Development and repair of the vertebrate skeleton requires the precise coordination of bone-forming osteoblasts and bone-resorbing osteoclasts. In diseases such as osteoporosis, bone resorption dominates over bone formation, suggesting a failure to harmonize osteoclast and osteoblast function. Here, we show that mice expressing a constitutively nuclear NFATc1 variant (NFATc1(nuc)) in osteoblasts develop high bone mass. NFATc1(nuc) mice have massive osteoblast overgrowth, enhanced osteoblast proliferation, and coordinated changes in the expression of Wnt signaling components. In contrast, viable NFATc1-deficient mice have defects in skull bone formation in addition to impaired osteoclast development. NFATc1(nuc) mice have increased osteoclastogenesis despite normal levels of RANKL and OPG, indicating that an additional NFAT-regulated mechanism influences osteoclastogenesis in vivo. Calcineurin/NFATc signaling in osteoblasts controls the expression of chemoattractants that attract monocytic osteoclast precursors, thereby coupling bone formation and bone resorption. Our results indicate that NFATc1 regulates bone mass by functioning in both osteoblasts and osteoclasts.

Jasmonoyl-isoleucine (JA-Ile), the active form of the plant hormone jasmonate (JA), is sensed by the F-box protein CORONATINE INSENSITIVE 1 (COI1), a component of a functional Skp-Cullin-F-box E3 ubiquitin ligase complex. Sensing of JA-Ile by COI1 rapidly triggers genome-wide transcriptional changes that are largely regulated by the basic helix-loop-helix transcription factor MYC2. However, it remains unclear how the JA-Ile receptor protein COI1 relays hormone-specific regulatory signals to the RNA polymerase II general transcriptional machinery. Here, we report that the plant transcriptional coactivator complex Mediator directly links COI1 to the promoters of MYC2 target genes. MED25, a subunit of the Mediator complex, brings COI1 to MYC2 target promoters and facilitates COI1-dependent degradation of jasmonate-ZIM domain (JAZ) transcriptional repressors. MED25 and COI1 influence each other's enrichment on MYC2 target promoters. Furthermore, MED25 physically and functionally interacts with HISTONE ACETYLTRANSFERASE1 (HAC1), which plays an important role in JA signaling by selectively regulating histone (H) 3 lysine (K) 9 (H3K9) acetylation of MYC2 target promoters. Moreover, the enrichment and function of HAC1 on MYC2 target promoters depend on COI1 and MED25. Therefore, the MED25 interface of Mediator links COI1 with HAC1-dependent H3K9 acetylation to activate MYC2-regulated transcription of JAresponsive genes. This study exemplifies how a single Mediator subunit integrates the actions of both genetic and epigenetic regulators into a concerted transcriptional program.is an oxylipin-derived plant hormone that regulates diverse aspects of plant immunity and development (1, 2). Decades of studies in the model plant Arabidopsis thaliana have revealed a core JA signaling module consisting of the F-box protein CORONATINE INSENSITIVE 1 (COI1) (3), a group of jasmonate-ZIM domain (JAZ) proteins (4-6), and the basic helixloop-helix transcription factor MYC2 (7, 8). COI1 forms a functional Skp-Cullin-F-box (SCF) E3 ubiquitin ligase SCF COI1 along with Cullin1 and Skp1-like1 (ASK1) (9, 10), MYC2 acts as a master transcription factor that differentially regulates diverse aspects of JA responses (11-13), and the JAZ proteins are substrates of SCF COI1 and serve as transcriptional repressors of MYC2 (4, 5, 14). The identification of jasmonoyl-isoleucine (JA-Ile) as the receptor-active form of the hormone, along with the discovery that sensing of JA-Ile involves formation of the SCF COI1 -JAZs coreceptor complex (4, 15-17), represented a breakthrough in our mechanistic understanding of JA signaling. In the absence of the hormone, JAZ repressors interact with and repress the activity of MYC2. In response to internal or external cues that trigger JAIle synthesis, elevated JA-Ile levels promote SCF COI1 -dependent degradation of JAZ repressors, and thereby activate (de-repress) the MYC2-directed transcriptional program. These discoveries imply that sensing of the active hormone is tightly linked to transcription of JA-responsive...

In tomato (Solanum lycopersicum), as in other plants, the immunity hormone jasmonate (JA) triggers genome-wide transcriptional changes in response to pathogen and insect attack. These changes are largely regulated by the basic helix-loop-helix (bHLH) transcription factor MYC2. The function of MYC2 depends on its physical interaction with the MED25 subunit of the Mediator transcriptional coactivator complex. Although much has been learned about the MYC2-dependent transcriptional activation of JA-responsive genes, relatively less studied is the termination of JA-mediated transcriptional responses and the underlying mechanisms. Here, we report an unexpected function of MYC2 in regulating the termination of JA signaling through activating a small group of JA-inducible bHLH proteins, termed MYC2-TARGETED BHLH1 (MTB1), MTB2, and MTB3. MTB proteins negatively regulate JA-mediated transcriptional responses via their antagonistic effects on the functionality of the MYC2-MED25 transcriptional activation complex. MTB proteins impair the formation of the MYC2-MED25 complex and compete with MYC2 to bind to its target gene promoters. Therefore, MYC2 and MTB proteins form an autoregulatory negative feedback circuit to terminate JA signaling in a highly organized manner. We provide examples demonstrating that gene editing tools such as CRISPR/Cas9 open up new avenues to exploit MTB genes for crop protection.

Histone H3-lysine79 (H3K79) methyl transferase DOT1L plays critical roles in normal cell differentiation as well as initiation of acute leukemia. We used structure and mechanism based design to discover several potent inhibitors of DOT1L with IC50 values as low as 38 nM. These inhibitors exhibit only weak or no activities against four other representative histone lysine and arginine methyltransferases, G9a, SUV39H1, PRMT1 and CARM1. The x-ray crystal structure of a DOT1L:inhibitor complex reveals that N6-methyl group of the inhibitor, located favorably in a predominantly hydrophobic cavity of DOT1L, provides the observed high selectivity. Structural analysis shows that it will disrupt at least one H-bond and/or have steric repulsion for other histone methyltransferases. These compounds represent novel chemical probes for biological function studies of DOT1L in health and disease.

Dietary anthocyanins are important health-promoting antioxidants that make a major contribution to the quality of fruits. It is intriguing that most tomato cultivars do not produce anthocyanins in fruit. However, the purple tomato variety Indigo Rose, which has the dominant Aft locus combined with the recessive atv locus from wild tomato species, exhibits light-dependent anthocyanin accumulation in the fruit skin.Here, we report that Aft encodes a functional anthocyanin activator named SlAN2-like, while atv encodes a nonfunctional version of the anthocyanin repressor SlMYBATV. The expression of SlAN2-like is responsive to light, and the functional SlAN2-like can activate the expression of both anthocyanin biosynthetic genes and their regulatory genes, suggesting that SlAN2-like acts as a master regulator in the activation of anthocyanin biosynthesis. We further showed that cultivated tomatoes contain nonfunctional alleles of SlAN2-like and therefore fail to produce anthocyanins. Consistently, expression of a functional SlAN2like gene driven by the fruit-specific promoter in a tomato cultivar led to the activation of the entire anthocyanin biosynthesis pathway and high-level accumulation of anthocyanins in both the peel and flesh. Taken together, our study exemplifies that efficient engineering of complex metabolic pathways could be achieved through tissue-specific expression of master transcriptional regulators.

box genes encode a highly conserved gene family of transcriptional factors that regulate numerous developmental processes in plants. In this study, a tomato (Solanum lycopersicum) MADS-box gene, SlMADS1, was cloned and its tissue-specific expression profile was analyzed. The real-time polymerase chain reaction results showed that SlMADS1 was highly expressed in sepals and fruits; its expression level was increased with the development of sepals, while the transcript of SlMADS1 decreased significantly in accordance with fruit ripening. To further explore the function of SlMADS1, an RNA interference (RNAi) expression vector targeting SlMADS1 was constructed and transformed into tomato plants. Shorter ripening time of fruit was observed in SlMADS1-silenced tomatoes. The accumulation of carotenoid and the expression of PHYTOENE SYNTHETASE1 were enhanced in RNAi fruits. Besides, ethylene biosynthetic genes, including 1-AMINOCYCLOPROPANE-1-CARBOXYLATE SYNTHASE1A, 1-AMINOCYCLOPROPANE-1-CARBOXYLATE SYNTHASE6, 1-AMINOCYCLOPROPANE-1-CARBOXYLATE OXIDASE1, and 1-AMINOCYCLOPROPANE-1-CARBOXYLATE OXIDASE3, and the ethylene-responsive genes E4 and E8, which were involved in fruit ripening, were also up-regulated in silenced plants. SlMADS1 RNAi fruits showed approximately 2-to 4-fold increases in ethylene production compared with the wild type. Furthermore, SlMADS1-silenced seedlings displayed shorter hypocotyls and were more sensitive to 1-aminocyclopropane-1-carboxylate than the wild type. Additionally, a yeast two-hybrid assay revealed a clear interaction between SlMADS1 and SlMADS-RIN. These results suggest that SlMADS1 plays an important role in fruit ripening as a repressive modulator.

Histone3-lysine79 (H3K79) methyltransferase DOT1L has been found to be a drug target for acute leukemia with MLL (mixed lineage leukemia) gene translocations. A total of 55 adenosine-containing compounds were designed and synthesized, among which several potent DOT1L inhibitors were identified with Ki values as low as 0.5 nM. These compounds also show high selectivity (>4,500-fold) over three other histone methyltransferases. Structure activity relationships (SAR) of these compounds for their inhibitory activities against DOT1L are discussed. Potent DOT1L inhibitors exhibit selective activity against the proliferation of MLL-translocated leukemia cell lines MV4;11 and THP1 with EC50 values of 4–11 μM. Isothermal titration calorimetry studies showed two representative inhibitors bind with a high affinity to the DOT1L:nucleosome complex, and only compete with the enzyme cofactor SAM (S-adenosyl-L-methionine), but not the substrate nucleosome.

cNorwalk virus (NV), the prototype human calicivirus, is the leading cause of nonbacterial acute gastroenteritis. The NV protease cleaves the polyprotein encoded by open reading frame 1 of the viral genome at five nonhomologous sites, releasing six nonstructural proteins that are essential for viral replication. The structural details of how NV protease recognizes multiple substrates are unclear. In our X-ray structure of an NV protease construct, we observed that the C-terminal tail, representing the native substrate positions P5 to P1, is inserted into the active site cleft of the neighboring protease molecule, providing atomic details of how NV protease recognizes a substrate. The crystallographic structure of NV protease with the C-terminal tail redesigned to mimic P4 to P1 of another substrate site provided further structural details on how the active site accommodates sequence variations in the substrates. Based on these structural analyses, substrate-based aldehyde inhibitors were synthesized and screened for inhibition potency. Crystallographic structures of the protease in complex with each of the three most potent inhibitors were determined. These structures showed concerted conformational changes in the S4 and S2 pockets of the protease to accommodate variations in the P4 and P2 residues of the substrate/inhibitor, which could be a mechanism for how the NV protease recognizes multiple sites in the polyprotein with differential affinities during virus replication. These structures further indicate that the mechanism of inhibition by these inhibitors involves covalent bond formation with the side chain of the conserved cysteine in the active site by nucleophilic addition, and such substrate-based aldehydes could be effective protease inhibitors.