RNA polymerase II pausing during development (original) (raw)

Modifications of RNA polymerase II are pivotal in regulating gene expression states

EMBO reports, 2009

The regulation of gene expression programmes is essential for the generation of diverse cell types during development and for adaptation to environmental signals. RNA polymerase II (RNAPII) transcribes genetic information and coordinates the recruitment of accessory proteins that are responsible for the establishment of active chromatin states and transcript maturation. RNAPII is posttranslationally modified at active genes during transcription initiation, elongation and termination, and thereby recruits specific histone and RNA modifiers. RNAPII complexes are also located at silent genes in promoter-proximal paused configurations that provide dynamic transcriptional regulation downstream from initiation. In embryonic stem cells, silent developmental regulator genes that are repressed by Polycomb are associated with a form of RNAPII that can elongate through coding regions but that lacks the post-translational modifications that are important for coupling RNA synthesis to co-transcriptional maturation. Here, we discuss the mechanisms through which the transcription of silent genes might be dissociated from productive expression, and the sophisticated interplay between the transcriptional machinery, Polycomb repression and RNA processing.

Control of Transcriptional Elongation by RNA Polymerase II: A Retrospective

The origins of our current understanding of control of transcription elongation lie in pioneering experiments that mapped RNA polymerase II on viral and cellular genes. These studies first uncovered the surprising excess of polymerase molecules that we now know to be situated at the at the 5' ends of most genes in multicellular organisms. The pileup of pol II near transcription start sites reflects a ubiquitous bottle-neck that limits elongation right at the start of the transcription elongation. Subsequent seminal work identified conserved protein factors that positively and negatively control the flux of polymerase through this bottle-neck, and make a major contribution to control of gene expression.

Regulating RNA polymerase pausing and transcription elongation in embryonic stem cells

Genes & Development, 2011

Transitions between pluripotent stem cells and differentiated cells are executed by key transcription regulators. Comparative measurements of RNA polymerase distribution over the genome's primary transcription units in different cell states can identify the genes and steps in the transcription cycle that are regulated during such transitions. To identify the complete transcriptional profiles of RNA polymerases with high sensitivity and resolution, as well as the critical regulated steps upon which regulatory factors act, we used genome-wide nuclear run-on (GRO-seq) to map the density and orientation of transcriptionally engaged RNA polymerases in mouse embryonic stem cells (ESCs) and mouse embryonic fibroblasts (MEFs). In both cell types, progression of a promoter-proximal, paused RNA polymerase II (Pol II) into productive elongation is a rate-limiting step in transcription of~40% of mRNA-encoding genes. Importantly, quantitative comparisons between cell types reveal that transcription is controlled frequently at paused Pol II's entry into elongation. Furthermore, ''bivalent'' ESC genes (exhibiting both active and repressive histone modifications) bound by Polycomb group complexes PRC1 (Polycomb-repressive complex 1) and PRC2 show dramatically reduced levels of paused Pol II at promoters relative to an average gene. In contrast, bivalent promoters bound by only PRC2 allow Pol II pausing, but it is confined to extremely 59 proximal regions. Altogether, these findings identify rate-limiting targets for transcription regulation during cell differentiation.

RNA polymerase stalling at developmental control genes in the Drosophila melanogaster embryo

Nature genetics, 2007

It is widely assumed that the key rate-limiting step in gene activation is the recruitment of RNA polymerase II (Pol II) to the core promoter. Although there are well-documented examples in which Pol II is recruited to a gene but stalls, a general role for Pol II stalling in development has not been established. We have carried out comprehensive Pol II chromatin immunoprecipitation microarray (ChIP-chip) assays in Drosophila embryos and identified three distinct Pol II binding behaviors: active (uniform binding across the entire transcription unit), no binding, and stalled (binding at the transcription start site). The notable feature of the approximately 10% genes that are stalled is that they are highly enriched for developmental control genes, which are either repressed or poised for activation during later stages of embryogenesis. We propose that Pol II stalling facilitates rapid temporal and spatial changes in gene activity during development.

RNA polymerase II pauses and associates with pre-mRNA processing factors at both ends of genes

Nature Structural & Molecular Biology, 2007

We investigated co-transcriptional recruitment of pre-mRNA processing factors to human genes. Capping factors associate with paused RNA pol II at the 5′ ends of quiescent genes. They also track throughout actively transcribed genes, and accumulate with paused polymerase in the 3′ flanking region. 3′ processing factors CstF and CPSF are maximally recruited 0.5-1.5 kb downstream of poly (A) sites where they coincide with capping factors, Spt5, and Ser2 hyperphosphorylated, paused pol II. 3′ end processing factors also localize at transcription start sites, and this early recruitment is enhanced after polymerase arrest with DRB. These results suggest that promoters may help specify recruitment of 3′ end processing factors. We propose a dual pausing model where elongation arrests near the transcription start site and in the 3′ flank to allow co-transcriptional processing by factors recruited to the pol II ternary complex.

RNA polymerase is poised for activation across the genome

Nature Genetics, 2007

Regulation of gene expression is integral to the development and survival of all organisms. Transcription begins with the assembly of a pre-initiation complex at the gene promoter 1 , followed by initiation of RNA synthesis and the transition to productive elongation 2-4 . In many cases, recruitment of RNA polymerase II (Pol II) to a promoter is necessary and sufficient for activation of genes. However, there are a few notable exceptions to this paradigm, including heat shock genes and several proto-oncogenes, whose expression is attenuated by regulated stalling of polymerase elongation within the promoter-proximal region 5-13 . To determine the importance of polymerase stalling for transcription regulation, we carried out a genome-wide search for Drosophila melanogaster genes with Pol II stalled within the promoter-proximal region. Our data show that stalling is widespread, occurring at hundreds of genes that respond to stimuli and developmental signals. This finding indicates a role for regulation of polymerase elongation in the transcriptional responses to dynamic environmental and developmental cues.

Transition from initiation to promoter proximal pausing requires the CTD of RNA polymerase II

Nucleic Acids Research, 2005

The C-terminal domain (CTD) of mammalian RNA polymerase II consists of 52 repeats of the consensus hepta-peptide YSPTSPS, and links transcription to the processing of pre-mRNA. Although Pol II with a CTD shortened to five repeats (Pol II D5) is transcriptionally inactive on chromatin templates, it is not clear whether CTD is required for promoter recognition in vivo. Here, we demonstrate that in the context of chromatin, Pol II D5 can bind to the c-myc promoter with the same efficiency as wild type Pol II. However, Pol II D5 does not form a stable initiation complex, and does not transcribe promoter proximal sequences. Fluorescence recovery after photobleaching (FRAP) experiments with cells expressing enhanced green fluorescent protein (EGFP)-tagged D5 or wildtype Pol II revealed a single, highly mobile Pol II D5 fraction whereas wildtype Pol II yielded less mobile fractions. These data suggest that CTD is not required for promoter recognition, but rather for subsequent formation of a stable initiation complex and isomerization to an elongation competent complex.

Transcription regulation during stable elongation by a reversible halt of RNA polymerase II

2014

Regulation of RNA polymerase II (RNAPII) during transcription is essential for controlling gene expression. Here we report that the transcriptional activity of RNAPII at the Balbiani ring 2.1 gene could be halted during stable elongation in salivary gland cells of Chironomus tentans larvae for extended time periods in a regulated manner. The transcription halt was triggered by heat shock and affected all RNAPII independently of their position in the gene. During the halt, incomplete transcripts and RNAPII remained at the transcription site, the phosphorylation state of RNAPII was unaltered, and the transcription bubbles remained open. The transcription of halted transcripts was resumed upon relief of the heat shock. The observed mechanism allows cells to interrupt transcription for extended time periods and rapidly reactivate it without the need to reinitiate transcription of the complete gene. Our results suggest a so-far-unknown level of transcriptional control in eukaryotic cells.