Additional file 1 of Activation-induced cytidine deaminase causes recurrent splicing mutations in diffuse large B-cell lymphoma

Supplementary Material 1

Analysis of the Transcriptomes Downstream of Eyeless and the Hedgehog, Decapentaplegic and Notch Signaling Pathways in <em>Drosophila melanogaster</em>

<div><p>Tissue-specific transcription factors are thought to cooperate with signaling pathways to promote patterned tissue specification, in part by co-regulating transcription. The <em>Drosophila melanogaster</em> Pax6 homolog Eyeless forms a complex, incompletely understood regulatory network with the Hedgehog, Decapentaplegic and Notch signaling pathways to control eye-specific gene expression. We report a combinatorial approach, including mRNAseq and microarray analyses, to identify targets co-regulated by Eyeless and Hedgehog, Decapentaplegic or Notch. Multiple analyses suggest that the transcriptomes resulting from co-misexpression of Eyeless+signaling factors provide a more complete picture of eye development compared to previous efforts involving Eyeless alone: (1) Principal components analysis and two-way hierarchical clustering revealed that the Eyeless+signaling factor transcriptomes are closer to the eye control transcriptome than when Eyeless is misexpressed alone; (2) more genes are upregulated at least three-fold in response to Eyeless+signaling factors compared to Eyeless alone; (3) based on gene ontology analysis, the genes upregulated in response to Eyeless+signaling factors had a greater diversity of functions compared to Eyeless alone. Through a secondary screen that utilized RNA interference, we show that the predicted gene <em>CG4721</em> has a role in eye development. <em>CG4721</em> encodes a neprilysin family metalloprotease that is highly up-regulated in response to Eyeless+Notch, confirming the validity of our approach. Given the similarity between <em>D. melanogaster</em> and vertebrate eye development, the large number of novel genes identified as potential targets of Ey+signaling factors will provide novel insights to our understanding of eye development in <em>D. melanogaster</em> and humans.</p> </div

Fuzzy-crisp comparison.

<p>The four first rows show the mean values of fuzzy/crisp support and <i>p</i>-value of the combinations respectively. The last two rows show the statistical significance returned by the ANOVA procedure.</p><p>Fuzzy-crisp comparison.</p

Post-processing of the results.

<p>The value indicates the membership degree of each binding site to its corresponding transaction. (a) Pairs of overlapping binding sites are directly removed. (b) The optimum way of fitting itemset {A, B, C} is found.</p

Comparison of Patser and Consite.

<p>Number of TFBSs from the dataset by Harbison et al. against the total number of TFBSs detected by Patser and Consite.</p

Co-misexpression of Ey together with Hh, Dpp or activated Notch results in larger ectopic eye fields that misexpression of Ey alone.

<p>(A–H) Wing discs from third instar larvae of the indicated genotypes stained with anti-Eya and anti-Elav.</p

Eye-antennal discs from otherwise wild-type <i>hh-lacZ</i> (A) or <i>dpp-lacZ</i> (B) larvae.

<p>(A) <i>hh-lacZ</i> is expressed in photoreceptors behind the furrow (white arrow) and in the ocellar precursors (black arrow). (B) <i>dpp-lacZ</i> is expressed in the furrow. (C) <i>dan>CG4721^IR/hh-lacZ</i> eye-antennal disc. <i>hh-lacZ</i> expression in ocelli is normal (black arrow), but expression in eye field is advanced relative to wild type (A). (D) <i>dan>CG4721^IR/dpp-lacZ</i> eye-antennal disc. <i>dpp-lacZ</i> is expressed in a broader domain compared to wild type (B). (E,F) Projection of confocal sections of eye-antennal discs containing clones expressing <i>CG4721^IR</i> (clones marked by GFP). (E) NICD is enriched in the furrow in wild-type tissue (white arrows), but is not enriched in the furrow in <i>CG4721^IR</i> tissue (yellow arrow). (F) pMAPK is enriched in intermediate groups in wild-type tissue (white arrows), but is enriched in all cells in <i>CG4721^IR</i> clones.</p

Parameter values.

<p>Second dataset. Summary of the input parameters used.</p><p>Parameter values.</p

Top-20 TF combinations.

<p>First dataset. The twenty TF combinations with the lowest p-value and highest support obtained when using the dataset by Harbison et al. Evidence column shows whether results were yielded when PubMed was queried for evidence in the literature (P), STRING <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0108065#pone.0108065-Franceschini1" target="_blank">[48]</a> yielded a connected graph for the given TFs (S), both conditions (SP) or none (-) were met.</p><p>Top-20 TF combinations.</p

More eye-specific genes are identified as ≥3 upregulated relative to the wing control when Ey+signaling factors are misexpressed compared to when only Ey is misexpressed.

<p>More eye-specific genes are identified as ≥3 upregulated relative to the wing control when Ey+signaling factors are misexpressed compared to when only Ey is misexpressed.</p