Tolga Cagatay

Architecture-Dependent Noise Discriminates Functionally Analogous Differentiation Circuits

Gene regulatory circuits with different architectures (patterns of regulatory interactions) can generate similar dynamics. This raises the question of why a particular circuit architecture is selected to implement a given cellular process. To investigate this problem, we compared the Bacillus subtilis circuit that regulates differentiation into the competence state to an engineered circuit with an alternative architecture (SynEx) in silico and in vivo. Time-lapse microscopy measurements showed that SynEx cells generated competence dynamics similar to native cells and reconstituted the physiology of differentiation. However, architectural differences between the circuits altered the dynamic distribution of stochastic fluctuations (noise) during circuit operation. This distinction in noise causes functional differences between the circuits by selectively controlling the timing of competence episodes and response of the system to various DNA concentrations. These results reveal a tradeoff between temporal precision and physiological response range that is controlled by distinct noise characteristics of alternative circuit architectures.

Localized cell death focuses mechanical forces during 3D patterning in a biofilm

From microbial biofilm communities to multicellular organisms, 3D macroscopic structures develop through poorly understood interplay between cellular processes and mechanical forces. Investigating wrinkled biofilms of Bacillus subtilis, we discovered a pattern of localized cell death that spatially focuses mechanical forces, and thereby initiates wrinkle formation. Deletion of genes implicated in biofilm development, together with mathematical modeling, revealed that ECM production underlies the localization of cell death. Simultaneously with cell death, we quantitatively measured mechanical stiffness and movement in WT and mutant biofilms. Results suggest that localized cell death provides an outlet for lateral compressive forces, thereby promoting vertical mechanical buckling, which subsequently leads to wrinkle formation. Guided by these findings, we were able to generate artificial wrinkle patterns within biofilms. Formation of 3D structures facilitated by cell death may underlie self-organization in other developmental systems, and could enable engineering of macroscopic structures from cell populations.

p53 mutation as a source of aberrant β-catenin accumulation in cancer cells

β-catenin is involved in both cell–cell interactions and wnt pathway–dependent cell fate determination through its interactions with E-cadherin and TCF/LEF transcription factors, respectively. Cytoplasmic/nuclear levels of β-catenin are important in regulated transcriptional activation of TCF/LEF target genes. Normally, these levels are kept low by proteosomal degradation of β-catenin through Axin1- and APC-dependent phosphorylation by CKI and GSK-3β. Deregulation of β-catenin degradation results in its aberrant accumulation, often leading to cancer. Accordingly, aberrant accumulation of β-catenin is observed at high frequency in many cancers. This accumulation correlates with either mutational activation of CTNNB1 (β-catenin) or mutational inactivation of APC and Axin1 genes in some tumors. However, there are many tumors that display β-catenin accumulation in the absence of a mutation in these genes. Thus, there must be additional sources for aberrant β-catenin accumulation in cancer cells. Here, we provide experimental evidence that wild-type β-catenin accumulates in hepatocellular carcinoma (HCC) cells in association with mutational inactivation of p53 gene. We also show that worldwide p53 and β-catenin mutation rates are inversely correlated in HCC. These data suggest that inactivation of p53 is an important cause of aberrant accumulation of β-catenin in cancer cells.

Temporal competition between differentiation programs determines cell fate choice

Multipotent differentiation, where cells adopt one of several possible fates, occurs in diverse systems ranging from bacteria to mammals. This decision‐making process is driven by multiple differentiation programs that operate simultaneously in the cell. How these programs interact to govern cell fate choice is poorly understood. To investigate this issue, we simultaneously measured activities of the competing sporulation and competence programs in single Bacillus subtilis cells. This approach revealed that these competing differentiation programs progress independently without cross‐regulation before the decision point. Cells seem to arrive at a fate choice through differences in the relative timing between the two programs. To test this proposed dynamic mechanism, we altered the relative timing by engineering artificial cross‐regulation between the sporulation and competence circuits. Results suggest a simple model that does not require a checkpoint or intricate cross‐regulation before cellular decision‐making. Rather, cell fate choice appears to be the outcome of a ‘molecular race’ between differentiation programs that compete in time, providing a simple dynamic mechanism for decision‐making.

Lithium-mediated downregulation of PKB/Akt and cyclin E with growth inhibition in hepatocellular carcinoma cells

We studied in vitro effects of glycogen synthase kinase 3β (GSK3β)‐inhibitor lithium on the growth of hepatocellular carcinoma (HCC) cells. Lithium induced strong growth inhibition (>70%) in 75% (n = 9 of 12) of cell lines, apparently independent from the status of major genes that are mutated in HCC including p53, p16INK4a, β‐catenin and Axin1. Comparative studies with a growth‐sensitive Huh7 and growth‐resistant Hep40 cell lines showed that lithium induces growth arrest in Huh7 cells but not in Hep40 cells. Lithium induced the accumulation of N‐terminally phosphorylated inactive form of GSK3β with concomitant increase in β‐catenin and β‐catenin/TCF transcriptional activity in both cell lines. This suggests that lithium‐mediated HCC growth inhibition is independent of its well‐known stimulatory effect on Wnt‐β‐catenin signaling. The main differences between Huh7 and Hep40 responses to lithium treatment were observed at the levels PKB/Akt and cyclin E proteins. Lithium induced depletion of both proteins in growth‐sensitive Huh7, but not in growth‐resistant Hep40 cells. PKB/Akt and Cyclin E are 2 major proteins that are known to be constitutively active in HCC. The targeting of both proteins with lithium may be the main reason why most HCC cells are responsive to lithium‐mediated growth inhibition, independent of their p53, retinoblastoma and Wnt‐β‐catenin pathways. The exploration of molecular mechanisms involved in lithium‐mediated growth inhibition in relation with PKB/Akt and cyclin E downregulation may provide new insights for therapy of liver tumors.

Saccharomyces cerevisiae C1D is implicated in both non-homologous DNA end joining and homologous recombination

C1D is a gamma‐irradiation inducible nuclear matrix protein that interacts with and activates the DNA‐dependent protein kinase (DNA‐PK) that is essential for the repair of the DNA double‐strand breaks and V(D)J recombination. Recently, it was demonstrated that C1D can also interact with TRAX and prevent the association of TRAX with Translin, a factor known to bind DNA break‐point junctions, and that over expression of C1D can induce p53‐dependent apoptosis. Taken together, these findings suggest that mammalian C1D could be involved in maintenance of genome integrity by regulating the activity of proteins involved in DNA repair and recombination. To obtain direct evidence for the biological function of C1D that we show is highly conserved between diverse species, we have analysed the Saccharomyces cerevisiae C1D homologue. We report that the disruption of the YC1D gene results in a temperature sensitivity and that yc1d mutant strains exhibit defects in non‐homologous DNA end joining (NHEJ) and accurate DNA repair. In addition, using a novel plasmid‐based in vivo recombination assay, we show that yc1d mutant strains are also defective in homologous recombination. These results indicate that YC1D is implicated in both homologous recombination and NHEJ pathways for the repair of DNA double‐strand breaks.

LocNES: a computational tool for locating classical NESs in CRM1 cargo proteins

MOTIVATION Classical nuclear export signals (NESs) are short cognate peptides that direct proteins out of the nucleus via the CRM1-mediated export pathway. CRM1 regulates the localization of hundreds of macromolecules involved in various cellular functions and diseases. Due to the diverse and complex nature of NESs, reliable prediction of the signal remains a challenge despite several attempts made in the last decade. RESULTS We present a new NES predictor, LocNES. LocNES scans query proteins for NES consensus-fitting peptides and assigns these peptides probability scores using Support Vector Machine model, whose feature set includes amino acid sequence, disorder propensity, and the rank of position-specific scoring matrix score. LocNES demonstrates both higher sensitivity and precision over existing NES prediction tools upon comparative analysis using experimentally identified NESs. AVAILABILITY AND IMPLEMENTATION LocNES is freely available at http://prodata.swmed.edu/LocNES CONTACT: yuhmin.chook@utsouthwestern.edu SUPPLEMENTARY INFORMATION Supplementary data are available at Bioinformatics online.

Mutations in the Human naked cuticle Homolog NKD1 Found in Colorectal Cancer Alter Wnt/Dvl/β-Catenin Signaling

Background Mutation of Wnt signal antagonists Apc or Axin activates β-catenin signaling in many cancers including the majority of human colorectal adenocarcinomas. The phenotype of apc or axin mutation in the fruit fly Drosophila melanogaster is strikingly similar to that caused by mutation in the segment-polarity gene, naked cuticle (nkd). Nkd inhibits Wnt signaling by binding to the Dishevelled (Dsh/Dvl) family of scaffold proteins that link Wnt receptor activation to β-catenin accumulation and TCF-dependent transcription, but human NKD genes have yet to be directly implicated in cancer. Methodology/Principal Findings We identify for the first time mutations in NKD1 - one of two human nkd homologs - in a subset of DNA mismatch repair-deficient colorectal tumors that are not known to harbor mutations in other Wnt-pathway genes. The mutant Nkd1 proteins are defective at inhibiting Wnt signaling; in addition, the mutant Nkd1 proteins stabilize β-catenin and promote cell proliferation, in part due to a reduced ability of each mutant Nkd1 protein to bind and destabilize Dvl proteins. Conclusions/Significance Our data raise the hypothesis that specific NKD1 mutations promote Wnt-dependent tumorigenesis in a subset of DNA mismatch-repair-deficient colorectal adenocarcinomas and possibly other Wnt-signal driven human cancers.

Viable Mice with Compound Mutations in the Wnt/Dvl Pathway Antagonists nkd1 and nkd2

ABSTRACT Gradients of Wnt/β-catenin signaling coordinate development and physiological homeostasis in metazoan animals. Proper embryonic development of the fruit fly Drosophila melanogaster requires the Naked cuticle (Nkd) protein to attenuate a gradient of Wnt/β-catenin signaling across each segmental anlage. Nkd inhibits Wnt signaling by binding the intracellular protein Dishevelled (Dsh). Mice and humans have two nkd homologs, nkd1 and nkd2, whose encoded proteins can bind Dsh homologs (the Dvl proteins) and inhibit Wnt signaling. To determine whether nkd genes are necessary for murine development, we replaced nkd exons that encode Dvl-binding sequences with IRES-lacZ/neomycin cassettes. Mutants homozygous for each nkdlacZ allele are viable with slightly reduced mean litter sizes. Surprisingly, double-knockout mice are viable, with subtle alterations in cranial bone morphology that are reminiscent of mutation in another Wnt/β-catenin antagonist, axin2. Our data show that nkd function in the mouse is dispensable for embryonic development.

Next-generation XPO1 inhibitor shows improved efficacy and in vivo tolerability in hematological malignancies

The nuclear export receptor, Exportin 1 (XPO1), mediates transport of growth-regulatory proteins, including tumor suppressors, and is overactive in many cancers, including chronic lymphocytic leukemia (CLL), acute myeloid leukemia (AML) and aggressive lymphomas. Oral selective inhibitor of nuclear export (SINE) compounds that block XPO1 function were recently identified and hold promise as a new therapeutic paradigm in many neoplasms. One of these compounds, KPT-330 (selinexor), has made progress in Phase I/II clinical trials, but systemic toxicities limit its administration to twice-per-week and requiring supportive care. We designed a new generation SINE compound, KPT-8602, with a similar mechanism of XPO1 inhibition and potency but considerably improved tolerability. Efficacy of KPT-8602 was evaluated in preclinical animal models of hematological malignancies, including CLL and AML. KPT-8602 shows similar in vitro potency compared with KPT-330 but lower central nervous system penetration, which resulted in enhanced tolerability, even when dosed daily, and improved survival in CLL and AML murine models compared with KPT-330. KPT-8602 is a promising compound for further development in hematological malignancies and other cancers in which upregulation of XPO1 is seen. The wider therapeutic window of KPT-8602 may also allow increased on-target efficacy leading to even more efficacious combinations with other targeted anticancer therapies.