF3-null prostate lesions. In line with these in vivo results, knockdown of ATF3 expression in human prostate cancer cells by sgRNA-mediated targeting activated AKT and increased matrix metalloproteinase-9 expression. Our results thus link ATF3 to the AKT signaling, and suggest that ATF3 is a tumor suppressor for the major subset of prostate cancers harboring dysfunctional Pten.

The phosphate transporter PHT4;1 is a salicylic acid regulator likely controlled by the circadian clock protein CCA1.

The small phenolic compound salicylic acid (SA) plays a critical role in plant defense against broad-spectrum of pathogens. The phosphate transporter gene PHT4;1 was previously shown to affect SA-mediated defense and its expression is regulated by the circadian clock. To further understand how PHT4;1 affects SA accumulation, here we analyzed the genetic interactions between the gain-of-function mutant pht4;1-1 and several known SA mutants, including sid2-1, ald1-1, eds5-3, and pad4-1. The genetic analysis was conducted in the acd6-1 background since the change of acd6-1 dwarfism can be used as a convenient readout for the change of defense levels caused by impairments in some SA genes. We found that compared with the corresponding double mutants, the triple mutants acd6-1pht4;1-1ald1-1, acd6-1pht4;1-1eds5-3, and acd6-1pht4;1-1pad4-1 accumulated lower levels of SA and PR1 transcripts, suggesting that PHT4;1 contributes to acd6-1-conferred defense phenotypes independently of these known SA regulators. Although some triple mutants had wild type (wt)-like levels of SA and PR1 transcripts, these plants were smaller than wt and displayed minor cell death, suggesting that additional regulatory pathways contribute to acd6-1-conferred dwarfism and cell death. Our data further showed that circadian expression of PHT4;1 was dependent on CIRCADIAN CLOCK ASSOCIATED 1 (CCA1), a central oscillator component of Arabidopsis circadian clock. Recombinant CCA1 protein was demonstrated to bind to the PHT4;1 promoter in electrophoretic mobility shift assays, suggesting a direct transcriptional regulation of PHT4;1 by CCA1. Together these results indicate that PHT4;1 is a SA regulator acting independently of several known SA genes and they also implicate a role of the circadian clock mediated by CCA1 in regulating phosphate transport and/or innate immunity in Arabidopsis.

Sox4 Is Required for the Formation and Maintenance of Multipotent Progenitors

IntroductionHematopoiesis is maintained by a hierarchical system, whereas aberrant control of hematopoiesis is the underlying cause of many diseases. Within the hematopoietic hierarchy, hematopoietic stem cells (HSCs) give rise to multipotent progenitors that have lost their self-renewal capacity but remain multipotent to differentiate into mature blood cells. However, the precise molecular mechanisms that modulate this transition are not fully understood yet.ResultsWe recently discovered that genetic ablation of SRY sex determining region Y-box 4 gene (Sox4) in the murine hematopoietic system resulted in dramatic loss of multipotent progenitor population (CD48+CD150-Lin-kit+Sca1+, or CD48+CD150-LSK) both relatively (to the total LSK population) and in absolute number. Interestingly, the absolute number of HSCs (CD48-CD150+Lin-kit+Sca1+, or SLAM+LSK) in these conditional Sox4-deficient mice was comparable to their wild-type counterparts. Transcriptional factor Sox4 belongs to the high-mobility group (HMG) domain superfamily which also includes other Sox proteins, TCF-1 (T-cell factor 1) and LEF-1 (lymphoid enhancer factor 1). Sox4 has been implicated in leukemogenesis and may potentially contribute to stem cell properties. Nevertheless, the precise roles of Sox4 in hematopoietic stem/progenitor cells and the underlying mechanisms have not been defined yet. Further analysis of stem/progenitor compartment defined by Flt3 and CD34 expression demonstrated a major loss in lymphoid-primed multipotent progenitors (LMPPs) (CD34+Flt3+LSK) with relatively normal formation of LT-HSCs (CD34-Flt3-LSK) and ST-HSCs (CD34+Flt3-LSK) upon the loss of Sox4, suggesting that Sox4 is essential for the development from HSCs to multipotent progenitors. Such observation is in line with the expression pattern of Sox4. Quantitative PCR (qPCR) analysis of wild-type mice revealed that expression of Sox4 increased from HSCs to multipotent progenitors which expressed Sox4 at the highest level among all the hematopoietic compartments.Studies of biological behaviors further indicateed that disruption of Sox4 had no effect on proliferative capacity of HSCs and multipotent progenitors, as evidenced by BrdU incorporation assay. However, Annexin V/propidium iodide staining revealed an increased frequency of apoptotic multipotent progenitors, but not that of HSCs upon the ablation of Sox4. In a transplantation setting, although Sox4-deficient LSKs homed appropriately to the bone marrow, they exhibited severely impaired ability to give rise to multipotent progenitors, but contributed normally to HSCs compared to the wild-type donors.Among a set of genes crucial to the biological properties of stem/progenitor cells, qPCR analysis revealed that upon the loss of Sox4, only the levels of Ikaros1 and Ikaros2, the two major Ikaros isoforms in stem/progenitor cells, were downregulated specifically in multipotent progenitors, but remained normal in HSCs. Intriguingly, in a reminiscent manner of Sox4-deficient mice, mice lacking both Ikaros 1 and Ikaros 2 proteins, also exhibited disrupted B cell development and selectively impaired LMPPs. Previous study identified an enhancer of Ikaros locus as the only cis-regulatory element that was capable of stimulating reporter expression in the LMPPs. Our sequence analysis revealed a highly conserved Sox4 binding motif within this enhancer, therefore potentially connecting Sox4 with the known regulatory networks that modulate the differentiation of HSCs. Currently, we are working on (1) confirming the direct transcriptional regulation of Ikaros by Sox4; (2) assessing whether Ikaros mediates the functions of Sox4 in the formation or maintenance of the multipotent progenitors population in vivo; and (3) delineating the downstream regulatory network of Sox4 in stem/progenitor cells.ConclusionIn summary, out study reveals a novel role for Sox4 gene in early hematopoiesis and brings important insights into the regulatory mechanisms underlying the commitment of HSCs toward multipotent progenitors. DisclosuresNo relevant conflicts of interest to declare.

Krüppel-like Factor 4 (KLF4) Suppresses T-Cell Acute Lymphoblastic Leukemia By Inhibiting Expression of MAP2K7 and Expansion of Leukemia Initiating Cells

Acute lymphoblastic leukemia (ALL) is the most common hematological malignancy in children. Although risk-adaptive therapy, CNS-directed chemotherapy and supportive care have improved the survival of ALL patients, disease relapse is still the leading cause of cancer-related death in children. Therefore, new drugs or novel multi-drug combinations are needed as frontline treatments for high-risk patients and as salvage agents for relapsed disease. T-cell ALL (T-ALL) is a subset of ALL that exhibits activating mutations of NOTCH1 in more than 50% of the patients. However, the use of gamma-secretase inhibitors to reduce NOTCH1 activity has not been successful in patients due to limited response and toxicity. Therefore, identification of genetic factors that cooperate with T-ALL leukemogenesis is needed for the development of alternative therapies.KLF4 is a transcription factor that functions as a tumor suppressor or an oncogene depending on cellular context. Our data showed significant reduction of KLF4 transcripts in lymphoblasts from T-ALL patients compared to blood and bone marrow cells from healthy individuals. In consistent with reduced KLF4 levels, these patients exhibit hyper-methylation of CpG islands located between nt -811 and +1190 relative to KLF4 transcription start site. From these findings we hypothesized that KLF4 has tumor suppressor function in T-ALL leukemogenesis. To test our hypothesis, we transduced 5-FU treated bone marrow (BM) cells from control (Klf4fl/fl), Klf4 null (Klf4fl/fl; Vav-iCre) and Klf4 heterozygous (Klf4fl/+; Vav-iCre) mice with retrovirus carrying a NOTCH1 activating mutant (L1601P-ΔP) and then transplanted these BM cells into irradiated recipient mice. In contrast to controls, mice transplanted with transduced Klf4-null BM cells developed T-ALL with significantly higher penetrance (Klf4 null 76.5% v.s. control 21.3%) and shorter latency (Klf4 null 93 days v.s. control 130 days). Interestingly, Klf4 heterozygous group shows similar survival kinetics as Klf4 null group, suggesting that Klf4 haploinsufficiency is enough to accelerate onset of leukemia. To investigate the effect of Klf4 deletion in established leukemia cells, we transplanted NOTCH1 L1601P-ΔP transduced BM cells from Klf4fl/fl; CreER+ mice to induce leukemia. Post-transplantation deletion of the Klf4 gene by tamoxifen administration was able to accelerate T-ALL development compared to mice injected with vehicle. On the cellular level, loss of KLF4 led to increased proliferation of leukemia cells as assessed by in vivo BrdU incorporation, which correlated with decreased levels of p21 protein. Limited dilution transplantation of primary leukemia cells into secondary recipients showed a 9-fold increase of leukemia initiating cells (LIC) frequency in Klf4null leukemia cells compared to controls, suggesting that KLF4 controls expansion of LIC in T-ALL.To elucidate molecular mechanism underlying KLF4 regulation in T-ALL cells, we performed microarray and ChIP-Seq in control and Klf4 null CD4+CD8+ leukemia cells. Combined analyses revealed 202 genes as KLF4 direct targets, of which 11 genes are also deregulated in human T-ALL cells by comparing with published microarray datasets. One of the top upregulated genes is Map2k7, which encodes a kinase upstream of the JNK pathway. Immunoblots in leukemia cells confirmed increased expression of MAP2K7 protein and enhanced phosphorylation of its downstream targets JNK and ATF2. To further investigate the role of JNK pathway in T-ALL, we tested JNK inhibitor SP600125 in human T-ALL cell lines (KOPTK1, DND41, CCRF-CEM, MOLT3). Interestingly, SP600125 showed dose-dependent cytotoxicity in all human T-ALL cell lines tested regardless of their NOTCH1 status.Overall our results showed for the first time that KLF4 functions as a tumor suppressor in T-ALL by regulating proliferation of leukemia cells and frequency of LIC. Additional study elucidated that KLF4 suppresses the JNK pathway via direct transcriptional regulation of MAP2K7. Moreover, the vulnerability of human T-ALL cell lines to JNK inhibition provides a novel target for future therapy in T-ALL patients. DisclosuresNo relevant conflicts of interest to declare.

The spliceosome factor SART1 exerts its anti-HCV action through mRNA splicing

The broadly used antiviral cytokine interferon-α (IFNα)'s mechanisms of action against HCV infection are not well understood. We previously identified SART1, a host protein involved in RNA splicing and pre-mRNA processing, as a regulator of IFN's antiviral effects. We hypothesized that SART1 regulates antiviral IFN effector genes (IEGs) through mRNA processing and splicing. We performed siRNA knockdown in HuH7.5.1 cells and mRNA-sequencing with or without IFN treatment. Selected gene mRNA variants and their proteins, together with HCV replication, were monitored by qRT-PCR and Western blot in HCV OR6 replicon cells and the JFH1 HCV infectious model. We identified 419 genes with a greater than 2-fold expression difference between Neg siRNA and SART1 siRNA treated cells in the presence or absence of IFN. Bioinformatic analysis identified at least 10 functional pathways. SART1 knockdown reduced classical IFN stimulating genes (ISG) mRNA transcription including MX1 and OAS3. However, SART1 did not affect JAK-STAT pathway gene mRNA expression and IFN stimulated response element (ISRE) signaling. We identified alternative mRNA splicing events for several genes, including EIF4G3, GORASP2, ZFAND6, and RAB6A that contribute to their antiviral effects. EIF4G3 and GORASP2 were also confirmed to have anti-HCV effect. The spliceosome factor SART1 is not IFN-inducible but is an IEG. SART1 exerts its anti-HCV action through direct transcriptional regulation for some ISGs and alternative splicing for others, including EIF4G3, GORASP2. SART1 does not have an effect on IFN receptor or canonical signal transduction components. Thus, SART1 regulates ISGs using a novel, non-classical mechanism.

Cancer progression by breast tumors with Pit-1-overexpression is blocked by inhibition of metalloproteinase (MMP)-13.

IntroductionThe POU class 1 homeobox 1 transcription factor (POU1F1, also known as Pit-1) is expressed in the mammary gland and its overexpression induces profound phenotypic changes in proteins involved in cell proliferation, apoptosis, and invasion. Patients with breast cancer and elevated expression of Pit-1 show a positive correlation with the occurrence of distant metastasis. In this study we evaluate the relationship between Pit-1 and two collagenases: matrix metalloproteinase-1 (MMP-1) and matrix metalloproteinase-13 (MMP-13), which have been related to metastasis in breast cancer.MethodsWe began by transfecting the MCF-7 and MDA-MB-231 human breast adenocarcinoma cell lines with the Pit-1 overexpression vector (pRSV-hPit-1). Afterward, the mRNA, protein, and transcriptional regulation of both MMP-1 and MMP-13 were evaluated by real-time PCR, Western blot, chromatin immunoprecipitation (ChIP), and luciferase reporter assays. We also evaluated Pit-1 overexpression with MMP-1 and MMP-13 knockdown in a severe combined immunodeficiency (SCID) mouse tumor xenograft model. Finally, by immunohistochemistry we correlated Pit-1 with MMP-1 and MMP-13 protein expression in 110 human breast tumors samples.ResultsOur data show that Pit-1 increases mRNA and protein of both MMP-1 and MMP-13 through direct transcriptional regulation. In SCID mice, knockdown of MMP-13 completely blocked lung metastasis in Pit-1-overexpressing MCF-7 cells injected into the mammary fat pad. In breast cancer patients, expression of Pit-1 was found to be positively correlated with the presence of both MMP-1 and MMP-13.ConclusionsOur data indicates that Pit-1 regulates MMP-1 and MMP-13, and that inhibition of MMP-13 blocked invasiveness to lung in Pit-1-overexpressed breast cancer cells.Electronic supplementary materialThe online version of this article (doi:10.1186/s13058-014-0505-8) contains supplementary material, which is available to authorized users.

SOX30, a novel epigenetic silenced tumor suppressor, promotes tumor cell apoptosis by transcriptional activating p53 in lung cancer.

Although members of SOX family have been well documented for their essential roles in embryonic development, cell proliferation and disease, the functional role and molecular mechanism of SOX30 in cancer are largely unexplored. Here, we first identified SRY-box containing gene 30 (SOX30) as a novel preferentially methylated gene using genome-wide methylation screening. SOX30 hypermethylation was detected in 100% of lung cancer cell lines (9/9) and 70.83% (85/120) of primary lung tumor tissues compared with none (0/20) of normal and 8.0% (2/25) of peri-tumoral lung tissues (P<0.01). SOX30 was expressed in normal and peri-tumoral lung tissues in which SOX30 was unmethylated, but was silenced or downregulated in lung cancer cell lines and primary lung tumor tissues harboring a hypermethylated SOX30. De-methylation experiments further confirmed that silence of SOX30 was regulated by its hypermethylation. Ectopic expression of SOX30 induces cancer cell apoptosis with inhibiting proliferation in vitro and represses tumor formation in vivo, whereas knockdown of SOX30 demonstrates a reversed effect both in vitro and in vivo. At the molecular level, the antitumorigenic effect of SOX30 is mediated by directly binding to CACTTTG (+115 to +121) of p53 promoter region and activating p53 transcription, suggesting that SOX30 is a novel transcriptional activating factor of p53. Indeed, blockade of p53 attenuates the tumor inhibition of SOX30. Overall, these findings demonstrate that SOX30 is a novel epigenetic silenced tumor suppressor acting through direct regulation of p53 transcription and expression. This study provides novel insights on the mechanism of tumorigenesis in lung cancer.

Hand2 Is an Essential Regulator for Two Notch-Dependent Functions within the Embryonic Endocardium

The basic-helix-loop-helix (bHLH) transcription factor Hand2 plays critical roles during cardiac morphogenesis via expression and function within myocardial, neural crest, and epicardial cell populations. Here, we show that Hand2 plays two essential Notch-dependent roles within the endocardium. Endocardial ablation of Hand2 results in failure to develop a patent tricuspid valve, intraventricular septum defects, and hypotrabeculated ventricles, which collectively resemble the human congenital defect tricuspid atresia. We show endocardial Hand2 to be an integral downstream component of a Notch endocardium-to-myocardium signaling pathway and a direct transcriptional regulator of Neuregulin1. Additionally, Hand2 participates in endocardium-to-endocardium-based cell signaling, with Hand2 mutant hearts displaying an increased density of coronary lumens. Molecular analyses further reveal dysregulation of several crucial components of Vegf signaling, including VegfA, VegfR2, Nrp1, and VegfR3. Thus, Hand2 functions as a crucial downstream transcriptional effector of endocardial Notch signaling during both cardiogenesis and coronary vasculogenesis.

Induction of Ras by SAF-1/MAZ through a feed-forward loop promotes angiogenesis in breast cancer.

In the majority of breast cancers, overexpression and hyperactivation of Ras in the tumor microenvironment play significant role in promoting cancer cell growth, angiogenesis, and metastasis. We have previously shown that vascular endothelial growth factor (VEGF) expression in triple negative breast cancer cells is regulated, at least in part, by SAF-1 (serum amyloid A activating factor 1) transcription factor. In this study we show that transformation of normal MCF-10A breast epithelial cells by constitutively active, oncogenic Ras, induces the DNA-binding activity and transcription function of SAF-1. Furthermore, we show that inhibition of MEK/MAPK-signaling pathway prevents Ras-mediated activation of SAF-1. Interestingly, silencing of SAF-1 expression in breast cancer cells by SAF-1-specific short hairpin RNAs (shRNAs) significantly reduced H-Ras and K-Ras mRNA level. We show that SAF-1 is a direct transcriptional regulator of H-Ras and K-Ras and overexpression of SAF-1 increases H-Ras and K-Ras gene expression. Chromatin immunoprecipitation (ChIP) analyses demonstrated in vivo interaction of SAF-1 at highly purine-rich sequences present at the proximal promoter region, upstream of the transcription start site, in H-Ras and K-Ras genes. Previous studies have shown that these sequences are nuclease hypersensitive and capable of forming G4 quadruplex structure. Together, our results show the presence of a novel transactivating loop, in which, Ras and SAF-1 are interconnected. These findings will help defining molecular mechanisms of abnormal overexpression of Ras in breast tumors, which seldom show genetic Ras mutations.

ChIP-seq and in vivo transcriptome analyses of the Aspergillus fumigatus SREBP SrbA reveals a new regulator of the fungal hypoxia response and virulence.

The Aspergillus fumigatus sterol regulatory element binding protein (SREBP) SrbA belongs to the basic Helix-Loop-Helix (bHLH) family of transcription factors and is crucial for antifungal drug resistance and virulence. The latter phenotype is especially striking, as loss of SrbA results in complete loss of virulence in murine models of invasive pulmonary aspergillosis (IPA). How fungal SREBPs mediate fungal virulence is unknown, though it has been suggested that lack of growth in hypoxic conditions accounts for the attenuated virulence. To further understand the role of SrbA in fungal infection site pathobiology, chromatin immunoprecipitation followed by massively parallel DNA sequencing (ChIP-seq) was used to identify genes under direct SrbA transcriptional regulation in hypoxia. These results confirmed the direct regulation of ergosterol biosynthesis and iron uptake by SrbA in hypoxia and revealed new roles for SrbA in nitrate assimilation and heme biosynthesis. Moreover, functional characterization of an SrbA target gene with sequence similarity to SrbA identified a new transcriptional regulator of the fungal hypoxia response and virulence, SrbB. SrbB co-regulates genes involved in heme biosynthesis and demethylation of C4-sterols with SrbA in hypoxic conditions. However, SrbB also has regulatory functions independent of SrbA including regulation of carbohydrate metabolism. Loss of SrbB markedly attenuates A. fumigatus virulence, and loss of both SREBPs further reduces in vivo fungal growth. These data suggest that both A. fumigatus SREBPs are critical for hypoxia adaptation and virulence and reveal new insights into SREBPs' complex role in infection site adaptation and fungal virulence.

Salt-induced stabilization of EIN3/EIL1 confers salinity tolerance by deterring ROS accumulation in Arabidopsis.

Ethylene has been regarded as a stress hormone to regulate myriad stress responses. Salinity stress is one of the most serious abiotic stresses limiting plant growth and development. But how ethylene signaling is involved in plant response to salt stress is poorly understood. Here we showed that Arabidopsis plants pretreated with ethylene exhibited enhanced tolerance to salt stress. Gain- and loss-of-function studies demonstrated that EIN3 (ETHYLENE INSENSITIVE 3) and EIL1 (EIN3-LIKE 1), two ethylene-activated transcription factors, are necessary and sufficient for the enhanced salt tolerance. High salinity induced the accumulation of EIN3/EIL1 proteins by promoting the proteasomal degradation of two EIN3/EIL1-targeting F-box proteins, EBF1 and EBF2, in an EIN2-independent manner. Whole-genome transcriptome analysis identified a list of SIED (Salt-Induced and EIN3/EIL1-Dependent) genes that participate in salt stress responses, including several genes encoding reactive oxygen species (ROS) scavengers. We performed a genetic screen for ein3 eil1-like salt-hypersensitive mutants and identified 5 EIN3 direct target genes including a previously unknown gene, SIED1 (At5g22270), which encodes a 93-amino acid polypeptide involved in ROS dismissal. We also found that activation of EIN3 increased peroxidase (POD) activity through the direct transcriptional regulation of PODs expression. Accordingly, ethylene pretreatment or EIN3 activation was able to preclude excess ROS accumulation and increased tolerance to salt stress. Taken together, our study provides new insights into the molecular action of ethylene signaling to enhance plant salt tolerance, and elucidates the transcriptional network of EIN3 in salt stress response.

Abstract 2945: Characterization of a novel protein-coding gene named TIHL (Translocated in Hodgkin's Lymphoma)

Abstract Previously, we described the CIITA-BX648577 gene fusion (Steidl C. et al., Nature 2011) was highly expressed in Hodgkin's Lymphoma cell line KM-H2. The Class II Transactivator (CIITA) has been described to function in the regulation of immune responses and its deregulation may serve as a mechanism through which tumor cells evade immunosurveillance. In contrast, the expression and function of the gene/protein encoded by the novel locus BX648577/FLJ27352/C15orf65 is unknown. In the present study, we report the endogenous expression and describe the biological function of the novel gene which we named TIHL (Translocated in Hodgkin's Lymphoma). Comparative analysis in various species revealed high evolutionary conservation (≥50%) between human, mouse, gorilla and Drosophila TIHL orthologs. Using quantitative real time-PCR, we detected expression of full-length TIHL in both healthy and malignant hematopoietic cells. By immunoblot, we detected robust expression of the 13-16 kDa protein in leukemia and lymphoma cell lines using a TIHL-specific antibody. Lentiviral expression of TIHL in human NB4 leukemia cells and murine hematopoietic progenitor HPC-7 cells led to enhanced clonogenicity and increased proliferative capacity with significant increases in the integration of EdU (5-ethynyl-2′-deoxyuridine) and the percentage of cells in S-phase. Likewise, TIHL knockdown using 2 independent lentiviral shRNAs, led to decreased proliferation of both KG1a and NB4 leukemia cells grown in either suspension culture or semi-solid media. Ingenuity Pathway Analysis (IPA) after microarray analysis of NB4 cells overexpressing TIHL highlights significantly altered functional categories including ‘cell growth and proliferation,’ ‘cell death and survival,' and ‘cell cycle,' in line with our previous observations. In silico studies, predicted transcriptional regulators of TIHL including the gene GLI1 (Glioma-Associated Oncogene Homolog 1). GLI1, a positive regulator of the Hedgehog signaling pathway, targets genes involved in cell cycle regulation, cell proliferation and cell death. Treatment of KG1a and NB4 leukemia cells with the small molecule GLI inhibitor GANT61 led to significant inhibition of cell proliferation. Additionally, TIHL mRNA expression was significantly downregulated at increasing concentrations of GANT61 by qRT-PCR. Preliminary data suggests that TIHL overexpression may partially rescue the leukemia inhibitory effects of GANT61 treatment. These data strongly suggest that the novel gene TIHL is involved in cell cycle regulation/modulation in hematopoietic cells. Further studies will involve analysis of the effects of TIHL overexpression or knockdown in healthy donor-derived bone marrow cells, chromatin immunoprecipitation (ChIP)-PCR to confirm its direct transcriptional regulation by GLI1 and co-IP coupled to mass spectrometry to identify direct protein partners of this novel gene. Citation Format: Ujunwa C. Okoye-Okafor, Laura Barreyro, Mario Pujato, Heng Rui Wang, Boris Bartholdy, Britta Will, Tihomira I. Todorova, Masahiro Kawahara, Bruce Woolcock, Andras Fiser, Randy Gascoyne, Christian Steidl, Ulrich Steidl. Characterization of a novel protein-coding gene named TIHL (Translocated in Hodgkin's Lymphoma). [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2945. doi:10.1158/1538-7445.AM2014-2945

Identification of target genes of the bZIP transcription factor OsTGAP1, whose overexpression causes elicitor-induced hyperaccumulation of diterpenoid phytoalexins in rice cells.

Phytoalexins are specialised antimicrobial metabolites that are produced by plants in response to pathogen attack. Momilactones and phytocassanes are the major diterpenoid phytoalexins in rice and are synthesised from geranylgeranyl diphosphate, which is derived from the methylerythritol phosphate (MEP) pathway. The hyperaccumulation of momilactones and phytocassanes due to the hyperinductive expression of the relevant biosynthetic genes and the MEP pathway gene OsDXS3 in OsTGAP1-overexpressing (OsTGAP1ox) rice cells has previously been shown to be stimulated by the chitin oligosaccharide elicitor. In this study, to clarify the mechanisms of the elicitor-stimulated coordinated hyperinduction of these phytoalexin biosynthetic genes in OsTGAP1ox cells, transcriptome analysis and chromatin immunoprecipitation with next-generation sequencing were performed, resulting in the identification of 122 OsTGAP1 target genes. Transcriptome analysis revealed that nearly all of the momilactone and phytocassane biosynthetic genes, which are clustered on chromosomes 4 and 2, respectively, and the MEP pathway genes were hyperinductively expressed in the elicitor-stimulated OsTGAP1ox cells. Unexpectedly, none of the clustered genes was included among the OsTGAP1 target genes, suggesting that OsTGAP1 did not directly regulate the expression of these biosynthetic genes through binding to each promoter region. Interestingly, however, several OsTGAP1-binding regions were found in the intergenic regions among and near the cluster regions. Concerning the MEP pathway genes, only OsDXS3, which encodes a key enzyme of the MEP pathway, possessed an OsTGAP1-binding region in its upstream region. A subsequent transactivation assay further confirmed the direct regulation of OsDXS3 expression by OsTGAP1, but other MEP pathway genes were not included among the OsTGAP1 target genes. Collectively, these results suggest that OsTGAP1 participates in the enhanced accumulation of diterpenoid phytoalexins, primarily through mechanisms other than the direct transcriptional regulation of the genes involved in the biosynthetic pathway of these phytoalexins.

Two C4-sterol methyl oxidases (Erg25) catalyse ergosterol intermediate demethylation and impact environmental stress adaptation in Aspergillus fumigatus.

The human pathogen Aspergillus fumigatus adapts to stress encountered in the mammalian host as part of its ability to cause disease. The transcription factor SrbA plays a significant role in this process by regulating genes involved in hypoxia and low-iron adaptation, antifungal drug responses and virulence. SrbA is a direct transcriptional regulator of genes encoding key enzymes in the ergosterol biosynthesis pathway, including erg25A and erg25B, and ΔsrbA accumulates C4-methyl sterols, suggesting a loss of Erg25 activity [C4-sterol methyl oxidase (SMO)]. Characterization of the two genes encoding SMOs in Aspergillus fumigatus revealed that both serve as functional C4-demethylases, with Erg25A serving in a primary role, as Δerg25A accumulates more C4-methyl sterol intermediates than Δerg25B. Single deletion of these SMOs revealed alterations in canonical ergosterol biosynthesis, indicating that ergosterol may be produced in an alternative fashion in the absence of SMO activity. A Δerg25A strain displayed moderate susceptibility to hypoxia and the endoplasmic reticulum stress-inducing agent DTT, but was not required for virulence in murine or insect models of invasive aspergillosis. Inducing expression of erg25A partially restored the hypoxia growth defect of ΔsrbA. These findings implicated Aspergillus fumigatus SMOs in the maintenance of canonical ergosterol biosynthesis and indicated an overall involvement in the fungal stress response.

Ascl1 as a Novel Player in the Ptf1a Transcriptional Network for GABAergic Cell Specification in the Retina

In contrast with the wealth of data involving bHLH and homeodomain transcription factors in retinal cell type determination, the molecular bases underlying neurotransmitter subtype specification is far less understood. Using both gain and loss of function analyses in Xenopus, we investigated the putative implication of the bHLH factor Ascl1 in this process. We found that in addition to its previously characterized proneural function, Ascl1 also contributes to the specification of the GABAergic phenotype. We showed that it is necessary for retinal GABAergic cell genesis and sufficient in overexpression experiments to bias a subset of retinal precursor cells towards a GABAergic fate. We also analysed the relationships between Ascl1 and a set of other bHLH factors using an in vivo ectopic neurogenic assay. We demonstrated that Ascl1 has unique features as a GABAergic inducer and is epistatic over factors endowed with glutamatergic potentialities such as Neurog2, NeuroD1 or Atoh7. This functional specificity is conferred by the basic DNA binding domain of Ascl1 and involves a specific genetic network, distinct from that underlying its previously demonstrated effects on catecholaminergic differentiation. Our data show that GABAergic inducing activity of Ascl1 requires the direct transcriptional regulation of Ptf1a, providing therefore a new piece of the network governing neurotransmitter subtype specification during retinogenesis.