Regulation of replicative histone RNA metabolism by the histone chaperone ASF1.

In S phase, duplicating and assembling the whole genome into chromatin requires upregulation of replicative histone gene expression. Here, we explored how histone chaperones control histone production in human cells to ensure a proper link with chromatin assembly. Depletion of the ASF1 chaperone specifically decreases the pool of replicative histones both at the protein and RNA levels. The decrease in their overall expression, revealed by total RNA sequencing (RNA-seq), contrasted with the increase in nascent/newly synthesized RNAs observed by 4sU-labeled RNA-seq. Further inspection of replicative histone RNAs showed a 3' end processing defect with an increase of pre-mRNAs/unprocessed transcripts likely targeted to degradation. Collectively, these data argue for a production defect of replicative histone RNAs in ASF1-depleted cells. We discuss how this regulation of replicative histone RNA metabolism by ASF1 as a "chaperone checkpoint" fine-tunes the histone dosage to avoid unbalanced situations deleterious for cell survival.

Enantioselective C-H Arylation for Axially Chiral Heterobiaryls.

An enantioselective palladium-catalyzed C-H arylation of functionalized pyrazoles/triazoles/imidazoles is developed, affording a variety of axially chiral ortho-nitro/formyl-substituted heterobiaryls with excellent enantioselectivities and good yields. The method features a deuterated P-chiral phosphorus ligand CD3-AntPhos, a broad substrate scope of functionalized heterobiaryls, mild reaction conditions, and low palladium loadings.

Cu(I)-Catalyzed Alkynylation of Quinolones.

Herein we report the first alkynylation of quinolones with terminal alkynes under mild reaction conditions. The reaction is catalyzed by Cu(I) salts in the presence of a Lewis acid, which is essential for the reactivity of the system. The enantioselective version of this transformation has also been explored, and the methodology has been applied in the synthesis of the enantioenriched tetrahydroquinoline alkaloid cuspareine.

Directed regioselective ortho,ortho'-magnesiations of aromatics and heterocycles using sBu2Mg in toluene.

Aryl azoles are ubiquitous as bioactive compounds and their regioselective functionalization is of utmost synthetic importance. Here, we report the development of a toluene-soluble dialkylmagnesium base sBu2Mg. This new reagent allows mild and regioselective ortho-magnesiations of various N-arylated pyrazoles and 1,2,3-triazoles as well as arenes bearing oxazoline, phosphorodiamidate or amide directing groups. The resulting diarylmagnesium reagents were further functionalized either by Pd-catalyzed arylation or by trapping reactions with a broad range of electrophiles (aldehydes, ketones, allylic halides, acyl chlorides, Weinreb amides, aryl halides, hydroxylamine benzoates, terminal alkynes). Furthermore, several double ortho,ortho'-magnesiations were realized in the case of aryl oxazolines, N-aryl pyrazoles as well as 2-aryl-2H-1,2,3-triazoles by simply repeating the magnesiation/electrophile trapping sequence allowing the preparation of valuable 1,2,3-functionalized arenes.

Regioselective functionalization of aryl azoles as powerful tool for the synthesis of pharmaceutically relevant targets.

Aryl azole scaffolds are present in a wide range of pharmaceutically relevant molecules. Their ortho-selective metalation at the aryl ring is challenging, due to the competitive metalation of the more acidic heterocycle. Seeking a practical access to a key Active Pharmaceutical Ingredient (API) intermediate currently in development, we investigated the metalation of 1-aryl-1H-1,2,3-triazoles and other related heterocycles with sterically hindered metal-amide bases. We report here a room temperature and highly regioselective ortho-magnesiation of several aryl azoles using a tailored magnesium amide, TMPMgBu (TMP = 2,2,6,6-tetramethylpiperidyl) in hydrocarbon solvents followed by an efficient Pd-catalyzed arylation. This scalable and selective reaction allows variation of the initial substitution pattern of the aryl ring, the nature of the azole moiety, as well as the nature of the electrophile. This versatile method can be applied to the synthesis of bioactive azole derivatives and complements existing metal-mediated ortho-functionalizations.

Intragenic recruitment of NF-κB drives splicing modifications upon activation by the oncogene Tax of HTLV-1.

Chronic NF-κB activation in inflammation and cancer has long been linked to persistent activation of NF-κB-responsive gene promoters. However, NF-κB factors also massively bind to gene bodies. Here, we demonstrate that recruitment of the NF-κB factor RELA to intragenic regions regulates alternative splicing upon NF-κB activation by the viral oncogene Tax of HTLV-1. Integrative analyses of RNA splicing and chromatin occupancy, combined with chromatin tethering assays, demonstrate that DNA-bound RELA interacts with and recruits the splicing regulator DDX17, in an NF-κB activation-dependent manner. This leads to alternative splicing of target exons due to the RNA helicase activity of DDX17. Similar results were obtained upon Tax-independent NF-κB activation, indicating that Tax likely exacerbates a physiological process where RELA provides splice target specificity. Collectively, our results demonstrate a physical and direct involvement of NF-κB in alternative splicing regulation, which significantly revisits our knowledge of HTLV-1 pathogenesis and other NF-κB-related diseases.

Characterizing the interplay between gene nucleotide composition bias and splicing.

BACKGROUND: Nucleotide composition bias plays an important role in the 1D and 3D organization of the human genome. Here, we investigate the potential interplay between nucleotide composition bias and the regulation of exon recognition during splicing. RESULTS: By analyzing dozens of RNA-seq datasets, we identify two groups of splicing factors that activate either about 3200 GC-rich exons or about 4000 AT-rich exons. We show that splicing factor-dependent GC-rich exons have predicted RNA secondary structures at 5' ss and are dependent on U1 snRNP-associated proteins. In contrast, splicing factor-dependent AT-rich exons have a large number of decoy branch points, SF1- or U2AF2-binding sites and are dependent on U2 snRNP-associated proteins. Nucleotide composition bias also influences local chromatin organization, with consequences for exon recognition during splicing. Interestingly, the GC content of exons correlates with that of their hosting genes, isochores, and topologically associated domains. CONCLUSIONS: We propose that regional nucleotide composition bias over several dozens of kilobase pairs leaves a local footprint at the exon level and induces constraints during splicing that can be alleviated by local chromatin organization at the DNA level and recruitment of specific splicing factors at the RNA level. Therefore, nucleotide composition bias establishes a direct link between genome organization and local regulatory processes, like alternative splicing.

Interplay between coding and exonic splicing regulatory sequences.

The inclusion of exons during the splicing process depends on the binding of splicing factors to short low-complexity regulatory sequences. The relationship between exonic splicing regulatory sequences and coding sequences is still poorly understood. We demonstrate that exons that are coregulated by any given splicing factor share a similar nucleotide composition bias and preferentially code for amino acids with similar physicochemical properties because of the nonrandomness of the genetic code. Indeed, amino acids sharing similar physicochemical properties correspond to codons that have the same nucleotide composition bias. In particular, we uncover that the TRA2A and TRA2B splicing factors that bind to adenine-rich motifs promote the inclusion of adenine-rich exons coding preferentially for hydrophilic amino acids that correspond to adenine-rich codons. SRSF2 that binds guanine/cytosine-rich motifs promotes the inclusion of GC-rich exons coding preferentially for small amino acids, whereas SRSF3 that binds cytosine-rich motifs promotes the inclusion of exons coding preferentially for uncharged amino acids, like serine and threonine that can be phosphorylated. Finally, coregulated exons encoding amino acids with similar physicochemical properties correspond to specific protein features. In conclusion, the regulation of an exon by a splicing factor that relies on the affinity of this factor for specific nucleotide(s) is tightly interconnected with the exon-encoded physicochemical properties. We therefore uncover an unanticipated bidirectional interplay between the splicing regulatory process and its biological functional outcome.

Preparation of 2'-Deoxy-2'-spirocyclopropylcytidine via an Alternative Cyclopropanation Reaction.

Herein we report the preparation of 2'-deoxy-2'-spirocyclopropylcytidine via an alternative cyclopropanation reaction starting from γ-silyl tertiary alcohols. Activation of the hydroxyl function with thionyl chloride in the presence of 4-DMAP allows the ring-closing step under mild conditions. Participation of the uracil moiety in the cyclization step is proposed.

Exploiting Synergistic Effects in Organozinc Chemistry for Direct Stereoselective C-Glycosylation Reactions at Room Temperature.

Pairing a range of bis(aryl) zinc reagents ZnAr2 with the stronger Lewis acidic [(ZnArF2 )] (ArF =C6 F5 ), enables highly stereoselective cross-coupling between glycosyl bromides and ZnAr2 without the use of a transition metal. Reactions occur at room temperature with excellent levels of stereoselectivity, where ZnArF2 acts as a non-coupling partner although its presence is crucial for the execution of the C(sp2 )-C(sp3 ) bond formation process. Mechanistic studies have uncovered a unique synergistic partnership between the two zinc reagents, which circumvents the need for transition-metal catalysis or forcing reaction conditions. Key to the success of the coupling is the avoidance of solvents that act as Lewis bases versus diarylzinc compounds (e.g. THF).

Nucleosome eviction in mitosis assists condensin loading and chromosome condensation.

Condensins associate with DNA and shape mitotic chromosomes. Condensins are enriched nearby highly expressed genes during mitosis, but how this binding is achieved and what features associated with transcription attract condensins remain unclear. Here, we report that condensin accumulates at or in the immediate vicinity of nucleosome-depleted regions during fission yeast mitosis. Two transcriptional coactivators, the Gcn5 histone acetyltransferase and the RSC chromatin-remodelling complex, bind to promoters adjoining condensin-binding sites and locally evict nucleosomes to facilitate condensin binding and allow efficient mitotic chromosome condensation. The function of Gcn5 is closely linked to condensin positioning, since neither the localization of topoisomerase II nor that of the cohesin loader Mis4 is altered in gcn5 mutant cells. We propose that nucleosomes act as a barrier for the initial binding of condensin and that nucleosome-depleted regions formed at highly expressed genes by transcriptional coactivators constitute access points into chromosomes where condensin binds free genomic DNA.

Cine Substitution with Arylzinc Reagents: Scope and Mechanistic Studies.

The unexpected ability of arylzinc reagents bearing electron-donating substituents to react in a Friedel-Crafts fashion (cine) with electrophiles like perpivaloylated glucoside bromide and benzhydryl bromides in competition with organometallic coupling (ipso) is shown. The stereoelectronic factors required to promote the cine reactivity versus the classical ipso, and the mechanism of this alternative pathway, have been investigated. The Wheland intermediate is deprotonated intramolecularly in a 1,2-shift but also in a longer-range shift, leaving in this case the C-Zn untouched. In the latter case, it is possible to take advantage of this result for further functionalization.

The proto-oncogenic protein TAL1 controls TGF-ß1 signaling through interaction with SMAD3.

TGF-ß1 is involved in many aspects of tissue development and homeostasis including hematopoiesis. The TAL1 transcription factor is also an important player of this latter process and is expressed very early in the myeloid and erythroid lineages. We previously established a link between TGF-ß1 signaling and TAL1 by showing that the cytokine was able to induce its proteolytic degradation by the ubiquitin proteasome pathway. In this manuscript we show that TAL1 interacts with SMAD3 that acts in the pathway downstream of TGF-ß1 association with its receptor. TAL1 expression strengthens the positive or negative effect of SMAD3 on various genes. Both transcription factors activate the inhibitory SMAD7 factor through the E box motif present in its transcriptional promoter. DNA precipitation assays showed that TAL1 present in Jurkat or K562 cells binds to this SMAD binding element in a SMAD3 dependent manner. SMAD3 and TAL1 also inhibit several genes including ID1, hTERT and TGF-ß1 itself. In this latter case TAL1 and SMAD3 can impair the positive effect exerted by E47. Our results indicate that TAL1 expression can modulate TGF-ß1 signaling by interacting with SMAD3 and by increasing its transcriptional properties. They also suggest the existence of a negative feedback loop between TAL1 expression and TGF-ß1 signaling.

α-C-Glycosides via syn Opening of 1,2-Anhydro Sugars with Organozinc Compounds in Toluene/n-Dibutyl Ether.

The diastereoselective addition of organozinc species to 1,2-anhydro sugars in toluene/n-dibutyl ether solvent is reported. Compared to the existing methods, the reaction proceeds at 0 °C, and only a slight excess of nucleophile is required to achieve good yields. Scope was assessed with different O-protected glycals along with various nucleophiles (aryl, alkynyl). This methodology was applied to the synthesis of the α-anomer of canagliflozin.

Stereoselective C-glycosylation reactions with arylzinc reagents.

A general, transition-metal-free, highly stereoselective cross-coupling reaction between glycosyl bromides and various arylzinc reagents leading to ß-arylated glycosides is reported. The stereoselectivity of the reaction is explained by invoking anchimeric assistance via a bicyclic intermediate. Stereochemical probes confirm the participation of the 2-pivaloyloxy group. Finally, this new method was applied to a short and efficient stereoselective synthesis of Dapagliflozin and Canagliflozin.

Practical synthesis of (2'R)-2'-deoxy-2'-C-methyluridine by highly diastereoselective homogeneous hydrogenation.

Diastereoselective hydrogenation of 2'-deoxy-2'-exo-methyleneuridine was carried out under homogeneous conditions using a low loading of a chiral Rh catalyst. This, coupled with improvements in the synthesis of the substrate, allowed the smooth pilot plant preparation of the title compound on >10 kg scale.