Sajad Hussain Syed

Single-base resolution mapping of H1–nucleosome interactions and 3D organization of the nucleosome

Despite the key role of the linker histone H1 in chromatin structure and dynamics, its location and interactions with nucleosomal DNA have not been elucidated. In this work we have used a combination of electron cryomicroscopy, hydroxyl radical footprinting, and nanoscale modeling to analyze the structure of precisely positioned mono-, di-, and trinucleosomes containing physiologically assembled full-length histone H1 or truncated mutants of this protein. Single-base resolution •OH footprinting shows that the globular domain of histone H1 (GH1) interacts with the DNA minor groove located at the center of the nucleosome and contacts a 10-bp region of DNA localized symmetrically with respect to the nucleosomal dyad. In addition, GH1 interacts with and organizes about one helical turn of DNA in each linker region of the nucleosome. We also find that a seven amino acid residue region (121–127) in the COOH terminus of histone H1 was required for the formation of the stem structure of the linker DNA. A molecular model on the basis of these data and coarse-grain DNA mechanics provides novel insights on how the different domains of H1 interact with the nucleosome and predicts a specific H1-mediated stem structure within linker DNA.

Remosomes: RSC generated non-mobilized particles with approximately 180 bp DNA loosely associated with the histone octamer

Chromatin remodelers are sophisticated nano-machines that are able to alter histone-DNA interactions and to mobilize nucleosomes. Neither the mechanism of their action nor the conformation of the remodeled nucleosomes are, however, yet well understood. We have studied the mechanism of Remodels Structure of Chromatin (RSC)-nucleosome mobilization by using high-resolution microscopy and biochemical techniques. Atomic force microscopy and electron cryomicroscopy (EC-M) analyses show that two types of products are generated during the RSC remodeling: (i) stable non-mobilized particles, termed remosomes that contain about 180 bp of DNA associated with the histone octamer and, (ii) mobilized particles located at the end of DNA. EC-M reveals that individual remosomes exhibit a distinct, variable, highly-irregular DNA trajectory. The use of the unique “one pot assays” for studying the accessibility of nucleosomal DNA towards restriction enzymes, DNase I footprinting and ExoIII mapping demonstrate that the histone-DNA interactions within the remosomes are strongly perturbed, particularly in the vicinity of the nucleosome dyad. The data suggest a two-step mechanism of RSC-nucleosome remodeling consisting of an initial formation of a remosome followed by mobilization. In agreement with this model, we show experimentally that the remosomes are intermediate products generated during the first step of the remodeling reaction that are further efficiently mobilized by RSC.

The docking domain of histone H2A is required for H1 binding and RSC-mediated nucleosome remodeling

Histone variants within the H2A family show high divergences in their C-terminal regions. In this work, we have studied how these divergences and in particular, how a part of the H2A COOH-terminus, the docking domain, is implicated in both structural and functional properties of the nucleosome. Using biochemical methods in combination with Atomic Force Microscopy and Electron Cryo-Microscopy, we show that the H2A-docking domain is a key structural feature within the nucleosome. Deletion of this domain or replacement with the incomplete docking domain from the variant H2A.Bbd results in significant structural alterations in the nucleosome, including an increase in overall accessibility to nucleases, un-wrapping of ∼10 bp of DNA from each end of the nucleosome and associated changes in the entry/exit angle of DNA ends. These structural alterations are associated with a reduced ability of the chromatin remodeler RSC to both remodel and mobilize the nucleosomes. Linker histone H1 binding is also abrogated in nucleosomes containing the incomplete docking domain of H2A.Bbd. Our data illustrate the unique role of the H2A-docking domain in coordinating the structural-functional aspects of the nucleosome properties. Moreover, our data suggest that incorporation of a ‘defective’ docking domain may be a primary structural role of H2A.Bbd in chromatin.

The multifunctional protein nucleophosmin (NPM1) is a human linker histone H1 chaperone.

Linker histone H1 plays an essential role in chromatin organization. Proper deposition of linker histone H1 as well as its removal is essential for chromatin dynamics and function. Linker histone chaperones perform this important task during chromatin assembly and other DNA-templated phenomena in the cell. Our in vitro data show that the multifunctional histone chaperone NPM1 interacts with linker histone H1 through its first acidic stretch (residues 120-132). Association of NPM1 with linker histone H1 was also observed in cells in culture. NPM1 exhibited remarkable linker histone H1 chaperone activity, as it was able to efficiently deposit histone H1 onto dinucleosomal templates. Overexpression of NPM1 reduced the histone H1 occupancy on the chromatinized template of HIV-1 LTR in TZM-bl cells, which led to enhanced Tat-mediated transactivation. These data identify NPM1 as an important member of the linker histone chaperone family in humans.

From crystal and NMR structures, footprints and cryo-electron-micrographs to large and soft structures: nanoscale modeling of the nucleosomal stem

The interaction of histone H1 with linker DNA results in the formation of the nucleosomal stem structure, with considerable influence on chromatin organization. In a recent paper [Syed,S.H., Goutte-Gattat,D., Becker,N., Meyer,S., Shukla,M.S., Hayes,J.J., Everaers,R., Angelov,D., Bednar,J. and Dimitrov,S. (2010) Single-base resolution mapping of H1-nucleosome interactions and 3D organization of the nucleosome. Proc. Natl Acad. Sci. USA, 107, 9620–9625], we published results of biochemical footprinting and cryo-electron-micrographs of reconstituted mono-, di- and tri-nucleosomes, for H1 variants with different lengths of the cationic C-terminus. Here, we present a detailed account of the analysis of the experimental data and we include thermal fluctuations into our nano-scale model of the stem structure. By combining (i) crystal and NMR structures of the nucleosome core particle and H1, (ii) the known nano-scale structure and elasticity of DNA, (iii) footprinting information on the location of protected sites on the DNA backbone and (iv) cryo-electron micrographs of reconstituted tri-nucleosomes, we arrive at a description of a polymorphic, hierarchically organized stem with a typical length of 20 ± 2 base pairs. A comparison to linker conformations inferred for poly-601 fibers with different linker lengths suggests, that intra-stem interactions stabilize and facilitate the formation of dense chromatin fibers.

The incorporation of the novel histone variant H2AL2 confers unusual structural and functional properties of the nucleosome

In this work we have studied the properties of the novel mouse histone variant H2AL2. H2AL2 was used to reconstitute nucleosomes and the structural and functional properties of these particles were studied by a combination of biochemical approaches, atomic force microscopy (AFM) and electron cryo-microscopy. DNase I and hydroxyl radical footprinting as well as micrococcal and exonuclease III digestion demonstrated an altered structure of the H2AL2 nucleosomes all over the nucleosomal DNA length. Restriction nuclease accessibility experiments revealed that the interactions of the H2AL2 histone octamer with the ends of the nucleosomal DNA are highly perturbed. AFM imaging showed that the H2AL2 histone octamer was complexed with only ∼130 bp of DNA. H2AL2 reconstituted trinucleosomes exhibited a type of a ‘beads on a string’ structure, which was quite different from the equilateral triangle 3D organization of conventional H2A trinucleosomes. The presence of H2AL2 affected both the RSC and SWI/SNF remodeling and mobilization of the variant particles. These unusual properties of the H2AL2 nucleosomes suggest a specific role of H2AL2 during mouse spermiogenesis.

Discovery of novel pyrazolopyrimidinone analogs as potent inhibitors of phosphodiesterase type-5.

Cyclic guanosine monophosphate (cGMP) specific phosphodiesterase type-5 (PDE5), a clinically proven target to treat erectile dysfunction and diseases associated with lower cGMP levels in humans, is present in corpus cavernosum, heart, lung, platelets, prostate, urethra, bladder, liver, brain, and stomach. Sildenafil, vardenafil, tadalafil and avanafil are FDA approved drugs in market as PDE5 inhibitors for treating erectile dysfunction. In the present study a lead molecule 4-ethoxy-N-(6-hydroxyhexyl)-3-(1-methyl-7-oxo-3-propyl-6,7-dihydro-1H-pyrazolo[4,3-d]pyrimidin-5-yl)benzenesulfonamide, that is, compound-4a, an analog of pyrazolopyrimidinone scaffold has been identified as selective PDE5 inhibitor. A series of compounds was synthesized by replacing N-methylpiperazine moiety (ring-C) of sildenafil structure with different N-substitutions towards sulfonamide end. Compound-4a showed lower IC₅₀ value (1.5 nM) against PDE5 than parent sildenafil (5.6 nM) in in vitro enzyme assay. The isoform selectivity of the compound-4a against other PDE isoforms was similar to that of the Sildenafil. In corroboration with the in vitro data, this molecule showed better efficacy in in vivo studies using the conscious rabbit model. Also compound-4a exhibited good physicochemical properties like solubility, caco-2 permeability, cLogP along with optimal PK profile having no significant CYP enzyme inhibitory liabilities. Discovery of these novel bioactive compounds may open a new alternative for developing novel preclinical candidates based on this drugable scaffold.

Erratum: Structure and Dynamics of a 197 bp Nucleosome in Complex with Linker Histone H1 (Molecular Cell (2017) 66 (384–397)(S109727651730268X)(10.1016/j.molcel.2017.04.012)

Jan Bednar, Isabel Garcia-Saez, Ramachandran Boopathi, Amber R. Cutter, Gabor Papai, Anna Reymer, Sajad H. Syed, Imtiaz Nisar Lone, Ognyan Tonchev, Corinne Crucifix, Hervé Menoni, Christophe Papin, Dimitrios A. Skoufias, Hitoshi Kurumizaka, Richard Lavery, Ali Hamiche,* Jeffrey J. Hayes,* Patrick Schultz,* Dimitar Angelov,* Carlo Petosa,* and Stefan Dimitrov* *Correspondence: hamiche@igbmc.fr (A.H.), jeffrey_hayes@URMC.rochester.edu (J.J.H.), patrick.schultz@igbmc.fr (P.S.), dimitar.anguelov@ens-lyon.fr (D.A.), carlo.petosa@ibs.fr (C.P.), stefan.dimitrov@univ-grenoble-alpes.fr (S.D.) http://dx.doi.org/10.1016/j.molcel.2017.05.018

Divergent synthesis of prenylated carbazole alkaloid (+)-S-mahanimbine led to the discovery of a notch activator

(+)-Mahanimbine (1), a prenylated carbazole alkaloid, was isolated from the stems of Murraya koenigii. Herein we developed a divergent synthesis by the Vilsmeier–Haack reaction, which involves unusual oxidative cyclization and transformation to several distinct tetracyclic carbazole natural products (2–9). The new chemical entities were identified by extensive 1D and 2D NMR data, HR-ESI-MS, and comparison with the known literature. Compounds 2 and 5 were further characterized by X-ray crystal analysis. Compounds (1–9) were screened against the notch pathway activation in which the new compound 8 exhibited prominent notch activation in a reporter gene assay with an EC50 value equal to 0.85 μM. It also inhibited the cell proliferation and colony formation of the K562 human leukemia cell line.

Identification of a unique loss-of-function mutation in IGF1R and a crosstalk between IGF1R and Wnt/β-catenin signaling pathways

IGF1R is a ubiquitous receptor tyrosine kinase that plays critical roles in cell proliferation, growth and survival. Clinical studies have demonstrated upregulation of IGF1R mediated signaling in a number of malignancies including colon, breast, and lung cancers. Overexpression of the IGF1R in these malignancies is associated with a poor prognosis and overall survival. IGF1R specific kinase inhibitors have failed in multiple clinical trials partly because of the complex nature of IGF1R signaling. Thus identifying new binding partners and allosteric sites on IGF1R are emerging areas of research. More recently, IGF1R has been shown to translocate into the nucleus and perform many functions. In this study, we generated a library of IGF1R deletion and point mutants to examine IGF1R subcellular localization and activation of downstream signaling pathways. We show that the nuclear localization of IGF1R is primarily defined by its cytoplasmic domain. We identified a cross-talk between IGF1R and Wnt/β-catenin signaling pathways and showed, for the first time, that IGF1R is associated with upregulation of TCF-mediated β-catenin transcriptional activity. Using loss-of-function mutants, deletion analysis and IGF1R specific inhibitor(s), we show that cytoplasmic and nuclear activities are two independent functions of IGF1R. Furthermore, we identified a unique loss-of-function mutation in IGF1R. This unique loss-of-function mutant retains only nuclear functions and sits in a pocket, outside ATP and substrate binding region, that is suited for designing allosteric inhibitors of IGF1R.