Widespread occurrence of non-canonical transcription termination by human RNA polymerase III

Human RNA polymerase (Pol) III-transcribed genes are thought to share a simple termination signal constituted by four or more consecutive thymidine residues in the coding DNA strand, just downstream of the RNA 3'-end sequence. We found that a large set of human tRNA genes (tDNAs) do not display any T(≥4) stretch within 50 bp of 3'-flanking region. In vitro analysis of tDNAs with a distanced T(≥4) revealed the existence of non-canonical terminators resembling degenerate T(≥5) elements, which ensure significant termination but at the same time allow for the production of Pol III read-through pre-tRNAs with unusually long 3' trailers. A panel of such non-canonical signals was found to direct transcription termination of unusual Pol III-synthesized viral pre-miRNA transcripts in gammaherpesvirus 68-infected cells. Genome-wide location analysis revealed that human Pol III tends to trespass into the 3'-flanking regions of tDNAs, as expected from extensive terminator read-through. The widespread occurrence of partial termination suggests that the Pol III primary transcriptome in mammals is unexpectedly enriched in 3'-trailer sequences with the potential to contribute novel functional ncRNAs.

Genomic Study of RNA Polymerase II and III SNAPc-Bound Promoters Reveals a Gene Transcribed by Both Enzymes and a Broad Use of Common Activators

Nouria Hernandez, David Paul Roger Romascano

SNAP(c) is one of a few basal transcription factors used by both RNA polymerase (pol) II and pol III. To define the set of active SNAP(c)-dependent promoters in human cells, we have localized genome-wide four SNAP(c) subunits, GTF2B (TFIIB), BRF2, pol II, and pol III. Among some seventy loci occupied by SNAP(c) and other factors, including pol II snRNA genes, pol III genes with type 3 promoters, and a few un-annotated loci, most are primarily occupied by either pol II and GTF2B, or pol III and BRF2. A notable exception is the RPPH1 gene, which is occupied by significant amounts of both polymerases. We show that the large majority of SNAP(c)-dependent promoters recruit POU2F1 and/or ZNF143 on their enhancer region, and a subset also recruits GABP, a factor newly implicated in SNAP(c)-dependent transcription. These activators associate with pol II and III promoters in G1 slightly before the polymerase, and ZNF143 is required for efficient transcription initiation complex assembly. The results characterize a set of genes with unique properties and establish that polymerase specificity is not absolute in vivo.

Public Library of Science2012

Genomic targets and molecular partners of the chromatin regulator KAP1

Annamaria Kauzlaric

KAP1 is an enigmatic regulatory protein, first described some twenty years ago, shown to be involved in multiple and diverse cellular functions. Specifically, it mediates tasks critical to cell growth and differentiation, pluripotency, apoptosis, gene silencing, DNA repair and genomic integrity maintenance. In line with these findings, Kap1 deletion results in embryonic lethality, and its conditional ablation impairs crucial processes such as erythropoiesis, hepatic metabolism homeostasis and stress response. Initially named also TIF1ðœ, transcriptional intermediary factor 1ðœ, KAP1 was thought to mediate mostly chromatin-related functions. Accordingly, global maps of genomic recruitment of KAP1, generated with the advent of high-throughput technologies, display binding of this protein to thousands of loci in a multiplicity of cell types. Coupling such maps with data of transcriptional perturbations detected upon Kap1 depletion, and with the protein complexes associated with KAP1, proved to be a highly efficient approach to elucidate the molecular functions of KAP1. In particular, such studies unveiled the mechanisms of KAP1-mediated repression of different classes of transposable elements (TEs), and led to important evolutionary considerations on the strategies evolved by higher organisms to restrict the activity of these entities. Through its multiple functional domains, KAP1 interacts with a broad range of proteins, and our first goal was to establish the complete set of such potential cofactors. We found marked enrichment of KAP1 in the chromatin fraction of the nucleus, and accordingly, among its associated proteins, a strong preponderance of nucleic-acid contacting proteins. We revealed that KAP1 is part of a variety of relatively low molecular weight complexes, co-existing at comparable abundances. Study of the molecular functions of the hits suggested that KAP1 could partake in processes thus far unexplored in this context, such as DNA replication and active transcription. We then pursued with the thorough characterization of genome-wide KAP1 binding sites, and found significant fractions of RNA polymerase II (PolII)-dependent promoters and RNA polymerase III (PolIII)-transcribed elements, for the majority enriched with euchromatic marks, to be targeted by KAP1. Using high-throughput techniques, we uncovered that KAP1 variably affected the PolII pausing index of defined sets of genes, and downregulated transcription of tRNA genes. Our analyses of KAP1 genomic recruitments further led us to the discovery of novel genetic entities in the mouse genome, highly related to KRAB zinc finger protein (KZFP) genes. We described possible mechanisms underlying the evolution and amplification of these sequences and more broadly of the KZFP genes family. Furthermore, our data supported the existence of a KAP1-dependent regulatory circuit taming TE-contained enhancers located in the vicinity of these genetic units, thereby regulating their expression. We explored the proposed models of KAP1 functions in euchromatic regions of the genome, and with our results we provided the most complete analysis of the interactome of KAP1 thus far reported. Moreover, we annotated new potentially protein-coding transcripts belonging to the biggest single family of transcription factors encoded in the murine and human genomes. In sum, this work brings novel pieces of information that complement the current knowledge in the field, opening the way to further molecular studies clarifying the physiological roles of KAP1 in higher vertebrates.

EPFL2017

Loss of the RNA polymerase III repressor MAF1 confers obesity resistance

Johan Auwerx, Nouria Hernandez, Norman Lionel Moullan, Rajat Singh, Gilles Willemin

MAF1 is a global repressor of RNA polymerase III transcription that regulates the expression of highly abundant noncoding RNAs in response to nutrient availability and cellular stress. Thus, MAF1 function is thought to be important for metabolic economy. Here we show that a whole-body knockout of Maf1 in mice confers resistance to diet-induced obesity and nonalcoholic fatty liver disease by reducing food intake and increasing metabolic inefficiency. Energy expenditure in Maf1(-/-) mice is increased by several mechanisms. Precursor tRNA synthesis was increased in multiple tissues without significant effects on mature tRNA levels, implying increased turnover in a futile tRNA cycle. Elevated futile cycling of hepatic lipids was also observed. Metabolite profiling of the liver and skeletal muscle revealed elevated levels of many amino acids and spermidine, which links the induction of autophagy in Maf1(-/-) mice with their extended life span. The increase in spermidine was accompanied by reduced levels of nicotinamide N-methyltransferase, which promotes polyamine synthesis, enables nicotinamide salvage to regenerate NAD(+), and is associated with obesity resistance. Consistent with this, NAD(+) levels were increased in muscle. The importance of MAF1 for metabolic economy reveals the potential for MAF1 modulators to protect against obesity and its harmful consequences.

Cold Spring Harbor Lab Press, Publications Dept2015

Genomic targets and molecular partners of the chromatin regulator KAP1

Annamaria Kauzlaric

EPFL2017