Putative candidate genes responsible for leaf rolling in rye (Secale cereale L.).

BACKGROUND: Rolling of leaves (RL) is a phenomenon commonly found in grasses. Morphology of the leaf is an important agronomic trait in field crops especially in rice; therefore, majority of the rice breeders are interested in RL. There are only few studies with respect to RL of wheat and barley; however, the information regarding the genetic base of RL with respect to the shape of leaf in rye is lacking. To the best of our knowledge, this is the first study on the localization of loci controlling RL on high density consensus genetic map of rye. RESULTS: Genotypic analysis led to the identification of 43 quantitative trait loci (QTLs) for RL, grouped into 28 intervals, which confirms the multigenic base of the trait stated for wheat and rice. Four stable QTLs were located on chromosomes 3R, 5R, and 7R. Co-localization of QTL for RL and for different morphological, biochemical and physiological traits may suggests pleiotropic effects of some QTLs. QTLs for RL were associated with QTLs for such morphological traits as: grain number and weight, spike number per plant, compactness of spike, and plant height. Two QTLs for RL were found to coincide with QTLs for drought tolerance (4R, 7R), two with QTLs for heading earliness (2R, 7R), one with α-amylase activity QTL (7R) and three for pre-harvest sprouting QTL (1R, 4R, 7R). The set of molecular markers strongly linked to RL was selected, and the putative candidate genes controlling the process of RL were identified. Twelve QTLs are considered as linked to candidate genes on the base of DArT sequences alignment, which is a new information for rye. CONCLUSIONS: Our results expand the knowledge about the network of QTLs for different morphological, biochemical and physiological traits and can be a starting point to studies on particular genes controlling RL and other important agronomic traits (yield, earliness, pre-harvest sprouting, reaction to water deficit) and to appoint markers useful in marker assisted selection (MAS). A better knowledge of the rye genome and genes could both facilitate rye improvement itself and increase the efficiency of utilizing rye genes in wheat breeding.

Iron deficiency triggered transcriptome changes in bread wheat.

A series of complex transport, storage and regulation mechanisms control iron metabolism and thereby maintain iron homeostasis in plants. Despite several studies on iron deficiency responses in different plant species, these mechanisms remain unclear in the allohexaploid wheat, which is the most widely cultivated commercial crop. We used RNA sequencing to reveal transcriptomic changes in the wheat flag leaves and roots, when subjected to iron limited conditions. We identified 5969 and 2591 differentially expressed genes (DEGs) in the flag leaves and roots, respectively. Genes involved in the synthesis of iron ligands i.e., nicotianamine (NA) and deoxymugineic acid (DMA) were significantly up-regulated during iron deficiency. In total, 337 and 635 genes encoding transporters exhibited altered expression in roots and flag leaves, respectively. Several genes related to MAJOR FACILITATOR SUPERFAMILY (MFS), ATP-BINDING CASSETTE (ABC) transporter superfamily, NATURAL RESISTANCE ASSOCIATED MACROPHAGE PROTEIN (NRAMP) family and OLIGOPEPTIDE TRANSPORTER (OPT) family were regulated, indicating their important roles in combating iron deficiency stress. Among the regulatory factors, the genes encoding for transcription factors of BASIC HELIX-LOOP-HELIX (bHLH) family were highly up-regulated in both roots and the flag leaves. The jasmonate biosynthesis pathway was significantly altered but with notable expression differences between roots and flag leaves. Homoeologs expression and induction bias analysis revealed subgenome specific differential expression. Our findings provide an integrated overview on regulated molecular processes in response to iron deficiency stress in wheat. This information could potentially serve as a guideline for breeding iron deficiency stress tolerant crops as well as for designing appropriate wheat iron biofortification strategies.

Sequences of Circadian Clock Proteins in the Nudibranch Molluscs Hermissenda crassicornis, Melibe leonina, and Tritonia diomedea.

While much is known about the genes and proteins that make up the circadian clocks in vertebrates and several arthropod species, much less is known about the clock genes in many other invertebrates, including nudibranchs. The goal of this project was to identify the RNA and protein products of putative clock genes in the central nervous system of three nudibranchs, Hermissenda crassicornis, Melibe leonina, and Tritonia diomedea. Using previously published transcriptomes (Hermissenda and Tritonia) and a new transcriptome (Melibe), we identified nudibranch orthologs for the products of five canonical clock genes: brain and muscle aryl hydrocarbon receptor nuclear translocator like protein 1, circadian locomotor output cycles kaput, non-photoreceptive cryptochrome, period, and timeless. Additionally, orthologous sequences for the products of five related genes-aryl hydrocarbon receptor nuclear translocator like, photoreceptive cryptochrome, cryptochrome DASH, 6-4 photolyase, and timeout-were determined. Phylogenetic analyses confirmed that the nudibranch proteins were most closely related to known orthologs in related invertebrates, such as oysters and annelids. In general, the nudibranch clock proteins shared greater sequence similarity with Mus musculus orthologs than Drosophila melanogaster orthologs, which is consistent with the closer phylogenetic relationships recovered between lophotrochozoan and vertebrate orthologs. The suite of clock-related genes in nudibranchs includes both photoreceptive and non-photoreceptive cryptochromes, as well as timeout and possibly timeless. Therefore, the nudibranch clock may resemble the one exhibited in mammals, or possibly even in non-drosopholid insects and oysters. The latter would be evidence supporting this as the ancestral clock for bilaterians.

E47 Governs the MYC-CDKN1B/p27KIP1-RB Network to Growth Arrest PDA Cells Independent of CDKN2A/p16INK4A and Wild-Type p53.

BACKGROUND & AIMS: Oncogenic mutations in KRAS, coupled with inactivation of p53, CDKN2A/p16INK4A, and SMAD4, drive progression of pancreatic ductal adenocarcinoma (PDA). Overexpression of MYC and deregulation of retinoblastoma (RB) further promote cell proliferation and make identifying a means to therapeutically alter cell-cycle control pathways in PDA a significant challenge. We previously showed that the basic helix-loop-helix transcription factor E47 induced stable growth arrest in PDA cells in vitro and in vivo. Here, we identified molecular mechanisms that underlie E47-induced growth arrest in low-passage, patient-derived primary and established PDA cell lines. METHODS: RNA sequencing was used to profile E47-dependent transcriptomes in 5 PDA cell lines. Gene Ontology analysis identified cell-cycle control as the most altered pathway. Small interfering RNA/short hairpin RNA knockdown, small-molecule inhibitors, and viral expression were used to examine the function of E47-dependent genes in cell-cycle arrest. Cell morphology, expression of molecular markers, and senescence-associated ß-galactosidase activity assays identified cellular senescence. RESULTS: E47 uniformly inhibited PDA cell-cycle progression by decreasing expression of MYC, increasing the level of CDKN1B/p27KIP1, and restoring RB tumor-suppressor function. The molecular mechanisms by which E47 elicited these changes included altering both RNA transcript levels and protein stability of MYC and CDKN1B/p27KIP1. At the cellular level, E47 elicited a senescence-like phenotype characterized by increased senescence-associated ß-galactosidase activity and altered expression of senescence markers. CONCLUSIONS: E47 governs a highly conserved network of cell-cycle control genes, including MYC, CDKN1B/p27KIP1, and RB, which can induce a senescence-like program in PDA cells that lack CDKN2A/p16INK4A and wild-type p53. RNA sequencing data are available at the National Center for Biotechnology Information GEO at https://www.ncbi.nlm.nih.gov/geo/; accession number: GSE100327.

Transcriptomic analyses of Hand2 transgenic embryos.

In this article, we further provide the data generated for the previously published research article "Specification of jaw identity by the Hand2 transcription factor." To better understand the downstream genes of the basic helix-loop-helix transcription factor Hand2, we generated double-transgenic mice (Hand2 (NC) ) by intercrossing CAG-floxed CAT-Hand2 mice with Wnt1-Cre mice for conditional activation of Hand2 expression in the neural crest. Altered expression of Hand2 induces transformation of the upper jaw to the lower jaw in Hand2 (NC) mutant mice. This data article provides Tables detailing the differentially expressed genes between wild-type and Hand2 (NC) mutant embryos. The raw array data of our transcriptomes as generated using Affymetrix microarrays are available on the NCBI Gene Expression Omnibus (GEO) browser (Reference number GSE75805).

Interacting mechanism of ID3 HLH domain towards E2A/E12 transcription factor - An Insight through molecular dynamics and docking approach.

Inhibitor of DNA binding protein 3 (ID3) has long been characterized as an oncogene that implicates its functional role through its Helix-Loop-Helix (HLH) domain upon protein-protein interaction. An insight into the dimerization brought by this domain helps in identifying the key residues that favor the mechanism behind it. Molecular dynamics (MD) simulations were performed for the HLH proteins ID3 and Transcription factor E2-alpha (E2A/E12) and their ensemble complex (ID3-E2A/E12) to gather information about the HLH domain region and its role in the interaction process. Further evaluation of the results by Principal Component Analysis (PCA) and Free Energy Landscape (FEL) helped in revealing residues of E2A/E12: Lys570, Ala595, Val598, and Ile599 and ID3: Glu53, Gln63, and Gln66 buried in their HLH motifs imparting key roles in dimerization process. Furthermore the T-pad analysis results helped in identifying the key fluctuations and conformational transitions using the intrinsic properties of the residues present in the domain region of the proteins thus specifying their crucial role towards molecular recognition. The study provides an insight into the interacting mechanism of the ID3-E2A/E12 complex and maps the structural transitions arising in the essential conformational space indicating the key structural changes within the helical regions of the motif. It thereby describes how the internal dynamics of the proteins might regulate their intrinsic structural features and its subsequent functionality.

The satiating hormone amylin enhances neurogenesis in the area postrema of adult rats.

OBJECTIVE: Adult neurogenesis in the subgranular zone and subventricular zone is generally accepted, but its existence in other brain areas is still controversial. Circumventricular organs, such as the area postrema (AP) have recently been described as potential neurogenic niches in the adult brain. The AP is the major site of action of the satiating hormone amylin. Amylin has been shown to promote the formation of neuronal projections originating from the AP in neonatal rodents but the role of amylin in adult neurogenesis remains unknown. METHODS: To test this, we first performed an RNA-sequencing of the AP of adult rats acutely injected with either amylin (20 µg/kg), amylin plus the amylin receptor antagonist AC187 (500 µg/kg) or vehicle. Second, animals were subcutaneously equipped with minipumps releasing either amylin (50 µg/kg/day) or vehicle for 3 weeks to assess cell proliferation and differentiation with the 5'-bromo-2-deoxyuridine (BrdU) technique. RESULTS: Acute amylin injections affected genes involved in pathways and processes that control adult neurogenesis. Amylin consistently upregulated NeuroD1 transcript and protein in the adult AP, and this effect was blocked by the co-administration of AC187. Further, chronic amylin treatment increased the number of newly proliferated AP-cells and significantly promoted their differentiation into neurons rather than astrocytes. CONCLUSION: Our findings revealed a novel role of the satiating hormone amylin in promoting neurogenesis in the AP of adult rats.

The epigenetic regulation of embryonic myogenesis and adult muscle regeneration by histone methylation modification.

Skeletal muscle formation in vertebrates is derived from the paraxial mesoderm, which develops into myogenic precursor cells and finally differentiates into mature myofibers. This myogenic program involves temporal-spatial molecular events performed by transcription regulators (such as members of the Pax, MRFs and Six families) and signaling pathways (such as Wnts, BMP and Shh signaling). Epigenetic regulation, including histone post-translational modifications is crucial for controlling gene expression through recruitment of various chromatin-modifying enzymes that alter chromatin dynamics during myogenesis. The chromatin modifying enzymes are also recruited at regions of muscle gene regulation, coordinating transcription regulators to influence gene expression. In particular, the reversible methylation status of histone N-terminal tails provides the important regulatory mechanisms in either activation or repression of muscle genes. In this report, we review the recent literatures to deduce mechanisms underlying the epigenetic regulation of gene expression with a focus on histone methylation modification during embryo myogenesis and adult muscle regeneration. Recent results from different histone methylation/demethylation modifications have increased our understanding about the highly intricate layers of epigenetic regulations involved in myogenesis and cross-talk of histone enzymes with the muscle-specific transcriptional machinery.

Overexpression of a CYP94 family gene CYP94C2b increases internode length and plant height in rice.

Plant growth is controlled by intrinsic developmental programmes and environmental cues. Jasmonate (JA) has important roles in both processes, by regulating cell division and differentiation, as well as in defense responses and senescence. We report an increase in rice plant height caused by overexpression of a gene encoding a cytochrome P450 enzyme, CYP94C2b, which promoted deactivation of JA-Ile. The height increase occurred through enhanced elongation of internodes in the absence of concomitant cell elongation, unlike previous findings with coi1 knock-down plants. Thus, modulating JA metabolism can increase the number of elongated cells in an internode. Based on these and previous findings, we discuss the difference in the effects of CYP94C2b overexpression vs. coi1 knock-down.

Recent insights into Groucho co-repressor recruitment and function.

Gene expression is often controlled by transcriptional repressors during development. Many transcription factors lack intrinsic repressive activity but recruit co-factors that inhibit productive transcription. Here we discuss new insights and models for repression mediated by the Groucho/Transducin-Like Enhancer of split (Gro/TLE) family of co-repressor proteins.

A new role for the PI3K/Akt signaling pathway in the epithelial-mesenchymal transition.

Tumor metastasis is not only a sign of disease severity but also a major factor causing treatment failure and cancer-related death. Therefore, studies on the molecular mechanisms of tumor metastasis are critical for the development of treatments and for the improvement of survival. The epithelial-mesenchymal transition (EMT) is an orderly, polygenic biological process that plays an important role in tumor cell invasion, metastasis and chemoresistance. The complex, multi-step process of EMT involves multiple regulatory mechanisms. Specifically, the PI3K/Akt signaling pathway can affect the EMT in a variety of ways to influence tumor aggressiveness. A better understanding of the regulatory mechanisms related to the EMT can provide a theoretical basis for the early prediction of tumor progression as well as targeted therapy.

Reprogramming during epithelial to mesenchymal transition under the control of TGFß.

Epithelial-mesenchymal transition (EMT) refers to plastic changes in epithelial tissue architecture. Breast cancer stromal cells provide secreted molecules, such as transforming growth factor ß (TGFß), that promote EMT on tumor cells to facilitate breast cancer cell invasion, stemness and metastasis. TGFß signaling is considered to be abnormal in the context of cancer development; however, TGFß acting on breast cancer EMT resembles physiological signaling during embryonic development, when EMT generates or patterns new tissues. Interestingly, while EMT promotes metastatic fate, successful metastatic colonization seems to require the inverse process of mesenchymal-epithelial transition (MET). EMT and MET are interconnected in a time-dependent and tissue context-dependent manner and are coordinated by TGFß, other extracellular proteins, intracellular signaling cascades, non-coding RNAs and chromatin-based molecular alterations. Research on breast cancer EMT/MET aims at delivering biomolecules that can be used diagnostically in cancer pathology and possibly provide ideas for how to improve breast cancer therapy.

Hes1: a key role in stemness, metastasis and multidrug resistance.

Hes1 is one mammalian counterpart of the Hairy and Enhancer of split proteins that play a critical role in many physiological processes including cellular differentiation, cell cycle arrest, apoptosis and self-renewal ability. Recent studies have shown that Hes1 functions in the maintenance of cancer stem cells (CSCs), metastasis and antagonizing drug-induced apoptosis. Pathways that are involved in the up-regulation of Hes1 level canonically or non-canonically, such as the Hedgehog, Wnt and hypoxia pathways are frequently aberrant in cancer cells. Here, we summarize the recent data supporting the idea that Hes1 may have an important function in the maintenance of cancer stem cells self-renewal, cancer metastasis, and epithelial-mesenchymal transition (EMT) process induction, as well as chemotherapy resistance, and conclude with the possible mechanisms by which Hes1 functions have their effect, as well as their crosstalk with other carcinogenic signaling pathways.

HIF-1α pathway: role, regulation and intervention for cancer therapy.

Hypoxia-inducible factor-1 (HIF-1) has been recognized as an important cancer drug target. Many recent studies have provided convincing evidences of strong correlation between elevated levels of HIF-1 and tumor metastasis, angiogenesis, poor patient prognosis as well as tumor resistance therapy. It was found that hypoxia (low O2 levels) is a common character in many types of solid tumors. As an adaptive response to hypoxic stress, hypoxic tumor cells activate several survival pathways to carry out their essential biological processes in different ways compared with normal cells. Recent advances in cancer biology at the cellular and molecular levels highlighted the HIF-1α pathway as a crucial survival pathway for which novel strategies of cancer therapy could be developed. However, targeting the HIF-1α pathway has been a challenging but promising progresses have been made in the past twenty years. This review summarizes the role and regulation of the HIF-1α in cancer, and recent therapeutic approaches targeting this important pathway.

Overexpression of the JAZ factors with mutated jas domains causes pleiotropic defects in rice spikelet development.

In a determinate meristem, such as a floral meristem, a genetically determined number of organs are produced before the meristem is terminated. In rice, iterative formation of organs during flower development with defects in meristem determinacy, classically called 'proliferation', is caused by several mutations and observed in dependence on environmental conditions. Here we report that overexpression of several JAZ proteins, key factors in jasmonate signaling, with mutations in the Jas domains causes an increase in the numbers of organs in florets, aberrant patterns of organ formation and repetitious organ production in spikelets. Our results imply that JAZ factors modulate mechanisms that regulate meristem functions during spikelet development.

The activation mechanism of the aryl hydrocarbon receptor (AhR) by molecular chaperone HSP90.

The aryl hydrocarbon receptor is a member of the nuclear receptor superfamily that associates with the molecular chaperone HSP90 in the cytoplasm. The activation mechanism of the AhR is not yet fully understood. It has been proposed that after binding of ligands such as 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), 3methylcholanthrene (3-MC), or ß-naphthoflavone (ß-NF), the AhR dissociates from HSP90 and translocates to the nucleus. It has also been hypothesized that the AhR translocates to the nucleus and forms a complex with HSP90 and other co-chaperones. There are a few reports about the direct association or dissociation of AhR and HSP90 due to difficulties in purifying AhR. We constructed and purified the PAS domain from AhR. Binding of the AhR-PAS domain to ß-NF affinity resin suggested that it possesses ligand-binding affinity. We demonstrated that the AhR-PAS domain binds to HSP90 and the association is not affected by ligand binding. The ligand 17-DMAG inhibited binding of HSP90 to GST-PAS. In an immunoprecipitation assay, HSP90 was co-immunoprecipitated with AhR both in the presence or absence of ligand. Endogenous AhR decreased in the cytoplasm and increased in the nucleus of HeLa cells 15 min after treatment with ligand. These results suggested that the ligand-bound AhR is translocated to nucleus while in complex with HSP90. We used an in situ proximity ligation assay to confirm whether AhR was translocated to the nucleus alone or together with HSP90. HSP90 was co-localized with AhR after the nuclear translocation. It has been suggested that the ligand-bound AhR was translocated to the nucleus with HSP90. Activated AhR acts as a transcription factor, as shown by the transcription induction of the gene CYP1A1 8 h after treatment with ß-NF.

Apotransferrin-induced recovery after hypoxic/ischaemic injury on myelination.

We have previously demonstrated that aTf (apotransferrin) accelerates maturation of OLs (oligodendrocytes) in vitro as well as in vivo. The purpose of this study is to determine whether aTf plays a functional role in a model of H/I (hypoxia/ischaemia) in the neonatal brain. Twenty-four hours after H/I insult, neonatal rats were intracranially injected with aTf and the effects of this treatment were evaluated in the CC (corpus callosum) as well as the SVZ (subventricular zone) at different time points. Similar to previous studies, the H/I event produced severe demyelination in the CC. Demyelination was accompanied by microglial activation, astrogliosis and iron deposition. Ferritin levels increased together with lipid peroxidation and apoptotic cell death. Histological examination after the H/I event in brain tissue of aTf-treated animals (H/I aTF) revealed a great number of mature OLs repopulating the CC compared with saline-treated animals (H/I S). ApoTf treatment induced a gradual increase in MBP (myelin basic protein) and myelin lipid staining in the CC reaching normal levels after 15 days. Furthermore, significant increase in the number of OPCs (oligodendroglial progenitor cells) was found in the SVZ of aTf-treated brains compared with H/I S. Specifically, there was a rise in cells positive for OPC markers, i.e. PDGFRα and SHH(+) cells, with a decrease in cleaved-caspase-3(+) cells compared with H/I S. Additionally, neurospheres from aTf-treated rats were bigger in size and produced more O4/MBP(+) cells. Our findings indicate a role for aTf as a potential inducer of OLs in neonatal rat brain in acute demyelination caused by H/I and a contribution to the differentiation/maturation of OLs and survival/migration of SVZ progenitors after demyelination in vivo.

The neurogenic basic helix-loop-helix transcription factor NeuroD6 confers tolerance to oxidative stress by triggering an antioxidant response and sustaining the mitochondrial biomass.

Preserving mitochondrial mass, bioenergetic functions and ROS (reactive oxygen species) homoeostasis is key to neuronal differentiation and survival, as mitochondria produce most of the energy in the form of ATP to execute and maintain these cellular processes. In view of our previous studies showing that NeuroD6 promotes neuronal differentiation and survival on trophic factor withdrawal, combined with its ability to stimulate the mitochondrial biomass and to trigger comprehensive antiapoptotic and molecular chaperone responses, we investigated whether NeuroD6 could concomitantly modulate the mitochondrial biomass and ROS homoeostasis on oxidative stress mediated by serum deprivation. In the present study, we report a novel role of NeuroD6 as a regulator of ROS homoeostasis, resulting in enhanced tolerance to oxidative stress. Using a combination of flow cytometry, confocal fluorescence microscopy and mitochondrial fractionation, we found that NeuroD6 sustains mitochondrial mass, intracellular ATP levels and expression of specific subunits of respiratory complexes upon oxidative stress triggered by withdrawal of trophic factors. NeuroD6 also maintains the expression of nuclear-encoded transcription factors, known to regulate mitochondrial biogenesis, such as PGC-1alpha (peroxisome-proliferator-activated receptor gamma co-activator-1alpha), Tfam (transcription factor A, mitochondrial) and NRF-1 (nuclear respiratory factor-1). Finally, NeuroD6 triggers a comprehensive antioxidant response to endow PC12-ND6 cells with intracellular ROS scavenging capacity. The NeuroD6 effect is not limited to the classic induction of the ROS-scavenging enzymes, such as SOD2 (superoxide dismutase 2), GPx1 (glutathione peroxidase 1) and PRDX5 (peroxiredoxin 5), but also to the recently identified powerful ROS suppressors PGC-1alpha, PINK1 (phosphatase and tensin homologue-induced kinase 1) and SIRT1. Thus our collective results support the concept that the NeuroD6-PGC-1alpha-SIRT1 neuroprotective axis may be critical in co-ordinating the mitochondrial biomass with the antioxidant reserve to confer tolerance to oxidative stress.

The neurogenic basic helix-loop-helix transcription factor NeuroD6 concomitantly increases mitochondrial mass and regulates cytoskeletal organization in the early stages of neuronal differentiation.

Mitochondria play a central role during neurogenesis by providing energy in the form of ATP for cytoskeletal remodelling, outgrowth of neuronal processes, growth cone activity and synaptic activity. However, the fundamental question of how differentiating neurons control mitochondrial biogenesis remains vastly unexplored. Since our previous studies have shown that the neurogenic bHLH (basic helix-loop-helix) transcription factor NeuroD6 is sufficient to induce differentiation of the neuronal progenitor-like PC12 cells and that it triggers expression of mitochondrial-related genes, we investigated whether NeuroD6 could modulate the mitochondrial biomass using our PC12-ND6 cellular paradigm. Using a combination of flow cytometry, confocal microscopy and mitochondrial fractionation, we demonstrate that NeuroD6 stimulates maximal mitochondrial mass at the lamellipodia stage, thus preceding axonal growth. NeuroD6 triggers remodelling of the actin and microtubule networks in conjunction with increased expression of the motor protein KIF5B, thus promoting mitochondrial movement in developing neurites with accumulation in growth cones. Maintenance of the NeuroD6-induced mitochondrial mass requires an intact cytoskeletal network, as its disruption severely reduces mitochondrial mass. The present study provides the first evidence that NeuroD6 plays an integrative role in co-ordinating increase in mitochondrial mass with cytoskeletal remodelling, suggestive of a role of this transcription factor as a co-regulator of neuronal differentiation and energy metabolism.