Decoding a Signature-Based Model of Transcription Cofactor Recruitment Dictated by Cardinal Cis-Regulatory Elements in Proximal Promoter Regions
Figure 4
Diversity of functional outcomes associated with NRF1/LSD1 recruitment at −150/+50 bp regions.
(A) Left: Western blot analysis of NRF1 and LSD1 in whole cell extracts obtained from MCF7 cells in which NRF1 or LSD1 (indicated on top) were depleted by siRNA. Actin is shown as loading control. Scrambled (CTL) siRNA is included as control of transfection. Panels: RT-qPCR analysis of genes that have been identified in this study as having promoters co-occupied by NRF1 and LSD1 (NRF1+ and LSD1+ targets). Gene names are indicated on top of each panel. Treatments are indicated at the bottom. Scrambled (CTL) siRNA was used as control. The y-axis refers to normalized expression to levels of ACTB mRNA. (B) Top: Western blot analysis as shown in A (left panel) but in U2OS cells. Bottom: Venn diagram depicting the overlap of genes affected by NRF1 (light red circle) and LSD1 (orange circle) siRNA treatments with respect to control (CTL) siRNA in U2OS cells, based on microarray. The number of total and category-wise genes affected by the treatments are indicated, as well as the statistical significance of the overlap. (C) Matrix of motif enrichment of cardinal motifs in −150/+50 bp regions of genes identified by microarray as affected by NRF1 (left) or LSD1 (right) knockdown in U2OS cells. Enrichment levels were determined with respect to background frequencies of the same motifs in −150/+50 bp regions. Motif enrichments higher than background are shown as a gradient of blue, while motif enrichments lower than background are shown as a gradient of red. No motif enrichment is shown as white. The number of promoters analyzed is also indicated. (D) Venn diagram depicting the overlap of microarray-identified genes classified based on their type of response to NRF1 or LSD1 siRNA treatments. The numbers of genes in the overlaps are indicated, as well as the numbers of genes that did not overlap and the total numbers. (E) Combined analysis of microarray and ChIP-based data. We combined microarray results identifying NRF1 and/or LSD1 siRNA-mediated effects and ChIP-DSL data identifying NRF1 and/or LSD1 occupied promoters. Gene classes based on (D). The y-axis refers to the relative enrichment over background in the number of genes that were affected by both NRF1 and LSD1 siRNA treatments and that had promoters occupied by LSD1 (orange) and/or NRF1 (red; see text for more details). A ratio of ‘fold over background’ higher or lower than 1 (>1 or <1, respectively) distinguishes when a gene class contains a higher or lower frequency of either LSD1- or NRF1-occupied promoters over the frequency observed in genes not affected by LSD1 or NRF1 siRNA treatments (which we defined as ‘background’). (F) As in E, but for genes that were affected only by either NRF1 or LSD1 siRNA treatments.