Analysis of <i>Plagl1</i> region in <i>Dnmt3l −/+.</i>

<p>(A) The methylation status of the <i>Plagl1</i> promoter regions in wild type +/+ and <i>Dnmt3l</i> −/+ embryos examined by bisulphite PCR. Each circle represents a single CpG dinucleotide on a DNA strand, a methylated cytosine (•) or an unmethylated cytosine (○). (B) RT-PCRs on cDNA generated with (+) and without (−) reverse transcriptase show an increase in the expression of the imprinted transcripts in <i>Dnmt3l</i>−/+ embryos as a result of reactivation of the maternal allele. (C) The histone modification signature of the <i>Plagl1</i>-DMR in wild type B×C embryos, and after targeted deletion of the <i>Dnmt3l</i> gene. DNA extracted from antibody bound (B) and unbound (U) chromatin fractions were subject to either qPCR or PCR and SSCP analysis with primers that can discriminate parental alleles.</p

Cellular localization and RNA stability of the ncRNAs.

<p>(A) Distribution of the various transcripts in the nuclear (dark grey) and cytoplasmic (black) fractions, compared to total RNA (light grey). <i>U937 snoRNA</i> and <i>Airn</i> are nuclear-retained controls, whereas <i>Igf2</i> is cytoplasm-exported control. (B) Abundance of the various transcripts after exposure to Actinomycin D to determine RNA stability. The relative expression values of the control untreated samples are set to 1 (light grey bars) for each transcript. <i>C-Myc</i> and <i>Airn</i> are control transcripts for with short half-life; <i>Gapdh</i> and <i>Igf2r</i> are long half-life controls.</p

Schematic overview of the mouse chromosome 10 imprinted domain.

<p>(A) Map of the <i>Plagl1</i> locus, showing the location of the various imprinted transcripts and CpG islands (paternally expressed transcripts are in blue; biallelically expressed transcripts are in grey). Arrows represent direction of transcription. (B) The allelic expression of the various transcripts in embryonic tissues in reciprocal mouse crosses (for clarity only (B×C) F1 tissues are shown).</p

Structural Basis for Highly Selective Class II Alpha Phosphoinositide-3-Kinase Inhibition

Class II phosphoinositide-3-kinases (PI3Ks) play central roles in cell signaling, division, migration, and survival. Despite evidence that all PI3K class II isoforms serve unique cellular functions, the lack of isoform-selective inhibitors severely hampers the systematic investigation of their potential relevance as pharmacological targets. Here, we report the structural evaluation and molecular determinants for selective PI3K-C2α inhibition by a structure–activity relationship study based on a pteridinone scaffold, leading to the discovery of selective PI3K-C2α inhibitors called PITCOINs. Cocrystal structures and docking experiments supported the rationalization of the structural determinants essential for inhibitor activity and high selectivity. Profiling of PITCOINs in a panel of more than 118 diverse kinases showed no off-target kinase inhibition. Notably, by addressing a selectivity pocket, PITCOIN4 showed nanomolar inhibition of PI3K-C2α and >100-fold selectivity in a general kinase panel. Our study paves the way for the development of novel therapies for diseases related to PI3K-C2α function

Structural Basis for Highly Selective Class II Alpha Phosphoinositide-3-Kinase Inhibition

Class II phosphoinositide-3-kinases (PI3Ks) play central roles in cell signaling, division, migration, and survival. Despite evidence that all PI3K class II isoforms serve unique cellular functions, the lack of isoform-selective inhibitors severely hampers the systematic investigation of their potential relevance as pharmacological targets. Here, we report the structural evaluation and molecular determinants for selective PI3K-C2α inhibition by a structure–activity relationship study based on a pteridinone scaffold, leading to the discovery of selective PI3K-C2α inhibitors called PITCOINs. Cocrystal structures and docking experiments supported the rationalization of the structural determinants essential for inhibitor activity and high selectivity. Profiling of PITCOINs in a panel of more than 118 diverse kinases showed no off-target kinase inhibition. Notably, by addressing a selectivity pocket, PITCOIN4 showed nanomolar inhibition of PI3K-C2α and >100-fold selectivity in a general kinase panel. Our study paves the way for the development of novel therapies for diseases related to PI3K-C2α function