FLOWERING LOCUS C (FLC) has a key role in the timing of the initiation of flowering in Arabidopsis. FLC binds and represses two genes that promote flowering, FT and SOC1. We show that FLC binds to many other genes, indicating that it has regulatory roles other than the repression of flowering. We identified 505 FLC binding sites, mostly located in the promoter regions of genes and containing at least one CArG box, the motif known to be associated with MADS-box proteins such as FLC. We examined 40 of the target genes, and 20 showed increased transcript levels in an flc mutant compared with the wild type. Five genes showed decreased expression in the mutant, indicating that FLC binding can result in either transcriptional repression or activation. The genes we identified as FLC targets are involved in developmental pathways throughout the life history of the plant, many of which are associated with reproductive development. FLC is also involved in vegetative development, as evidenced by its binding to SPL15, delaying the progression from juvenile to adult phase. Some of the FLC target genes are also bound by two other MADSbox proteins, AP1 and SEP3, suggesting that MADS-box genes may operate in a network of control at different stages of the life cycle, many ultimately contributing to the development of the reproductive phase of the plant.reproductive transition | phase change | environmental response | floral morphology | ChIP sequencing E ncoding a MADS-box transcription factor, FLOWERING LOCUS C (FLC) is a major repressor of flowering in Arabidopsis (1, 2). The regulatory role of FLC in the control of flowering initiation is of special significance in vernalization (2), a period of low temperature that stimulates flowering. Before vernalization, FLC represses the initiation of flowering, preventing the changes that convert the apical meristem to one producing the reproductive structures. After the prolonged period of low temperature, FLC expression is repressed and plants are able to initiate flowering. The repression of FLC is associated with modifications to FLC chromatin, which prevent transcriptional activity of the gene (3, 4). The state of reduced transcriptional activity is maintained through the subsequent cell divisions of the developing plant when growing under normal temperature conditions (5). Two loci regulating FLC are FRIGIDA (FRI) (6) and VERNALIZATION INSENSITIVE 3 (VIN3) (4). FRI is responsible for a high level of production of the FLC protein and VIN3, which is induced by low temperature, reduces FLC transcriptional activity during vernalization. The vernalization process overrides the FRI-mediated control of FLC, resulting in the repression of transcriptional activity and the promotion of flowering initiation (7).The transition of the vegetative apical meristem to one producing reproductive structures involves the interaction of FLC with a small number of key genes. Three flowering-time genes,

Carcinoma-associated fibroblasts (CAFs) play a critical role in malignant progression. Loss of TGF-ϐ receptor II (TGFϐR2) in the prostate stroma is correlated with prostatic tumorigenesis. To determine the mechanisms by which stromal heterogeneity due to loss of TGFϐR2 might contribute to cancer progression, we attenuated TGF-ϐ signaling in a subpopulation of immortalized human prostate fibroblasts in a model of tumor progression. In a tissue recombination model, loss of TGFϐR2 function in 50% of the stromal cell population resulted in malignant transformation of the non-tumorigenic human prostate epithelial cell line BPH1. Mixing fibroblasts expressing the empty vector and dominant negative TGFϐR2 increased the expression of markers of myofibroblast differentiation [co-expression of vimentin and alpha smooth muscle actin (αSMA)] through elevation of TGF-ϐ1 and activation of the Akt pathway. In combination, these two populations of stromal cells recapitulated the tumor inductive activity of CAFs. TGFϐR2 activity in mixed stromal cell populations cultured in vitro caused secretion of factors that are known to promote tumor progression, including TGF-ϐ1, SDF1/CXCL12, and members of the FGF and BMP families. In vivo, tissue recombination of fibroblasts overexpressing TGF-ϐ1 and SDF1/CXCL12 not only induced transformation of BPH1 cells, but also promoted a robust growth of highly invasive cells, similar to effects produced by CAFs. While the precise nature and/or origin of the particular stromal cell populations in vivo remain unknown, these findings strongly link heterogeneity in TGF-ϐ signaling to tumor promotion by tumor stromal cells.

The roles of protein-tyrosine phosphatases (PTPs) in processes such as cell growth and adhesion are poorly understood. To explore the ability of specific PTPs to regulate cell signaling pathways initiated by stimulation of growth factor receptors, we expressed the receptor-like PTP, PTP␣, in A431 epidermoid carcinoma cells. These cells express high levels of the epidermal growth factor (EGF) receptor and proliferate in response to the autocrine production of transforming growth factor-␣. Conversely, EGF stimulation of A431 cells in vitro leads to growth inhibition and triggers the rapid detachment of these cells from the substratum. Although PTP␣ expression did not alter the growth characteristics of either unstimulated or EGF-stimulated cells, this phosphatase was associated with increased cell-substratum adhesion. Furthermore, PTP␣-expressing A431 cells were strikingly resistant to EGF-induced cell rounding. Overexpression of PTP␣ in A431 cells was associated with the dephosphorylation/activation of specific Src family kinases, suggesting a potential mechanism for the observed alteration in A431 cell-substratum adhesion. Src kinase activation was dependent on the D1 catalytic subunit of PTP␣, and there was evidence of association between PTP␣ and Src kinase(s). PTP␣ expression also led to increased association of Src kinase with the integrin-associated focal adhesion kinase, pp125FAK . In addition, paxillin, a Src and/or pp125 FAK substrate, displayed increased levels of tyrosine phosphorylation in PTP␣-expressing cells and was associated with elevated amounts of Csk. In view of these alterations in focal adhesion-associated molecules in PTP␣-expressing A431 cells, as well as the changes in adhesion demonstrated by these cells, we propose that PTP␣ may have a role in regulating cell-substratum adhesion.