Cell signaling that culminates in posttranslational modifications directs protein activity. Here we report how multiple Ca 2+ -dependent phosphorylation sites within the transcription activator Ets-1 act additively to produce graded DNA binding affinity. Nuclear magnetic resonance spectroscopic analyses show that phosphorylation shifts Ets-1 from a dynamic conformation poised to bind DNA to a well-folded inhibited state. These phosphates lie in an unstructured flexible region that functions as the allosteric effector of autoinhibition. Variable phosphorylation thus serves as a “rheostat” for cell signaling to fine-tune transcription at the level of DNA binding.

Membraneless organelles like stress granules are active liquid-liquid phase-separated droplets that are involved in many intracellular processes. Their active and dynamic behavior is often regulated by ATP-dependent reactions. However, how exactly membraneless organelles control their dynamic composition remains poorly understood. Herein, we present a model for membraneless organelles based on RNA-containing active coacervate droplets regulated by a fuel-driven reaction cycle. These droplets emerge when fuel is present, but decay without. Moreover, we find these droplets can transiently up-concentrate functional RNA which remains in its active folded state inside the droplets. Finally, we show that in their pathway towards decay, these droplets break apart in multiple droplet fragments. Emergence, decay, rapid exchange of building blocks, and functionality are all hallmarks of membrane-less organelles, and we believe that our work could be powerful as a model to study such organelles.

IntroductionThe Isodon plant, Rabdosia rubescens (RR), and its extracts, were shown in China to be able to suppress disease progress, reduce tumor burden, alleviate syndrome, and prolong survival in patients with esophageal, gastric carcinoma or liver cancer. [1][2][3][4][5] Of interest, other Isodon plants including Isodon japonicus Hara (IJ) and I trichocarpus (IT) were also applied as home remedies for similar disorders in Japan and Korea. 6 Oridonin ( Figure 1A), a bitter tetracycline diterpenoid compound, was isolated from RR, IJ, and IT separately, 7,8 suggesting oridonin should be an essential antitumor component of Isodon plants. Studies showed that oridonin induced apoptosis in a variety of cancer cells including those from prostate, breast, non-small cell lung cancers, acute leukemia (NB4, HL-60 cells), glioblastoma multiforme, and human melanoma cells. [9][10][11][12] Oridonin could also increase lifespan of mice bearing Ehrlich ascites or P388 lymphocytic leukemia. 13,14 However, though studies showed that caspase-3 (casp-3), casp-8, P53, Bcl-2/Bax, cytochrome c (cyt C), 10,15,16 and nuclear factor kappa B (NFB) 17 were involved in apoptosis induced by oridonin, mechanisms underlying the antitumor activity of oridonin remain largely unknown, and whether oridonin can find clinical application still needs more investigation.Genetic abnormalities have been shown to play a key role in leukemogenesis, 18 and treatment strategies interfering with oncoproteins involved in leukemia pathogenesis have been reported to have high therapeutic efficacy with low adverse effects. The BCR-ABL-targeting STI-571, in the treatment of chronic myeloid leukemia (CML), 19 and the PML-RAR␣-targeting agents all-trans retinoic acid (ATRA) and arsenic trioxide (ATO), in taming acute promyelocytic leukemia (APL), 20,21 can serve as paradigms. Hence molecular target-based therapies should be developed for leukemias with poor prognosis. The AML1-ETO (AE) fusion gene is the result of translocation t(8;21)(q22;q22), which is seen in 40% to 80% of M2-type acute myeloid leukemia (AML M2) and 12% to 20% of all cases of AML. 22,23 The AE fusion protein recruits the nuclear receptor corepressor (NCoR)-mammalian Sin3 (mSin3)-histone deacetylase (HDAC) complex, 24,25 inhibits transcription of AML1 target genes 22,24 including interleukin-3 (IL3), 26 activates transcription of apoptotic antagonist Bcl-2, 27 up-regulates protein tyrosine kinase C-KIT, 28 induces the expression of granulocyte colony-stimulating factor receptor (G-CSFR) as well as myeloperoxidase (MPO), 29 and blocks transactivation of the GM-CSF promoter. 30 The AE oncoprotein enhances self-renewal of hematopoietic stem/progenitor cells, blocks hematopoietic differentiation, disturbs normal cell proliferation, 31 and immortalizes murine hematopoietic progenitors. 32,33 Although reports suggest that additional mutations are required to cooperate with AE to cause murine The online version of this article contains a data supplement.The publication costs of this article were defr...

The transcription factor Ets-1 is regulated by the allosteric coupling of DNA binding with the unfolding of an ␣-helix (HI-1) within an autoinhibitory module. To understand the structural and dynamic basis for this autoinhibition, we have used NMR spectroscopy to characterize Ets-1⌬N301, a partially inhibited fragment of Ets-1. The NMR-derived Ets-1⌬N301 structure reveals that the autoinhibitory module is formed predominantly by the hydrophobic packing of helices from the N-terminal (HI-1, HI-2) and C-terminal (H4, H5) inhibitory sequences, along with H1 of the intervening DNA binding ETS domain. The intramolecular interactions made by HI-1 in Ets-1⌬N301 are similar to the intermolecular contacts observed in the crystal structure of an Ets-1⌬N300 dimer, confirming that the latter represents a domain-swapped species.15 N relaxation studies demonstrate that the backbone of the N-terminal inhibitory sequence is mobile on the nanosecond-picosecond and millisecond-microsecond time scales. Furthermore, hydrogen exchange measurements reveal that amide protons in helices HI-1 and HI-2 exchange with water at rates only ϳ15-and ϳ75-fold slower, respectively, than predicted for an unfolded polypeptide. These findings indicate that inhibitory helices are only marginally stable even in the absence of DNA. The energetic coupling of DNA binding with the facile unfolding of the labile HI-1 provides a mechanism for modulating Ets-1 DNA binding activity via protein partnerships, post-translational modifications, or mutations. Ets-1 autoinhibition illustrates how conformational equilibria within structural domains can regulate macromolecular interactions.Gene expression can be controlled by modulating the DNA binding affinity of sequence specific transcription factors. Similar to several other transcription factors, the DNA binding of Ets-1 is modulated by an autoinhibitory module that provides a route to biological regulation (1). The Ets-1 inhibitory module is composed of sequences flanking the winged helix-turn-helix (HTH) 1 DNA binding ETS domain (2, 3). When these sequences are deleted, as in an alternatively spliced isoform of Ets-1, or when their structural elements are disrupted by mutations, as in the case of the oncogenic v-Ets, the affinity of Ets-1 for its target DNA sites is enhanced by 10-to 20-fold (4 -6). In a cellular context, this module is essential for response to different regulatory signals. DNA binding of Ets-1 is enhanced 10-to 20-fold through a partnership with the transcription factor RUNX1 (CBF␣2/AML1) (7). Conversely, in activated T-cells, phosphorylation of a serine-rich region (residues 244 -300) inhibits the DNA binding of Ets-1 by another ϳ50-fold (8). Importantly, these two effects require an intact inhibitory module.Mechanistic insight into autoinhibition has come from the observation that the Ets-1 inhibitory module changes conformation upon binding to DNA. Initial secondary structural studies performed in our laboratories demonstrated that this module is composed of four coupled ␣-helices, locat...

Mammalian mesenchymal stem cells (MSCs) have been shown to be strongly immunosuppressive in both animal disease models and human clinical trials. We have reported that the key molecule mediating immunosuppression by MSCs is species dependent: indoleamine 2,3-dioxygenase (IDO) in human and inducible nitric oxide synthase (iNOS) in mouse. In the present study, we isolated MSCs from several mammalian species, each of a different genus, and investigated the involvement of IDO and iNOS during MSC-mediated immunosuppression. The characterization of MSCs from different species was by adherence to tissue culture plastic, morphology, specific marker expression, and differentiation potential. On the basis of the inducibility of IDO and iNOS by inflammatory cytokines in MSCs, the tested mammalian species fall into two distinct groups: IDO utilizers and iNOS utilizers. MSCs from monkey, pig, and human employ IDO to suppress immune responses, whereas MSCs from mouse, rat, rabbit, and hamster utilize iNOS. Interestingly, based on the limited number of species tested, the iNOS-utilizing species all belong to the phylogenetic clade, Glires. Although the evolutionary significance of this divergence is not known, we believe that this study provides critical guidance for choosing appropriate animal models for preclinical studies of MSCs.

Sam68 and T-STAR are members of the STAR family of proteins that directly link signal transduction with post-transcriptional gene regulation. Sam68 controls the alternative splicing of many oncogenic proteins. T-STAR is a tissue-specific paralogue that regulates the alternative splicing of neuronal pre-mRNAs. STAR proteins differ from most splicing factors, in that they contain a single RNA-binding domain. Their specificity of RNA recognition is thought to arise from their property to homodimerize, but how dimerization influences their function remains unknown. Here, we establish at atomic resolution how T-STAR and Sam68 bind to RNA, revealing an unexpected mode of dimerization different from other members of the STAR family. We further demonstrate that this unique dimerization interface is crucial for their biological activity in splicing regulation, and suggest that the increased RNA affinity through dimer formation is a crucial parameter enabling these proteins to select their functional targets within the transcriptome.

Summary Binding of the transcription factor Ets-1 to DNA is allosterically regulated by a serine rich region (SRR) that modulates the dynamic character of the adjacent structured DNA-binding ETS domain and its flanking autoinhibitory elements. Multi-site phosphorylation of the flexible SRR in response to Ca+2 signaling mediates variable regulation of Ets-1 DNA-binding affinity. In this study, we further investigated the mechanism of this regulation. First, thermal and urea denaturation experiments demonstrated that phosphorylation of the predominantly unstructured SRR imparts enhanced thermodynamic stability on the well-folded ETS domain and its inhibitory module. We next identified a minimal fragment (residues 279–440) that exhibits both enhanced autoinhibition of Ets-1 DNA-binding and allosteric reinforcement by phosphorylation. To test for intramolecular interactions between the SRR and the rest of the fragment that were not detectable by 1H-1H NOE measurements, paramagnetic relaxation enhancements were performed using a Cu2+ bound to the N-terminal ATCUN motif. Increased relaxation detected for specific amides and methyls revealed a preferential interaction surface for the flexible SRR extending from the inhibitory module to the DNA binding interface. Phosphorylation enhanced the localization of the SRR to this surface. We therefore hypothesize that the positioning of the SRR at the DNA binding interface and its role in shifting Ets-1 to an inhibited conformation are linked. In particular, transient interactions dampen the conformational flexibility of the ETS domain and inhibitory module required for high affinity binding, as well as possibly occlude the DNA interaction site. Surprisingly, the phosphorylation-dependent effects were relatively insensitive to changes in ionic strength, suggesting that electrostatic forces are not the dominant mechanism for mediating these interactions. The results of this study highlight the role of flexibility and transient binding in the variable regulation of Ets-1 activity.

Ras/MAPK signaling is often aberrantly activated in human cancers. The downstream effectors are transcription factors, including those encoded by the ETS gene family. Using cell-based assays and biophysical measurements, we have determined the mechanism by which Ras/MAPK signaling affects the function of Ets1 via phosphorylation of Thr38 and Ser41. These ERK2 phosphoacceptors lie within the unstructured N-terminal region of Ets1, immediately adjacent to the PNT domain. NMR spectroscopic analyses demonstrated that the PNT domain is a four-helix bundle (H2-H5), resembling the SAM domain, appended with two additional helices (H0-H1). Phosphorylation shifted a conformational equilibrium, displacing the dynamic helix H0 from the core bundle. The affinity of Ets1 for the TAZ1 (or CH1) domain of the coactivator CBP was enhanced 34-fold by phosphorylation, and this binding was sensitive to ionic strength. NMR-monitored titration experiments mapped the interaction surfaces of the TAZ1 domain and Ets1, the latter encompassing both the phosphoacceptors and PNT domain. Charge complementarity of these surfaces indicate that electrostatic forces act in concert with a conformational equilibrium to mediate phosphorylation effects. We conclude that the dynamic helical elements of Ets1, appended to a conserved structural core, constitute a phospho-switch that directs Ras/MAPK signaling to downstream changes in gene expression. This detailed structural and mechanistic information will guide strategies for targeting ETS proteins in human disease.MAP kinase | protein structure/dynamics | transcriptional regulation | protein-protein interaction | Ets2