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/SUMMARYProtein-protein interactions (PPIs) form the underpinnings of any cellular signaling network. PPIs are highly dynamic processes and often, cell-based assays can be essential for their study as they closely mimic the biological intricacies of cellular environments. Since no sole platform can perform all needed experiments to gain a thoroughly comprehensive understanding into these processes, developing a versatile toolkit is much needed to address this longstanding gap. The use of small peptide tags, such as the V5-tag, has been extensively used in biological and biomedical research, including labeling the C-termini of one of the largest human genome-wide open-reading frame collections. However, these small peptide tags have been primarily usedin vitroand lack thein vivotraceability and functionality of larger specialized tags. In this study, we combined structural studies and computer-aided maturation to generate an intracellular nanobody, interacting with the V5-tag. Suitable for assays commonly used to study protein-protein interactions, our nanobody has been applied herein to interrogate G protein-coupled receptor signalling. This novel serviceable intrabody is the cornerstone of a multipurpose intracellular nanobody-based biosensors toolkit, named iBodyV5, which will be available for the scientific community at large.

The tail of replication-dependent histone H3.1 varies from that of replication-independent H3.3 at the amino acid located at position 31 in plants and animals, but no function has been assigned to this residue to demonstrate a unique and conserved role for H3.1 during replication. We found that TONSOKU (TSK/TONSL), which rescues broken replication forks, specifically interacts with H3.1 via recognition of alanine 31 by its tetratricopeptide repeat domain. Our results indicate that genomic instability in the absence of ATXR5/ATXR6-catalyzed histone H3 lysine 27 monomethylation in plants depends on H3.1, TSK, and DNA polymerase theta (Pol θ). This work reveals an H3.1-specific function during replication and a common strategy used in multicellular eukaryotes for regulating post-replicative chromatin maturation and TSK, which relies on histone monomethyltransferases and reading of the H3.1 variant.

The ATP-binding cassette (ABC) sterol transporters are responsible for maintaining cholesterol homeostasis in mammals by participating in reverse cholesterol transport (RCT) or transintestinal cholesterol efflux (TICE). The heterodimeric ABCG5/G8 carries out selective sterol excretion, preventing the abnormal accumulation of plant sterols in human bodies, while homodimeric ABCG1 contributes to the biogenesis and metabolism of high-density lipoproteins. A sterol-binding site on ABCG5/G8 was proposed at the interface of the transmembrane domain and the core of lipid bilayers. In this study, we have determined the crystal structure of ABCG5/G8 in a cholesterol-bound state. The structure combined with amino acid sequence analysis shows that in the proximity of the sterol-binding site, a highly conserved phenylalanine array supports functional implications for ABCG cholesterol/sterol transporters. Lastly, in silico docking analysis of cholesterol and stigmasterol (a plant sterol) suggests sterol-binding selectivity on ABCG5/G8, but not ABCG1. Together, our results provide a structural basis for cholesterol binding on ABCG5/G8 and the sterol selectivity by ABCG transporters.

Ribonuclease 6 (RNase 6) is one of eight catalytically active human pancreatic-type RNases that belong to a superfamily of rapidly evolving enzymes. Like some of its human homologs, RNase 6 exhibits host defense properties such as antiviral and antibacterial activities. Recently solved crystal structures of this enzyme in its nucleotide-free form show conservation of the prototypical kidney-shaped fold preserved among vertebrate RNases, in addition to revealing the presence of a unique secondary active site. In this study, we determine the structural and conformational properties experienced by RNase 6 upon binding to substrate and product analogs. We present the first crystal structures of RNase 6 bound to a nucleotide ligand (adenosine 5’-monophosphate), in addition to RNase 6 bound to phosphate ions. While the enzyme preserves B(2) subsite ligand preferences, our results show lack of typical B(2) subsite interactions normally observed in homologous ligand-bound RNases. Comparison of the dynamical properties of RNase 6 in its apo, substrate-, and product-bound states highlight unique dynamical properties experienced on time scales ranging from nano- to milliseconds. Overall, our results confirm the specific evolutionary adaptation of RNase 6 relative to its unique catalytic and biological activities.

COMPlex ASsociating with SET1 (COMPASS) is a histone H3 Lys-4 methyltransferase that typically marks the promoter region of actively transcribed genes. COMPASS is a multi-subunit complex in which the catalytic unit, SET1, is required for H3K4 methylation. An important subunit known to regulate SET1 methyltransferase activity is the CxxC zinc finger protein 1 (Cfp1). Cfp1 binds to COMPASS and is critical to maintain high level of H3K4me3 in cells but the mechanisms underlying its stimulatory activity is poorly understood. In this study, we show that Cfp1 only modestly activates COMPASS methyltransferase activity in vitro. Binding of Cfp1 to COMPASS is in part mediated by a new type of monovalent zinc finger (ZnF). This ZnF interacts with the COMPASS’s subunits RbBP5 and disruption of this interaction blunts its methyltransferase activity in cells and in vivo. Collectively, our studies reveal that a novel form of ZnF on Cfp1 enables its integration into COMPASS and contributes to epigenetic signaling.

Crohn's disease (CD) is characterized by the chronic inflammation of the gastrointestinal tract. A dysbiotic microbiome and a defective immune system are linked to CD, where hydrogen sulfide (H

CXXC Zinc finger protein 1 (CFP1) is a multitasking protein playing essential roles during various developmental processes. Its ability to interact with several proteins contribute to several epigenetic events. Here, we review CFP1's functions and its impact on DNA methylation and the post-translational modification of histone proteins such as lysine acetylation and methylation. We will also discuss the potential role of CFP1 in carcinogenesis and the impact of the mutations identified in patients suffering from various cancers.

The proliferating cell nuclear antigen (PCNA) is a sliding clamp associated with DNA polymerases and serves as a binding platform for the recruitment of regulatory proteins linked to DNA damage repair, cell cycle regulation, and epigenetic signaling. The histone H3 lysine-27 (H3K27) mono-methyltransferase Arabidopsis trithorax-related protein 5/6 (ATXR5/6) associates with PCNA, and this interaction has been proposed to act as a key determinant controlling the reestablishment of H3K27 mono-methylation following replication. In this study, we provide biochemical evidence showing that PCNA inhibits ATXR6 enzymatic activity. The structure of the ATXR6 PCNA-interacting peptide (PIP) in complex with PCNA indicates that a trio of hydrophobic residues contributes to the binding of the enzyme to the sliding clamp. Finally, despite the presence of three PIP binding clefts, only two molecules of ATXR6 bind to PCNA likely enabling the recruitment of a third protein to the sliding clamp. Collectively, these results rule out the model wherein PCNA-bound ATXR6 actively reestablishes H3K27 mono-methylation following DNA replication and provides insights into the role of ATXR6 PIP motif in its interaction with PCNA.

The post-translational modifications (PTMs) of histone proteins play essential functions in shaping the chromatin environment. Alone or in combination, these PTMs create templates recognized by dedicated proteins or change the chemistry of chromatin, enabling a myriad of nuclear processes to occur. Referred to as cross-talk, the positive or negative impact of a PTM on another post-translational modification has rapidly emerged as a mechanism controlling nuclear transactions. One of those includes the stimulatory functions of histone H2B ubiquitylation on the methylation of histone H3 on K79 and K4 by Dot1L and COMPASS, respectively. While these findings were established early on, the structural determinants underlying the positive impact of H2B ubiquitylation on H3K79 and H3K4 methylation were resolved only recently. We will review the molecular features controlling these cross-talks and the impact of H3K27 tri-methylation on EZH2, when embedded in the PRC complex, activity.

Aromatic d-amino acids are key precursors for the production of many small molecule therapeutics. Therefore, the development of biocatalytic methods for their synthesis is of great interest. An enzyme that has great potential as a biocatalyst for the synthesis of d-amino acids is the stereoinverting d-phenylglycine aminotransferase (DPAT) from Pseudomonas stutzeri ST-201. This enzyme catalyzes a unique l to d transamination reaction that produces d-phenylglycine and α-ketoglutarate from benzoylformate and l-glutamate, via a mechanism that is poorly understood. Here, we present the crystal structure of DPAT, which shows that the enzyme folds into a two-domain structure representative of class III aminotransferases. Guided by the crystal structure, we performed saturation mutagenesis to probe the substrate binding pockets of the enzyme. These experiments helped us identify two arginine residues (R34 and R407), one in each binding pocket, that are essential to catalysis. Together with kinetic analyses using a library of amino acid substrates, our mutagenesis and structural studies allow us to propose a binding model that explains the dual l/d specificity of DPAT. Our kinetic analyses also demonstrate that DPAT can catalyze the transamination of β- and γ-amino acids, reclassifying this enzyme as an ω-aminotransferase. Collectively, our studies highlight that the DPAT active site is amenable to protein engineering for expansion of its substrate scope, which offers the opportunity to generate new biocatalysts for the synthesis of a variety of valuable optically pure d-amino acids from inexpensive and abundant l-amino acids.

Mutations in proteins like FUS which cause Amyotrophic Lateral Sclerosis (ALS) result in the aberrant formation of stress granules while ALS-linked mutations in other proteins impede elimination of stress granules. Repeat expansions in C9ORF72, the major cause of ALS, reduce C9ORF72 levels but how this impacts stress granules is uncertain. Here, we demonstrate that C9ORF72 associates with the autophagy receptor p62 and controls elimination of stress granules by autophagy. This requires p62 to associate via the Tudor protein SMN with proteins, including FUS, that are symmetrically methylated on arginines. Mice lacking p62 accumulate arginine-methylated proteins and alterations in FUS-dependent splicing. Patients with C9ORF72 repeat expansions accumulate symmetric arginine dimethylated proteins which co-localize with p62. This suggests that C9ORF72 initiates a cascade of ALS-linked proteins (C9ORF72, p62, SMN, FUS) to recognize stress granules for degradation by autophagy and hallmarks of a defect in this process are observable in ALS patients., Many Amyotrophic Lateral Sclerosis (ALS)-linked mutations cause accumulation of stress granules, and most ALS cases are caused by repeat expansions in C9ORF72. Here the authors show that C9ORF72 and the autophagy receptor p62 interact to associate with proteins symmetrically dimethylated on arginines such as FUS, to eliminate stress granules by autophagy.

We investigated two critical aspects of rose Bengal (RB) photosensitized protein cross-linking that may underlie recently developed medical applications. Our studies focused on the binding of RB to collagen by physical interaction and the effect of this binding and certain amino acids on RB photochemistry. Molecular dynamics simulations and free-energy calculation techniques, complemented with isothermal titration calorimetry, provided insight into the binding between RB and a collagen-like peptide (CLP) at the atomic level. Electrostatic interactions dominated, which is consistent with the finding that RB bound equally well to triple helical and single chain collagen. The binding free energy ranged from −5.7 to −3 kcal/mol and was strongest near the positively charged amino groups at the N-terminus and on lysine side chains. At high RB concentration, a maximum of 16 ± 3 bound dye molecules per peptide was found, which is consistent with spectroscopic evidence for aggregated RB bound to collagen or the CLP. Within a tissue-mimetic collagen matrix, RB photobleached rapidly, probably due to electron transfer to certain protein amino acids, as was demonstrated in solutions of free RB and arginine. In the presence of arginine and low oxygen concentrations, a product absorbing at 510 nm formed, presumably due to dehalogenation after electron transfer to RB. In the collagen matrix without arginine, the dye generated singlet oxygen as well as the 510 nm product. These results provide the first evidence of the effects of a tissue-like environment on the photochemical mechanisms of rose Bengal.

During DNA replication, proliferating cell nuclear antigen (PCNA) adopts a ring-shaped structure to promote processive DNA synthesis, acting as a sliding clamp for polymerases. Known posttranslational modifications function at the outer surface of the PCNA ring to favor DNA damage bypass. Here, we demonstrate that acetylation of lysine residues at the inner surface of PCNA is induced by DNA lesions. We show that cohesin acetyltransferase Eco1 targets lysine 20 at the sliding surface of the PCNA ring in vitro and in vivo in response to DNA damage. Mimicking constitutive acetylation stimulates homologous recombination and robustly suppresses the DNA damage sensitivity of mutations in damage tolerance pathways. In comparison to the unmodified trimer, structural differences are observed at the interface between protomers in the crystal structure of the PCNA-K20ac ring. Thus, acetylation regulates PCNA sliding on DNA in the presence of DNA damage, favoring homologous recombination linked to sister-chromatid cohesion.

The DNA damage response (DDR) is central to the cell survival and it requires post-translational modifications, in part, to sense the damage, amplify the signaling response and recruit and regulate DNA repair enzymes. Lysine methylation of histones such as H4K20 and non-histone proteins including p53 has been shown to be essential for the mounting of the DDR. It is well-known that the lysine methyltransferase SET7 regulates the DDR, as cells lacking this enzyme are hypersensitive to chemotherapeutic drugs. To define addition substrates of SET7 involved in the DDR, we screened a peptide array encompassing potential lysine methylation sites from >100 key DDR proteins and identified peptides from 58 proteins to be lysine methylated defining a methylation consensus sequence of [S>K-2; S>R-1; K0] consistent with previous findings. We focused on K377 methylation of the Flap endonuclease 1 (FEN1), a structure specific endonuclease with important functions in Okazaki fragment processing during DNA replication as a substrate of SET7. FEN1 was monomethylated by SET7 in vivo in a cell cycle dependent manner with levels increasing as cells progressed through S phase and decreasing as they exited S phase, as detected using K377me1 specific antibodies. Although K377me1 did not affect the enzymatic activity of FEN1, it was required for the cellular response to replicative stress by FEN1. These finding define FEN1 as a new substrate of SET7 required for the DDR.

The effect of accounting for the total surface in the association of thiol-containing molecules to nanosilver was assessed using isothermal titration calorimetry, along with a new open access algorithm that calculates the total surface area for samples of different polydispersity. Further, we used advanced molecular dynamic calculations to explore the underlying mechanisms for the interaction of the studied molecules in the presence of a nanosilver surface in the form of flat surfaces or as three-dimensional pseudospherical nanostructures. Our data indicate that not only is the total surface area available for binding but also the supramolecular arrangements of the molecules in the near proximity of the nanosilver surface strongly affects the affinity of thiol-containing molecules to nanosilver surfaces.

The ferric uptake regulator (Fur) is a superfamily of transcription factors found in bacteria which control the expression of a myriad of genes. In this study, we report a simple protocol for the purification of recombinant untagged Campylobacter jejuni Fur (CjFur). CjFur was isolated using a combination of three ion exchange chromatography steps followed by size exclusion chromatography on a Superdex 75. ESI–MS analysis shows that our method yields pure CjFur and that this tag-free version incorporates metal more efficiently than recombinant CjFur harboring a tag or tag remnants. Finally, electrophoretic mobility shift assays show that this new purification method yields a CjFur preparation that binds DNA more efficiently. These results suggest that adding a N-terminus tag onto CjFur is detrimental to its activity. Overall, the approaches detailed in this study offer an alternative strategy for the purification of CjFur, and likely other metalloregulators, for future biochemical and biophysical studies.

T-cell acute lymphoblastic leukemia (T-ALL) is a heterogeneous group of hematological tumors composed of distinct subtypes that vary in their genetic abnormalities, gene expression signatures, and prognoses. However, it remains unclear whether T-ALL subtypes differ at the functional level, and, as such, T-ALL treatments are uniformly applied across subtypes, leading to variable responses between patients. Here we reveal the existence of a subtype-specific epigenetic vulnerability in T-ALL by which a particular subgroup of T-ALL characterized by expression of the oncogenic transcription factor TAL1 is uniquely sensitive to variations in the dosage and activity of the histone 3 Lys27 (H3K27) demethylase UTX/KDM6A. Specifically, we identify UTX as a coactivator of TAL1 and show that it acts as a major regulator of the TAL1 leukemic gene expression program. Furthermore, we demonstrate that UTX, previously described as a tumor suppressor in T-ALL, is in fact a pro-oncogenic cofactor essential for leukemia maintenance in TAL1-positive (but not TAL1-negative) T-ALL. Exploiting this subtype-specific epigenetic vulnerability, we propose a novel therapeutic approach based on UTX inhibition through in vivo administration of an H3K27 demethylase inhibitor that efficiently kills TAL1-positive primary human leukemia. These findings provide the first opportunity to develop personalized epigenetic therapy for T-ALL patients.

Red fluorescent proteins (RFPs) are used extensively in chemical biology research as fluorophores for live cell imaging, as partners in FRET pairs, and as signal transducers in biosensors. For all of these applications, brighter RFP variants are desired. Here, we used rational design to increase the quantum yield of monomeric RFPs in order to improve their brightness. We postulated that we could increase quantum yield by restricting the conformational degrees of freedom of the RFP chromophore. To test our hypothesis, we introduced aromatic residues above the chromophore of mRojoA, a dim RFP containing a π-stacked Tyr residue directly beneath the chromophore, in order to reduce chromophore conformational flexibility via improved packing and steric complementarity. The best mutant identified displayed an absolute quantum yield increase of 0.07, representing an over 3-fold improvement relative to mRojoA. Remarkably, this variant was isolated following the screening of only 48 mutants, a library size that is several orders of magnitude smaller than those previously used to achieve equivalent gains in quantum yield in other RFPs. The crystal structure of the highest quantum yield mutant showed that the chromophore is sandwiched between two Tyr residues in a triple-decker motif of aromatic rings. Presence of this motif increases chromophore rigidity, as evidenced by the significantly reduced temperature factors compared to dim RFPs. Overall, the approach presented here paves the way for the rapid development of fluorescent proteins with higher quantum yield and overall brightness.

International audience; Normal aging is characterized by cognitive, functional, and neuroanatomic changes. Executive functions (EF), linked to the autonomy in the realization of instrumental Activity of Daily Living (iADL) are particularly sensitive to the effect of aging. To date, the best way to describe the cognitive profile of an individual is using traditional neuropsychological assessment. However, these tasks showed a week ecological validity that compromise the prediction of iADL functioning. Fortunately, virtual reality (VR) seems to be an interesting alternative to assess the functional capacities by reproducing everyday life situations accurately. Nevertheless, we know that the elderly could have some difficulty when interacting with technology (Banville et al. 2017). The aims of this study were (1) to differentiate performance of older adult compared to young people in a sorting task implemented in a virtual environment (VE) and (2) to analyze psychometric properties of that novel task. To do this, 30 participants were recruited in the general population and were divided in 2 groups based on their age. The results indicated that older subjects take more time than younger to compete the sorting task. Then, the scores obtained correlated significantly with some neuropsychological tasks, particularly with those assessing EF. To conclude, the VMT-2 could offer the opportunity to make a valid assessment of functional capacities during aging. Further work could validate the relevance to use it when the individual facing with mild cognitive impairment or dementia.

In every living organism, the control of metal homoeostasis is a tightly regulated process coordinated by several intertwined biological pathways. In many bacteria, the ferric uptake regulator (Fur) family of transcriptional factors (TFs) are key factors in controlling the expression of genes involved in metal homeostasis and can also regulate the expression of genes involved in responses to oxidative stresses. Since the crystallization of Escherichia coli Fur DNA binding domain, the crystal structure of several metalloregulators have been reported. While the Fur family of proteins adopt similar structures, each contains unique structural features relating to their specific biological functions. Moreover, recent groundbreaking studies have provided additional insights into the mechanisms underlying the binding of DNA by these metalloregulators. In this review, we present a comprehensive overview of the crystal structure of Fur family metalloregulators with a specific focus on the new structures of these TFs bound to DNA.

In Campylobacter jejuni (Cj), the metal-cofactored peroxide response regulator (PerR) transcription factor allows C. jejuni to respond to oxidative stresses. The crystal structure of the metalated form of CjPerR shows that the protein folds as an asymmetric dimer displaying structural differences in the orientation of its DNA-binding domain. Comparative analysis shows that such asymmetry is a conserved feature among crystallized PerR proteins, and mutational analysis reveals that residues found in the first α-helix of CjPerR contribute to DNA binding. These studies present the structure of CjPerR protein and highlight structural heterogeneity in the orientation of the metalated PerR DNA-binding domain which may underlie the ability of PerR to recognize DNA, control gene expression, and contribute to bacterial pathogenesis.

Ferric uptake regulators (Fur) are a family of transcription factors coupling gene regulatory events to metal concentration. Recent evidence has expanded the mechanistic repertoires employed by Fur to activate or repress gene expression in the presence or absence of regulatory metals. However, the mechanistic basis underlying this extended repertoire has remained largely unexplored. In this study, we used an extensive set of mutations to demonstrate that Campylobacter jejuni Fur (CjFur) employs the same surface to positively and negatively control gene expression regardless of the presence or absence of metals. Moreover, the crystal structure determination of a CjFur devoid of any regulatory metals shows that subtle reorientation of the transcription factor DNA binding domain negatively impacts DNA binding, gene expression and gut colonization in chickens. Overall, these results highlight the versatility of the CjFur DNA binding domain in mediating all gene regulatory events controlled by the metalloregulator and that the full metalation of CjFur is critical to the Campylobacter jejuni life cycle in vivo.

SummaryCharacterization of lysine methylation has proven challenging despite its importance in biological processes such as gene transcription, protein turnover, and cytoskeletal organization. In contrast to other key posttranslational modifications, current proteomics techniques have thus far shown limited success at characterizing methyl-lysine residues across the cellular landscape. To complement current biochemical characterization methods, we developed a multistate computational protein design procedure to probe the substrate specificity of the protein lysine methyltransferase SMYD2. Modeling of substrate-bound SMYD2 identified residues important for substrate recognition and predicted amino acids necessary for methylation. Peptide- and protein- based substrate libraries confirmed that SMYD2 activity is dictated by the motif [LFM]−1-K∗-[AFYMSHRK]+1-[LYK]+2 around the target lysine K∗. Comprehensive motif-based searches and mutational analysis further established four additional substrates of SMYD2. Our methodology paves the way to systematically predict and validate posttranslational modification sites while simultaneously pairing them with their associated enzymes.

SummaryDPY-30 is a subunit of mammalian COMPASS-like complexes (complex of proteins associated with Set1) and regulates global histone H3 Lys-4 trimethylation. Here we report structural evidence showing that the incorporation of DPY-30 into COMPASS-like complexes is mediated by several hydrophobic interactions between an amphipathic α helix located on the C terminus of COMPASS subunit ASH2L and the inner surface of the DPY-30 dimerization/docking (D/D) module. Mutations impairing the interaction between ASH2L and DPY-30 result in a loss of histone H3K4me3 at the β locus control region and cause a delay in erythroid cell terminal differentiation. Using overlay assays, we defined a consensus sequence for DPY-30 binding proteins and found that DPY-30 interacts with BAP18, a subunit of the nucleosome remodeling factor complex. Overall, our results indicate that the ASH2L/DPY-30 complex is important for cell differentiation and provide insights into the ability of DPY-30 to associate with functionally divergent multisubunit complexes.

An α-helix bundle is a small and compact protein fold always composed of more than 2 α-helices that typically run nearly parallel or antiparallel to each other. The repertoire of arrangements of α-helix bundle is such that these domains bind to a myriad of molecular entities including DNA, RNA, proteins and small molecules. A special instance of α-helical bundle is the X-type in which the arrangement of two α-helices interact at 45° to form an X. Among those, some X-helix bundle proteins bind to the hydrophobic section of an amphipathic α-helix in a seemingly orientation and sequence specific manner. In this review, we will compare the binding mode of amphipathic α-helices to X-helix bundle and α-helical bundle proteins. From these structures, we will highlight potential regulatory paradigms that may control the specific interactions of X-helix bundle proteins to amphipathic α-helices. This article is part of a Special Issue entitled: Biophysics in Canada, edited by Lewis Kay, John Baenziger, Albert Berghuis and Peter Tieleman.

International audience; Prospective memory (PM) is defined be the capacity to remember to realize an intended action in the future. This is a very important cognitive function that permit to maximize autonomy in everyday life. Unfortunately, few assessment tool, valid, reliable and ecological is accessible for clinicians. To obtain a verisimilar and ecologically prospective memory assessment tool, virtual reality seems to be a promising way. A specific and sensible tool could help the clinician to detect subtle changes in the cognition of the elderly and, ideally detect pathological aging soon before the beginning of decline. Because older adults are not really at ease with technology, these (dis)abilities could be confounded with cognitive inefficacy and lead to false positives diagnostics. To avoid this, the psychometrician must consider the impact of human-computer interfaces (HMI) on cognition. This paper present three experiments that show the impact of HMI on stress, capacity to achieve a task and on cognitive load. The first pilot study shown that a “heavy to use” HMI generated stress and difficulty to achieve the task with healthy adults. The second pilot study revealed that VMT-2 is judged moderately challenging cognitively and it seems to be more for older participants. The third pilot study shown that a complex virtual environment (in terms of navigation and interaction) is more cognitively challenging than a simple virtual environment for older peoples compared to young participants. These results indicated the importance of considering HMI as a potential variable that could create bias in the cognitive measurement.

Making a Histone Mark The covalent marks on histones (the principal components of chromatin) play a critical role in the regulation of gene expression. Somehow these marks are preserved when a cell in a tissue divides so that the daughter cells maintain the gene expression program and tissue identity of the parent cell. Jacob et al. (p. 1249 ) show that the Arabidopsis histone methylase ATXR5 is specific for the replication-dependent histone variant H3.1 and maintains the repressive histone H3 lysine-27 methyl mark on the H3.1 variant during genome replication, thus, preserving cell-type–specific regions of heterochromatin and gene repression through cell division and beyond.

The Ferric Uptake Regulator (FUR) is a transcription factor (TF) regulating the expression of several genes to control iron levels in prokaryotes. Members of this family of TFs share a common structural scaffold that typically comprises two regions that include a DNA binding and dimerization domains. While this structural organization is conserved, FUR proteins employ different mechanisms to bind divergent DNA binding elements and regulate gene expression in the absence or presence of regulatory metals. These findings, combined with the observations that FUR proteins display different geometries in regard to the relative orientation of the DNA binding and dimerization domains, have highlighted an expanding repertoire of molecular mechanisms controlling the activity of this family of TFs. In this chapter, we present an overview of the methods to purify, crystallize, and solve the structure of Campylobacter jejuni FUR.

The Ferric Uptake Regulator (FUR) is a transcription factor (TF) regulating the expression of several genes to control iron levels in prokaryotes. Members of this family of TFs share a common structural scaffold that typically comprises two regions that include a DNA binding and dimerization domains. While this structural organization is conserved, FUR proteins employ different mechanisms to bind divergent DNA binding elements and regulate gene expression in the absence or presence of regulatory metals. These findings, combined with the observations that FUR proteins display different geometries in regard to the relative orientation of the DNA binding and dimerization domains, have highlighted an expanding repertoire of molecular mechanisms controlling the activity of this family of TFs. In this chapter, we present an overview of the methods to purify, crystallize, and solve the structure of Campylobacter jejuni FUR.

Summary Dpy-30 is a regulatory subunit controlling the histone methyltransferase activity of the KMT2 enzymes in vivo. Paradoxically, in vitro methyltransferase assays revealed that Dpy-30 only modestly participates in the positive heterotypic allosteric regulation of these methyltransferases. Detailed genome-wide, molecular and structural studies reveal that an extensive network of interactions taking place at the interface between Dpy-30 and Ash2L are critical for the correct placement, genome-wide, of H3K4me2 and H3K4me3 but marginally contribute to the methyltransferase activity of KMT2 enzymes in vitro. Moreover, we show that H3K4me2 peaks persisting following the loss of Dpy-30 are found in regions of highly transcribed genes, highlighting an interplay between Complex of Proteins Associated with SET1 (COMPASS) kinetics and the cycling of RNA polymerase to control H3K4 methylation. Overall, our data suggest that Dpy-30 couples its modest positive heterotypic allosteric regulation of KMT2 methyltransferase activity with its ability to help the positioning of SET1/COMPASS to control epigenetic signaling.

APOBEC3G (A3G) is a host-encoded protein that potently restricts the infectivity of a broad range of retroviruses. This can occur by mechanisms dependent on catalytic activity, resulting in the mutagenic deamination of nascent viral cDNA, and/or by other means that are independent of its catalytic activity. It is not yet known to what extent deamination-independent processes contribute to the overall restriction, how they exactly work or how they are regulated. Here, we show that alanine substitution of either tryptophan 94 (W94A) or 127 (W127A) in the non-catalytic N-terminal domain of A3G severely impedes RNA binding and alleviates deamination-independent restriction while still maintaining DNA mutator activity. Substitution of both tryptophans (W94A/W127A) produces a more severe phenotype in which RNA binding and RNA-dependent protein oligomerization are completely abrogated. We further demonstrate that RNA binding is specifically required for crippling late reverse transcript accumulation, preventing proviral DNA integration and, consequently, restricting viral particle release. We did not find that deaminase activity made a significant contribution to the restriction of any of these processes. In summary, this work reveals that there is a direct correlation between A3G’s capacity to bind RNA and its ability to inhibit retroviral infectivity in a deamination-independent manner.

WDR5 (WD40 repeat protein 5) is an essential component of the human trithorax-like family of SET1 [Su(var)3–9 enhancer-of-zeste trithorax 1] methyltransferase complexes that carry out trimethylation of histone 3 Lys4 (H3K4me3), play key roles in development and are abnormally expressed in many cancers. In the present study, we show that the interaction between WDR5 and peptides from the catalytic domain of MLL (mixed-lineage leukaemia protein) (KMT2) can be antagonized with a small molecule. Structural and biophysical analysis show that this antagonist binds in the WDR5 peptide-binding pocket with a Kd of 450 nM and inhibits the catalytic activity of the MLL core complex in vitro. The degree of inhibition was enhanced at lower protein concentrations consistent with a role for WDR5 in directly stabilizing the MLL multiprotein complex. Our data demonstrate inhibition of an important protein–protein interaction and form the basis for further development of inhibitors of WDR5-dependent enzymes implicated in MLL-rearranged leukaemias or other cancers.

Conditional gene expression systems that enable inducible and reversible transcriptional control are essential research tools and have broad applications in biomedicine and biotechnology. The reverse tetracycline transcriptional activator is a canonical system for engineered gene expression control that enables graded and gratuitous modulation of target gene transcription in eukaryotes from yeast to human cell lines and transgenic animals. However, the system has a tendency to activate transcription even in the absence of tetracycline and this leaky target gene expression impedes its use. Here, we identify single amino-acid substitutions that greatly enhance the dynamic range of the system in yeast by reducing leaky transcription to undetectable levels while retaining high expression capacity in the presence of inducer. While the mutations increase the inducer concentration required for full induction, additional sensitivity-enhancing mutations can compensate for this effect and confer a high degree of robustness to the system. The novel transactivator variants will be useful in applications where tight and tunable regulation of gene expression is paramount.

Methylation of arginine and lysine (RK) residues play essential roles in epigenetics and the regulation of gene expression. However, research in this area is often hindered by the lack of effective tools for probing the protein methylation. Here, we present an antibody-free strategy to capture protein methylation on RK residues by using chemical reactions to eliminate the charges on un-modified RK residues and peptide N-termini. Peptides containing methylated RK residues remain positively charged and are then enriched by strong cation exchange chromatography, followed by high-resolution mass spectrometry identification.

Since its initial discovery by Allfrey and colleagues, methylation of histone proteins on lysine residues has emerged as an important posttranslational modification controlling a myriad of nuclear events, such as DNA damage repair, transcription, and replication. Following the biochemical characterization of the first histone K methyltransferase in 2000, efforts have uncovered the mechanistic basis underlying the activity of several histone lysine methyltransferases (HKMTs) and the impact of the reactions they catalyzed. Moreover, high-throughput sequencing and genome-wide association studies demonstrated that histone methyltransferases are linked to aggressive forms of cancers, including, but not limited to, lymphomas, leukemias, and gliomas, thereby opening new avenues for therapeutic interventions. In this chapter, we focus on histone H3 Lys-4, Lys-9, Lys-27, Lys-36, and Lys-79, as well as histone H4 Lys-20 methyltransferases.

The full regulatory potential of the ferric uptake regulator (Fur) family of proteins remains undefined despite over 20 years of study. We report herein an integrated approach that combines both genome-wide technologies and structural studies to define the role of Fur in Campylobacter jejuni (Cj). CjFur ChIP-chip assays identified 95 genomic loci bound by CjFur associated with functions as diverse as iron acquisition, flagellar biogenesis, and non-iron ion transport. Comparative analysis with transcriptomic data revealed that CjFur regulation extends beyond solely repression and also includes both gene activation and iron-independent regulation. Computational analysis revealed the presence of an elongated holo-Fur repression motif along with a divergent holo-Fur activation motif. This diversity of CjFur DNA-binding elements is supported by the crystal structure of CjFur, which revealed a unique conformation of its DNA-binding domain and the absence of metal in the regulatory site. Strikingly, our results indicate that the apo-CjFur structure retains the canonical V-shaped dimer reminiscent of previously characterized holo-Fur proteins enabling DNA interaction. This conformation stems from a structurally unique hinge domain that is poised to further contribute to CjFur’s regulatory functions by modulating the orientation of the DNA-binding domain upon binding of iron. The unique features of the CjFur crystal structure rationalize the binding sequence diversity that was uncovered during ChIP-chip analysis and defines apo-Fur regulation.

Campylobacter jejuni is a prevalent cause of bacterial gastroenteritis in humans worldwide. The mechanisms by which C. jejuni survives stomach acidity remain undefined. In the present study, we demonstrated that the C. jejuni ferric uptake regulator (Fur) plays an important role in C. jejuni acid survival and acid-induced cross-protection against oxidative stress. A C. jejuni Δ fur mutant was more sensitive to acid than the wild-type strain. Profiling of the acid stimulon of the C. jejuni Δ fur mutant allowed us to uncover Fur-regulated genes under acidic conditions. In particular, Fur was found to upregulate genes involved in flagellar and cell envelope biogenesis upon acid stress, and mutants with deletions of these genes were found to be defective in surviving acid stress. Interestingly, prior acid exposure of C. jejuni cross-protected against oxidative stress in a catalase (KatA)- and Fur-dependent manner. Western blotting and reverse transcription-quantitative PCR revealed increased expression of KatA upon acid stress. Electrophoretic mobility shift assays (EMSAs) demonstrated that the binding affinity between Fur and the katA promoter is reduced in vitro under conditions of low pH, rationalizing the higher levels of expression of katA under acidic conditions. Strikingly, the Δ fur mutant exhibited reduced virulence in both human epithelial cells and the Galleria mellonella infection model. Altogether, this is the first study showing that, in addition to its role in iron metabolism, Fur is an important regulator of C. jejuni acid responses and this function cross-protects against oxidative stress. Moreover, our results clearly demonstrate Fur's important role in C. jejuni pathogenesis.

The SMYD (SET and MYND domain) family of lysine methyltransferases (KMTs) plays pivotal roles in various cellular processes, including gene expression regulation and DNA damage response. Initially identified as genuine histone methyltransferases, specific members of this family have recently been shown to methylate non-histone proteins such as p53, VEGFR, and the retinoblastoma tumor suppressor (pRb). To gain further functional insights into this family of KMTs, we generated the protein interaction network for three different human SMYD proteins (SMYD2, SMYD3, and SMYD5). Characterization of each SMYD protein network revealed that they associate with both shared and unique sets of proteins. Among those, we found that HSP90 and several of its co-chaperones interact specifically with the tetratrico peptide repeat (TPR)-containing SMYD2 and SMYD3. Moreover, using proteomic and biochemical techniques, we provide evidence that SMYD2 methylates K209 and K615 on HSP90 nucleotide-binding and dimerization domains, respectively. In addition, we found that each methylation site displays unique reactivity in regard to the presence of HSP90 co-chaperones, pH, and demethylation by the lysine amine oxidase LSD1, suggesting that alternative mechanisms control HSP90 methylation by SMYD2. Altogether, this study highlights the ability of SMYD proteins to form unique protein complexes that may underlie their various biological functions and the SMYD2-mediated methylation of the key molecular chaperone HSP90.

Dans son plan stratégique, le ministère de la Sécurité publique du Québec souligne les problèmes actuels de surpopulation carcérale au Québec. Ce phénomène peut contribuer à une augmentation des conflits vécus par les détenus et, par extension, à l’émergence des incidents disciplinaires. Dans un tel contexte, il semble nécessaire d’améliorer la gestion du risque lié à ce type d’incidents. Dans le cadre de cet article, l’objectif principal visé est de mettre à l’épreuve la validité prédictive du LS/CMI sur l’occurrence des incidents disciplinaires. L’échantillon consiste en 934 détenus incarcérés au Service correctionnel du Québec (SCQ) entre janvier et décembre 2007. Durant cette même période, tous les détenus ont été évalués dans les semaines suivant leur admission avec la version québécoise du LS/CMI. Les résultats permettent de confirmer l’efficacité du LS/CMI dans la prédiction des incidents disciplinaires, et ce, indépendamment de leur nature et de leur cible., The purpose of this study was to address the critical need to improve accuracy in risk assessment for prison misconducts and hence improve decision-making. With the increased prison crowding in the correctional facilities in Quebec, there is a critical need for the development of better instruments to assist clinicians in their task. Since February 2007, all offenders sentenced for a violent crime or to six month or more are now evaluated with the French-Canadian version of the LS/CMI. Although the LS/CMI is generally good at predicting recidivism, it was not specifically designed to predict prison misconducts. The present study investigates the potential predictive validity of the LS/CMI. The sample consisted of 934 male offenders sentenced for a violent crime or to 6 month or more in the Correctional Services of Quebec between February 2007 and December 2007. In general, results indicate that the LS/CMI can predict violent and non violent misconducts., En su plan estratégico, el ministerio de Seguridad pública de Quebec destaca los problemas actuales de sobrepoblación en las cárceles de Quebec. Dicho fenómeno corre el riesgo de contribuir al aumento de conflictos entre los detenidos y, por extensión, a la emergencia de incidentes disciplinarios. En ese contexto, se vuelve necesario mejorar la gestión de riesgos asociados a ese tipo de incidentes. El objetivo principal del presente estudio fue poner a prueba la validez predictiva del LS/CMI sobre ocurrencia de incidentes disciplinarios. La muestra consistió en 934 detenidos encarcelados en el Servicio correccional de Quebec (SCQ) entre enero y diciembre de 2007. Durante ese periodo, todos los detenidos fueron evaluados en las semanas que siguieron a su admisión con la versión canadiense del LS/CMI. Los resultados permiten confirmar la eficacia del LS/CMI en la predicción de incidentes disciplinarios independientemente de su naturaleza.

SummaryHistone H3 Lys-4 methylation is predominantly catalyzed by a family of methyltransferases whose enzymatic activity depends on their interaction with a three-subunit complex composed of WDR5, RbBP5, and Ash2L. Here, we report that a segment of 50 residues of RbBP5 bridges the Ash2L C-terminal domain to WDR5. The crystal structure of WDR5 in ternary complex with RbBP5 and MLL1 reveals that both proteins binds peptide-binding clefts located on opposite sides of WDR5′s β-propeller domain. RbBP5 engages in several hydrogen bonds and van der Waals contacts within a V-shaped cleft formed by the junction of two blades on WDR5. Mutational analyses of both the WDR5 V-shaped cleft and RbBP5 residues reveal that the interactions between RbBP5 and WDR5 are important for the stimulation of MLL1 methyltransferase activity. Overall, this study provides the structural basis underlying the formation of the WDR5-RbBP5 subcomplex and further highlight the crucial role of WDR5 in scaffolding the MLL1 core complex.

The SET1 family of methyltransferases carries out the bulk of histone H3 Lys-4 methylation in vivo. One of the common features of this family is the regulation of their methyltransferase activity by a tripartite complex composed of WDR5, RbBP5, and Ash2L. To selectively probe the role of the SET1 family of methyltransferases, we have developed a library of histone H3 peptide mimetics and report herein the characterization of an Nα acetylated form of histone H3 peptide (NαH3). Binding and inhibition studies reveal that the addition of an acetyl moiety to the N terminus of histone H3 significantly enhances its binding to WDR5 and prevents the stimulation of MLL1 methyltransferase activity by the WDR5-RbBP5-Ash2L complex. The crystal structure of NαH3 in complex with WDR5 reveals that a high-affinity hydrophobic pocket accommodates the binding of the acetyl moiety. These results provide the structural basis to control WDR5-RbBP5-Ash2L-MLL1 activity and a tool to manipulate stem cell differentiation programs.