Revisiting Global Gene Expression Analysis

Lovén, Jakob; Orlando, David A.; Sigova, Alla A.; Lin, Charles Y.; Rahl, Peter B.; Burge, Christopher B.; Levens, David; Lee, Tong Ihn; Young, Richard A.

doi:10.1016/j.cell.2012.10.012

Cited by 540 publications

(566 citation statements)

References 45 publications

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“…As reported here, it was remarkable how few mRNAs were significantly dysregulated at least twofold by the CTD mutations S2A, Y1F, S7A, and T4A, which affected only 4.4%, 1.4%, 1.2%, and 0.14% of the annotated fission yeast protein-coding RNAs, respectively. We note that the method of RNA-seq analysis used here, whereby equal amounts of RNA from different fission yeast strains are subjected to high-throughput sequencing, assumes that the fission yeast strains analyzed produce similar levels of RNA per cell; we cannot exclude the possibility that a CTD mutation might elicit a global increase or decrement in the steady-state levels of all, or most, RNAs per yeast cell, an issue that has been discussed in relation to gene expression analyses in metazoan cells (28). Nonetheless, the analysis is highly instructive with respect to the selective effect of CTD mutations.…”

Section: Discussionmentioning

confidence: 99%

Individual letters of the RNA polymerase II CTD code govern distinct gene expression programs in fission yeast

Schwer

Bitton

Sánchez

et al. 2014

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

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The primary structure and phosphorylation pattern of the tandem Y 1 S 2 P 3 T 4 S 5 P 6 S 7 repeats of the RNA polymerase II carboxyl-terminal domain (CTD) comprise an informational code that coordinates transcription, chromatin modification, and RNA processing. To gauge the contributions of individual CTD coding "letters" to gene expression, we analyzed the poly(A) + transcriptomes of fission yeast mutants that lack each of the four inessential CTD phosphoacceptors: Tyr1, Ser2, Thr4, and Ser7. There was a hierarchy of CTD mutational effects with respect to the number of dysregulated protein-coding RNAs, with S2A (n = 227) >> Y1F (n = 71) > S7A (n = 58) >> T4A (n = 7). The majority of the protein-coding RNAs affected in Y1F cells were coordinately affected by S2A, suggesting that Tyr1-Ser2 constitutes a two-letter code "word." Y1F and S2A elicited increased expression of genes encoding proteins involved in iron uptake (Frp1, Fip1, Fio1, Str3, Str1, Sib1), without affecting the expression of the genes that repress the iron regulon, implying that Tyr1-Ser2 transduces a repressive signal. Y1F and S2A cells had increased levels of ferric reductase activity and were hypersensitive to phleomycin, indicative of elevated intracellular iron. The T4A and S7A mutations had opposing effects on the phosphate response pathway. T4A reduced the expression of two genes encoding proteins involved in phosphate acquisition (the Pho1 acid phosphatase and the phosphate transporter SPBC8E4.01c), without affecting the expression of known genes that regulate the phosphate response pathway, whereas S7A increased pho1 + expression. These results highlight specific cellular gene expression programs that are responsive to distinct CTD cues.iron homeostasis | phosphate homeostasis | transcription profiling T he carboxyl-terminal domain (CTD) of the Rpb1 subunit of RNA polymerase II (Pol II) consists of tandemly repeated heptapeptides of consensus sequence Y 1 S 2 P 3 T 4 S 5 P 6 S 7 . The inherently plastic CTD structure is modulated by phosphorylation of the Tyr1, Ser2, Thr4, Ser5, and Ser7 residues and by cis-trans isomerization of the prolines (1, 2). With as many as 128 n potential CTD primary structures (where n is the number of heptads), the CTD provides information about the state of the transcription machinery-a CTD code-that is "read" by CTD receptor proteins that control transcription, modify chromatin structure, and catalyze or regulate mRNA capping, splicing, and polyadenylation (1, 2).Basic informational rules that govern the CTD code have been elucidated by genetically manipulating the composition and structure of the Rpb1 CTD in the fission yeast Schizosaccharomyces pombe (3-7). By introducing alanines and conservative mutations in lieu of Tyr1, Ser2, Pro3, Thr4, Ser5, Pro6, and Ser7 of every consensus heptad of a fully functional Rpb1 CTD array (comprising 14 consensus heptad repeats linked to the body of Rpb1 by a "rump" consisting of four degenerate heptads), we determined that: (i) Tyr1, Pro3, Ser5, and Pro6 are essential ...

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Section: Discussionmentioning

confidence: 99%

Individual letters of the RNA polymerase II CTD code govern distinct gene expression programs in fission yeast

Schwer

Bitton

Sánchez

et al. 2014

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

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“…The internal reference is mixed with the experimental sample and undergoes all experimental steps following fragmentation of the chromatin, i.e., immunoprecipitation, library preparation, and sequencing. The method is related, in its principle of introducing an internal reference into each sample, to the method recently described by Loven et al (2012) to normalize RNAseq data. In that case, a synthetic RNA standard is added to each RNA sample to be analyzed in proportion to the starting number of cells, thus allowing quantification of RNA relative to starting cell number .…”

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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“…2 In some biological contexts, such as the mitogenic stimulation of resting B-cells, MYC enhances expression of all genes with an open chromatin structure. [3][4][5] In contrast, deregulation of MYC activates and represses more restricted sets of target genes during tumorigenesis. 6 For example, complex formation with MIZ1 (ZBTB17), which occurs predominantly at oncogenic MYC levels, mediates binding to low-affinity sites and shifts the direction of transcriptional responses at these "newly acquired" target genes toward repression.…”

Section: Introductionmentioning

confidence: 99%

MYC-induced apoptosis in mammary epithelial cells is associated with repression of lineage-specific gene signatures

et al. 2016

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Apoptosis caused by deregulated MYC expression is a prototype example of intrinsic tumor suppression. However, it is still unclear how supraphysiological MYC expression levels engage specific sets of target genes to promote apoptosis. Recently, we demonstrated that repression of SRF target genes by MYC/MIZ1 complexes limits AKT-dependent survival signaling and contributes to apoptosis induction. Here we report that supraphysiological levels of MYC repress gene sets that include markers of basal-like breast cancer cells, but not luminal cancer cells, in a MIZ1-dependent manner. Furthermore, repressed genes are part of a conserved gene signature characterizing the basal subpopulation of both murine and human mammary gland. These repressed genes play a role in epithelium and mammary gland development and overlap with genes mediating cell adhesion and extracellular matrix organization. Strikingly, acute activation of oncogenic MYC in basal mammary epithelial cells is sufficient to induce luminal cell identity markers. We propose that supraphysiological MYC expression impacts on mammary epithelial cell identity by repressing lineage-specific target genes. Such abrupt cell identity switch could interfere with adhesiondependent survival signaling and thus promote apoptosis in pre-malignant epithelial tissue.

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Revisiting Global Gene Expression Analysis

Cited by 540 publications

References 45 publications

Individual letters of the RNA polymerase II CTD code govern distinct gene expression programs in fission yeast

Individual letters of the RNA polymerase II CTD code govern distinct gene expression programs in fission yeast

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

MYC-induced apoptosis in mammary epithelial cells is associated with repression of lineage-specific gene signatures

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