RIG-I is a cytosolic helicase that senses 5’-ppp-RNA contained in negative strand RNA viruses and triggers innate antiviral immune responses. Calorimetric binding studies establish that the RIG-I C-terminal regulatory domain (CTD) binds to blunt-end double-stranded 5’-ppp-RNA a factor of 17 more tightly than to its single-stranded counterpart. Here we report on the crystal structure of RIG-I CTD domain bound to both blunt-ends of a self-complementary 5’-ppp-dsRNA 12-mer, with interactions involving 5’-pp clearly visible in the complex. The structure, supported by mutation studies, defines how a lysine-rich basic cleft within the RIG-I CTD domain sequesters the observable 5’-pp of the bound RNA, with a stacked Phe capping the terminal base pair. Key intermolecular interactions observed in the crystalline state are retained in the complex of 5’-ppp-dsRNA 24-mer and full-length RIG-I under in vivo conditions, as evaluated from the impact of binding pocket RIG-I mutations and 2’-OCH3 RNA modifications on the interferon response.

Human L3MBTL1, which contains three malignant brain tumor (MBT) repeats, binds monomethylated and dimethylated lysines, but not trimethylated lysines, in several histone sequence contexts. In crystal structures of L3MBTL1 complexes, the monomethyl- and dimethyllysines insert into a narrow and deep cavity of aromatic residue-lined pocket 2, while a proline ring inserts into shallower pocket 1. We have also engineered a single Y to E substitution within the aromatic cage of the BPTF PHD finger, resulting in a reversal of binding preference from trimethyl- to dimethyllysine in an H3K4 sequence context. In both the "cavity insertion" (L3MBTL1) and "surface groove" (PHD finger) modes of methyllysine recognition, a carboxylate group both hydrogen bonds and ion pairs to the methylammonium proton. Our structural and binding studies of these two modules provide insights into the molecular principles governing the decoding of lysine methylation states, thereby highlighting a methylation state-specific layer of histone mark readout impacting on epigenetic regulation.

The histone variant macroH2A generally associates with transcriptionally inert chromatin; however, the factors that regulate its chromatin incorporation remain elusive. Here, we identify the SWI/SNF helicase ATRX (a-thalassemia/ MR, X-linked) as a novel macroH2A-interacting protein.Unlike its role in assisting H3.3 chromatin deposition, ATRX acts as a negative regulator of macroH2A's chromatin association. In human erythroleukemic cells deficient for ATRX, macroH2A accumulates at the HBA gene cluster on the subtelomere of chromosome 16, coinciding with the loss of a-globin expression. Collectively, our results implicate deregulation of macroH2A's distribution as a contributing factor to the a-thalassemia phenotype of ATRX syndrome. The replacement of canonical histones with histone variants contributes to the dynamic nature of chromatin.Due to amino acid differences and, in turn, unique posttranslational modifications, histone variants can alter nucleosome structure, stability, and binding of effector proteins. Histone variants have unique genomic localization patterns, and thus specialized roles such as regulating gene expression or chromosome segregation during cell division . Therefore, the differential genomic incorporation of histone variants directly impacts critical cellular functions.The histone variant macroH2A (mH2A) is a vertebratespecific member of the H2A family and is unusual due to the presence of a C-terminal macro domain (Pehrson and Fried 1992). Two different genes encode mH2A1 and mH2A2 (H2AFY and H2AFY2, respectively), and two splice forms of mH2A1 exist: mH2A1.1 and mH2A1.2 (Costanzi and Pehrson 2001). mH2A is abundant in heterochromatin, including senescence-associated heterochromatic foci (SAHF) and the inactivated X chromosome (Xi) (Costanzi and Pehrson 1998;Zhang et al. 2005). In vitro studies suggest that the macro domain sterically hinders access of transcription factors to DNA, while mH2A's L1 loop produces inflexible nucleosomes (Angelov et al. 2003;Chakravarthy et al. 2005).Our group has recently demonstrated a role for mH2A isoforms in suppressing melanoma progression, and others have linked mH2A expression or its splice patterns to breast and lung cancer (Sporn et al. 2009;Kapoor et al. 2010;Novikov et al. 2011). However, the factors that regulate the association of mH2A with chromatin remain obscure. Therefore, identifying regulators of the incorporation of histone variants at distinct genomic loci is key to understanding how chromatin domains are established and maintained and how these may go awry in disease.A second group of factors contributing to chromatin dynamics are ATP-dependent chromatin remodeling complexes that rearrange or mobilize nucleosomes. Deregulation of members of the SWI/SNF family is implicated in various cancers and mental retardation (MR) syndromes, including ATRX (a-thalassemia/MR, X-linked), (Wilson and Roberts 2011). Mutations in ATRX, predominantly found in the H3K9me3-binding ADD (ATRX-DNMT3-DNMT3L) and/or helicase domains, are associated with ATRX sy...

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Berberine, an isoquinoline alkaloid, is a traditional oriental medicine used to treat diarrhea and gastroenteritis. Recently, we reported that it could inhibit the growth of intestinal polyp in animals and in patients with the familial adenomatous polyposis by downregulating β-catenin signaling. However, the intracellular target mediating the effects of berberine remains elusive. Here, we provide evidence that berberine inhibits β-catenin function via directly binding to a unique region comprising residues Gln275, Arg316 and Arg371 in nuclear receptor retinoid X receptor alpha (RXRα), where berberine concomitantly binding to and synergistically activating RXRα with 9-cis-retinoic acid (9-cis-RA), a natural ligand binding to the classical ligand-binding pocket of RXRα. Berberine binding promotes RXRα interaction with nuclear β-catenin, leading to c-Cbl mediated degradation of β-catenin, and consequently inhibits the proliferation of colon cancer cells. Furthermore, berberine suppresses the growth of human colon carcinoma xenograft in nude mice in an RXRα-dependent manner. Together, our study not only identifies RXRα as a direct protein target for berberine but also dissects their binding mode and validates that berberine indeed suppresses β-catenin signaling and cell growth in colon cancer via binding RXRα, which provide new strategies for the design of new RXRα-based antitumor agents and drug combinations.