A FRET-based sensor reports the spatiotemporal dynamics of the protease TACE in live cells.

Identifying cis-regulatory regions in mammalian genomes is a key challenge toward understanding transcriptional regulation. However, identification and functional characterization of those regulatory elements governing differential gene expression has been hampered by the limited understanding of their organization and locations in genomes. We hypothesized that genes that are conserved across species will also display conservation at the level of their transcriptional regulation and that this will be reflected in the organization of cis-elements mediating this regulation. Using a computational approach, clusters of transcription factor binding sites that are absolutely conserved in order and in spacing across human, rat, and mouse genomes were identified. We term these regions pattern-defined regulatory islands (PRIs). We discovered that these sequences are frequently active sites of transcriptional regulation. These PRIs occur in ≈1.1% of the half-billion base pairs covered in the search and are located mainly in noncoding regions of the genome. We show that the premise of PRIs can be used to identify previously known and novel cis-regulatory regions controlling genes regulated by myogenic differentiation. Thus, PRIs may represent a fundamental property of the architecture of cis-regulatory elements in mammalian genomes, and this feature can be exploited to pinpoint critical transcriptional regulatory elements governing cell type-specific gene expression.

TGF- is a multifunctional growth factor whose best-known function is to inhibit cell growth and suppress tumor formation. TGF- causes cells to accumulate in mid-to-late G1 phase by blocking the transition from G1 to S. It has been shown that TGF- inhibits Cdk2-cyclin E kinase activity by promoting the binding of cell cycle inhibitor p27Kip1 to the kinase complexes. Here, we show that TGF- treatment leads to stabilization of p27Kip1 during G1 to S transition. We found that TGF- negatively regulates components of the SCF complex, which degrades the p27Kip1 during the G1 to S transition, through two distinct mechanisms. Using a pulse-chase analysis, we demonstrated that the stability of Skp2 decreases in the presence of TGF-. Destabilization of Skp2 by ubiquitin-mediated proteolysis was also demonstrated that in an in vitro degradation system, using cell extracts prepared from TGF--treated cultured cells. In addition, TGF- treatment decreases the levels of Cks1 mRNA. The deficiency of Cks1 in TGF--treated cells likely contributes to the stabilization of p27Kip1 and destabilization of Skp2, because in the absence of Cks1, SCFSkp2 cannot ubiquitinate p27Kip1; instead, self-ubiquitination of Skp2 occurs. Thus, stabilization of the cell cycle inhibitor p27Kip1 and cell growth inhibition in response to TGF- occur in part through limiting the threshold of the SCFSkp2 ubiquitin ligase by transcriptional and post-transcriptional mechanisms.

It is known that excess amounts of Ski, or any member of its proto-oncoprotein family, causes disruption of the transforming growth factor beta signal transduction pathway, thus causing oncogenic transformation of cells. Previous studies indicate that Ski is a relatively unstable protein whose expression levels can be regulated by ubiquitin-mediated proteolysis. Here, we investigate the mechanism by which the stability of Ski is regulated. We show that the steady-state levels of Ski protein are controlled post-translationally by cell cycle-dependent proteolysis, wherein Ski is degraded during the interphase of the cell cycle but is relatively stable during mitosis. Furthermore, we demonstrate that the ubiquitin-conjugating enzyme Cdc34 mediates cell cycle-dependent Ski degradation both in vitro and in vivo. Overexpression of dominant-negative Cdc34 stabilizes Ski and enhances its ability to antagonize TGF- signaling. Our data suggest that regulated proteolysis of Ski is one of the key mechanisms that control the threshold levels of this proto-oncoprotein, and thus prevents epithelial cells from becoming TGF- resistant.

Thymic stromal lymphopoietin (TSLP) is an IL-7-like cytokine that requires a heterodimeric receptor complex composed of the interleukin-7 receptor α chain and the TSLP receptor, which is related to the common gamma chain. TSLP has been shown to play an important role in the development of allergic inflammation, such as asthma and atopic dermatitis. Chimeric receptors composed of the cytoplasmic region of the TSLP receptor fused to the extracellular regions of homodimeric receptors, such as erythropoietin (Epo) receptor and thrombopoietin receptor have been used to dissect signaling events induced by the TSLP receptor. Intriguingly, studies using such chimeric TSLP receptors revealed that the human, but not mouse, TSLP receptor cytoplasmic domain can support proliferation of growth factor-dependent cells after homodimerization. Here, we used a systematic approach to investigate the mechanistic basis of this difference. Our studies revealed that induced homodimerization of receptor chimeras containing the transmembrane and cytoplasmic domains of both human and mouse TSLP receptors is not sufficient for driving cell proliferation. However, chimeric receptors with the transmembrane and juxtamembrane domains of Epo receptor fused to the cytoplasmic domain of human TSLP receptor signal like the Epo receptor and induce the activation of Jak2. Site-directed mutagenesis showed that the lone tyrosine residue in human TSLP receptor is not required for transmitting proliferative signals in receptor chimeras, which is consistent with the observation that none of the tyrosine residues are required for Epo receptor to support proliferation. Our data suggests that in the chimeric receptor context, the transmembrane and juxtamembrane domains of mouse Epo receptor are essential for the cytoplasmic domain of human TSLPR to achieve the strong proliferative ability and can modulate signaling pathway transmitted by the cytoplasmic domains of these chimeras.

The physiological responses to TGF-β stimulation are diverse and vary amongst different cell types and environmental conditions. Even though the principal molecular components of the canonical and the non-canonical TGF-β signaling pathways have been largely identified, the mechanism that underlies the well-established context dependent physiological responses remains a mystery. Understanding how the components of TGF-β signaling function as a system and how this system functions in the context of the global cellular regulatory network requires a more quantitative and systematic approach. Here, we review the recent progress in understanding TGF-β biology using integration of mathematical modeling and quantitative experimental analysis. These studies reveal many interesting dynamics of TGF-β signaling and how cells quantitatively decode variable doses of TGF-β stimulation.