The RNA polymerase III transcriptome revealed by genome-wide localization and activity–occupancy relationships

Roberts, Douglas; Stewart, Allen; Huff, Jason T.; Cairns, Bradley R.

doi:10.1073/pnas.2435566100

Cited by 165 publications

(231 citation statements)

References 38 publications

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“…Tfc6 is a subunit of the recognition factor TFIIC, which has been well characterized with respect to its chromatin immunoprecipitation (ChIP) occupancy (Harismendy et al, 2003;Moqtaderi and Struhl, 2004;Roberts et al, 2003). To compare the efficacies of the four epitope tags in ChIP experiments, we performed ChIP on the tagged Tfc6 strains.…”

Section: Resultsmentioning

confidence: 99%

Expanding the repertoire of plasmids for PCR‐mediated epitope tagging in yeast

Moqtaderi

Struhl

2008

Yeast

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Epitope tagging of yeast proteins provides a convenient means of tracking proteins of interest in Western blots and immunoprecipitation experiments without the need to raise and test specific antibodies. We have constructed four plasmids for use as templates in PCR-based epitope tagging in the yeast Saccharomyces cerevisiae. These plasmids expand the range of epitopes available in a tag-URA3-tag context to include the FLAG, HSV, V5 and VSV-G epitopes. The cloning strategy used would be easily applicable to the construction of a similar tag-URA3-tag molecule for essentially any desired epitope. Oligonucleotides designed for PCR from one plasmid may be used interchangeably with any of the other template molecules to allow tagging with different epitopes without the need for new primer synthesis. We have tagged Tfc6 with each of the triple epitope tags and assessed the efficiency of these epitopes for chromatin immunoprecipitation (ChIP). For all the tagged alleles, ChIP occupancy signals are easily detectable at known Tfc6 target genes. These new tags provide additional options in experimental schemes requiring multiple tagged proteins.

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Section: Resultsmentioning

confidence: 99%

Expanding the repertoire of plasmids for PCR‐mediated epitope tagging in yeast

Moqtaderi

Struhl

2008

Yeast

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“…Indeed, Pol III transcription is, for example, elevated in cancer cells, and is globally diminished under certain conditions such as nutrient deprivation or other kinds of stress (for reviews, see White 2004;Goodfellow and White 2007;Gjidoda and Henry 2013). In yeast, a global Pol III transcription decrease upon nutrient deprivation is accompanied by a general decrease in Pol III occupancy at Pol III loci (Roberts et al 2003(Roberts et al , 2006Oficjalska-Pham et al 2006). Consistent with such global regulation, most known regulators of Pol III transcription act on general transcription factors used by all Pol III promoters such as TFIIIB or, in the case of the general Pol III repressor MAF1, on the polymerase itself (for reviews, see Geiduschek and Kassavetis 2006;Willis and Moir 2007;Ciesla and Boguta 2008).…”

Section: A Spiking Methods To Normalize Chip-seq Experimentsmentioning

confidence: 99%

Quantifying ChIP-seq data: a spiking method providing an internal reference for sample-to-sample normalization

et al. 2014

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Chromatin immunoprecipitation followed by deep sequencing (ChIP-seq) experiments are widely used to determine, within entire genomes, the occupancy sites of any protein of interest, including, for example, transcription factors, RNA polymerases, or histones with or without various modifications. In addition to allowing the determination of occupancy sites within one cell type and under one condition, this method allows, in principle, the establishment and comparison of occupancy maps in various cell types, tissues, and conditions. Such comparisons require, however, that samples be normalized. Widely used normalization methods that include a quantile normalization step perform well when factor occupancy varies at a subset of sites, but may miss uniform genome-wide increases or decreases in site occupancy. We describe a spike adjustment procedure (SAP) that, unlike commonly used normalization methods intervening at the analysis stage, entails an experimental step prior to immunoprecipitation. A constant, low amount from a single batch of chromatin of a foreign genome is added to the experimental chromatin. This “spike” chromatin then serves as an internal control to which the experimental signals can be adjusted. We show that the method improves similarity between replicates and reveals biological differences including global and largely uniform changes.

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“…ChIP was performed on livers from naïve males. Samples were crosslinked in 1% formaldehyde, neutralized with glycine, Dounce homogenized, and chromatin-immunoprecipated as described (54).…”

Section: Methodsmentioning

confidence: 99%

Reintroducing domesticated wild mice to sociality induces adaptive transgenerational effects on MUP expression

Nelson

Cauceglia

Merkley

et al. 2013

Proc. Natl. Acad. Sci. U.S.A.

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When brought into captivity, wild animals can adapt to domestication within 10 generations. Such adaptations may decrease fitness in natural conditions. Many selective pressures are disrupted in captivity, including social behavioral networks. Although lack of sociality in captivity appears to mediate domestication, the underlying mechanisms are not well understood. Additionally, determining the contribution of genetic inheritance vs. transgenerational effects during relaxed selection may provide insight into the flexibility of adaptation. When wild-derived mice kept under laboratory conditions for eight generations were reintroduced to sociality and promiscuity (free mate choice), they adapted within two generations. Fitness assessments between this promiscuous lineage and a monogamous laboratory lineage revealed malespecific effects. Promiscuous-line males had deficits in viability, but a striking advantage in attracting mates, and their scent marks were also more attractive to females. Here, we investigate mechanistic details underlying this olfactory signal and identify a role of major urinary protein (MUP) pheromones. Promiscuous-line males inherit higher MUP expression than monogamous-line males through transgenerational inheritance. Sociality-driven maternal and paternal effects reveal intriguing conflicts among parents and offspring over pheromone expression. MUP up-regulation is not driven by hormone-driven transduction pathways, but rather is associated with reduction in DNA methylation of a CpG dinucleotide in the promoter. This reduction in methylation could enhance transcription by promoting the binding of transcription factor USF1 (upstream stimulatory factor 1). Finally, we experimentally demonstrate that increased MUP expression is a female attractant. These results identify molecular mechanisms guiding domestication and adaptive responses to fluctuating sociality.social selection | sexy sons | epigenetics W ild animals bred in captivity have been observed to adapt to domestication within 10 generations (1-4). As a result, captive bred animals often have reduced fitness when reintroduced to natural conditions (5). Selective pressures disrupted by captivity include inbreeding avoidance, effective population size, disease exposure, predation, and sexual selection. For the latter example, evidence suggests that lack of social context for mate choice mediates adaptation to captivity (6). This context is especially relevant for social animals because the opportunity for mating success is regulated by hierarchical behavioral networks (i.e., social selection) (7), and this information is missing in captivity. The molecular mechanisms underlying phenotypes affected by lack of social selection are poorly understood. Furthermore, rapid adaptation to captivity raises questions about the roles of genetic inheritance vs. transgenerational effects (i.e., inheritance independent of genetic variation) (8). Resolving the mechanistic and hereditary basis of these transitions can provide insight into the flexibilit...

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The RNA polymerase III transcriptome revealed by genome-wide localization and activity–occupancy relationships

Cited by 165 publications

References 38 publications

Expanding the repertoire of plasmids for PCR‐mediated epitope tagging in yeast

Expanding the repertoire of plasmids for PCR‐mediated epitope tagging in yeast

Quantifying ChIP-seq data: a spiking method providing an internal reference for sample-to-sample normalization

Reintroducing domesticated wild mice to sociality induces adaptive transgenerational effects on MUP expression

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