Structural insight into the TFIIE–TFIIH interaction: TFIIE and p53 share the binding region on TFIIH

RNA polymerase II and general transcription factors (GTFs) assemble on a promoter to form a transcription preinitiation complex (PIC). Among the GTFs, TFIIE recruits TFIIH to complete the PIC formation and regulates enzymatic activities of TFIIH. However, the mode of binding between TFIIE and TFIIH is poorly understood. Here, we demonstrate the specific binding of the C-terminal acidic domain (AC-D) of the human TFIIEα subunit to the pleckstrin homology domain (PH-D) of the human TFIIH p62 subunit and describe the solution structures of the free and PH-D-bound forms of AC-D. Although the flexible N-terminal acidic tail from AC-D wraps around PH-D, the core domain of AC-D also interacts with PH-D. AC-D employs an entirely novel binding mode, which differs from the amphipathic helix method used by many transcriptional activators. So the binding surface between PH-D and AC-D is much broader than the specific binding surface between PH-D and the p53 acidic fragments. From our in vitro studies, we demonstrate that this interaction could be a switch to replace p53 with TFIIE on TFIIH in transcription

Novel Self-Transmissible and Broad-Host-Range Plasmids Exogenously Captured From Anaerobic Granules or Cow Manure

Novel self-transmissible plasmids were exogenously captured from environmental samples by triparental matings with pBBR1MCS-2 as a mobilizable plasmid and Pseudomonas resinovorans as a recipient. A total of 272 recipients were successfully obtained as plasmid host candidates from granules of an anaerobic methane fermentation plant and from cow manure. The whole nucleotide sequences of six plasmids were determined, including one IncP-1 plasmid (pSN1104-59), four PromA-like plasmids (pSN1104-11, pSN1104-34, pSN0729-62, and pSN0729-70), and one novel plasmid (pSN1216-29), whose incompatibility group has not been previously identified. No previously known antibiotic resistance genes were found in these plasmids. In-depth phylogenetic analyses showed that the PromA-like plasmids belong to subgroups of PromA (designated as PromAγ and PromAδ) different from previously proposed subgroups PromAα and PromAβ. Twenty-four genes were identified as backbone genes by comparisons with other PromA plasmids. The nucleotide sequences of pSN1216-29 share high identity with those found in clinical isolates. A minireplicon of pSN1216-29 was successfully constructed from repA encoding a replication initiation protein and oriV. All the captured plasmids were found to have a broad host range and could be transferred to and replicated in different classes of Proteobacteria. Notably, repA and oriV of pSN1216-29 showed high similarity with one of two replication systems of pSRC119-A/C, known as a plasmid with multidrug resistance genes found in Salmonella enterica serovar Senftenberg. Our findings suggest that these “cryptic” but broad-host-range plasmids may be important for spreading several genes as “vehicles” in a wider range of bacteria in natural environments

CDK19-related disorder results from both loss-of-function and gain-of-function de novo missense variants

Purpose To expand the recent description of a new neurodevelopmental syndrome related to alterations in CDK19. Methods Individuals were identified through international collaboration. Functional studies included autophosphorylation assays for CDK19 Gly28Arg and Tyr32His variants and in vivo zebrafish assays of the CDK19(G28R) and CDK19(Y32H). Results We describe 11 unrelated individuals (age range: 9 months to 14 years) with de novo missense variants mapped to the kinase domain of CDK19, including two recurrent changes at residues Tyr32 and Gly28. In vitro autophosphorylation and substrate phosphorylation assays revealed that kinase activity of protein was lower for p.Gly28Arg and higher for p.Tyr32His substitutions compared with that of the wild-type protein. Injection of CDK19 messenger RNA (mRNA) with either the Tyr32His or the Gly28Arg variants using in vivo zebrafish model significantly increased fraction of embryos with morphological abnormalities. Overall, the phenotype of the now 14 individuals with CDK19-related disorder includes universal developmental delay and facial dysmorphism, hypotonia (79%), seizures (64%), ophthalmologic anomalies (64%), and autism/autistic traits (56%). Conclusion CDK19 de novo missense variants are responsible for a novel neurodevelopmental disorder. Both kinase assay and zebrafish experiments showed that the pathogenetic mechanism may be more diverse than previously thought.Peer reviewe

The HIRA complex subunit Hip3 plays important roles in the silencing of meiosis-specific genes in Schizosaccharomyces pombe.

BACKGROUND: The control of gene expression is essential for growth and responses to environmental changes in various organisms. It is known that some meiosis-specific genes are silenced during mitosis and expressed upon nitrogen starvation in Schizosaccharomyces pombe. When the factors responsible for this regulation were studied, a hip3 mutant was isolated via discovery of a defect in the transcriptional repression of meiosis-specific genes. Hip3 is a subunit of the HIRA (histone regulatory complex A) complex, which consists of four subunits (Hip1, Hip3, Hip4 and Slm9) and acts as a histone chaperone that is independent of DNA replication. METHODOLOGY/PRINCIPAL FINDINGS: In a search for mutants, the meiosis-specific gene SPCC663.14c(+) was identified by screening for genes that are silenced during mitosis and induced upon nitrogen starvation. A reporter plasmid that expresses the ura4(+) gene driven by the SPCC663.14c(+) promoter was constructed. Screening for suppressor mutants was then carried out in nitrogen-rich medium without uracil. A mutant with a mutation in the hip3(+) gene was isolated and named hip3-1. This mutation alleviated the transcriptional repression of the ura4(+) gene on the reporter plasmid and of the endogenous SPCC663.14c(+) gene in the presence of nitrogen. A ChIP assay revealed that RNA polymerase II (Pol II) and TFIIE were enriched at the SPCC663.14c(+) locus, whereas the levels of histone H3 were decreased in hip3-1 cells. Intriguingly, histone H3 was heavily modified at the SPCC663.14c(+) locus in hip3-1 cells; these modifications included tri-methylation and acetylation of H3 lysine 9 (H3K9), mono-methylation of H3 arginine 2 (H3R2), and tri-methylation of H3 lysine 4 (H3K4). In addition, the tri-methylation of H3K9 and H3K4 were strongly elevated in hip3-1 mutants. CONCLUSIONS: Taken together, these results indicate that Hip3 plays important roles in the control of histone modifications at meiosis-specific gene loci and induces their transcriptional repression

<i>S. pombe</i> strains used in this study.

<p><i>S. pombe</i> strains used in this study.</p

Interaction between HA-Asf1 and Hip3-1<i>-</i>flag.

<p>The pREP42-HA-Asf1 plasmid was transfected into Hip3-flag- and hip3-1-flag-expressing strains. Whole-cell extract (WCE) was immunoprecipitated using anti-FLAG M2 agarose (anti-flag IP) and analyzed by western blotting with anti-HA (3F10) and horseradish peroxidase (HRP)-conjugated anti-rat antibodies.</p