Three distinct proliferative signals for multiple myeloma (MM) cell lines induce enhancer of zeste homolog 2 (ezh2) transcript expression. EZH2 is a polycomb group protein that mediates repression of gene transcription at the chromatin level through its methyltransferase activity. Normal bone marrow plasma cells do not express ezh2; however, gene expression is induced and correlates with tumor burden during progression of this disease. We therefore investigated how EZH2 expression is deregulated in MM cell lines and determined the consequence of this activity on proliferation and transformation. We found that EZH2 protein expression is induced by interleukin 6 (IL-6) in growth factor-dependent cell lines and is constitutive in IL-6-independent cell lines. Furthermore, EZH2 expression correlates with proliferation and B-cell terminal differentiation. Significantly, EZH2 protein inhibition by short interference RNA treatment results in MM cell growth arrest. Conversely, EZH2 ectopic overexpression induces growth factor independence. We found that the growth factor-independent proliferative phenotype in MM cell lines harboring a mutant N-or K-ras gene requires EZH2 activity. Finally, this is the first report to demonstrate that EZH2 has oncogenic activity in vivo, and that cell transformation and tumor formation require histone methyltransferase activity.

Biological signaling generally involves the activation of a receptor protein by an external stimulus followed by protein-protein interactions between the activated receptor and its downstream signal transducer. The current paradigm for the relay of signals along a signal transduction chain is that it occurs by highly specific interactions between fully folded proteins. However, recent results indicate that many regulatory proteins are intrinsically unstructured, providing a serious challenge to this paradigm and to the nature of structurefunction relationships in signaling. Here we study the structural changes that occur upon activation of the blue light receptor photoactive yellow protein (PYP). Activation greatly reduces the tertiary structure of PYP but leaves the level secondary structure largely unperturbed. In addition, activated PYP exposes previously buried hydrophobic patches and allows significant solvent penetration into the core of the protein. These traits are the distinguishing hallmarks of molten globule states, which have been intensively studied for their role in protein folding. Our results show that receptor activation by light converts PYP to a molten globule and indicate stimulus-induced unfolding to a partially unstructured molten globule as a novel theme in signaling. PYP1 displays rhodopsin-like photochemistry (1, 2) based on the trans to cis photoisomerization (3-6) of its unique p-coumaric acid chromophore (7,8). PYP is a prototypical PAS domain (9) involved in photosensory processes in purple bacteria (10,11). PAS domains are a ubiquitous signaling module involved in regulation, sensing, the circadian rhythm, and a number of human diseases, which were first identified in the proteins Per, Arnt, and Sim (12). Photoexcitation of PYP triggers a series of processes that result in the formation of a long-lived blue-shifted photocycle intermediates (1, 2). This intermediate is considered to be the functionally active signaling state of PYP (1,4,6,10). The three-dimensional structure of PYP has been determined at very high resolution (5,13,14), providing a unique opportunity to study photosensory signaling at the atomic level. Previous results have indicated a link between formation of the signaling state and protein unfolding in PYP. This partial unfolding was detected by: (i) the nonArrhenius temperature dependence of the photocycle kinetics (15, 16); (ii) the loss of amide-proton NMR HSQC cross-peaks (17); (iii) the solvent exposure of amide backbone sites (16); and (iv) the exchange broadening of NMR HSQC signals, particularly in the N-terminal 28 residues of PYP (18). Here we examine the hypothesis that the PYP signaling state is a molten globule state. To this end, we determine to what extent the PYP signaling state possesses the following set of specific properties widely used as the operational definition of a molten globule state (19 -21): (i) a large decrease in tertiary structure but only slight reduction in secondary structure as probed by circular dichroism (CD) spectroscopy; (ii...

The photoreceptor photoactive yellow protein (PYP) was used as a model system to study receptor activation and protein folding. Refolding was studied by stopped-flow absorbance spectroscopy for PYP with either a trans or a cis chromophore. Chromophore trans to cis isomerization, the mechanism of light detection by PYP, greatly affects the protein folding process. When the cis chromophore is present, refolding from the unfolded state proceeds through the putative signaling state of PYP as an on-pathway intermediate. In addition, moderate denaturant concentrations result in the specific unfolding of the signaling state of PYP. Thus, the signaling state is common to the pathways of folding and signaling. This result provides an avenue for the study of protein folding. We demonstrate how this approach can be used to establish whether a folding intermediate is on-pathway or offpathway. The results also reveal transient partial unfolding as a molecular mechanism for signaling.T o become functionally active, a protein needs to fold into its native three-dimensional structure. Protein folding has been investigated extensively, but many of its aspects remain unresolved. The properties of folding intermediates and their role in the folding mechanism as either productive on-pathway intermediates or dispensable off-pathway intermediates have proven to be difficult to unambiguously determine for many proteins (see refs. 1-4). Here evidence is reported for the hypothesis that protein folding and signaling are directly related. We demonstrate that this provides an approach to experimental studies on protein folding, using a small water soluble receptor protein that is activated by an external stimulus: unfolding and refolding reactions can be triggered not only by using traditional rapid mixing methods, but also by activation of the receptor. Thus, two independent pathways are available to populate the same folding intermediate. The existence, properties, and position in the folding pathway of the intermediate as deduced from rapid mixing studies therefore can be directly and independently tested. The use of a photoreceptor in this approach provides the additional benefit that signaling is triggered by exposure of the receptor to light, allowing for time-resolved measurements on the receptor activation process.To study the relationship between protein folding and signaling, we used photoactive yellow protein (PYP) as a model system. PYP is a small water-soluble photoreceptor from purple bacteria (5-7) that displays rhodopsin-like photochemistry (5, 8) based on its unique p-coumaric acid chromophore (9, 10). PYP is a member of the PAS domain family that is involved in regulation, sensing, and the circadian rhythm (11,12). The involvement of PAS domains in a wide range of biological responses has triggered studies on PYP aimed at understanding the signaling mechanism of this ubiquitous signaling module. Absorbance of a photon initiates a photocycle in PYP by the trans to cis photoisomerization of its chromophore (13)(14)(15)(16)...

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A hallmark of the research experience is encountering difficulty and working through those challenges to achieve success. This ability is essential to being a successful scientist, but replicating such challenges in a teaching setting can be difficult. The Genomics Education Partnership (GEP) is a consortium of faculty who engage their students in a genomics Course-Based Undergraduate Research Experience (CURE). Students participate in genome annotation, generating gene models using multiple lines of experimental evidence. Our observations suggested that the students' learning experience is continuous and recursive, frequently beginning with frustration but eventually leading to success as they come up with defendable gene models. In order to explore our "formative frustration" hypothesis, we gathered data from faculty via a survey, and from students via both a general survey and a set of student focus groups. Upon analyzing these data, we found that all three datasets mentioned frustration and struggle, as well as learning and better understanding of the scientific process. Bioinformatics projects are particularly well suited to the process of iteration and refinement because iterations can be performed quickly and are inexpensive in both time and money. Based on these findings, we suggest that a dynamic of "formative frustration" is an important aspect for a successful CURE.