Gene network modeling via TopNet reveals functional dependencies between diverse tumor-critical mediator genes.

Malignant cell transformation and the underlying reprogramming of gene expression require the cooperation of multiple oncogenic mutations. This cooperation is reflected in the synergistic regulation of non-mutant downstream genes, so-called cooperation response genes (CRGs). CRGs affect diverse hallmark features of cancer cells and are not known to be functionally connected. However, they act as critical mediators of the cancer phenotype at an unexpectedly high frequency >50%, as indicated by genetic perturbations. Here, we demonstrate that CRGs function within a network of strong genetic interdependencies that are critical to the malignant state. Our network modeling methodology, TopNet, takes the approach of incorporating uncertainty in the underlying gene perturbation data and can identify non-linear gene interactions. In the dense space of gene connectivity, TopNet reveals a sparse topological gene network architecture, effectively pinpointing functionally relevant gene interactions. Thus, among diverse potential applications, TopNet has utility for identification of non-mutant targets for cancer intervention.

SoxB1 downregulation in vegetal lineages of sea urchin embryos is achieved by both transcriptional repression and selective protein turnover.

Patterning of cell fates along the sea urchin animal-vegetal embryonic axis requires the opposing functions of nuclear beta-catenin/TCF-Lef, which activates the endomesoderm gene regulatory network, and SoxB1, which antagonizes beta-catenin and limits its range of function. A crucial aspect of this interaction is the temporally controlled downregulation of SoxB1, first in micromeres and then in macromere progeny. We show that SoxB1 is regulated at the level of protein turnover in these lineages. This mechanism is dependent on nuclear beta-catenin function. It can be activated by Pmar1, but not by Krl, both of which function downstream of beta-catenin/TCF-Lef. At least partially distinct, lineage-specific mechanisms operate, as turnover in the macromeres depends on entry of SoxB1 into nuclei, and on redundant destruction signals, neither of which is required in micromeres. Neither of these turnover mechanisms operates in mesomere progeny, which give rise to ectoderm. However, in mesomeres, SoxB1 appears to be subject to negative autoregulation that helps to maintain tight regulation of SoxB1 mRNA levels in presumptive ectoderm. Between the seventh and tenth cleavage stages, beta-catenin not only promotes degradation of SoxB1, but also suppresses accumulation of its message in macromere-derived blastomeres. Collectively, these different mechanisms work to regulate precisely the levels of SoxB1 in the progeny of different tiers of blastomeres arrayed along the animal-vegetal axis.

Tight regulation of SpSoxB factors is required for patterning and morphogenesis in sea urchin embryos.

Previous studies in sea urchin embryos have demonstrated that nuclearization of beta-catenin is essential for initial steps in the specification of endoderm and mesenchyme, which are derived from vegetal blastomeres. This process begins at the 4th and extends through the 9th cleavage stage, an interval in which the SpSoxB1 transcription regulator is downregulated by beta-catenin-dependent gene products that include the transcription repressor SpKrl. These observations raise the possibility that SpSoxB1 removal is required to allow vegetal development to proceed. Here we show that elevated and ectopic expression of this factor suppresses differentiation of all vegetal cell types, a phenotype that is very similar to that caused by the suppression of beta-catenin nuclear function by cadherin overexpression. Suppression of vegetal fates involves interference at the protein-protein level because a mutation of SpSoxB1 that prevents its binding to DNA does not significantly reduce this activity. Reduction in SpSoxB1 level results in elevated TCF/Lef-beta-catenin-dependent expression of a luciferase reporter gene in vivo, indicating that in the normal embryo this protein suppresses the primary vegetal signaling mechanism that is required for specification of mesenchyme and endoderm. Surprisingly, normal expression of SpSoxB1 is required for gastrulation and endoderm differentiation, as shown by both morpholino-mediated translational interference and expression of a dominant negative protein. Similar gain-of-function and loss-of-function assays of a closely related factor, SpSoxB2, demonstrate that it, too, is required for gastrulation and that its overexpression can suppress vegetal development. However, significant phenotypic differences are apparent in the two perturbations, indicating that SpSoxB1 and SpSoxB2 have at least some distinct developmental functions. The results of all these studies support a model in which the concentration of SpSoxB factors must be tightly regulated along the animal-vegetal axis of the early sea urchin embryo to allow beta-catenin-dependent specification of endoderm and mesenchyme cell fates as well as to activate target genes required for gastrulation.