GAD2 on chromosome 10p12 is a candidate gene for human obesity

The gene GAD2 encoding the glutamic acid decarboxylase enzyme (GAD65) is a positional candidate gene for obesity on Chromosome 10p11&ndash;12, a susceptibility locus for morbid obesity in four independent ethnic populations. GAD65 catalyzes the formation of &gamma;-aminobutyric acid (GABA), which interacts with neuropeptide Y in the paraventricular nucleus to contribute to stimulate food intake. A case-control study (575 morbidly obese and 646 control subjects) analyzing GAD2 variants identified both a protective haplotype, including the most frequent alleles of single nucleotide polymorphisms (SNPs) +61450 C&gt;A and +83897 T&gt;A (OR = 0.81, 95% CI [0.681&ndash;0.972], p = 0.0049) and an at-risk SNP (&minus;243 A&gt;G) for morbid obesity (OR = 1.3, 95% CI [1.053&ndash;1.585], p = 0.014). Furthermore, familial-based analyses confirmed the association with the obesity of SNP +61450 C&gt;A and +83897 T&gt;A haplotype (&chi;2 = 7.637, p = 0.02). In the murine insulinoma cell line &beta;TC3, the G at-risk allele of SNP &minus;243 A&gt;G increased six times GAD2 promoter activity (p &lt; 0.0001) and induced a 6-fold higher affinity for nuclear extracts. The &minus;243 A&gt;G SNP was associated with higher hunger scores (p = 0.007) and disinhibition scores (p = 0.028), as assessed by the Stunkard Three-Factor Eating Questionnaire. As GAD2 is highly expressed in pancreatic &beta; cells, we analyzed GAD65 antibody level as a marker of &beta;-cell activity and of insulin secretion. In the control group, &minus;243 A&gt;G, +61450 C&gt;A, and +83897 T&gt;A SNPs were associated with lower GAD65 autoantibody levels (p values of 0.003, 0.047, and 0.006, respectively). SNP +83897 T&gt;A was associated with lower fasting insulin and insulin secretion, as assessed by the HOMA-B% homeostasis model of &beta;-cell function (p = 0.009 and 0.01, respectively). These data support the hypothesis of the orexigenic effect of GABA in humans and of a contribution of genes involved in GABA metabolism in the modulation of food intake and in the development of morbid obesity.<br /

Genetic and Physical Interactions between Tel2 and the Med15 Mediator Subunit in Saccharomyces cerevisiae

International audienceBACKGROUND: In budding yeast, the highly conserved Tel2 protein is part of several complexes and its main function is now believed to be in the biogenesis of phosphatidyl inositol 3-kinase related kinases. PRINCIPAL FINDINGS: To uncover potentially novel functions of Tel2, we set out to isolate temperature-sensitive (ts) mutant alleles of TEL2 in order to perform genetic screenings. MED15/GAL11, a subunit of Mediator, a general regulator of transcription, was isolated as a suppressor of these mutants. The isolated tel2 mutants exhibited a short telomere phenotype that was partially rescued by MED15/GAL11 overexpression. The tel2-15 mutant was markedly deficient in the transcription of EST2, coding for the catalytic subunit of telomerase, potentially explaining the short telomere phenotype of this mutant. In parallel, a two-hybrid screen identified an association between Tel2 and Rvb2, a highly conserved member of the AAA+ family of ATPases further found by in vivo co-immunoprecipitation to be tight and constitutive. Transiently overproduced Tel2 and Med15/Gal11 associated together, suggesting a potential role for Tel2 in transcription. Other Mediator subunits, as well as SUA7/TFIIB, also rescued the tel2-ts mutants. SIGNIFICANCE: Altogether, the present data suggest the existence of a novel role for Tel2, namely in transcription, possibly in cooperation with Rvb2 and involving the existence of physical interactions with the Med15/Gal11 Mediator subunit

Increased dosage of several Mediator genes partially rescue the temperature sensitivity of the <i>tel2</i>-<i>19</i> mutant.

<p>(<b>A</b>) Overexpression of the indicated genes, under the control of the <i>GAL1</i>-<i>10</i> promoter, was continuously induced by growing on solid media containing galactose as the sole carbon source. Cells transformed with the indicated plasmid were first grown in liquid culture in selective minimal medium containing glucose as the carbon source before being re-streaked on agar-based selective minimal medium containing galactose as the carbon source. Growth was assessed after 3 days at the indicated temperature in the serially diluted cells (ten-fold dilutions from left to right in each condition). At 35°C, <i>MED16</i>, <i>CDK8</i> and <i>MED18</i> overexpression rescued <i>tel2</i>-<i>19</i>, although slightly less efficiently than <i>MED15</i>. (<b>B</b>) Tel2 and Med15 physically interact <i>in vivo</i>. Putative physical interactions between Tel2-Myc₃ and Med15-HA₂ were measured upon immunoprecipitation (IP) with anti-HA or anti-Myc monoclonal antibodies, followed by Western blotting (West.), from crude extracts from strains having transiently overexpressed the indicated constructs after incubation in galactose-based liquid selective media, for 2 hr at 29°C (same conditions for panels B–E). All experiments were conducted in parallel in strains expressing both constructs and in strains with the single constructs to take into account possible background signals. Full length Tel2-Myc₃ specifically binds the first half of Med15 (Med15^1–540-HA₂) in both directions, that is to say that a positive signal was detected whether one or the other of the two proteins was immunoprecipitated. (<b>C</b>) In contrast, the second half of Med15 (Med15^541–1081-HA₂) did not associate <i>in vivo</i> with full length Tel2-Myc₃. (<b>D</b>) Finally, the first quarter of Med15 (Med15^1–270-HA₂) efficiently bound the first half of Tel2 (Tel2^1–343-HA₂). (<b>E</b>) At the maximal permissive temperature for growth for <i>tel2</i>-<i>15</i> of 29°C, the Tel2-15-Myc₃ protein still physically interacted with Med15^1–540-HA₂, as was also the case at 34°C (data not shown).</p

Temperature-sensitive <i>Saccharomyces cerevisiae tel2</i> mutants.

<p>(<b>A</b>) Growth characteristics of the temperature-sensitive <i>tel2</i> mutants. Ten-fold serial dilutions (from left to right in each condition) of cultures of the indicated relevant genotype were grown for 3 days on YEPD agar at the indicated temperature and photographed. (<b>B</b>) Temperature-sensitive <i>tel2</i>-<i>15</i> and <i>tel2</i>-<i>19</i> mutants fail to arrest at a specific cell cycle stage when incubated at the restrictive temperature for growth of 34°C. Cell lysis was evident under the microscope upon extended incubation at restrictive temperatures (not shown). (<b>C</b>) Overexpression of <i>MED15</i> from a multi-copy plasmid (2 µ, YEp) does not result in an increase in <i>TEL2</i> transcription. (<b>D</b>) Western blot showing that overexpression of <i>MED15</i>, either from a low-copy (CEN) or a multi-copy (2 µ) plasmid, does not result in an increase in the amount of endogenous Tel2 (HA₂-Tel2 or Myc₂-Tel2) in the cell (the lanes labeled “HA₂-Tel2/Myc₂-Tel2+plasmid” represent the controls with plasmid alone). Endogenous Tel2, tagged with either Myc₂ or HA₂ in its N-terminus, was immunoprecipitated from crude extracts with the corresponding monoclonal antibody and Western blotting with the same antibody. Therefore, overexpression of <i>MED15</i> does not rescue the <i>tel2</i>-<i>ts</i> mutants by increasing either <i>TEL2</i> transcription or even indirectly by increasing Tel2 protein levels.</p

Sequence analysis of the amino acid changes in the <i>tel2</i> mutant proteins.

<p>Sequence analysis of the amino acid changes in the <i>tel2</i> mutant proteins.</p

Tel2 controls transcription of <i>EST2</i>/telomerase (but not that of the telomerase RNA subunit <i>TLC1</i>) and is regulated by the Med15 Mediator subunit.

<p>(<b>A</b>) Northern blot analysis of endogenous <i>TLC1</i> RNA levels indicates an absence of deregulation in the indicated <i>tel2</i> mutants. <i>rvb2</i> mutants that exhibit slight and stable telomere shortening (NG, MC, submitted for publication) were also used in that experiment. A <i>tlc1</i> null strain was used to ascertain that the highlighted band indeed corresponds to TLC1 RNA. <i>ACT1</i> and <i>SCR1</i> RNA levels were measured to serve as loading controls. (<b>B</b>) Immunoprecipitation-Western experiments aiming at assessing Myc₁₈-Est2 levels (construct integrated at <i>EST2</i> genomic locus, under the control of native promoter) indicate that <i>EST2</i>/telomerase levels are depressed in the <i>tel2</i>-<i>15</i> ts mutant strain grown at the semi-permissive temperature for growth of 29°C for 2 hr or overnight (ON). The <i>rvb2</i>-<i>2</i> and <i>rvb2</i>-<i>24</i> mutants (NG, MC, submitted for publication) were used as controls. (<b>C</b>) Levels of <i>EST2</i> mRNA, coding for the protein subunit of budding yeast telomerase, were measured relative to those of <i>ACT1</i>, in wild-type cells (wt, normalized to 1.0) and in the temperature-sensitive <i>tel2</i>-<i>15</i> (<b>left panel</b>) and <i>tel2</i>-<i>19</i> (<b>right panel</b>) mutants. The mean values ± standard error correspond to 4 experiments performed with the <i>tel2</i>-<i>15</i> mutant and 5 experiments with the <i>tel2</i>-<i>19</i> mutant, each sample being performed in triplicate. (<b>D</b>) <i>MAE1</i> and <i>MEP2</i> mRNA levels in the <i>tel2</i>-<i>15</i> ts mutant, at the indicated temperatures. Results are from two experiments, each sample being performed in triplicate. (<b>E</b>) Continuous overexpression of the <i>MED15</i> Mediator subunit, achieved by transforming the strains of the indicated relevant genotype with an episomal (2 µ) vector harboring <i>MED15</i> ORF flanked by upstream and downstream natural genomic sequences, resulted in a dramatic increase in <i>EST2</i> mRNA levels. Data are from two experiments using the wild type and the two mutants, each sample being performed in triplicate.</p

Menin controls the concentration of retinoblastoma protein.

International audienceMenin, the protein encoded by the Multiple Endocrine Neoplasia type 1 gene, is involved in the cell cycle control through its participation in functional dynamics of chromatin and regulation of transcription. RB, the protein of the retinoblastoma gene RB1, controls the progression of the cell cycle and is regulated in its activity by means of a feedback by phosphorylation. Studies in double heterozygous knockout mice for Men1 and the Retinoblastoma gene Rb1 have recently indicated that both genes may be implicated in the same pathways. In the course of our studies on Menin, we found that after suppression or in absence of Menin, RB1 expression was strongly reduced in a posttranscriptional manner. Under conditions of growth arrest, the hyperphosphorylated form of RB was most strongly affected, whereas its hypophosphorylated form was less or not at all reduced. Our findings confirm the hypothesis that the pathways of two tumor suppressor genes are connected

The temperature-sensitive <i>tel2</i>-<i>15</i> and <i>tel2</i>-<i>19</i> mutants exhibit short telomeres.

<p>(<b>A</b>) <i>tel2</i>-<i>15</i> confers a more severe telomere length defect than <i>tel2</i>-<i>19</i>, compared here with the previously described <i>tel2</i>-<i>1</i> mutant <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0030451#pone.0030451-Lustig1" target="_blank">[2]</a>, all grown at 24°C. (<b>B</b>) The shortened telomeres in the <i>tel2</i>-<i>19</i> mutant were not furthered shortened when combined with a <i>tel1</i> null mutation. These strains were grown at 29°C. (<b>C</b>). In contrast, both the <i>tel2</i>-<i>19 yku70</i> null and <i>tel2</i>-<i>15 yku70</i> null double mutants, grown here at 24°C, exhibited shorter telomeres than either one of the corresponding single mutants. Average telomere length (corresponding to the DNA smear migrating at around 1.3 kb) of <i>tel2</i> mutant strains with the indicated relevant genotype, grown for at least 22 days to allow telomere length to attain a stable value, was detected by Southern blotting with a TG_1–3 telomeric P³²-labeled probe. The dashed horizontal lines, drawn between the mean length values of two or more wild-type (wt) or mutant strains, as indicated in the margin, allow to get a better appreciation of the variations in telomere length in the mutants.</p

<i>MED15</i>, coding for a subunit of Mediator, found in the present study to be an extragenic suppressor of <i>tel2</i>-<i>ts</i> mutations, also partially rescues their telomere length defect.

<p>(<b>A</b>) The temperature-sensitive <i>tel2</i>-<i>15</i> and <i>tel2</i>-<i>19</i> mutants, as well as a wild-type (wt) strain,were re-transformed, here, with the <i>MED15</i> or <i>TEL2</i> plasmids, either low-copy (1 or 2 copies) <i>CEN</i> (YCplac) or multi-copy 2 µ (YEplac), both originally isolated from the genetic screens performed in the present study, or with the corresponding empty plasmids (YCp or YEp), and their growth characteristics evaluated after spotting ten-fold dilutions (from left to right in each condition) on selective, -Ura, minimal medium at the indicated temperatures. YCp-<i>MED15</i> rescued <i>tel2</i>-<i>15</i> at up to 29°C, while YEp-<i>MED15</i> rescued <i>tel2</i>-<i>19</i> at up to 34°C. (<b>B</b>) Overexpression of <i>MED15</i>, from the plasmids described above, in A, partially rescued the short telomere phenotype of the <i>tel2</i>-<i>15</i> and <i>tel2</i>-<i>19</i> mutant strains. Compare the extent of the rescue with that provided by <i>TEL2</i> overexpression. Telomere length was measured as described in the legend to <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0030451#pone-0030451-g003" target="_blank">Figure 3</a> after the strains had been propagated at 27.5°C (<i>tel2</i>-<i>15</i>) or 29°C (<i>tel2</i>-<i>19</i>) on minimal medium for 30 days. (<b>C</b>) Overexpression of <i>MED15</i> had no elongation effect on telomere length in wild-type cells, grown here at 29°C. In fact, a slight shortening of telomeres was observed following <i>MED15</i> overexpression compared with expression of vector alone.</p

SK4 oncochannels regulate calcium entry and promote cell migration in KRAS-mutated colorectal cancer

International audienceBackgroundColorectal cancer (CRC) metastases are the main cause of CRC mortality. Intracellular Ca2+ regulates cell migration and invasion, key factors for metastases. Ca2+ also activates Ca2+-dependent potassium channels which in turn affect Ca2+ driving force. We have previously reported that the expression of the Ca2+ activated potassium channel KCNN4 (SK4) is higher in CRC primary tumors compared to normal tissues. Here, we aimed to investigate the role of SK4 in the physiology of CRC.ResultsSK4 protein expression is enhanced in CRC tissues compared to normal colon tissues, with a higher level of KCNN4 in CRC patients with KRAS mutations. At the cellular level, we found that SK4 regulates the membrane potential of HCT116 cells. We also found that its inhibition reduced store operated Ca2+ entry (SOCE) and constitutive Ca2+ entry (CCE), while reducing cell migration. We also found that the activity of SK4 is linked to resistance pathways such as KRAS mutation and the expression of NRF2 and HIF-1α. In addition, the pharmacological inhibition of SK4 reduced intracellular reactive oxygen species (ROS) production, NRF2 expression and HIF1α stabilization.ConclusionOur results suggest that SK4 contributes to colorectal cancer cell migration and invasion by modulating both Ca2+ entry and ROS regulation. Therefore, SK4 could be a potential target to reduce metastasis in KRAS-mutated CRC