Volume 10, Issue 10, Pages 1349-1352 (October 2017) Alternative Polyadenylation of the Sense Transcript Controls Antisense Transcription of DELAY OF GERMINATION 1 in Arabidopsis  Justyna Kowalczyk, Malgorzata Palusinska, Agata Wroblewska-Swiniarska, Zbigniew Pietras, Lukasz Szewc, Jakub Dolata, Artur Jarmolowski, Szymon Swiezewski  Molecular Plant  Volume 10, Issue 10, Pages 1349-1352 (October 2017) DOI: 10.1016/j.molp.2017.07.011 Copyright © 2017 The Author Terms and Conditions

Figure 1 Alternative Polyadenylation of DOG1 Sense Transcript Controls the Antisense DOG1 Transcript Level, Presumably through Histone H2B Monoubiquitylation. (A–C) Germination efficiency shown as percentage (%) of germinated seeds: (A) freshly harvested from Col-0 and cpl1 mutant plants with statistical analysis performed against Col-0; (B) Col-0 and the cpl1 mutants stratified for two days after harvesting; (C) freshly harvested from a cpl1-9 dog1-4 double mutant and single mutants, with Col-0 as a control. Bars represent mean values from at least four biological replicates. For germination efficiency curves, see Supplemental Figure 2. (D) Relative expression of shDOG1 and lgDOG1 mRNA and the ratio of these two isoforms in freshly harvested seeds of Col-0 and cpl1 mutants, measured by RT–qPCR, and double normalized against the UBC transcript level and Col-0. (E) Relative expression level of asDOG1 in freshly harvested seeds of Col-0 and cpl1 mutants measured by strand-specific adapter-mediated RT–qPCR (Fedak et al., 2016), double normalized against UBC mRNA level and Col-0. Bars represent the mean values from at least three biological replicates. (F) Schematic diagram of the structure of the DOG1 gene showing regions included in LUC gene fusion constructs used to produce transgenic lines. Black bars and the gray bar indicate exons and the alternative exon, respectively; the white bar indicates the shDOG1 3′ UTR; arrows mark the transcription start sites; TTS, transcription termination site. (G–I) Luciferase activity assay on transgenic seeds containing the LUC gene fused with (G) the asDOG1 promoter, (H) the whole DOG1 gene, or (I) the sense DOG1 fragment. Upper panels show the light signals produced by equal seed aliquots, with each column containing seeds from individual plants and rows containing technical replicates (the full set of results is presented in Supplemental Figure 5). The colored scale next to each image shows the light intensity in counts per second (CPS). The lower panels show luciferase activity values normalized to the signal area and then against the Col-0 signal. Error bars show 95% confidence intervals. The Wilcoxon test was used for statistical analysis: ***p < 0.001; n.s., non-significant. (J) Relative expression level of shDOG1 and lgDOG1 and the ratio of the two isoforms in freshly harvested seeds of Col-0 and the fy-2 mutant measured by RT–qPCR, double normalized against UBC mRNA level and Col-0. Bars represent mean values from at least four biological replicates. (K) Relative expression level of asDOG1 in freshly harvested seeds of Col-0 and the fy-2 mutant, measured by strand-specific adapter-mediated RT–PCR, quantified using ImageJ (gel images are shown in Supplemental Figure 6). Double normalization was performed against the UBC mRNA level and Col-0. Bars represent mean values from at least six biological replicates. (L) DOG1 alternative polyadenylation (APA) site selection controls asDOG1 expression. Preferential proximal APA site selection in the cpl1 mutant results in enhanced asDOG1 transcription. More frequent distal APA site usage in the fy mutant leads to a reduction in the asDOG1 level. (M) Histone H2B monoubiquitylation level, measured by chromatin immunoprecipitation (ChIP), on the DOG1 gene in Col-0 and the fy-2 mutant. Data were double normalized against a control gene (ACTIN 7, AT5G09810) and Col-0. Bars represent mean values from four independent experiments. A representative ChIP experiment without Col-0 normalization and regions amplified by primers used in its analysis are shown in Supplemental Figure 7. (N) Relative expression of shDOG1 and lgDOG1 mRNA and the ratio of these two isoforms in freshly harvested seeds of Col-0 and hub1-5 mutant, measured by RT–qPCR, and double normalized against the UBC transcript level and Col-0. Bars represent mean values from at least three biological replicates. (O) Relative expression level of asDOG1 in freshly harvested seeds of Col-0 and a hub1-5 mutant measured by strand-specific, adapter-mediated RT–qPCR, double normalized against the UBC mRNA level and Col-0. Bars represent mean values from at least three biological replicates. For all graphs except (G), (H), and (I), error bars show the SD and Student's t-test was used for statistical analysis: *p < 0.05; **p < 0.01; ***p < 0.001; n.s., non-significant. Molecular Plant 2017 10, 1349-1352DOI: (10.1016/j.molp.2017.07.011) Copyright © 2017 The Author Terms and Conditions