Giuseppe Macino

Role of a white collar-1-white collar-2 complex in blue-light signal transduction

Mutations in either white collar‐1 (wc‐1) or white collar‐2 (wc‐2) lead to a loss of most blue‐light‐induced phenomena in Neurospora crassa. Sequence analysis and in vitro experiments show that wc‐1 and wc‐2 are transcription factors regulating the expression of light‐induced genes. The WC proteins form homo‐ and heterodimers in vitro; this interaction could represent a fundamental step in the control of their activity. We demonstrate in vivo that the WC proteins are assembled in a white collar complex (WCC) and that wc‐1 undergoes a change in mobility due to light‐induced phosphorylation events. The phosphorylation level increases progressively upon light exposure, producing a hyperphosphorylated form that is degraded and apparently replaced in the complex by a newly synthesized wc‐1. wc‐2 is unmodified and also does not change quantitatively in the time frame examined. Light‐dependent phosphorylation of wc‐1 also occurs in a wc‐2 mutant, suggesting that a functional wc‐2 is dispensable for this light‐specific event. These results suggest that light‐induced phosphorylation and degradation of wc‐1 could play a role in the transient expression of blue‐light‐regulated genes. Our findings suggest a mechanism by which wc‐1 and wc‐2 mediate light responses in Neurospora.

Molecular cloning of a Neurospora crassa carotenoid biosynthetic gene (albino-3) regulated by blue light and the products of the white collar genes.

The albino-3 (al-3) gene of Neurospora crassa, which probably encodes the carotenoid biosynthetic enzyme geranylgeranyl pyrophosphate synthetase, was cloned. The N. crassa triple mutant al-3 qa-2 aro-9 was transformed to qa-2+ with mixtures of plasmids bearing N. crassa DNA inserts, and the transformants were screened for the al-3+ phenotype. One al-3+ qa-2+ transformant (AL3-1) was examined in detail and shown to contain intact vector sequences integrated into the N. crassa genome. The vector and some flanking sequences were recovered from AL3-1 after restriction, ligation, and selection of chloramphenicol-resistant transformants of Escherichia coli. The flanking sequences were subsequently used to detect the al-3-containing plasmid in the mixture of about 1,800 plasmids. Restriction fragment length polymorphism mapping was carried out to confirm the identity of the cloned fragment. The level of the al-3 mRNA was shown to be increased 15-fold in light-induced (compared with that in dark-grown) wild-type mycelia. The light-dependent increase in al-3 mRNA levels was not observed in presumed regulatory mutant (white collar) strains.

Characterization of B‐ and T‐lineage acute lymphoblastic leukemia by integrated analysis of MicroRNA and mRNA expression profiles

Acute lymphoblastic leukemia (ALL) is an heterogeneous disease comprising several subentities that differ for both immunophenotypic and molecular characteristics. Over the years, the biological understanding of this neoplasm has largely increased. Gene expression profiling has allowed to identify specific signatures for the different ALL subsets and permitted the identification of pathways deregulated by a given lesion. MicroRNAs (miRNAs) are small noncoding RNAs, which play a pivotal role in several cellular functions. In this study, we investigated miRNAs expression profiles in a series of adult ALL cases by microarray analysis. Unsupervised hierarchical clustering largely recapitulated ALL subgroups. Furthermore, we identified miR‐148, miR‐151, and miR‐424 as discriminative of T‐lineage versus B‐lineage ALL; ANOVA highlighted a set of six miRNAs—namely miR‐425‐5p, miR‐191, miR‐146b, miR‐128, miR‐629, and miR‐126—that can discriminate B‐lineage ALL subgroups harboring specific molecular lesions. These results were confirmed and extended by quantitative‐PCR on a further cohort of cases. Finally, we used Pearson correlation analysis to combine miRNA and gene expression profiles. The distribution of correlation coefficients generated by comparing the expression of every miRNA/gene pair in our data set shows enrichment of both positively and negatively correlated pairs over background distributions obtained using randomized data. Moreover, a clear enrichment for predicted miRNA:target pairs is observed at negative correlation coefficient intervals. Signal‐to‐noise ratio highlighted several miRNA/gene pairs with a possible role in the disease. In fact, gene set enrichment analysis of genes composing the selected miRNA/gene pairs displays over‐representation of functional categories related to cancer and cell‐cycle regulation.

Involvement of protein kinase C in the response of Neurospora crassa to blue light

Abstract As a first step towards understanding the process of blue light perception, and the signal transduction mechanisms involved, in Neurospora crassa we have used a pharmacological approach to screen a wide range of second messengers and chemical compounds known to interfere with the activity of well-known signal transducing molecules in vivo. We tested the influence of these compounds on the induction of the al-3 gene, a key step in light-induced carotenoid biosynthesis. This approach has implicated protein kinase C (PKC) as a component of the light transduction machinery. The conclusion is based on the effects of specific inhibitors (calphostin C and chelerythrine chloride) and activators of PKC (1,2-dihexanoyl-sn-glycerol). During vegetative growth PKC may be responsible for desensitization to light because inhibitors of the enzyme cause an increase in the total amount of mRNA transcribed after illumination. PKC is therefore proposed here to be an important regulator of transduction of the blue light signal, and may act through modification of the protein White Collar-1, which we show to be a substrate for PKC in N. crassa.

Light induction of the clock-controlled geneccg-1 is not transduced through the circadian clock inNeurospora crassa

Ambient light and the circadian clock have been shown to be capable of acting either independently or in an interrelated fashion to regulate the expression of conidiation in the ascomycete fungusNeurospora crassa. Recently several molecular correlates of the circadian clock have been identified in the form of the morning-specific clock-controlled genesccg-1 andccg-2. In this paper we report studies on the regulation ofccg-1, an abundantly expressed gene displaying complex regulation. Consistent with an emerging consensus for clock-controlled genes and conidiation genes inNeurospora, we report thatccg-1 expression is induced by light, and show that this induction is independent of the direct effects of light on the circadian clock. Although circadian regulation of the gene is lost in strains lacking a functional clock, expression ofccg-1 is still not constitutive, but rather fluctuates in concert with changes in developmental potential seen in such strains. Light induction ofccg-1 requires the products of theNeurospora wc-1 andwc-2 genes, but surprisingly the requirement forwc-2 is suppressed in conditional mutants ofcot-1, a gene that encodes a cAMP-dependent protein kinase. These data provide insight into a complex regulatory web, involving at least circadian clock control, light control, metabolic control, and very probably developmental regulation, that governs the expression ofccg-1.

Protein kinase C modulates light responses in Neurospora by regulating the blue light photoreceptor WC-1

The Neurospora protein kinase C (NPKC) is a regulator of light responsive genes. We have studied the function of NPKC in light response by investigating its biochemical and functional interaction with the blue light photoreceptor white‐collar 1 (WC‐1), showing that activation of NPKC leads to a significant decrease in WC‐1 protein levels. Furthermore, we show that WC‐1 and NPKC interact in a light‐regulated manner in vivo, and that protein kinase C (PKC) phosphorylates WC‐1 in vitro. We designed dominant negative and constitutively active forms of PKC which are able to induce either a large increase of WC‐1 protein level or a strong reduction respectively. Moreover, these changes in PKC activity result in an altered light response. As WC‐1 is a key component of Neurospora circadian clock and regulates the clock oscillator component FRQ we investigated the effect of NPKC‐mutated forms on FRQ levels. We show that changes in PKC activity affect FRQ levels and the robustness of the circadian clock. Together these data identify NPKC as a novel component of the Neurospora light signal transduction pathway that modulates the circadian clock.

Cloning of a yeast gene coding for the glutamate synthase small subunit (GUS2) by complementation of Saccharomyces cerevisiae and Escherichia coli glutamate auxotrophs

A Saccharomyces cerevisiae glutamate auxotroph, lacking NADP‐glutamate dehydrogenase (NADP‐GDH) and glutamate synthase (GOGAT) activities, was complemented with a yeast genomic library. Clones were obtained which still lacked NADP‐GDH but showed GOGAT activity. Northern analysis revealed that the DNA fragment present in the complementing plasmids coded for a 1.5kb mRNA. Since the only GOGAT enzyme so far purified from S. cerevisiae is made up of a small and a large subunit, the size of the mRNA suggested that the cloned DNA fragment could code for the GOGAT small subunit. Plasmids were purified and used to transform Escherichia coli glutamate auxotrophs. Transformants were only recovered when the recipient strain was an E. coli GDH‐less mutant lacking the small GOGAT subunit. These data show that we have cloned the structural gene coding for the yeast small subunit (GUS2). Evidence is also presented indicating that the GOGAT enzyme which is synthesized in the E. coli transformants is a hybrid comprising the large E. coli subunit and the small S. cerevisiae subunit.

Characterization of HuH6, Hep3B, HepG2 and HLE liver cancer cell lines by WNT/β - catenin pathway, microRNA expression and protein expression profile.

Somatic mutations in the genes members of WNT/β-catenin pathway, especially in CTNNB1 codifying for β-catenin, have been found to play an important role in hepatocarcinogenesis. The purpose of this work is to characterize alterations of the WNT/β-catenin signalling pathway, and to study the expression pattern of a panel of microRNAs and proteins potentially involved in the pathogenesis of liver cancer. In this respect, the molecular characterization of the most used liver cancer cell lines HuH6, Hep3B, HepG2, and HLE, could represent a useful tool to identify novel molecular markers for hepatic tumour. A significant modulation of FZD7, NLK, RHOU, SOX17, TCF7L2, TLE1, SLC9A3R1 and WNT10A transcripts was observed in all the four liver cancer cell lines. The analysis of selected microRNAs showed that miR-122a, miR-125a and miR-150 could be suitable candidates to discriminate tumoural versus normal human primary hepatocytes. Finally, Grb-2 protein expression resulted to be increased more than two-fold in liver cancer cell lines in comparison to normal human primary hepatocytes. These advances in the knowledge of molecular mechanisms involved in the pathogenesis of liver cancer may provide new potential biomarkers and molecular targets for the diagnosis and therapy.