Histone posttranslational modifications and cell fate determination: Lens induction requires the lysine acetyltransferases CBP and p300

Lens induction is a classical embryologic model to study cell fate determination. It has been proposed earlier that specific changes in core histone modifications accompany the process of cell fate specification and determination. The lysine acetyltransferases CBP and p300 function as principal enzymes that modify core histones to facilitate specific gene expression. Herein, we performed conditional inactivation of both CBP and p300 in the ectodermal cells that give rise to the lens placode. Inactivation of both CBP and p300 resulted in the dramatic discontinuation of all aspects of lens specification and organogenesis, resulting in aphakia. The CBP/p300(−/−) ectodermal cells are viable and not prone to apoptosis. These cells showed reduced expression of Six3 and Sox2, while expression of Pax6 was not upregulated, indicating discontinuation of lens induction. Consequently, expression of αB- and αA-crystallins was not initiated. Mutant ectoderm exhibited markedly reduced levels of histone H3 K18 and K27 acetylation, subtly increased H3 K27me3 and unaltered overall levels of H3 K9ac and H3 K4me3. Our data demonstrate that CBP and p300 are required to establish lens cell-type identity during lens induction, and suggest that posttranslational histone modifications are integral to normal cell fate determination in the mammalian lens

Bilateral blockade of MEK- and PI3K-mediated pathways downstream of mutant KRAS as a treatment approach for peritoneal mucinous malignancies - Fig 4

<p><b>A.</b> Resistance to PI3Ki is correlated to increased pERK. Western blots of proteins isolated from LS174T cells treated with either MEKi, PI3Ki or both were probed with anti-phosphoERK1/2 (pERK1/2), anti-total ERK1/2, anti-phosphoAKT (pAKT) or anti-total AKT. Elevated levels of pERK1/2 (A, green arrow) and pAKT (A, green arrow) were seen 72 hours after exposure to PI3Ki coincident with the development of resistance to this inhibitor. <b>B.</b> Increased receptor tyrosine kinase (RTK) phosphorylation upon prolonged exposure to PI3K inhibitors. Protein lysates from PI3Ki-treated LS174T or RW7213 cells (72 hours) were incubated with human phospho-RTK array containing 49 RTKs, washed and probed with anti-phospho-tyrosine antibodies. Antigen-antibody complexes were detected by chemiluminescence and the results were quantified by densitometry. Numbers within brackets indicate fold increase in RTKs in PI3Ki-treated MCA cells relative to vehicle-treated controls. <b>C.</b> Synergistic reduction in viability of MCA cells treated with PI3K inhibitor and Linsitinib (inhibitor of IR and IGFR1). LS174T and RW7213 cells were treated with Linsitinib (blue) and PI3Ki (red) as single agents or in combination (green) in a fixed ratio for 72 hours. Each data point is the average of an n = 6. Error bars indicate standard error of the mean.</p

MCA cells are sensitive to MEK inhibition in vitro.

<p><b>A.</b> LS174T, RW7213 and RW2982 cells were treated with increasing concentrations of the MEK inhibitor, Cobimetinib, for 96 hours. <b>B.</b> Mutant KRAS knockdown sensitizes MCA cells to MEK inhibition. LS174T and RW7213 cells, either untreated (blue) or induced with DOX (green) to knockdown mutant KRAS were treated with increasing concentrations of Cobimetinib (MEKi). The half-maximal concentration (IC50) of Cobimetinib was reduced from 0.28 μM to 0.042 μM (6.7 fold) in LS174T cells and from 0.24 μM to 0.022 μM (10.7 fold) in RW7213 cells. <b>C.</b> MEKi and PI3Ki combination treatment synergistically reduces viability of MCA in vitro. LS174T and RW7213 cells were treated with MEKi (blue) and PI3Ki (red) as single agents or in combination (green) in a fixed ratio for 72 hours. Each data point on all graphs is the average of an n = 6. Error bars indicate standard error of the mean.</p

Inducible and reversible knockdown of mutant and wildtype KRAS in vitro.

<p><b>A.</b> The pINDUCER lentiviral vector was used for targeted knockdown KRAS in MCA cells in vitro. Addition (+) or withdrawal (-) of DOX induced or extinguished respectively, the expression of KRAS shRNA (Fig adapted from Meerbrey et al. 2011). <b>B.</b> Western blots showing reduction of KRAS protein expression after induction of shRNA targeting wildtype and mutant KRAS in LS174T cells (red arrow) and restoration of KRAS protein expression within 2 days after DOX withdrawal (green arrow). Note that the KRAS western blot shown in panel B was probed with an anti-KRAS antibody that binds to both wildtype and mutant KRAS. <b>C-E.</b> KRAS knockdown reduces MUC2 protein expression in MCA in vitro. Knockdown of wildtype and mutant KRAS in LS174T (B) and RW7213 (D) cells reduces MUC2 protein expression (C, E). Note that the KRAS western blot shown in panel D was probed with an anti-KRAS antibody that binds to both wildtype and mutant KRAS. <b>F, G.</b> Knockdown of mutant but not wild type KRAS reduces MUC2. Western blots of mutant KRAS (F), wildtype KRAS (G) and MUC2 protein (F, G, bottom panels) expression in LS174T cells. Mutant KRAS knockdown in LS174T cells (F, top panel, arrows) substantially reduces MUC2 protein levels (F, bottom panel, arrows). In contrast, knockdown of wildtype KRAS (G, top panel, arrow) did not reduce MUC2 protein expression (G, bottom panel, arrow). Note that the KRAS western blot presented in panel F was probed with an anti-KRASG12D specific antibody that binds only to mutant KRAS protein while the western blot in panel G was probed with an antibody that binds both wildtype and mutant KRAS proteins. Knockdown of wildtype KRAS did not alter mutant KRAS protein levels (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0179510#pone.0179510.s003" target="_blank">S3 Fig</a>). <b>H, I.</b> HRAS and NRAS are dispensable for MUC2 expression. Quantitative RTPCR results show reduction of HRAS and NRAS in LS174T cells (H). Error bars indicate standard error of the mean. Knockdown of HRAS or NRAS did not reduce MUC2 protein expression (I, green arrows) in contrast to KRAS knockdown (I, red arrow). Expression levels of proteins on western blots were quantified by densitometry and percent protein (MUC2 or KRAS) expression in DOX-treated (+) samples relative to untreated (-) controls (normalized to actin) are shown below the blots. Abbreviations: d, days; 5d+/2d- indicates that cells were cultured for 5 days in the presence of DOX and for 2 days after the removal of DOX.</p

Combined treatment of Cobimetinib and Pictilisib is effective in reducing tumor growth in MCA xenograft mouse models.

<p><b>A.</b> LS174T cell line-derived tumors in xenograft mouse models were treated with Cobimetinib (20 mg/kg) or Pictilisib (150 mg/kg) alone or in combination. Drugs were delivered Q3D by oral gavage. Tumor growth rates between different groups were compared by two-way ANOVA (two tailed). In the Fig, one, two and three asterisks indicate p<0.05, p<0.01 and p<0.001 respectively. <b>B.</b> Proteins from two tumors in each treatment group were resolved by SDS-PAGE, blotted and probed with antibodies to antigens indicated to the left of the blots. For detection of MUC2, proteins were resolved by 1.5% agarose gel. Combined treatment of MEKi and PI3Ki was effective in reducing MUC2 protein levels compared to single agent treatment.</p

Novel homozygous, heterozygous and hemizygous FRMD7 gene mutations segregated in the same consanguineous family with congenital X-linked nystagmus

Congenital nystagmus (NYS) is characterized by bilateral, spontaneous, and involuntary movements of the eyeballs that most commonly presents between 2 and 6 months of life. To date, 44 different FRMD7 gene mutations have been found to be etiological factors for the NYS1 locus at Xq26-q27. The aim of this study was to find the FRMD7 gene mutations in a large eleven-generation Indian pedigree with 71 members who are affected by NYS. Mutation analysis of the entire coding region and splice junctions of the FRMD7 gene revealed a novel missense mutation, c.A917G, predicts a substitution of Arg for Gln at codon 305 (Q305R) within exon 10 of FRMD7. The mutation was detected in hemizygous males, and in homozygous and heterozygous states in affected female members of the family. This mutation was not detected in unaffected members of the family or in 100 unrelated control subjects. This mutation was found to be at a highly conserved residue within the FERM-adjacent domain in affected members of the family. Structure prediction and energetic analysis of wild-type FRMD7 compared with mutant (Q305R) revealed that this change in amino acid led to a change in secondary structure predicted to be an energetically unstable protein. The present study represents the first confirmation of FRMD7 gene mutations in a multigenerational Indian family and expands the mutation spectrum for this locus