Ovarian carcinoma CDK12 mutations misregulate expression of DNA repair genes via deficient formation and function of the Cdk12/CycK complex

The Cdk12/CycK complex promotes expression of a subset of RNA polymerase II genes, including those of the DNA damage response. CDK12 is among only nine genes with recurrent somatic mutations in high-grade serous ovarian carcinoma. However, the influence of these mutations on the Cdk12/CycK complex and their link to cancerogenesis remain ill-defined. Here, we show that most mutations prevent formation of the Cdk12/CycK complex, rendering the kinase inactive. By examining the mutations within the Cdk12/CycK structure, we find that they likely provoke structural rearrangements detrimental to Cdk12 activation. Our mRNA expression analysis of the patient samples containing the CDK12 mutations reveals coordinated downregulation of genes critical to the homologous recombination DNA repair pathway. Moreover, we establish that the Cdk12/CycK complex occupies these genes and promotes phosphorylation of RNA polymerase II at Ser2. Accordingly, we demonstrate that the mutant Cdk12 proteins fail to stimulate the faithful DNA double strand break repair via homologous recombination. Together, we provide the molecular basis of how mutated CDK12 ceases to function in ovarian carcinoma. We propose that CDK12 is a tumor suppressor of which the loss-of-function mutations may elicit defects in multiple DNA repair pathways, leading to genomic instability underlying the genesis of the cancer.     

TM-25659-Induced Activation of FGF21 Level Decreases Insulin Resistance and Inflammation in Skeletal Muscle via GCN2 Pathways

The TAZ activator 2-butyl-5-methyl-6-(pyridine-3-yl)-3-[2′-(1H-tetrazole-5-yl)-biphenyl-4-ylmethyl]-3H-imidazo[4,5-b]pyridine] (TM-25659) inhibits adipocyte differentiation by interacting with peroxisome proliferator-activated receptor gamma. TM-25659 was previously shown to decrease weight gain in a high fat (HF) diet-induced obesity (DIO) mouse model. However, the fundamental mechanisms underlying the effects of TM-25659 remain unknown. Therefore, we investigated the effects of TM-25659 on skeletal muscle functions in C2 myotubes and C57BL/6J mice. We studied the molecular mechanisms underlying the contribution of TM-25659 to palmitate (PA)-induced insulin resistance in C2 myotubes. TM-25659 improved PA-induced insulin resistance and inflammation in C2 myotubes. In addition, TM-25659 increased FGF21 mRNA expression, protein levels, and FGF21 secretion in C2 myotubes via activation of GCN2 pathways (GCN2-phosphoeIF2α-ATF4 and FGF21). This beneficial effect of TM-25659 was diminished by FGF21 siRNA. C57BL/6J mice were fed a HF diet for 30 weeks. The HF-diet group was randomly divided into two groups for the next 14 days: the HF-diet and HF-diet + TM-25659 groups. The HF diet + TM-25659-treated mice showed improvements in their fasting blood glucose levels, insulin sensitivity, insulin-stimulated Akt phosphorylation, and inflammation, but neither body weight nor food intake was affected. The HF diet + TM-25659-treated mice also exhibited increased expression of both FGF21 mRNA and protein. These data indicate that TM-25659 may be beneficial for treating insulin resistance by inducing FGF21 in models of PA-induced insulin resistance and HF diet-induced insulin resistance.     

Poly(ADP-ribosyl)ation of p53 Contributes to TPEN-Induced Neuronal Apoptosis

Depletion of intracellular zinc by N,N,N′,N′-tetrakis(2-pyridylmethyl) ethylenediamine (TPEN) induces p53-mediated protein synthesis-dependent apoptosis of mouse cortical neurons. Here, we examined the requirement for poly(ADP-ribose) polymerase (PARP)-1 as an upstream regulator of p53 in zinc depletion-induced neuronal apoptosis. First, we found that chemical inhibition or genetic deletion of PARP-1 markedly attenuated TPEN-induced apoptosis of cultured mouse cortical neurons. Poly(ADP-ribosyl)ation of p53 occurred starting 1 h after TPEN treatment. Suggesting the critical role of PARP-1, the TPEN-induced increase of stability and activity of p53 as well as poly(ADP-ribosyl)ation of p53 was almost completely blocked by PARP inhibition. Consistent with this, the induction of downstream proapoptotic proteins PUMA and NOXA was noticeably reduced by chemical inhibitors or genetic deletion of PARP-1. TPEN-induced cytochrome C release into the cytosol and caspase-3 activation were also blocked by inhibition of PARP-1. Taken together, these findings indicate that PARP-1 is essential for TPEN-induced neuronal apoptosis.     

LTR retrotransposon dynamics in the evolution of the olive (Olea europaea) genome

Improved knowledge of genome composition, especially of its repetitive component, generates important information for both theoretical and applied research. The olive repetitive component is made up of two main classes of sequences: tandem repeats and retrotransposons (REs). In this study, we provide characterization of a sample of 254 unique full-length long terminal repeat (LTR) REs. In the sample, Ty1-Copia elements were more numerous than Ty3-Gypsy elements. Mapping a large set of Illumina whole-genome shotgun reads onto the identified retroelement set revealed that Gypsy elements are more redundant than Copia elements. The insertion time of intact retroelements was estimated based on sister LTR’s divergence. Although some elements inserted relatively recently, the mean insertion age of the isolated retroelements is around 18 million yrs. Gypsy and Copia retroelements showed different waves of transposition, with Gypsy elements especially active between 10 and 25 million yrs ago and nearly inactive in the last 7 million yrs. The occurrence of numerous solo-LTRs related to isolated full-length retroelements was ascertained for two Gypsy elements and one Copia element. Overall, the results reported in this study show that RE activity (both retrotransposition and DNA loss) has impacted the olive genome structure in more ancient times than in other angiosperms.