Regulación Transcripciones del Gen GLS2 y Obtención de Células Madre Embrionarias Transformadas para Generación de Ratones Knockout

La enzima glutaminasa activada por fosfato (GA) cataliza la hidrólisis del grupo amido de la glutamina (Gln) para rendir glutamato (Glu) y amonio. Esta enzima es esencial para el ciclo Gln-Glu entre neuronas y astrocitos y, por tanto, fundamental para la generación del importante neurotransmisor Glu. En humanos, la familia GA está constituida por dos miembros principales localizados en genes distintos y situados en cromosomas diferentes: el gen GLS (cromosoma 2), codifica a las isoenzimas KGA y GAC; mientras que el gen GLS2 (cromosoma 12) codifica para las isoenzimas LGA y GAB. Genes ortólogos a los genes humanos GLS y GLS2 se han descrito en otras especies de mamíferos como ratón y rata. El hecho de que existan dos genes para la GA y de que se hayan identificado diferentes sublocalizaciones celulares para las glutaminasas GLS (mitocondria) y GLS2 (mitocondria y núcleo) nos hace plantearnos el papel que desempeñan en cada sublocalización, además del meramente catalítico, especialmente en cerebro. Un modelo transgénico nulo para el gen Gls2 podría ser una herramienta fundamental para elucidar la función cerebral de esta(s) isoenzima(s). Por otra parte, los mecanismos moleculares que controlan la transcripción son esenciales para comprender la expresión tejido-específica de GA y su alteración en condiciones patológicas, como el cáncer y enfermedades neurológicas. En la presente tesis doctoral se han llevado a cabo estudios relacionados con los dos objetivos principales indicados, llegando a los resultados y las conclusiones que mencionamos a continuación. Se ha diseñado y generado una construcción transgénica para el silenciamiento génico condicional del gen Gls2 murino. La construcción rendirá animales KO nulos para la isoenzima GAB y condicionales para LGA. Además, se ha adquirido una construcción transgénica del consorcio EUCOMM para el silenciamiento génico completo del gen Gls2 murino. Mediante microinyección en blastocistos se han obtenido animales quiméricos que se cruzarán para seleccionar aquellos donde la mutación se haya incorporado a la línea germinal, permitiendo obtener una colonia de animales fundadores. Mediante una combinación de técnicas de biología molecular, bioquímicas y bioinformáticas, hemos obtenido evidencia experimental directa que demuestra, por vez primera, la existencia de transcritos alternativos de los genes ortólogos Gls2 de ratón, rata y humano. Existen al menos dos transcritos sentido de mRNA codificados por el gen GLS2: un transcrito largo denominado GAB y un transcrito corto, LGA, que posee un sitio alternativo de inicio de la transcripción que omite el exón 1 del gen GLS2. También hemos identificado que la variante LGA posee un promotor alternativo situado al final del intrón 1 del gen GLS2 y separado una distancia de 7 kb del promotor canónico del transcrito GAB humano. El promotor LGA posee secuencias de reconocimiento de importantes factores de transcripción. Ambos promotores del gen GLS2 difieren notablemente en su grado de metilación y contenido en islas CpG, lo que permite especular que poseen una regulación transcripcional diferencial. En los estudios de RT-PCR empleando poli(A+)-mRNA hemos aislado transcritos sin sentido correspondientes a tres pseudogenes: uno en ratón y dos en humano. También se ha conseguido clonar y expresar el cDNA del transcrito LGA humano. Aunque dos polipéptidos diferentes fueron trascritos y traducidos con igual eficiencia en un sistema de expresión in vitro, solo el polipéptido completo traducido desde el primer codón de iniciación mostró actividad enzimática. Las isoenzimas GAB y LGA se expresan diferencialmente en cerebro, hígado y en células de neuroblastoma humano. Los cambios de expresión fueron tejido- y especie-específicos, aunque se desconoce su implicación funcional en estados normales y patológicos, fundamentalmente cáncer y alteraciones neurológicas. Con los presentes resultados nos hemos acercado a la obtención de modelos de ratones nulos para Gls2, así como hemos conseguido describir mecanismos de regulación transcripcional y caracterizar los transcritos del gen GLS2

Mammalian Glutaminase Gls2 Gene Encodes Two Functional Alternative Transcripts by a Surrogate Promoter Usage Mechanism

Glutaminase is expressed in most mammalian tissues and cancer cells, but the regulation of its expression is poorly understood. An essential step to accomplish this goal is the characterization of its species- and cell-specific isoenzyme pattern of expression. Our aim was to identify and characterize transcript variants of the mammalian glutaminase Gls2 gene.We demonstrate for the first time simultaneous expression of two transcript variants from the Gls2 gene in human, rat and mouse. A combination of RT-PCR, primer-extension analysis, bioinformatics, real-time PCR, in vitro transcription and translation and immunoblot analysis was applied to investigate GLS2 transcripts in mammalian tissues. Short (LGA) and long (GAB) transcript forms were isolated in brain and liver tissue of human, rat and mouse. The short LGA transcript arises by a combination of two mechanisms of transcriptional modulation: alternative transcription initiation and alternative promoter. The LGA variant contains both the transcription start site (TSS) and the alternative promoter in the first intron of the Gls2 gene. The full human LGA transcript has two in-frame ATGs in the first exon, which are missing in orthologous rat and mouse transcripts. In vitro transcription and translation of human LGA yielded two polypeptides of the predicted size, but only the canonical full-length protein displayed catalytic activity. Relative abundance of GAB and LGA transcripts showed marked variations depending on species and tissues analyzed.This is the first report demonstrating expression of alternative transcripts of the mammalian Gls2 gene. Transcriptional mechanisms giving rise to GLS2 variants and isolation of novel GLS2 transcripts in human, rat and mouse are presented. Results were also confirmed at the protein level, where catalytic activity was demonstrated for the human LGA protein. Relative abundance of GAB and LGA transcripts was species- and tissue-specific providing evidence of a differential regulation of GLS2 transcripts in mammals

Design and generation of a glutaminase GLS2 conditional knockout mice

Mammalian glutaminase (GA; EC 3.5.1.2) is the main enzyme involved in brain generation of glutamate (Glu). This amino acid acts as an excitatory neurotransmitter within the CNS, and it is also implicated in behavioral sensitization through the mesolimbic pathway. Two different GA genes have been described: Gls that encodes the isozymes KGA and GAC, and Gls2, which encodes GAB and LGA isozymes. Gls and Gls2 isoforms are co-expressed in different brain regions and cells. Of note, location of Gls2-encoded isoforms in neuronal nuclei suggests a novel role in the regulation of gene expression. The co-expression of different GA isoforms in mammalian brain is so far unexplained. Our objective is to study the cerebral function of Gls2; for this purpose, we develop a conditional knockout (KO) mouse model to silence GAB and LGA expression in brain. A vector carrying the Gls2 gene from exon 1 to 12 (obtained from the EUCOMM consortium) was transfected by electroporation into B6D2F1 murine embryonic stem cells (ES). These ES were selected by geneticin and PCR-genotyped before their microinjection in 8-cell stage embryos (Swiss strain). Embryo implantation was performed in pseudopregnant state mice, which leads to chimeric pups. This vector targets chromosome 10 and will yield a conditional KO mouse model, since exons 2 to 7 are included between loxP sites. The chimeric pups carrying this modification within their germ line were used to generate the homozygous Gls2 (-/-) mice. After integration of the vector in both alleles, the mice will be mated with mutant Cre mice, which express this recombinase enzyme under control of the synapsin specific promoter. This will result in a deletion of the exons 2 to 7 giving rise to null Gls2 mutants mainly in the following brain areas: cortex, hippocampus, amygdala and cerebellum, which are essential for glutamatergic transmission and related to the mesolimbic pathway.Universidad de Málaga. Campus de Excelencia Internacional Andalucía Tech

Structure of the human <i>GLS2</i> gene and predicted GA transcripts.

<p>The GLS2 gene is located in the 12q13 region of human chromosome 12, as shown in the upper part of this figure. The gene has a length of approximately 18 kb and split into 18 exons. Exon sequences are indicated as numbered light green boxes; intron sequences are shown as solid blue lines. Two GA transcripts are encoded by GLS2: the canonical GAB mRNA formed by joining the full 18 exons of the gene and the short LGA transcript that lacks exon 1. Dotted lines comprise exons involved in the generation of both transcript variants. The transcription start site is marked by an arrow and numbered as +1.</p

ENCODE Enhancer- and Promoter-Associated Histone Marks, CpG islands and methylation status of the <i>GLS2</i> gene locus on human chromosome 12.

<p>For simplicity, only the four first exons of human <i>GLS2</i> gene and the 5′-flanking genomic region are shown on top of the Figure. Below the <i>GLS2</i> gene, six plots are represented, as follows: first panel: Enhancer- and Promoter-Associated Histone mark (H3K4Me1) from 9 human cell lines; second panel: Promoter-Associated Histone mark (H3K4Me3) from 8 human cell lines; third panel: Enhancer- and Promoter-Associated Histone mark (H3K27Ac) from 8 human cell lines; fourth panel: CpG island shown as a solid green bar; fifth panel: Promoter-Associated Histone mark (H3K4Me3) from chromatin immunoprecipitation of DNA and sequencing data (ChIP-seq) and; sixth panel: Methylation-dependent immunoprecipitation of DNA and sequencing data from human brain (MeDIP-seq).</p

Comparison of the sequences of RT-PCR products demonstrating co-expression of LGA and GAB transcripts in mouse, rat and human tissues.

<p>The identity of each amplified fragment was assessed by sequence alignment with known sequences for rat liver LGA (GenBank# J05499) and human GAB (GenBank #AF348119) using Blast program. Top panel: the 5′-sequences of mouse brain and human liver LGA cDNA fragments obtained by RT-PCR were aligned with the sequence of rat liver LGA cDNA; bottom panel: the 5′-sequences of rat liver and mouse brain GAB cDNA fragments obtained by RT-PCR were aligned with the human GAB sequence from ZR-75 breast cancer cells. Identical nucleotides are indicated in red, different nucleotides are labeled in blue. Sequence alignment was done using Multalin program (<a href="http://multalin.toulouse.inra.fr/multalin/" target="_blank">http://multalin.toulouse.inra.fr/multalin/</a>).</p

Glutaminase activity assay.

<p>The <i>in vitro</i> transcribed and translated human LGA (hLGA) protein and a deletion mutant (Mut hLGA) protein, starting at the second ATG initiation codon, were assayed for GA activity as described in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0038380#s2" target="_blank">Materials and Methods</a> section. Results are mean ± S.E.M. of three independent experiments: protein hLGA (pink bar); protein Mut hLGA (blue bar). Background activity was measured by using the pGEM-T[hLGA] construct cloned in the antisense orientation and was always subtracted from activity values obtained with the hLGA proteins. Values are expressed as milliunits of enzyme activity per ml.</p

Immunoblot analysis of human SHSY-5Y neuroblastoma cells and rat liver and brain mitochondria.

<p>Cell extracts of human neuroblastoma SHSY-5Y cells (left and center panel) and mitochondria isolated from rat liver and brain (right panel) were analyzed by SDS-PAGE and Western blotting. Blots were revealed by chemiluminescence using polyclonal antibodies raised against the exclusive first exon of human GAB protein (left panel) or against the whole human GAB protein which recognize both GAB and LGA proteins (center and right panel). M, lanes containing the molecular mass markers indicated on the left margin; SHSY-5Y, lanes loaded with protein extracts isolated from human neuroblastoma cells; Liver 1 and 2: lanes containing total liver protein extracts and mitochondrial protein extracts, respectively; Brain 3 and 4: lanes containing total brain protein extracts and mitochondrial protein extracts, respectively.</p

In vitro transcription and translation of human LGA cDNA.

<p>The ORF of human LGA (hLGA), deletion mutant hLGA (Mut-hLGA) starting at the second ATG, and Δ4- and Δ10-LGA deletion mutants were cloned into the pGEM-T vector and transcribed and translated <i>in vitro</i> in the presence of ³⁵S-Met. The reaction mixtures were then analyzed by SDS-PAGE and autoradiography. Lane M, standard protein markers with the relative positions of prestained molecular mass markers indicated on the left; lane Mut-hLGA, aliquot of the translation mixture using the human LGA cDNA starting at the second in-frame ATG; lane hLGA, full-length human LGA; lane Mut-Δ4, aliquot of the translation mixture using the Δ4-LGA deletion mutant cDNA; lane Mut-Δ10, aliquot of the translation mixture using the Δ10-LGA deletion mutant cDNA; lane C-, negative control (pGEM-T with the hLGA cloned in the antisense orientation); lane C+, luciferase positive control. The radioactive LGA protein bands are indicated on the left using black arrows.</p

Quantification of mRNA levels of KGA, GAB and LGA transcripts in rat and mouse tissues by qPCR.

<p>The histogram shows the absolute copy number of mRNA transcripts for the KGA (dark blue), GAB (pink) and LGA (light blue) isoforms. Real time RT-PCR was performed as indicated in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0038380#s2" target="_blank">Materials and Methods</a> section. Results are mean ± S.E.M. of three independent experiments done in triplicate. Values were determined for liver and brain tissues from rat and mouse and are shown as copy number per ng of total RNA. Insets: GAB and LGA mRNA values in mouse and rat brains are depicted without KGA mRNA levels to appreciate differences among them.</p