Monitoring global messenger RNA changes in externally controlled microarray experiments

Peppel, Jeroen van de; Kemmeren, Patrick; Bakel, Harm van; Radonjić, Marijana; Leenen, Dik van; Holstege, Frank C.P.

doi:10.1038/sj.embor.embor798

Cited by 150 publications

(118 citation statements)

References 22 publications

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“…Cell-state transitions such as maturation can dramatically alter both total RNA and mRNA content, limiting reliability of normalization with internal standards. 49,53 Using spikein controls, we measured a greatly reduced ratio of GATA1 activated to repressed genes compared with previous estimates. 35,36,51,55 This enabled us to more accurately gauge the role of BETs during GATA1-induced changes in transcription, and established BETs chiefly as transcriptional coactivators.…”

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confidence: 64%

“…As dramatic alterations in cell size and RNA content occur during erythroid maturation, we added external spike-in RNA controls to each sample in proportion to cell number for normalization. 49,53 Focusing first on GATA1's impact on gene expression, we plotted all transcripts from most repressed to most activated (Figure 2A). Following GATA1 addition, 5094 transcripts decreased whereas only 220 increased using stringent differential expression criteria (twofold change and Bonferroni-corrected, P , .05) (supplemental Figure 4A).…”

Section: Org Frommentioning

confidence: 99%

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Functions of BET proteins in erythroid gene expression

Stonestrom

Hsu

Jahn

et al. 2015

Blood

109

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• BETs promote GATA1 chromatin occupancy and subsequently activate transcription; they are generally not required for repression.• BRD2 and BRD4 are essential for full GATA1 activity whereas BRD3 function overlaps with BRD2.Inhibitors of bromodomain and extraterminal motif proteins (BETs) are being evaluated for the treatment of cancer and other diseases, yet much remains to be learned about how BET proteins function during normal physiology. We used genomic and genetic approaches to examine BET function in a hematopoietic maturation system driven by GATA1, an acetylated transcription factor previously shown to interact with BETs. We found that BRD2, BRD3, and BRD4 were variably recruited to GATA1-regulated genes, with BRD3 binding the greatest number of GATA1-occupied sites. Pharmacologic BET inhibition impaired GATA1-mediated transcriptional activation, but not repression, genome-wide. Mechanistically, BETs promoted chromatin occupancy of GATA1 and subsequently supported transcriptional activation. Using a combination of CRISPRCas9-mediated genomic engineering and shRNA approaches, we observed that depletion of either BRD2 or BRD4 alone blunted erythroid gene activation. Surprisingly, depletion of BRD3 only affected erythroid transcription in the context of BRD2 deficiency. Consistent with functional overlap among BET proteins, forced BRD3 expression substantially rescued defects caused by BRD2 deficiency. These results suggest that pharmacologic BET inhibition should be interpreted in the context of distinct steps in transcriptional activation and overlapping functions among BET family members. (Blood. 2015;125(18):2825-2834 IntroductionThe mammalian bromodomain and extraterminal motif proteins (BETs) have drawn widespread interest as pharmacologic targets for the treatment of various diseases, including hematologic malignancies and solid tumors. [1][2][3][4] Within the BET family, BRD2, BRD3, and BRD4 are ubiquitously expressed in mammalian tissues, whereas BRDT is testis-specific. BETs contain 2 tandem bromodomains that mediate association with chromatin by binding to acetylated histones and transcription factors. [5][6][7][8][9] BETs function in regulatory complexes that impact messenger RNA (mRNA) production at multiple steps of the transcription cycle, such as modifying and remodeling chromatin and promoting transcription elongation. [10][11][12][13][14][15][16][17] Both BRD2 and BRD4 are essential for normal development.18-20 A BRD3 knockout mouse has not been reported.Promising results obtained with pharmacologic BET inhibitors in animal models of malignancy have sparked clinical trials and intensified efforts to better understand BET function. 1,2,4,21 Given the widespread expression and essential functions of BETs, it was initially surprising that BET inhibitors like JQ1 elicit cell-and genespecific responses. These inhibitors block the acetyl-lysine-binding pockets specifically of BET family bromodomains triggering their release from acetylated lysine residues on histones and transcription factors....

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mentioning

confidence: 64%

Section: Org Frommentioning

confidence: 99%

Functions of BET proteins in erythroid gene expression

Stonestrom

Hsu

Jahn

et al. 2015

Blood

109

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“…Whereas DTA accurately measures the relative rates for different RNAs within a single sample, it cannot compare rates from different samples, since the samples differ by an unknown global factor (Miller et al 2011). In standard transcriptomics, comparison between samples with different mRNA levels may be achieved by counting cells and spiking RNA standards into the samples (Holstege et al 1998;Wang et al 2002;van de Peppel et al 2003). However, such normalization does not take into account differences in cell lysis and RNA extraction efficiency, which can vary so strongly that no conclusions are possible.…”

Section: [Supplemental Materials Is Available For This Article]mentioning

confidence: 99%

Comparative dynamic transcriptome analysis (cDTA) reveals mutual feedback between mRNA synthesis and degradation

Sun

Schwalb²,

Schulz³

et al. 2012

Genome Res.

271

377

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To monitor eukaryotic mRNA metabolism, we developed comparative dynamic transcriptome analysis (cDTA). cDTA provides absolute rates of mRNA synthesis and decay in Saccharomyces cerevisiae (Sc) cells with the use of Schizosaccharomyces pombe (Sp) as an internal standard. cDTA uses nonperturbing metabolic labeling that supersedes conventional methods for mRNA turnover analysis. cDTA reveals that Sc and Sp transcripts that encode orthologous proteins have similar synthesis rates, whereas decay rates are fivefold lower in Sp, resulting in similar mRNA concentrations despite the larger Sp cell volume. cDTA of Sc mutants reveals that a eukaryote can buffer mRNA levels. Impairing transcription with a point mutation in RNA polymerase (Pol) II causes decreased mRNA synthesis rates as expected, but also decreased decay rates. Impairing mRNA degradation by deleting deadenylase subunits of the Ccr4-Not complex causes decreased decay rates as expected, but also decreased synthesis rates. Extended kinetic modeling reveals mutual feedback between mRNA synthesis and degradation that may be achieved by a factor that inhibits synthesis and enhances degradation.

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“…RNA amplification and labeling were performed18 on an automated system (Caliper Life Sciences, Waltham, MA) with 3 μg total RNA from each sample. Hybridization was performed on an HS4800PRO system supplemented with QuadChambers (Tecan Benelux B.V.B.A., Giessen, the Netherlands), using 500–1000 ng labeled cRNA per channel 19. One half of the RNA was labeled with cy5 against cy3 labeled common reference RNA on dual‐channel arrays; the second half was analyzed in dye swap.…”

Section: Methodsmentioning

confidence: 99%

Growth plate expression profiling: Large and small breed dogs provide new insights in endochondral bone formation

Teunissen

Riemers

Leenen

et al. 2017

Journal Orthopaedic Research

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The difference in the adult height of mammals, and hence in endochondral bone formation, is not yet fully understood and may serve to identify targets for bone and cartilage regeneration. In line with this hypothesis, the intra‐species disparity between the adult height of Great Danes and Miniature Poodles was investigated at a transcriptional level. Microarray analysis of the growth plate of five Great Danes and five Miniature Poodles revealed 2,981 unique genes that were differentially expressed, including many genes with an unknown role in skeletal development. A signaling pathway impact analysis indicated activation of the cell cycle, extracellular matrix receptor interaction and the tight junction pathway, and inhibition of pathways associated with inflammation and the complement cascade. In additional validation steps, the gene expression profile of the separate growth plate zones for both dog breeds were determined. Given that the BMP signaling is known for its crucial role in skeletal development and fracture healing, and BMP‐2 is used in orthopaedic and spine procedures for bone augmentation, further investigations concentrated on the BMP pathway.The canonical BMP‐2 and BMP‐6 signaling pathway was activated in the Great Danes compared to Miniature Poodles. In conclusion, investigating the differential expression of genes involved in endochondral bone formation in small and large breed dogs, could be a game changing strategy to provide new insights in growth plate development and identify new targets for bone and cartilage regeneration. © 2017 The Authors. Journal of Orthopaedic Research® published by Wiley Periodicals, Inc. on behalf of the Orthopaedic Research Society. J Orthop Res 36:138–148, 2018.

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Monitoring global messenger RNA changes in externally controlled microarray experiments

Cited by 150 publications

References 22 publications

Functions of BET proteins in erythroid gene expression

Functions of BET proteins in erythroid gene expression

Comparative dynamic transcriptome analysis (cDTA) reveals mutual feedback between mRNA synthesis and degradation

Growth plate expression profiling: Large and small breed dogs provide new insights in endochondral bone formation

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