Bryan J. Venters

Nucleosome organization in the Drosophila genome

Comparative genomics of nucleosome positions provides a powerful means for understanding how the organization of chromatin and the transcription machinery co-evolve. Here we produce a high-resolution reference map of H2A.Z and bulk nucleosome locations across the genome of the fly Drosophila melanogaster and compare it to that from the yeast Saccharomyces cerevisiae. Like Saccharomyces, Drosophila nucleosomes are organized around active transcription start sites in a canonical -1, nucleosome-free region, +1 arrangement. However, Drosophila does not incorporate H2A.Z into the -1 nucleosome and does not bury its transcriptional start site in the +1 nucleosome. At thousands of genes, RNA polymerase II engages the +1 nucleosome and pauses. How the transcription initiation machinery contends with the +1 nucleosome seems to be fundamentally different across major eukaryotic lines.

NELF and GAGA Factor Are Linked to Promoter-Proximal Pausing at Many Genes in Drosophila

ABSTRACT Recent analyses of RNA polymerase II (Pol II) revealed that Pol II is concentrated at the promoters of many active and inactive genes. NELF causes Pol II to pause in the promoter-proximal region of the hsp70 gene in Drosophila melanogaster. In this study, genome-wide location analysis (chromatin immunoprecipitation-microarray chip [ChIP-chip] analysis) revealed that NELF is concentrated at the 5′ ends of 2,111 genes in Drosophila cells. Permanganate genomic footprinting was used to determine if paused Pol II colocalized with NELF. Forty-six of 56 genes with NELF were found to have paused Pol II. Pol II pauses 30 to 50 nucleotides downstream from transcription start sites. Analysis of DNA sequences in the vicinity of paused Pol II identified a conserved DNA sequence that probably associates with TFIID but detected no evidence of RNA secondary structures or other conserved sequences that might directly control elongation. ChIP-chip experiments indicate that GAGA factor associates with 39% of the genes that have NELF. Surprisingly, NELF associates with almost one-half of the most highly expressed genes, indicating that NELF is not necessarily a repressor of gene expression. NELF-associated pausing of Pol II might be an obligatory but sometimes transient checkpoint during the transcription cycle.

How eukaryotic genes are transcribed

Regulation of eukaryotic gene expression is far more complex than one might have imagined 30 years ago. However, progress towards understanding gene regulatory mechanisms has been rapid and comprehensive, which has made the integration of detailed observations into broadly connected concepts a challenge. This review attempts to integrate the following concepts: (1) a well-defined organization of nucleosomes and modification states at most genes; (2) regulatory networks of sequence-specific transcription factors; (3) chromatin remodeling coupled to promoter assembly of the general transcription factors and RNA polymerase II; and (4) phosphorylation states of RNA polymerase II coupled to chromatin modification states during transcription. The wealth of new insights arising from the tools of biochemistry, genomics, cell biology, and genetics is providing a remarkable view into the mechanics of gene regulation.

Histone Arg Modifications and p53 Regulate the Expression of OKL38, a Mediator of Apoptosis

Protein Arg methyltransferases function as coactivators of the tumor suppressor p53 to regulate gene expression. Peptidylarginine deiminase 4 (PAD4/PADI4) counteracts the functions of protein Arg methyltransferases in gene regulation by deimination and demethylimination. Here we show that the expression of a tumor suppressor gene, OKL38, is activated by the inhibition of PAD4 or the activation of p53 following DNA damage. Chromatin immunoprecipitation assays showed a dynamic change of p53 and PAD4 occupancy and histone Arg modifications at the OKL38 promoter during DNA damage, suggesting a direct role of PAD4 and p53 in the expression of OKL38. Furthermore, we found that OKL38 induces apoptosis through localization to mitochondria and induction of cytochrome c release. Together, our studies identify OKL38 as a novel p53 target gene that is regulated by PAD4 and plays a role in apoptosis.

Genomic organization of human transcription initiation complexes

The human genome is pervasively transcribed, yet only a small fraction is coding. Here we address whether this non-coding transcription arises at promoters, and detail the interactions of initiation factors TATA box binding protein (TBP), transcription factor IIB (TFIIB) and RNA polymerase (Pol) II. Using ChIP-exo (chromatin immunoprecipitation with lambda exonuclease digestion followed by high-throughput sequencing), we identify approximately 160,000 transcription initiation complexes across the human K562 genome, and more in other cancer genomes. Only about 5% associate with messenger RNA genes. The remainder associates with non-polyadenylated non-coding transcription. Regardless, Pol II moves into a transcriptionally paused state, and TBP and TFIIB remain at the promoter. Remarkably, the vast majority of locations contain the four core promoter elements— upstream TFIIB recognition element (BREu), TATA, downstream TFIIB recognition element (BREd), and initiator element (INR)—in constrained positions. All but the INR also reside at Pol III promoters, where TBP makes similar contacts. This comprehensive and high-resolution genome-wide detection of the initiation machinery produces a consolidated view of transcription initiation events from yeast to humans at Pol II/III TATA-containing/TATA-less coding and non-coding genes.

Discovery of antibacterial cyclic peptides that inhibit the ClpXP protease

A method to rapidly screen libraries of cyclic peptides in vivo for molecules with biological activity has been developed and used to isolate cyclic peptide inhibitors of the ClpXP protease. Fluorescence activated cell sorting was used in conjunction with a fluorescent reporter to isolate cyclic peptides that inhibit the proteolysis of tmRNA‐tagged proteins in Escherichia coli. Inhibitors shared little sequence similarity and interfered with unexpected steps in the ClpXP mechanism in vitro. One cyclic peptide, IXP1, inhibited the degradation of unrelated ClpXP substrates and has bactericidal activity when added to growing cultures of Caulobacter crescentus, a model organism that requires ClpXP activity for viability. The screen used here could be adapted to identify cyclic peptide inhibitors of any enzyme that can be expressed in E. coli in conjunction with a fluorescent reporter.

p73 Is Required for Multiciliogenesis and Regulates the Foxj1-Associated Gene Network

We report that p73 is expressed in multiciliated cells (MCCs), is required for MCC differentiation, and directly regulates transcriptional modulators of multiciliogenesis. Loss of ciliary biogenesis provides a unifying mechanism for many phenotypes observed in p73 knockout mice including hydrocephalus; hippocampal dysgenesis; sterility; and chronic inflammation/infection of lung, middle ear, and sinus. Through p73 and p63 ChIP-seq using murine tracheal cells, we identified over 100 putative p73 target genes that regulate MCC differentiation and homeostasis. We validated Foxj1, a transcriptional regulator of multiciliogenesis, and many other cilia-associated genes as direct target genes of p73 and p63. We show p73 and p63 are co-expressed in a subset of basal cells and suggest that p73 marks these cells for MCC differentiation. In summary, p73 is essential for MCC differentiation, functions as a critical regulator of a transcriptome required for MCC differentiation, and, like p63, has an essential role in development of tissues.

Proteolytic Adaptor for Transfer-Messenger RNA-Tagged Proteins from α-Proteobacteria

We have identified an analog of SspB, the proteolytic adaptor for transfer-messenger RNA (tmRNA)-tagged proteins, in Caulobacter crescentus. C. crescentus SspB shares limited sequence similarity with Escherichia coli SspB but binds the tmRNA tag in vitro and is required for optimal proteolysis of tagged proteins in vivo.

Genomic Organization of Human Transcription Initiation Complexes

A repertoire of transcription initiation factors engage the core promoter of mRNA genes to recruit RNA polymerase (Pol) II to initiate transcription, yet their precise spatial organization remains unclear. Using ChIP-exo, here we detail the interactions and genomic organization of initiation factors TBP, TFIIB, and Pol II at mRNA genes and within CpG islands. We find that when Pol II moves into a transcriptionally paused state, TBP/TFIIB remain at the promoter. We show that TBP and TFIIB bound to the core promoter at two separate, resolvable locations that coincided with sites of divergent transcription initiation. We also examine the precise binding of TBP at Pol III transcribed tRNA genes. We find that TBP crosslinked to tRNA genes in a similar manner as at Pol II transcribed genes. This comprehensive and high resolution genome-wide detection of the initiation machinery produces a consolidated view of transcription initiation events humans at Pol II coding and Pol III transcribed tRNA genes.

Genome-Wide Transcriptional Dependence on Conserved Regions of Mot1

ABSTRACT TATA binding protein (TBP) plays a central role in transcription complex assembly and is regulated by a variety of transcription factors, including Mot1. Mot1 is an essential protein in Saccharomyces cerevisiae that exerts both negative and positive effects on transcription via interactions with TBP. It contains two conserved regions important for TBP interactions, another conserved region that hydrolyzes ATP to remove TBP from DNA, and a fourth conserved region with unknown function. Whether these regions contribute equally to transcriptional regulation genome-wide is unknown. Here, we employ a transient-replacement assay using deletion derivatives in the conserved regions of Mot1 to investigate their contributions to gene regulation throughout the S. cerevisiae genome. These four regions of Mot1 are essential for growth and are generally required for all Mot1-regulated genes. Loss of the ATPase region, but not other conserved regions, caused TBP to redistribute away from a subset of Mot1-inhibited genes, leading to decreased expression of those genes. A corresponding increase in TBP occupancy and expression occurred at another set of genes that are normally Mot1 independent. The data suggest that Mot1 uses ATP hydrolysis to redistribute accessible TBP away from intrinsically preferred sites to other sites of intrinsically low preference.