Subfamily Of Transcription Factors Research Articles

Expression of a wheat MYB gene in transgenic tobacco enhances resistance to Ralstonia solanacearum, and to drought and salt stresses

MYB transcription factors play diverse roles in plant growth, developmental processes and stress responses. A full-length cDNA sequence of a MYB gene, namely TaPIMP1, was isolated from wheat (Triticum aestivum L.). The TaPIMP1 transcript level was significantly up-regulated by inoculation with a fungal pathogen Bipolaris sorokiniana and by drought treatment. TaPIMP1 encodes the MYB protein TaPIMP1 consisting of 323 amino acids. TaPIMP1 contains two MYB DNA binding domains (R2, R3), two putative nuclear localization sites and two putative transcription activation domains. TaPIMP1 is a new member of the R2R3-MYB transcription factor subfamily. Transient expression in onion epidermal cells of GFP fused with TaPIMP1 proved that subcellular localization of TaPIMP1 occurred in the nucleus. The TaPIMP1 gene was transferred into tobacco (Nicotiana tabacum L.) cultivar W38 by Agrobacterium-mediated transformation. After screening through PCR and RT-PCR analyses, transgenic tobacco lines expressing TaPIMP1 were identified and evaluated for pathogen resistance, and drought and salt tolerance. Compared to untransformed tobacco host plants, TaPIMP1 expressing plants displayed significantly enhanced resistance to Ralstonia solanacearum and exhibited improved tolerances to drought and salt stresses. In these transgenic lines, the activities of phenylalanine ammonia-lyase (PAL) and superoxide dismutase (SOD) were significantly increased relative to wild-type tobacco plants. The results suggested that the wheat R2R3-MYB transcription factor plays an important role in modulating responses to biotic and abiotic stresses.

Transcription factor fork head regulates the promoter of diapause hormone gene in the cotton bollworm, Helicoverpa armigera, and the modification of SUMOylation

The transcription factor fork head (FoxA) plays important roles in development and metabolism. Here, we cloned a fork head gene in Helicoverpa armigera, and found that the fork head protein is mainly located in the nucleus. This fork head gene belongs to the FoxA subfamily of the Fox transcription factors. The diapause hormone and pheromone biosynthesis-activating neuropeptide (DH-PBAN), which are two well-documented insect neuropeptides that regulate insect development and pheromone biosynthesis, are encoded by a single mRNA. In the present study, fork head was shown to bind strongly to the promoter of H. armigera DH-PBAN gene, and regulate its promoter activity. Furthermore, the effect of SUMOylation of the FH protein on the regulation of Har-DH-PBAN gene was investigated, and we show that the SUMO can modify Har-FH protein and cause down-regulation of DH-PBAN gene expression. These results suggest that SUMOylated FH plays a key role in insect diapause in H. armigera.

Complex ovarian defects lead to infertility in Etv5-/- female mice

Etv5 is a member of the Etv4 subfamily of Ets transcription factors. In female mice, Etv5 was previously shown to be expressed in the mouse ovary. In this work, we show that Etv5-/- female mice are infertile due to a complex reproductive phenotype. Defects in the ovarian tissue architecture were noted as early as 2 weeks of age in Etv5-/- mice. Adult Etv5-/- female mice show decreased ovulation and no interest in mating even after gonadotrophin treatment. Histological abnormalities were also noted in Etv5-/- ovaries. Injection of 17β-estradiol to gonadotrophin-treated Etv5-/- mice significantly increased ovulation, mating and fertilization rates. However, 2-cell embryos of Etv5-/- females show compromised development, suggesting a role for Etv5 in the developmental competence of embryos. Expression of aromatase (CYP11a1), 17α-hydroxylase/17,20 lyase/17,20 desmolase (CYP17a1), side-chain-cleaving enzyme (CYP19a1) and luteinizing hormone/choriogonadotropin receptor mRNAs was not significantly altered in Etv5-/- ovaries. Collectively, our results suggest that Etv5 is important for the developmental competence of germ cells and the regulation of responses to steroid hormones in female mice.

CREB3 subfamily transcription factors are not created equal: Recent insights from global analyses and animal models

The CREB3 subfamily of membrane-bound bZIP transcription factors has five members in mammals known as CREB3 and CREB3L1-L4. One current model suggests that CREB3 subfamily transcription factors are similar to ATF6 in regulated intramembrane proteolysis and transcriptional activation. Particularly, they were all thought to be proteolytically activated in response to endoplasmic reticulum (ER) stress to stimulate genes that are involved in unfolded protein response (UPR). Although the physiological inducers of their proteolytic activation remain to be identified, recent findings from microarray analyses, RNAi screens and gene knockouts not only demonstrated their critical roles in regulating development, metabolism, secretion, survival and tumorigenesis, but also revealed cell type-specific patterns in the activation of their target genes. Members of the CREB3 subfamily show differential activity despite their structural similarity. The spectrum of their biological function expands beyond ER stress and UPR. Further analyses are required to elucidate the mechanism of their proteolytic activation and the molecular basis of their target recognition.

IdentifICATION OF E3 ubiquitin ligase STUB1 as a negative regulator of FOXP3

The level and activity of the forkhead family transcription factor FOXP3 determine the immune function of FOXP3+Tregs. At the beginning of infectious processes, FOXP3+Tregs may regulate effector immune cell responses and lead to failure to control infection. FOXP3+Tregs may also help to limit collateral tissue damage when the antiviral immune responses are too vigorous. Understanding the regulation of FOXP3 and the dynamic ensemble of FOXP3 with enzymatic cofactors in Tregs will provide therapeutic applications for major human viral infectious diseases including HIV, hepatitis B and C viruses. How FOXP3 protein is negatively regulated in CD4+ regulatory T cells during viral infection and inflammation is currently unknown. Here we report that a stress-signal activated E3 ubiquitin ligase STUB1 appears as a negative regulator of FOXP3. Reciprocal co-immunoprecipitation studies indicate that STUB1 interacts with FOXP3 in vivo. Overexpression of STUB1 specifically promotes the ubiquitination of FOXP3, but not other subfamily transcription factor FOXP1. MG132 treatment increased the ubiquitination level of FOXP3, and overexpression of STUB1 induced ubiquitin-mediated degradation of FOXP3. In contrast to the wild type STUB1, ectopic expression of H260Q mutant STUB1, which disrupts its interaction with E2 conjugation enzymes, didn't lead to FOXP3 degradation. Thus, FOXP3 degradation is mediated by enzymatically active STUB1. Moreover, FOXP3 degradation by STUB1 is also depend on its chaperoned binding, since overexpression of the K30A mutant of STUB1, which is incapable of interacting with chaperone proteins, also fails to promote FOXP3 degradation. Knockdown of endogenous STUB1 by shRNA could increase FOXP3 level in FOXP3 expressing T cells. Functionally, ectopic expression of STUB1 dramatically relieves FOXP3 mediated transcriptional suppression. Our studies identified a novel signal pathway to downregulate FOXP3 activity at posttranslational level by ubiquitin mediated protein degradation.

Involvement of LMO4 in tumorigenesis associated epithelial-mesenchymal transition

LMO4 is a novel member of the LIM-only (LMO) subfamily of LIM domain-containing transcription factors, so named because they are composed almost entirely of two tandem LIM domains. This subgroup of LIM proteins has 4 members: LMO-1, LMO-2, LMO-3 and LMO-4. They all play important roles in the normal mammalian development, functioning as an important regulator of cell proliferation. LMO4 is highly expressed in the epithelial compartments at locations of active epithelial-mesenchymal interactions, and can interact with some signaling pathways involved in epithelial-mesenchymal signaling. Thus the disregulation of LMO4 expression may be involved in tumorigenesis. In this paper, we will at first expound LMO4 in detail, based on which the possible mechanisms for its interaction with TGF-β signaling and the roles of this cross-talk between them in the vital process of cell will be introduced. All of those will add to our understanding of tumorigenesis and contribute to the search of new targets for the treatment of cancer.

Expression and function of the Ets transcription factor pea3 during formation of zebrafish pronephros

Polyomavirus enhancer activator 3 (Pea3), belonging to the PEA3 subfamily of Ets transcription factors, is essential for certain organogenesis in mammals. Previously, we found that pea3 correlated with wt1 expression and may contribute to nephrogenesis in rats. Here, we observed that pea3 was mainly expressed in the zebrafish pronephric glomerulus. We further performed functional analyses by in situ hybridization of pea3 in zebrafish embryos after pea3 messenger RNA (mRNA) overexpression and inhibition of double-target genes (pea3 and erm, another member of the PEA3 subfamily) by antisense morpholino-oligonucleotides (MO). Overexpression of pea3 induced abnormal pronephrogenesis. However, MO-pea3 coinjected with MO-erm, but not alone, inhibited zebrafish pronephros development, and these defects were rescued by overexpression of the zebrafish wt1a gene. Thus, pea3 and erm are required for zebrafish pronephrogenesis and can functionally complement each other, and the wt1a gene may be one of their downstream targets.

The ERK MAP kinase-PEA3/ETV4-MMP-1 axis is operative in oesophageal adenocarcinoma

BackgroundMany members of the ETS-domain transcription factor family are important drivers of tumourigenesis. In this context, their activation by Ras-ERK pathway signaling is particularly relevant to the tumourigenic properties of many ETS-domain transcription factors. The PEA3 subfamily of ETS-domain transcription factors have been implicated in tumour metastasis in several different cancers.ResultsHere, we have studied the expression of the PEA3 subfamily members PEA3/ETV4 and ER81/ETV1 in oesophageal adenocarcinomas and determined their role in oesophageal adenocarcinoma cell function. PEA3 plays an important role in controlling both the proliferation and invasive properties of OE33 oesophageal adenocarcinoma cells. A key target gene is MMP-1. The ERK MAP kinase pathway activates PEA3 subfamily members and also plays a role in these PEA3 controlled events, establishing the ERK-PEA3-MMP-1 axis as important in OE33 cells. PEA3 subfamily members are upregulated in human adenocarcinomas and expression correlates with MMP-1 expression and late stage metastatic disease. Enhanced ERK signaling is also more prevalent in late stage oesophageal adenocarcinomas.ConclusionsThis study shows that the ERK-PEA3-MMP-1 axis is upregulated in oesophageal adenocarcinoma cells and is a potentially important driver of the metastatic progression of oesophageal adenocarcinomas.

Isolation and characterization of an AP2/ERF-RAV transcription factor BnaRAV-1-HY15 in Brassica napus L. HuYou15

Transcriptional regulation is thought to be important for stress tolerance and response of transcription factors. RAV subfamily transcription factor contains an AP2- and B3-DNA binding domain, which belongs to the AP2/ERF family. It encodes transcriptional regulators with a variety of functions involved in the developmental and physiological processes in plants. Here, a RAV-like gene, BnaRAV-1-HY15, was isolated from Brassica napus L. cv HuYou15. Sequence homology analysis revealed that the BnaRAV-1-HY15 factor belongs to the RAV subfamily of the AP2/ERF family, and it shares high identity with the AtRAV2 of Arabidopsis. Sequence and three-dimensional structural analyses revealed that BnaRAV-1-HY15 contains two distinct DNA-binding domains, one AP2 domain together with one B3 domain. The AP2 domain composed of 54 amino acids and present in N-terminal region. In addition to AP2 domain, 117 amino acids show significant sequence similarity to the B3 domain present in C-terminal region. Semi-quantitative RT-PCR analysis indicated that the BnaRAV-1-HY15 gene is induced by cold, NaCl and PEG treatments. Under ABA stress, the expression of BnaRAV-1-HY15 gene was not detected. The gene expression was also not traceable from the tissues of pod, bud, petal, leaf, stem and root of normally grown B. napus L. HuYou15 plant at the period of flowering and seed development.

MicroRNA-486-5p Targets Foxo1 and Regulates Human Hematopoietic Stem Cell Proliferation and Erythroid Differentiation

AbstractAbstract 3871Previous studies have supported a critical role for specific miRNA in regulating hematopoiesis. However the relative abundance and specificity for most miRNAs remains to be investigated, and the role of expressed miRNA in regulating cell fate and function remains poorly understood. Using microRNA microarrays we identified increased expression of miR-486 in chronic myeloid leukemia (CML) compared to normal CD34+ cells. miR-486 is located within the last intron of the Ankyrin-1 gene on chromosome 8 and is reported to be enriched in muscle cells. The expression pattern of miR-486 in hematopoietic cells and its roles in hematopoietic regulation are not known. In CB cells, miR-486 expression level was highest in MEP and was low in HSC. There was 16-fold increased expression of miR-486 during in vitro erythroid differentiation of CB Lin-CD34+CD38– cells, associated with 5-fold increase in Ankyrin-1 gene expression. To explore the role of miR-486 in growth and differentiation of hematopoietic stem and progenitor cells (HSPC), we first expressed hsa-miR-486-5p in CB CD34+ cells using lentiviral vectors. CB CD34+ cells transduced with this vector demonstrated 2–3 fold increased expression of miRNA-486-5p compared to cells transduced with a control vector (Ctrl). CB CD34+ cells expressing miR-486-5p generated modestly increased numbers of cells (1.22 fold) in culture with SCF, IL-3, GM-CSF, G-CSF and EPO for 6 days. Increased numbers of erythroid cells and reduced numbers of myeloid cells were generated in culture (GPA+ cells: Ctrl 58% and miR-486-5p 72.2%; CD33+ cells: Ctrl 30.7% and miR-486-5p 21.9%;, CD11b cells: Ctrl 33.5% and miR-486-5p 21.5%). To further investigate the effect of inhibition of miR-486-5p on growth and differentiation of HSPC, we inhibited miR-486 expression in CB CD34+ cells using a modified miRZip anti-miRNA lentivirus vectors (SBI) expressing anti-miR-486-5p and compared to cells expressing a control scrambled anti-miRNA sequence. Anti-miR-486-5p expressing CB CD34+ cells generated reduced number of cells in growth factor (GF) culture (67.5% inhibition) compared to controls. Greater inhibition of erythroid compared to myeloid cells was seen (GPA+ cells: 62.5% inhibition; CD33+ cells: 37.1% inhibition compared to controls at day 6). Anti-miR-486-5p expressing CB CD34+ cells also demonstrated reduced colony formation (BFU-E: 67% inhibition;, CFU-GM 16% inhibition), and reduced proliferation (43.88% inhibition of proliferation index) compared to controls. Similar results were observed with CB Lin-CD34+CD38- cells transduced with anti-486-5p virus (GPA+ cells: 67% inhibition; CD33+ cells: 30 % inhibition). The number of CD34+ cells was however maintained after culture (117% for miR-486-5p compared to scramble). These results indicate an important role for miR-486-5p in preservation, proliferation and erythroid differentiation of HSC. A search for evolutionarily conserved miR-486-5p targets using Targetscan 5.1 identified Foxo1, a member of the Foxo subfamily of forkhead transcription factors which play negative regulatory roles in hematopoiesis, as the highest ranking target. To demonstrate that Foxo1 is a direct target of miR-486-5p, we generated pMIR-REPORT™ constructs containing two miR-486-5p seed sites (182 and 658) within the Foxo1 3′-UTR. These constructs were cotransfected into HEK293T cells along with a miR-486-5p expression plasmid or empty control vector. Expression of miR-486-5p resulted in a 65% reduction in luciferase activity. Expression of anti-miR-486-5p resulted in increased Foxo1 protein expression in CB CD34+ cells. Expression of miR-486-5p also resulted in 50% decrease of Foxo1 protein expression. Using a Fas-L promoter-luciferase reporter we found that inhibition of miR486-5p increased Foxo1 transactivation activity in HEK293T cells. These results demonstrate that Foxo1 is a direct target of miR-486-5p. We conclude that miR-486-5p expression is modulated during normal hematopoietic differentiation and in leukemic hematopoiesis. Our results indicate a regulatory role for miR-486-5p in the growth hematopoietic stem cells and their erythroid differentiation. We show that miR-486-5p directly inhibits Foxo1 expression, which may potentially play an important role in its hematopoietic regulatory function. Disclosures:No relevant conflicts of interest to declare.

Expression and Function of Apoptosis Initiator FOXO3 in Granulosa Cells During Follicular Atresia in Pig Ovaries

In mammalian ovaries, most follicles are lost by atresia before ovulation. It has become apparent that the apoptosis of granulosa cells induces follicular atresia. Forkhead box O3 (FOXO3), also called FKHRL1 (forkhead in rhabdomyosarcoma-like 1), is a proapoptotic molecule that belongs to the FOXO subfamily of forkhead transcription factors. Foxo3-deficient female mice were reported to be infertile because of abnormal ovarian follicular development, but the precise influences of FOXO3 on follicular atresia of mature ovary have not been determined. Therefore, we examined the expression and function of FOXO3 in porcine ovarian follicles and granulosa-derived cells. FOXO3 mRNA levels in granulosa cells of porcine ovaries increased during atresia, while FOXO3 protein was abundant in granulosa cells of early atretic follicles. By immunohistochemistry, the inner surface area of the granulosa layer in early atretic follicles was strongly stained with anti-FOXO3 antibody. The granulosa cells expressing FOXO3 coincided with apoptotic cells, indicating a role of FOXO3 as a proapoptotic factor in granulosa cells of porcine ovaries. In porcine (JC-410) and human (KGN) granulosa-derived cells, cell death was induced by transfection of FOXO3 expression vectors. Expression of the proapoptotic factors Fas ligand (FASLG) and BCL2-like 11 (BCL2L11) was upregulated by FOXO3 in KGN cells. In conclusion, FOXO3 is expressed in porcine ovarian follicles and induces apoptosis in granulosa cells, suggesting that it is a candidate for the initiator of follicular atresia.

Optimizing the binding activity of the AP2/ERF transcription factor with the GCC box element from Brassica napus by directed evolution

In this study, we cloned the ERF-B3 subfamily transcription factor gene BnaERF-B3-hy15 from Brassica napus L. Huyou15. This 600 bp gene encodes a 199 amino acid classic ethylene responsive factor (ERF), which shown no binding or very weak binding GCC box-binding activity by the yeast one-hybrid assay. We used gene shuffling and the yeast one-hybrid system to obtain three mutated sequences that can bind to the GCC box. Sequence analysis indicated that two residues, Gly156 in the AP2 domain and Phe62 at the N-terminal domain were mutated to arginine and serine, respectively. Changes of Gly156 to arginine and Phe62 to serine increased the GCCbinding activity of BnaERF-B3-hy15 and the alter of Gly156 to arginine changed the AP2-domain structure of BnaERF-B3- hy15.

Conserved and Distinct Modes of CREB/ATF Transcription Factor Regulation by PP2A/B56γ and Genotoxic Stress

Activating transcription factor 1 (ATF1) and the closely related proteins CREB (cyclic AMP resonse element binding protein) and CREM (cyclic AMP response element modulator) constitute a subfamily of bZIP transcription factors that play critical roles in the regulation of cellular growth, metabolism, and survival. Previous studies demonstrated that CREB is phosphorylated on a cluster of conserved Ser residues, including Ser-111 and Ser-121, in response to DNA damage through the coordinated actions of the ataxia-telangiectasia-mutated (ATM) protein kinase and casein kinases 1 and 2 (CK1/2). Here, we show that DNA damage-induced phosphorylation by ATM is a general feature of CREB and ATF1. ATF1 harbors a conserved ATM/CK cluster that is constitutively and stoichiometrically phosphorylated by CK1 and CK2 in asynchronously growing cells. Exposure to DNA damage further induced ATF1 phosphorylation on Ser-51 by ATM in a manner that required prior phosphorylation of the upstream CK residues. Hyperphosphorylated ATF1 showed a 4-fold reduced affinity for CREB-binding protein. We further show that PP2A, in conjunction with its targeting subunit B56γ, antagonized ATM and CK1/2-dependent phosphorylation of CREB and ATF1 in cellulo. Finally, we show that CK sites in CREB are phosphorylated during cellular growth and that phosphorylation of these residues reduces the threshold of DNA damage required for ATM-dependent phosphorylation of the inhibitory Ser-121 residue. These studies define overlapping and distinct modes of CREB and ATF1 regulation by phosphorylation that may ensure concerted changes in gene expression mediated by these factors.

Alternative Splicing and Gene Duplication in the Evolution of the FoxP Gene Subfamily

The FoxP gene subfamily of transcription factors is defined by its characteristic 110 amino acid long DNA-binding forkhead domain and plays essential roles in vertebrate biology. Its four members, FoxP1–P4, have been extensively characterized functionally. FoxP1, FoxP2, and FoxP4 are involved in lung, heart, gut, and central nervous system (CNS) development. FoxP3 is necessary and sufficient for the specification of regulatory T cells (Tregs) of the adaptive immune system.In Drosophila melanogaster, in silico predictions identify one unique FoxP subfamily gene member (CG16899) with no described function. We characterized this gene and established that it generates by alternative splicing two isoforms that differ in the forkhead DNA-binding domain. In D. melanogaster, both isoforms are expressed in the embryonic CNS, but in hemocytes, only isoform A is expressed, hinting to a putative modulation through alternative splicing of FoxP1 function in immunity and/or other hemocyte-dependent processes. Furthermore, we show that in vertebrates, this novel alternative splicing pattern is conserved for FoxP1. In mice, this new FoxP1 isoform is expressed in brain, liver, heart, testes, thymus, and macrophages (equivalent in function to hemocytes). This alternative splicing pattern has arisen at the base of the Bilateria, probably through exon tandem duplication. Moreover, our phylogenetic analysis suggests that in vertebrates, FoxP1 is more related to the FoxP gene ancestral form and the other three paralogues, originated through serial duplications, which only retained one of the alternative exons. Also, the newly described isoform differs from the other in amino acids critical for DNA-binding specificity. The integrity of its fold is maintained, but the molecule has lost the direct hydrogen bonding to DNA bases leading to a putatively lower specificity and possibly affinity toward DNA.With the present comparative study, through the integration of experimental and in silico studies of the FoxP gene subfamily across the animal kingdom, we establish a new model for the FoxP gene in invertebrates and for the vertebrate FoxP1 paralogue. Furthermore, we present a scenario for the structural evolution of this gene class and reveal new previously unsuspected levels of regulation for FoxP1 in the vertebrate system.

FoxO, Autophagy, and Cardiac Remodeling

In response to changes in workload, the heart grows or shrinks. Indeed, the myocardium is capable of robust and rapid structural remodeling. In the setting of normal, physiological demand, the heart responds with hypertrophic growth of individual cardiac myocytes, a process that serves to maintain cardiac output and minimize wall stress. However, disease-related stresses, such as hypertension or myocardial infarction, provoke a series of changes that culminate in heart failure and/or sudden death. At the other end of the spectrum, cardiac unloading, such as occurs with prolonged bed rest or weightlessness, causes the heart to shrink. In recent years, considerable strides have been made in deciphering the molecular and cellular events governing pro- and anti-growth events in the heart. Prominent among these mechanisms are those mediated by FoxO (Forkhead box-containing protein, O subfamily) transcription factors. In many cell types, these proteins are critical regulators of cell size, viability, and metabolism, and their importance in the heart is just emerging. Also in recent years, evidence has emerged for a pivotal role for autophagy, an evolutionarily conserved pathway of lysosomal degradation of damaged proteins and organelles, in cardiac growth and remodeling. Indeed, evidence for activated autophagy has been detected in virtually every form of myocardial disease. Now, it is clear that FoxO is an upstream regulator of both autophagy and the ubiquitin-proteasome system. Here, we discuss recent advances in our understanding of cardiomyocyte autophagy, its governance by FoxO, and the roles each of these plays in cardiac remodeling.

Hydrogen peroxide stress stimulates phosphorylation of FoxO1 in rat aortic endothelial cells

To examine whether the phosphorylation of the O subfamily of forkhead transcription factors (FoxO) is involved in response to oxidative stress in rat aortic endothelial cells (RAECs). RAECs were treated with H(2)0(2) and phosphorylation status of proteins were evaluated by Western blot analysis. The subcellular localization of FoxO1 was determined by nuclear and cytosolic fractionation followed by Western blot analysis as well as immunocytochemistry. The transcriptional activity of FoxO1 in H(2)0(2) stress was assessed by luciferase reporter assay. Expression of FoxO1 target gene was determined by real-time PCR analysis. H(2)0(2) stress stimulated phosphorylation of FoxO1 at Thr24 and Ser256 in a concentration and time dependent manner in RAECs. Pretreatment of RAECs with PI-3K inhibitors abolished the activation of Akt and prevented the phosphorylation of FoxO1. Akt-mediated phosphorylation promoted nuclear exclusion of FoxO1. An IRS-driven luciferase activity transactivated by exogenous FoxO1 was modestly suppressed by hydrogen peroxide stress. The expression of Bim, a target gene of FoxO factors, was negatively regulated by Akt-mediated phosphorylation in response to hydrogen peroxide stimulation. Our data demonstrate that phosphorylation of FoxO1 by PI-3K/Akt signaling is implicated in response to oxidative stress in vascular endothelial cells.

Pea3 Transcription Factors and Wnt1-Induced Mouse Mammary Neoplasia

The role of the PEA3 subfamily of Ets transcription factors in breast neoplasia is controversial. Although overexpression of PEA3 (E1AF/ETV4), and of the related factors ERM (ETV5) and ER81 (ETV1), have been observed in human and mouse breast tumors, PEA3 factors have also been ascribed a tumor suppressor function. Here, we utilized the MMTV/Wnt1 mouse strain to further interrogate the role of PEA3 transcription factors in mammary tumorigenesis based on our previous observation that Pea3 is highly expressed in MMTV/Wnt1 mammary tumors. Pea3 expression in mouse mammary tissues was visualized using a Pea3NLSlacZ reporter strain. In normal mammary glands, Pea3 expression is predominantly confined to myoepithelial cells. Wnt1 transgene expression induced marked amplification of this cell compartment in nontumorous mammary glands, accompanied by an apparent increase in Pea3 expression. The pattern of Pea3 expression in MMTV/Wnt1 mammary glands recapitulated the cellular profile of activated β-catenin/TCF signaling, which was visualized using both β-catenin immunohistochemistry and the β-catenin/TCF-responsive reporter Axin2NLSlacZ. To test the requirement for PEA3 factors in Wnt1-induced tumorigenesis, we employed a mammary-targeted dominant negative PEA3 transgene, ΔNPEA3En. Expression of ΔNPEA3En delayed early-onset tumor formation in MMTV/Wnt1 virgin females (P = 0.03), suggesting a requirement for PEA3 factor function for Wnt1-driven tumor formation. Consistent with this observation, expression of the ΔNPEA3En transgene was profoundly reduced in mammary tumors compared to nontumorous mammary glands from bigenic MMTV/Wnt1, MMTV/ΔNPEA3En mice (P = 0.01). Our data provide the first description of Wnt1-mediated expansion of the Pea3-expressing myoepithelial compartment in nontumorous mammary glands. Consistent with this observation, mammary myoepithelium was selectively responsive to Wnt1. Together these data suggest the MMTV/Wnt1 strain as a potential model of basal breast cancer. Furthermore, this study provides evidence for a protumorigenic role of PEA3 factors in breast neoplasia, and supports targeting the PEA3 transcription factor family in breast cancer.

Stable Chromatin Binding Prevents FoxA Acetylation, Preserving FoxA Chromatin Remodeling

FoxA1-3 (formerly HNF3alpha, -beta, and -gamma), members of the FoxA subfamily of forkhead transcription factors, function as initial chromatin-binding and chromatin-remodeling factors in a variety of tissues, including liver and pancreas. Despite essential roles in development and metabolism, regulation of FoxA factors is not well understood. This study examines a potential role for acetylation in the regulation of FoxA chromatin binding and remodeling. Using in silico analysis, we have identified 11 putative p300 acetylation sites within FoxA1, five of which are located within wings 1 and 2 of its winged-helix DNA-binding domain. These polypeptide structures stabilize FoxA DNA and chromatin binding, and we have demonstrated that acetylation attenuates FoxA binding to DNA and diminishes its ability to remodel chromatin. FoxA acetylation is inhibited by chromatin binding. We propose a model whereby stable chromatin binding protects the FoxA DNA-binding domain from acetylation to preserve chromatin binding and remodeling by FoxA factors in the absence of extracellular cues.

Transcriptional regulation of secondary wall formation controlled by NAC domain proteins

Woody cells develop secondary wall structure that mainly consists of polysaccharides (cellulose and hemicellulose) and lignin. These components are expected to be new sources of biofuels and biomaterials. Therefore, it is important to understand the molecular mechanism underlying secondary wall formation and how it contributes to plant biomass. Plant-specific NAC domain transcription factor family has been shown to be involved in diverse biological functions. Recently, several studies reported that a subfamily of the NAC domain transcription factors plays pivotal roles in secondary wall formation. In this review, we have summarized the role of NAC domain transcription factors in controlling the secondary wall formation.

Nucleotide Polymorphism in the Wheat Transcriptional ActivatorSpaInfluences Its Pattern of Expression and Has Pleiotropic Effects on Grain Protein Composition, Dough Viscoelasticity, and Grain Hardness

Storage protein activator (SPA) is a key regulator of the transcription of wheat (Triticum aestivum) grain storage protein genes and belongs to the Opaque2 transcription factor subfamily. We analyzed the sequence polymorphism of the three homoeologous Spa genes in hexaploid wheat. The level of polymorphism in these genes was high particularly in the promoter. The deduced protein sequences of each homoeolog and haplotype show greater than 93% identity. Two major haplotypes were studied for each Spa gene. The three Spa homoeologs have similar patterns of expression during grain development, with a peak in expression around 300 degree days after anthesis. On average, Spa-B is 10 and seven times more strongly expressed than Spa-A and Spa-D, respectively. The haplotypes are associated with significant quantitative differences in Spa expression, especially for Spa-A and Spa-D. Significant differences were found in the quantity of total grain nitrogen allocated to the gliadin protein fractions for the Spa-A haplotypes, whereas the synthesis of glutenins is not modified. Genetic association analysis between Spa and dough viscoelasticity revealed that Spa polymorphisms are associated with dough tenacity, extensibility, and strength. Except for Spa-A, these associations can be explained by differences in grain hardness. No association was found between Spa markers and the average single grain dry mass or grain protein concentration. These results demonstrate that in planta Spa is involved in the regulation of grain storage protein synthesis. The associations between Spa and dough viscoelasticity and grain hardness strongly suggest that Spa has complex pleiotropic functions during grain development.