A Polycomb domain found in committed cells impairs differentiation when introduced into PRC1 in pluripotent cells.

The CBX family of proteins is central to proper mammalian development via key roles in Polycomb-mediated maintenance of repression. CBX proteins in differentiated lineages have chromatin compaction and phase separation activities that might contribute to maintaining repressed chromatin. The predominant CBX protein in pluripotent cells, CBX7, lacks the domain required for these activities. We inserted this functional domain into CBX7 in embryonic stem cells (ESCs) to test the hypothesis that it contributes a key epigenetic function. ESCs expressing this chimeric CBX7 were impaired in their ability to properly form embryoid bodies and neural progenitor cells and showed reduced activation of lineage-specific genes across differentiation. Neural progenitors exhibited a corresponding inappropriate maintenance of Polycomb binding at neural-specific loci over the course of differentiation. We propose that a switch in the ability to compact and phase separate is a central aspect of Polycomb group function during the transition from pluripotency to differentiated lineages.

Polycomb Repressive Complex 2 Methylates Elongin A to Regulate Transcription.

Polycomb repressive complex 2 (PRC2-EZH2) methylates histone H3 at lysine 27 (H3K27) and is required to maintain gene repression during development. Misregulation of PRC2 is linked to a range of neoplastic malignancies, which is believed to involve methylation of H3K27. However, the full spectrum of non-histone substrates of PRC2 that might also contribute to PRC2 function is not known. We characterized the target recognition specificity of the PRC2 active site and used the resultant data to screen for uncharacterized potential targets. The RNA polymerase II (Pol II) transcription elongation factor, Elongin A (EloA), is methylated by PRC2 in vivo. Mutation of the methylated EloA residue decreased repression of a subset of PRC2 target genes as measured by both steady-state and nascent RNA levels and perturbed embryonic stem cell differentiation. We propose that PRC2 modulates transcription of a subset of low expression target genes in part via methylation of EloA.

The Ras-like GTPase Rem2 is a potent inhibitor of calcium/calmodulin-dependent kinase II activity.

Ca2+/calmodulin-dependent protein kinase II (CaMKII) is a well-characterized, abundant protein kinase that regulates a diverse set of functions in a tissue-specific manner. For example, in heart muscle, CaMKII regulates Ca2+ homeostasis, whereas in neurons, CaMKII regulates activity-dependent dendritic remodeling and long-term potentiation (LTP), a neurobiological correlate of learning and memory. Previously, we identified the GTPase Rem2 as a critical regulator of dendrite branching and homeostatic plasticity in the vertebrate nervous system. Here, we report that Rem2 directly interacts with CaMKII and potently inhibits the activity of the intact holoenzyme, a previously unknown Rem2 function. Our results suggest that Rem2 inhibition involves interaction with both the CaMKII hub domain and substrate recognition domain. Moreover, we found that Rem2-mediated inhibition of CaMKII regulates dendritic branching in cultured hippocampal neurons. Lastly, we report that substitution of two key amino acid residues in the Rem2 N terminus (Arg-79 and Arg-80) completely abolishes its ability to inhibit CaMKII. We propose that our biochemical findings will enable further studies unraveling the functional significance of Rem2 inhibition of CaMKII in cells.

Plasticity of the RNA kink turn structural motif.

The kink turn (K-turn) is an RNA structural motif found in many biologically significant RNAs. While most examples of the K-turn have a similar fold, the crystal structure of the Azoarcus group I intron revealed a novel RNA conformation, a reverse kink turn bent in the direction opposite that of a consensus K-turn. The reverse K-turn is bent toward the major grooves rather than the minor grooves of the flanking helices, yet the sequence differs from the K-turn consensus by only a single nucleotide. Here we demonstrate that the reverse bend direction is not solely defined by internal sequence elements, but is instead affected by structural elements external to the K-turn. It bends toward the major groove under the direction of a tetraloop-tetraloop receptor. The ability of one sequence to form two distinct structures demonstrates the inherent plasticity of the K-turn sequence. Such plasticity suggests that the K-turn is not a primary element in RNA folding, but instead is shaped by other structural elements within the RNA or ribonucleoprotein assembly.

Riboswitch effectors as protein enzyme cofactors.

The recently identified glmS ribozyme revealed that RNA enzymes, like protein enzymes, are capable of using small molecules as catalytic cofactors to promote chemical reactions. Flavin mononucleotide (FMN), S-adenosyl methionine (SAM), adenosyl cobalamin (AdoCbl), and thiamine pyrophosphate (TPP) are known ligands for RNA riboswitches in the control of gene expression, but are also catalytically powerful and ubiquitous cofactors in protein enzymes. If RNA, instead of just binding these molecules, could harness the chemical potential of the cofactor, it would significantly expand the enzymatic repertoire of ribozymes. Here we review the chemistry of AdoCbl, SAM, FMN, and TPP in protein enzymology and speculate on how these cofactors might have been used by ribozymes in the prebiotic RNA World or may still find application in modern biology.

Structural and chemical basis for glucosamine 6-phosphate binding and activation of the glmS ribozyme.

The glmS ribozyme is the first naturally occurring catalytic RNA that relies on an exogenous, nonnucleotide cofactor for reactivity. From a biochemical perspective, the glmS ribozyme derived from Bacillus anthracis is the best characterized. However, much of the structural work to date has been done on a variant glmS ribozyme, derived from Thermoanaerobacter tengcongensis. Here we present structures of the B. anthracis glmS ribozyme in states before the activating sugar, glucosamine 6-phosphate (GlcN6P), has bound and after the reaction has occurred. These structures show an active site preorganized to bind GlcN6P that retains some affinity for the sugar even after cleavage of the RNA backbone. A structure of an inactive glmS ribozyme with a mutation distal from the ligand-binding pocket highlights a nucleotide critical to the reaction that does not affect GlcN6P binding. Structures of the glmS ribozyme bound to a naturally occurring inhibitor, glucose 6-phosphate (Glc6P), and a nonnatural activating sugar, mannosamine 6-phosphate (MaN6P), reveal a binding mode similar to that of GlcN6P. Kinetic analyses show a pH dependence of ligand binding that is consistent with titration of the cofactor's phosphate group and support a model in which the major determinant of activity is the sugar amine independent of its stereochemical presentation.

Catalytic strategies of self-cleaving ribozymes.

[Structure: see text]. Five naturally occurring nucleolytic ribozymes have been identified: the hammerhead, hairpin, glmS, hepatitis delta virus (HDV), and Varkud satellite (VS) ribozymes. All of these RNA enzymes catalyze self-scission of the RNA backbone using a chemical mechanism equivalent to that of RNase A. RNase A uses four basic strategies to promote this reaction: geometric constraints, activation of the nucleophile, transition-state stabilization, and leaving group protonation. In this Account, we discuss the current thinking on how nucleolytic ribozymes harness RNase A's four sources of catalytic power. The geometry of the phosphodiester cleavage reaction constrains the nucleotides flanking the scissile phosphate so that they are unstacked from a canonical A-form helix and thus require alternative stabilization. Crystal structures and mutational analysis reveal that cross-strand base pairing, along with unconventional stacking and tertiary hydrogen-bonding interactions, work to stabilize the splayed conformation in nucleolytic ribozymes. Deprotonation of the 2'-OH nucleophile greatly increases its nucleophilicity in the strand scission reaction. Crystal structures of the hammerhead, hairpin, and glmS ribozymes reveal the N1 of a G residue within hydrogen-bonding distance of the 2'-OH. In each case, this residue has also been shown to be important for catalysis. In the HDV ribozyme, a hydrated magnesium has been implicated as the general base. Catalysis by the VS ribozyme requires both an A and a G, but the precise role of either has not been elucidated. Enzymes can lower the energy of a chemical reaction by binding more tightly to the transition state than to the ground states. Comparison of the hairpin ground- and transition-state mimic structures reveal greater hydrogen bonding to the transition-state mimic structure, suggesting transition-state stabilization as a possible catalytic strategy. However, the hydrogen-bonding pattern in the glmS ribozyme transition-state mimic structure and the ground-state structures are equivalent. Protonation of the 5'-O leaving group by a variety of functional groups can promote the cleavage reaction. In the HDV ribozyme, the general acid is a conserved C residue. In the hairpin ribozyme, a G residue has been implicated in protonation of the leaving group. An A in the hammerhead ribozyme probably plays a similar role. In the glmS ribozyme, an exogenous cofactor may provide the general acid. This diversity is in contrast to the relatively small number of functional groups that serve as a general base, where at least three of the nucleolytic ribozymes may use the N1 of a G.

Xist RNA antagonizes the SWI/SNF chromatin remodeler BRG1 on the inactive X chromosome.

The noncoding RNA Xist recruits silencing factors to the inactive X chromosome (Xi) and facilitates re-organization of Xi structure. Here, we examine the mouse epigenomic landscape of Xi and assess how Xist alters chromatin accessibility. Xist deletion triggers a gain of accessibility of select chromatin regions that is regulated by BRG1, an ATPase subunit of the SWI/SNF chromatin-remodeling complex. In vitro, RNA binding inhibits nucleosome-remodeling and ATPase activities of BRG1, while in cell culture Xist directly interacts with BRG1 and expels BRG1 from the Xi. Xist ablation leads to a selective return of BRG1 in cis, starting from pre-existing BRG1 sites that are free of Xist. BRG1 re-association correlates with cohesin binding and restoration of topologically associated domains (TADs) and results in the formation of de novo Xi 'superloops'. Thus, Xist binding inhibits BRG1's nucleosome-remodeling activity and results in expulsion of the SWI/SNF complex from the Xi.

RNA catalysis: ribozymes, ribosomes, and riboswitches.

The catalytic mechanisms employed by RNA are chemically more diverse than initially suspected. Divalent metal ions, nucleobases, ribosyl hydroxyl groups, and even functional groups on metabolic cofactors all contribute to the various strategies employed by RNA enzymes. This catalytic breadth raises intriguing evolutionary questions about how RNA lost its biological role in some cases, but not in others, and what catalytic roles RNA might still be playing in biology.

Structural investigation of the GlmS ribozyme bound to Its catalytic cofactor.

The GlmS riboswitch is located in the 5'-untranslated region of the gene encoding glucosamine-6-phosphate (GlcN6P) synthetase. The GlmS riboswitch is a ribozyme with activity triggered by binding of the metabolite GlcN6P. Presented here is the structure of the GlmS ribozyme (2.5 A resolution) with GlcN6P bound in the active site. The GlmS ribozyme adopts a compact double pseudoknot tertiary structure, with two closely packed helical stacks. Recognition of GlcN6P is achieved through coordination of the phosphate moiety by two hydrated magnesium ions as well as specific nucleobase contacts to the GlcN6P sugar ring. Comparison of this activator bound and the previously published apoenzyme complex supports a model in which GlcN6P does not induce a conformational change in the RNA, as is typical of other riboswitches, but instead functions as a catalytic cofactor for the reaction. This demonstrates that RNA, like protein enzymes, can employ the chemical diversity of small molecules to promote catalytic activity.

ATRX ADD domain links an atypical histone methylation recognition mechanism to human mental-retardation syndrome.

ATR-X (alpha-thalassemia/mental retardation, X-linked) syndrome is a human congenital disorder that causes severe intellectual disabilities. Mutations in the ATRX gene, which encodes an ATP-dependent chromatin-remodeler, are responsible for the syndrome. Approximately 50% of the missense mutations in affected persons are clustered in a cysteine-rich domain termed ADD (ATRX-DNMT3-DNMT3L, ADD(ATRX)), whose function has remained elusive. Here we identify ADD(ATRX) as a previously unknown histone H3-binding module, whose binding is promoted by lysine 9 trimethylation (H3K9me3) but inhibited by lysine 4 trimethylation (H3K4me3). The cocrystal structure of ADD(ATRX) bound to H3(1-15)K9me3 peptide reveals an atypical composite H3K9me3-binding pocket, which is distinct from the conventional trimethyllysine-binding aromatic cage. Notably, H3K9me3-pocket mutants and ATR-X syndrome mutants are defective in both H3K9me3 binding and localization at pericentromeric heterochromatin; thus, we have discovered a unique histone-recognition mechanism underlying the ATR-X etiology.

Consolidating critical binding determinants by noncyclic rearrangement of protein secondary structure.

We designed a single-chain variant of the Arc repressor homodimer in which the beta strands that contact operator DNA are connected by a hairpin turn and the alpha helices that form the tetrahelical scaffold of the dimer are attached by a short linker. The designed protein represents a noncyclic permutation of secondary structural elements in another single-chain Arc molecule (Arc-L1-Arc), in which the two subunits are fused by a single linker. The permuted protein binds operator DNA with nanomolar affinity, refolds on the sub-millisecond time scale, and is as stable as Arc-L1-Arc. The crystal structure of the permuted protein reveals an essentially wild-type fold, demonstrating that crucial folding information is not encoded in the wild-type order of secondary structure. Noncyclic rearrangement of secondary structure may allow grouping of critical active-site residues in other proteins and could be a useful tool for protein design and minimization.

Probing RNA structure and function by nucleotide analog interference mapping.

Nucleotide analog interference mapping (NAIM) can be used to simultaneously, yet individually, identify structurally or catalytically important functional groups within an RNA molecule. Phosphorothioate-tagged nucleotides and nucleotide analogs are randomly incorporated into an RNA of interest by in vitro transcription. The phosphorothioate tag marks the site of substitution and identifies sites at which the modification affects the structure or function of the RNA molecule. This technique has been expanded to include identification of hydrogen bonding pairs (NAIS), ionizable functional groups, metal ion ligands, and the energetics of protein binding (QNAIM). The analogs, techniques, and data analysis used in NAIM are described here.