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

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

Risk Factors for Introduction of Bovine Herpesvirus 1 (BoHV-1) Into Cattle Herds: A Systematic European Literature Review

Given that bovine herpesvirus 1 (BoHV-1) -the causative agent of Infectious Bovine Rhinotracheitis (IBR)- is still endemic in most European countries, BoHV-1 free herds are subject to a considerable risk of (re)introduction of the virus. The aim of this literature review was to describe published, quantified risk factors that are relevant for the introduction of BoHV-1. The risk factors described in this study can be used as input for modeling eradication scenarios and for communication on biosecurity measures to stakeholders. A literature search was conducted in November 2020 in two major online search databases, PubMed and Web of Science. The search criteria “risk factor” combined with different synonyms for BoHV-1 were explored, which resulted in 564 hits. Only studies performed in Europe, written in Dutch, English, French, German or Spanish with an English summary and that quantified risk factors for introduction of BoHV-1 into cattle herds were included. Studies had to quantify the risk factors with crude odds ratios (OR), an estimate of the chance of a particular event occurring in an exposed group to a non-exposed group. After checking for duplicates and excluding articles that did not meet the inclusion criteria, 12 publications remained for this review. Risk factors were classified into seven groups, i.e., herd characteristics, management, animal characteristics, purchase, direct animal contact, neighborhood and indirect transmission routes. Most relevant factors for introduction of BoHV-1 into cattle herds include herd size, purchase of cattle, cattle density, age of cattle, distance to neighboring cattle herds and professional visitors. Together with other direct and indirect animal contacts, these factors are important when elimination of BoHV-1 is considered. A closed farming system and protective clothing for professional visitors can eliminate the major routes of introduction of BoHV-1 in cattle herds. To the best of our knowledge, this is the first systematic review solely focussing on measures that can be taken to control introduction of BoHV-1 into cattle herds. Besides testing, focus on managing these (biosecurity) factors will decrease the risk of introducing the virus

Presence of H2AZ, H3K9/K14ac and a TATA sequence enhances binding of endogenous TFIID to recombinant nucleosomes.

<p>(A) Analysis of pulldowns with recombinant nucleosomes immobilized on magnetic streptavidin coated beads and GST-TAF3 (PHD). Proteins are visualized by silver stain. (B) Scatter plot of SILAC ratios for H3K4_Cme3/K27_Cme3 versus non-modified nucleosome interacting proteins. In the upper right corner significant outliers are depicted and labeled in grey based on box plots analysis. (C) Immunoblot analysis of recombinant nucleosomes with the indicated antibodies showing the presence of modifications or variants. (D) TFIID binds to H3K4_Cme3 nucleosomes independently of H2A.Z. Immunoblot analysis of eluted proteins using indicated antibodies. (E) TFIID binding is enhanced by histone H3 acetylation. Immunoblot analysis of eluted proteins using indicated antibodies. TAF5 antibody signal is quantified relative to the H3K_C4me3 pulldown. (F) Schematic representation of the nucleosome with the introduced TATA sequence and biotin group indicated. (G) Combination of TATA DNA and H3K14 acetylation on H3K4_Cme3 nucleosomes increases the interaction with TFIID. Immunoblot analysis of eluted proteins using indicated antibodies. TAF5 antibody signal is quantified relative to the H3K_C4me3 pulldown.</p

Stoichiometry analysis of endogenous TFIID.

<p>(A) Immunoblot analysis of GFP-TAF3, GFP-TAF3 M882A and GFP-TAF5 expression after 24 hours of doxycycline induction with the indicated antibodies (right). Endogenous proteins and GFP-fusions are indicated on the left. (B) Identification of interacting proteins for GFP-TAF5 by volcano plot. The ratio of identified proteins in all fusion lines over wildtype in label-free quantification are plotted against the -log10 of the false discovery rate (FDR) calculated by a permutation-based FDR adapted t-test. In all experiments FDR was set to <0.05 and S₀ = 1.5. Significant outliers are labeled. (C) Bar graphs indicate the stoichiometry of TFIID subunits (indicated at bottom) relative to TAF6. Black dashed line indicates a ratio to the total TAF6 protein. Error bars indicate the standard deviations of the technical triplicate. (D) Identification of interacting proteins for GFP-TAF3 by volcano plot. (E) Bar graphs indicate the stoichiometry of TFIID subunits (indicated at bottom) relative to TAF6 in GFP-TAF3 (E) and GFP-TAF3 M882A (F) purifications.</p

H3K4_Cme3 nucleosomes bind endogenous TFIID and recombinant TAF3.

<p>(A) Pulldown with the indicated biotinylated peptides using streptavidin coated beads incubated with GST-TAF3 PHD protein lysates. Proteins are visualized using Coomassie blue staining. (B) Immunoblot analysis of endogenous TAF5 binding to immobilized recombinant nucleosomes with the indicated MLA modification. Histones are visualized using Coomassie blue staining. (C) Workflow as applied for quantitative analysis of nucleosome interactors. In brief, heavy and light labeled extracts are used for pull-downs with immobilized, differentially modified nucleosomes. Experiments are also performed with a label swap. Eluted proteins are measured using LC-MS/MS. Enriched proteins in both experiments are selected based on box plot statistics. (D) Scatter plot of SILAC ratios for H3K4_Cme3 versus non-modified nucleosome interacting proteins. Upper right corner significant outliers are depicted and labeled based on the box plot analysis.</p

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

The role of histone modifications in regulating transcriptional activation remains incompletely understood. Here, the mitotic phosphorylation of H3T3 by haspin kinase results in the release of H3K4me3-mediated binding of TFIID from chromosomes thereby inhibiting transcription

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