Neuronal-specific microexon splicing of TAF1 mRNA is directly regulated by SRRM4/nSR100.

Neuronal microexons represent the most highly conserved class of alternative splicing events and their timed expression shapes neuronal biology, including neuronal commitment and differentiation. The six-nt microexon 34' is included in the neuronal form of TAF1 mRNA, which encodes the largest subunit of the basal transcription factor TFIID. In this study, we investigate the tissue distribution of TAF1-34' mRNA and protein and the mechanism responsible for its neuronal-specific splicing. Using isoform-specific RNA probes and antibodies, we observe that canonical TAF1 and TAF1-34' have different distributions in the brain, which distinguish proliferating from post-mitotic neurons. Knockdown and ectopic expression experiments demonstrate that the neuronal-specific splicing factor SRRM4/nSR100 promotes the inclusion of microexon 34' into TAF1 mRNA, through the recognition of UGC sequences in the poly-pyrimidine tract upstream of the regulated microexon. These results show that SRRM4 regulates temporal and spatial expression of alternative TAF1 mRNAs to generate a neuronal-specific TFIID complex.

Functional characterisation of a novel class of in-frame insertion variants of KRAS and HRAS.

Mutations in the RAS genes are identified in a variety of clinical settings, ranging from somatic mutations in oncology to germline mutations in developmental disorders, also known as 'RASopathies', and vascular malformations/overgrowth syndromes. Generally single amino acid substitutions are identified, that result in an increase of the GTP bound fraction of the RAS proteins causing constitutive signalling. Here, a series of 7 in-frame insertions and duplications in HRAS (n = 5) and KRAS (n = 2) is presented, resulting in the insertion of 7-10 amino acids residues in the switch II region. These variants were identified in routine diagnostic screening of 299 samples for somatic mutations in vascular malformations/overgrowth syndromes (n = 6) and in germline analyses for RASopathies (n = 1). Biophysical characterization shows the inability of Guanine Nucleotide Exchange Factors to induce GTP loading and reduced intrinsic and GAP-stimulated GTP hydrolysis. As a consequence of these opposing effects, increased RAS signalling is detected in a cellular model system. Therefore these in-frame insertions represent a new class of weakly activating clinically relevant RAS variants.

Multivalent engagement of TFIID to nucleosomes.

The process of eukaryotic transcription initiation involves the assembly of basal transcription factor complexes on the gene promoter. The recruitment of TFIID is an early and important step in this process. Gene promoters contain distinct DNA sequence elements and are marked by the presence of post-translationally modified nucleosomes. The contributions of these individual features for TFIID recruitment remain to be elucidated. Here, we use immobilized reconstituted promoter nucleosomes, conventional biochemistry and quantitative mass spectrometry to investigate the influence of distinct histone modifications and functional DNA-elements on the binding of TFIID. Our data reveal synergistic effects of H3K4me3, H3K14ac and a TATA box sequence on TFIID binding in vitro. Stoichiometry analyses of affinity purified human TFIID identified the presence of a stable dimeric core. Several peripheral TAFs, including those interacting with distinct promoter features, are substoichiometric yet present in substantial amounts. Finally, we find that the TAF3 subunit of TFIID binds to poised promoters in an H3K4me3-dependent manner. Moreover, the PHD-finger of TAF3 is important for rapid induction of target genes. Thus, fine-tuning of TFIID engagement on promoters is driven by synergistic contacts with both DNA-elements and histone modifications, eventually resulting in a high affinity interaction and activation of transcription.

A phospho/methyl switch at histone H3 regulates TFIID association with mitotic chromosomes.

Histone methylation patterns are correlated with eukaryotic gene transcription. High-affinity binding of the plant homeodomain (PHD) of TFIID subunit TAF3 to trimethylated lysine-4 of histone H3 (H3K4me3) is involved in promoter recruitment of this basal transcription factor. Here, we show that for transcription activation the PHD of TAF3 can be replaced by PHDs of other high-affinity H3K4me3 binders. Interestingly, H3K4me3 binding of TFIID and the TAF3-PHD is decreased by phosphorylation of the adjacent threonine residue (H3T3), which coincides with mitotic inhibition of transcription. Ectopic expression of the H3T3 kinase haspin repressed TAF3-mediated transcription of endogenous and of reporter genes and decreased TFIID association with chromatin. Conversely, immunofluorescence and live-cell microscopy studies showed an increased association of TFIID with mitotic chromosomes upon haspin knockdown. Based on our observations, we propose that a histone H3 phospho-methyl switch regulates TFIID-mediated transcription during mitotic progression of the cell cycle.

PHF8 targets histone methylation and RNA polymerase II to activate transcription.

Mutations in PHF8 are associated with X-linked mental retardation and cleft lip/cleft palate. PHF8 contains a plant homeodomain (PHD) in its N terminus and is a member of a family of JmjC domain-containing proteins. While PHDs can act as methyl lysine recognition motifs, JmjC domains can catalyze lysine demethylation. Here, we show that PHF8 is a histone demethylase that removes repressive histone H3 dimethyl lysine 9 marks. Our biochemical analysis revealed specific association of the PHF8 PHD with histone H3 trimethylated at lysine 4 (H3K4me3). Chromatin immunoprecipitation followed by high-throughput sequencing indicated that PHF8 is enriched at the transcription start sites of many active or poised genes, mirroring the presence of RNA polymerase II (RNAPII) and of H3K4me3-bearing nucleosomes. We show that PHF8 can act as a transcriptional coactivator and that its activation function largely depends on binding of the PHD to H3K4me3. Furthermore, we present evidence for direct interaction of PHF8 with the C-terminal domain of RNAPII. Importantly, a PHF8 disease mutant was defective in demethylation and in coactivation. This is the first demonstration of a chromatin-modifying enzyme that is globally recruited to promoters through its association with H3K4me3 and RNAPII.

Human Ccr4-Not complexes contain variable deadenylase subunits.

The Ccr4-Not complex is evolutionarily conserved and important for regulation of mRNA synthesis and decay. The composition of the yeast complex has been well described. Orthologues of the yeast Ccr4-Not components have been identified in human cells including multiple subunits with mRNA deadenylase activity. In the present study, we examine the composition of the human Ccr4-Not complex in an in-depth proteomic approach using stable cell lines expressing tagged CNOT proteins. We find at least four different variants of the human complex, consisting of seven stable core proteins and mutually exclusive associated mRNA deadenylase subunits. Interestingly, human CNOT4 is in a separate approximately 200 kDa complex. Furthermore, analyses of associated proteins indicate involvement of Ccr4-Not complexes in splicing, transport and localization of RNA molecules. Taken together, human Ccr4-Not complexes are heterogeneous in composition owing to differences in their deadenylase subunits, which may reflect the multi-functionality of these complexes in cellular processes.

Structural insight into the recognition of the H3K4me3 mark by the TFIID subunit TAF3.

Trimethylation of lysine residue K4 of histone H3 (H3K4me3) strongly correlates with active promoters for RNA polymerase II-transcribed genes. Several reader proteins, including the basal transcription factor TFIID, for this nucleosomal mark have been identified. Its TAF3 subunit specifically binds the H3K4me3 mark via its conserved plant homeodomain (PHD) finger. Here, we report the solution structure of the TAF3-PHD finger and its complex with an H3K4me3 peptide. Using a combination of NMR, mutagenesis, and affinity measurements, we reveal the structural basis of binding affinity, methylation-state specificity, and crosstalk with asymmetric dimethylation of R2. A unique local structure rearrangement in the K4me3-binding pocket of TAF3 due to a conserved sequence insertion underscores the requirement for cation-pi interactions by two aromatic residues. Interference by asymmetric dimethylation of arginine 2 suggests that a H3R2/K4 "methyl-methyl" switch in the histone code dynamically regulates TFIID-promoter association.

Selective anchoring of TFIID to nucleosomes by trimethylation of histone H3 lysine 4.

Trimethylation of histone H3 at lysine 4 (H3K4me3) is regarded as a hallmark of active human promoters, but it remains unclear how this posttranslational modification links to transcriptional activation. Using a stable isotope labeling by amino acids in cell culture (SILAC)-based proteomic screening we show that the basal transcription factor TFIID directly binds to the H3K4me3 mark via the plant homeodomain (PHD) finger of TAF3. Selective loss of H3K4me3 reduces transcription from and TFIID binding to a subset of promoters in vivo. Equilibrium binding assays and competition experiments show that the TAF3 PHD finger is highly selective for H3K4me3. In transient assays, TAF3 can act as a transcriptional coactivator in a PHD finger-dependent manner. Interestingly, asymmetric dimethylation of H3R2 selectively inhibits TFIID binding to H3K4me3, whereas acetylation of H3K9 and H3K14 potentiates TFIID interaction. Our experiments reveal crosstalk between histone modifications and the transcription factor TFIID. This has important implications for regulation of RNA polymerase II-mediated transcription in higher eukaryotes.

Involvement of the SMRT/NCoR-HDAC3 complex in transcriptional repression by the CNOT2 subunit of the human Ccr4-Not complex.

In eukaryotic cells, the Ccr4-Not complex can regulate mRNA metabolism at various levels. Previously, we showed that promoter targeting of the CNOT2 subunit resulted in strong repression of RNA polymerase II transcription, which was sensitive to the HDAC (histone deacetylase) inhibitor, trichostatin A [Zwartjes, Jayne, van den Berg and Timmers (2004) J. Biol. Chem. 279, 10848-10854]. In the present study, the cofactor requirement for CNOT2-mediated repression was investigated. We found that coexpression of SMRT (silencing mediator for retinoic acid receptor and thyroid-hormone receptor) or NCoR (nuclear hormone receptor co-repressor) in combination with HDAC3 (or HDAC5 and HDAC6) augmented the repression by CNOT2. This repressive effect is mediated by the conserved Not-Box, which resides at the C-terminus of CNOT2 proteins. We observed physical interactions of CNOT2 with several subunits of the SMRT/NCoR-HDAC3 complex. Our results show that the SMRT/NCoR-HDAC3 complex is a cofactor of CNOT2-mediated repression and suggest that transcriptional regulation by the Ccr4-Not complex involves regulation of chromatin modification.

Mutational analysis of BTAF1-TBP interaction: BTAF1 can rescue DNA-binding defective TBP mutants.

The BTAF1 transcription factor interacts with TATA-binding protein (TBP) to form the B-TFIID complex, which is involved in RNA polymerase II transcription. Here, we present an extensive mapping study of TBP residues involved in BTAF1 interaction. This shows that residues in the concave, DNA-binding surface of TBP are important for BTAF1 binding. In addition, BTAF1 interacts with residues in helix 2 on the convex side of TBP as assayed in protein-protein and in DNA-binding assays. BTAF1 drastically changes the TATA-box binding specificity of TBP, as it is able to recruit DNA-binding defective TBP mutants to both TATA-containing and TATA-less DNA. Interestingly, other helix 2 interacting factors, such as TFIIA and NC2, can also stabilize mutant TBP binding to DNA. In contrast, TFIIB which interacts with a distinct surface of TBP does not display this activity. Since many proteins contact helix 2 of TBP, this provides a molecular basis for mutually exclusive TBP interactions and stresses the importance of this structural element for eukaryotic transcription.

Solution structure of the ubiquitin-conjugating enzyme UbcH5B.

The ubiquitination pathway is the main pathway for protein degradation in eukaryotic cells. The attachment of ubiquitin to a substrate protein is catalyzed by three types of enzymes, namely a ubiquitin activating enzyme (E1), a ubiquitin-conjugating enzyme (E2), and a ubiquitin ligase (E3). Here, the structure of the human ubiquitin-conjugating enzyme (E2) UbcH5B has been solved by a combination of homology modeling, NMR relaxation data and automated NOE assignments. Comparison to E2 structures solved previously by X-ray crystallography or NMR shows in all cases the same compact fold, but differences are observed in the orientation of both N and C-terminal alpha-helices. The N-terminal helix that is involved in binding to ubiquitin ligases (E3) displays a different position, which could have consequences for precise E2-E3 recognition. In addition, multiple conformations of the side-chain of Asn77 are found in solution, which contrasts the single hydrogen-bonded conformation in the crystal structures of E2 enzymes. The possible implication of this conformational freedom of Asn77 for its catalytic function is discussed.

Structural model of the UbcH5B/CNOT4 complex revealed by combining NMR, mutagenesis, and docking approaches.

The protein CNOT4 possesses an N-terminal RING finger domain that acts as an E3 ubiquitin ligase and specifically interacts with UbcH5B, a ubiquitin-conjugating enzyme. The structure of the CNOT4 RING domain has been solved and the amino acids important for the binding to UbcH5B have been mapped. Here, the residues of UbcH5B important for the binding to CNOT4 RING domain were identified by NMR chemical shift perturbation experiments, and these data were used to generate structural models of the complex with the program HADDOCK. Together with the NMR data, additional biochemical data were included in a second docking, and comparisons of the resulting model with the structure of the c-Cbl/UbcH7 complex reveal some significant differences, notably at specific residues, and give structural insights into the E2/E3 specificity.