Technical Comments

Comment on “Drosophila Dosage Compensation Involves Enhanced Pol II Recruitment to Male X-Linked Promoters”

Science  19 Apr 2013:
Vol. 340, Issue 6130, pp. 273
DOI: 10.1126/science.1231895

Abstract

Conrad et al. (Reports, 10 August 2012, p. 742) reported that Drosophila dosage compensation might largely be due to increased recruitment of RNA polymerase II to promoters. A reassessment of the numerical operations revealed that the authors’ calculations are severely confounded by an inappropriate numerical procedure. A rectified analysis strongly suggests that the authors’ conclusions are not supported by their data.

X chromosome dosage compensation in male Drosophila flies involves a transcriptional activation of X-linked genes in the 2-fold range (1). The underlying mechanism is of general interest to the field. Conrad et al. (2) conclude that dosage compensation involves an increased recruitment of RNA polymerase II (Pol II) to promoters. The authors calculate that Pol II is about 2-fold enriched on the promoters and transcribed gene bodies of X-chromosomal genes in male flies in comparison with females. Furthermore, RNA interference–based knockdown of the key compensation protein MSL2 led to a 1.8-fold decrease in Pol II loading. Hence, the authors suggest that compensation is mainly due to enhanced transcription initiation through increased Pol II recruitment. This model is in stark contrast to the previous notion that compensated transcription is largely due to regulation at the level of transcription elongation (3).

In trying to recapitulate the results of Conrad et al., we discovered that the analysis pipeline applied in this study contains a numerical transformation that distorts the calculated Pol II enrichments. These values are the basis for essentially all computational analyses provided in the paper. Specifically, the authors apply a corrective multiplication on smoothed log2 enrichments in order to emphasize the enrichment values in regions that are covered well with sequencing reads (the numerical procedures are described in detail in an online protocol at www.epigenesys.eu/index.php/en/protcols/bio-informatics/411-a-pipeline-for-chip-seq-data-analysis). As a consequence of this transformation, the enrichment values get exponentially scaled. The enormous so-called “log2 IP/input” values in many of the accompanying figures clearly highlight this distortion. Usually, native log2(IP/input) enrichments in chromatin immunoprecipitation sequencing (ChIP-Seq) studies rarely exceed a log2 value of 5, whereas the reported values shown in this study reach average values of 15 and higher at binding sites [see, e.g., figure 2A in (2)]. In single loci, these “log2 enrichments” reach 30, reflecting a billion-fold enrichment of target protein over unbound regions, which is not plausible.

A more serious problem arises because the mathematical transformation affects the quantitative comparison between different enrichment profiles by ratio calculations—e.g., when comparing males and females or X chromosomes and autosomes. Because log ratios are obtained by subtraction, a previous multiplication of minuend and subtrahend by a correction factor will not be eliminated. Consequently, the ratios derived are scaled by this factor and the differences displayed in various figures [figure 2, A and E; figure 3, A and B; figure 4F; and plenty of supplementary figures in (2)] are severely exaggerated.

When the data are reanalyzed omitting the multiplication step, the differences between X and autosomal loading of Pol II as well as distribution differences between males, females, and MSL2-knockdown males are attenuated. Only a 1.17-fold enrichment of Pol II on male X-linked promoters remains (Fig. 1, A and B) and, even more importantly, only a 1.12-fold MSL2-dependent Pol II loading is seen (Fig. 2, A and B).

Fig. 1 Bar plots depicting the mean ratios of Pol II enrichments on X and autosomes in males and females according to figure 2E in (2).

(A) Calculation including the incorrect data transformation as performed by the authors. (B) Calculation without the transformation.

Fig. 2 Bar plots depicting the mean ratios of Pol II enrichments on X and autosomes in males, females, and MSL2-knockdown males according to figure 4F in (2).

(A) Calculation including the incorrect data transformation as performed by the authors. (B) Calculation without the transformation.

The statistical tests for differential loading of Pol II—as, for example, performed in the authors’ figure 2C—might still yield low P values, suggesting that there is in fact a robust enrichment of Pol II on male X-linked genes. However, the impact of the study for the chromatin/transcription field is primarily due to the magnitude of the difference (about 2-fold) rather than the numerical significance of a very small difference in Pol II loading. Accordingly, an ~10% difference between wild-type and MSL2-knockdown flies cannot be considered a major effect.

Taken together, the data presented by Conrad et al. do not provide sufficient evidence for a model in which enhanced Pol II loading and transcription initiation constitute a key mechanism for compensated gene expression in Drosophila.

References and Notes

  1. Acknowledgments: This work was supported by a grant from the European Research Council under the European Union’s Seventh Framework Programme (FP7/2007-2013)/ERC grant agreement 293948.
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