RNA polymerase III (Pol III) is tightly controlled in response to environmental cues, yet a genomic-scale picture of Pol III regulation and the role played by its repressor MAF1 is lacking. Here, we ...describe genome-wide studies in human fibroblasts that reveal a dynamic and gene-specific adaptation of Pol III recruitment to extracellular signals in an mTORC1-dependent manner. Repression of Pol III recruitment and transcription are tightly linked to MAF1, which selectively localizes at Pol III loci, even under serum-replete conditions, and increasingly targets transcribing Pol III in response to serum starvation. Combining Pol III binding profiles with EU-labeling and high-throughput sequencing of newly synthesized small RNAs, we show that Pol III occupancy closely reflects ongoing transcription. Our results exclude the long-term, unproductive arrest of Pol III on the DNA as a major regulatory mechanism and identify previously uncharacterized, differential coordination in Pol III binding and transcription under different growth conditions.
RNA polymerase I is unique among the nuclear RNA polymerases in transcribing only one set of genes, the large, tandemly repeated, ribosomal RNA genes, and thus in having to recognize a single ...promoter structure. RNA polymerase II transcribes the protein-coding genes (mRNA genes) as well as some small nuclear RNA (snRNA) genes. The RNA polymerase II promoters can be divided into a core region, defined as the minimal region capable of directing transcription in vitro, and a regulatory region. The regulatory regions are highly varied in structure, reflecting the highly varied synthesis patterns of cellular proteins and the need for exquisite and complex regulation of these patterns. The core promoters themselves come in different types that, in mRNA-encoding genes, can contain a TATA box, an initiator, a downstream promoter element, or various combinations thereof. The assembly of a functional RNA polymerase II transcription complex on a promoter consisting of just a TATA box has been extensively studied. All the factors involved in the process have been identified, and much is known about how these factors interact with DNA and with each other to recruit, eventually, RNA polymerase II (for reviews, see Orphanides et al. 1996; Woychik and Hampsey 2002). How RNA polymerase II transcription complexes assemble on TATA-less promoters is, however, not as well understood.
TFIIB-related factor 2 (Brf2) is a member of the family of TFIIB-like core transcription factors. Brf2 recruits RNA polymerase (Pol) III to type III gene-external promoters, including the U6 ...spliceosomal RNA and selenocysteine tRNA genes. Found only in vertebrates, Brf2 has been linked to tumorigenesis but the underlying mechanisms remain elusive. We have solved crystal structures of a human Brf2-TBP complex bound to natural promoters, obtaining a detailed view of the molecular interactions occurring at Brf2-dependent Pol III promoters and highlighting the general structural and functional conservation of human Pol II and Pol III pre-initiation complexes. Surprisingly, our structural and functional studies unravel a Brf2 redox-sensing module capable of specifically regulating Pol III transcriptional output in living cells. Furthermore, we establish Brf2 as a central redox-sensing transcription factor involved in the oxidative stress pathway and provide a mechanistic model for Brf2 genetic activation in lung and breast cancer.
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•Architectural conservation of TFIIB and TFIIB-related factors•Brf2 is a redox-sensing RNA polymerase III core transcription factor•Brf2 regulates cellular responses to oxidative stress•Brf2 amplification enables cancer cells to evade oxidative stress-induced apoptosis
Direct redox-sensing by the RNA polymerase III core transcription factor Brf2 couples cellular responses to oxidative stress and regulation of transcriptional output, contributing to the ability of cancer cells to evade death induced by reactive oxygen species.
Our view of the RNA polymerase III (Pol III) transcription machinery in mammalian cells arises mostly from studies of the RN5S (5S) gene, the Ad2 VAI gene, and the RNU6 (U6) gene, as paradigms for ...genes with type 1, 2, and 3 promoters. Recruitment of Pol III onto these genes requires prior binding of well-characterized transcription factors. Technical limitations in dealing with repeated genomic units, typically found at mammalian Pol III genes, have so far hampered genome-wide studies of the Pol III transcription machinery and transcriptome. We have localized, genome-wide, Pol III and some of its transcription factors. Our results reveal broad usage of the known Pol III transcription machinery and define a minimal Pol III transcriptome in dividing IMR90hTert fibroblasts. This transcriptome consists of some 500 actively transcribed genes including a few dozen candidate novel genes, of which we confirmed nine as Pol III transcription units by additional methods. It does not contain any of the microRNA genes previously described as transcribed by Pol III, but reveals two other microRNA genes, MIR886 (hsa-mir-886) and MIR1975 (RNY5, hY5, hsa-mir-1975), which are genuine Pol III transcription units.
DNA sequence variation has been associated with quantitative changes in molecular phenotypes such as gene expression, but its impact on chromatin states is poorly characterized. To understand the ...interplay between chromatin and genetic control of gene regulation, we quantified allelic variability in transcription factor binding, histone modifications, and gene expression within humans. We found abundant allelic specificity in chromatin and extensive local, short-range, and long-range allelic coordination among the studied molecular phenotypes. We observed genetic influence on most of these phenotypes, with histone modifications exhibiting strong context-dependent behavior. Our results implicate transcription factors as primary mediators of sequence-specific regulation of gene expression programs, with histone modifications frequently reflecting the primary regulatory event.
Chromatin state variation at gene regulatory elements is abundant across individuals, yet we understand little about the genetic basis of this variability. Here, we profiled several histone ...modifications, the transcription factor (TF) PU.1, RNA polymerase II, and gene expression in lymphoblastoid cell lines from 47 whole-genome sequenced individuals. We observed that distinct cis-regulatory elements exhibit coordinated chromatin variation across individuals in the form of variable chromatin modules (VCMs) at sub-Mb scale. VCMs were associated with thousands of genes and preferentially cluster within chromosomal contact domains. We mapped strong proximal and weak, yet more ubiquitous, distal-acting chromatin quantitative trait loci (cQTL) that frequently explain this variation. cQTLs were associated with molecular activity at clusters of cis-regulatory elements and mapped preferentially within TF-bound regions. We propose that local, sequence-independent chromatin variation emerges as a result of genetic perturbations in cooperative interactions between cis-regulatory elements that are located within the same genomic domain.
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•Modules of correlated molecular phenotypes represent inter-individual chromatin variation•Variable chromatin modules (VCMs) are embedded within chromosomal contact domains•VCMs are orchestrated by cis-acting genetic variation•VCMs rationalize chromatin state changes that are independent of local DNA sequence
Spatially defined chromosome regions, termed variable chromatin modules, exhibit coordinated chromatin state changes across cis-regulatory elements. Within these modules, genetic changes distal from the regulatory element itself can induce variation in chromatin patterns between individuals.
Many organisms exhibit temporal rhythms in gene expression that propel diurnal cycles in physiology. In the liver of mammals, these rhythms are controlled by transcription-translation feedback loops ...of the core circadian clock and by feeding-fasting cycles. To better understand the regulatory interplay between the circadian clock and feeding rhythms, we mapped DNase I hypersensitive sites (DHSs) in the mouse liver during a diurnal cycle. The intensity of DNase I cleavages cycled at a substantial fraction of all DHSs, suggesting that DHSs harbor regulatory elements that control rhythmic transcription. Using chromatin immunoprecipitation followed by DNA sequencing (ChIP-seq), we found that hypersensitivity cycled in phase with RNA polymerase II (Pol II) loading and H3K27ac histone marks. We then combined the DHSs with temporal Pol II profiles in wild-type (WT) and Bmal1-/- livers to computationally identify transcription factors through which the core clock and feeding-fasting cycles control diurnal rhythms in transcription. While a similar number of mRNAs accumulated rhythmically in Bmal1-/- compared to WT livers, the amplitudes in Bmal1-/- were generally lower. The residual rhythms in Bmal1-/- reflected transcriptional regulators mediating feeding-fasting responses as well as responses to rhythmic systemic signals. Finally, the analysis of DNase I cuts at nucleotide resolution showed dynamically changing footprints consistent with dynamic binding of CLOCK:BMAL1 complexes. Structural modeling suggested that these footprints are driven by a transient heterotetramer binding configuration at peak activity. Together, our temporal DNase I mappings allowed us to decipher the global regulation of diurnal transcription rhythms in the mouse liver.
Maf1
mice are lean, obesity-resistant and metabolically inefficient. Their increased energy expenditure is thought to be driven by a futile RNA cycle that reprograms metabolism to meet an increased ...demand for nucleotides stemming from the deregulation of RNA polymerase (pol) III transcription. Metabolic changes consistent with this model have been reported in both fasted and refed mice, however the impact of the fasting-refeeding-cycle on pol III function has not been examined. Here we show that changes in pol III occupancy in the liver of fasted versus refed wild-type mice are largely confined to low and intermediate occupancy genes; high occupancy genes are unchanged. However, in Maf1
mice, pol III occupancy of the vast majority of active loci in liver and the levels of specific precursor tRNAs in this tissue and other organs are higher than wild-type in both fasted and refed conditions. Thus, MAF1 functions as a chronic repressor of active pol III loci and can modulate transcription under different conditions. Our findings support the futile RNA cycle hypothesis, elaborate the mechanism of pol III repression by MAF1 and demonstrate a modest effect of MAF1 on global translation via reduced mRNA levels and translation efficiencies for several ribosomal proteins.
RNA polymerase (Pol) III is responsible for transcription of different noncoding genes in eukaryotic cells, whose RNA products have well-defined functions in translation and other biological ...processes for some, and functions that remain to be defined for others. For all of them, however, new functions are being described. For example, Pol III products have been reported to regulate certain proteins such as protein kinase R (PKR) by direct association, to constitute the source of very short RNAs with regulatory roles in gene expression, or to control microRNA levels by sequestration. Consistent with these many functions, deregulation of Pol III transcribed genes is associated with a large variety of human disorders. Here we review different human diseases that have been linked to defects in the Pol III transcription apparatus or to Pol III products imbalance and discuss the possible underlying mechanisms.
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