Gene expression profiling by RNA-seq of wild-type, fpa mutant, bdr1 mutant, bdr2 mutant, bdr3 mutant and bdrs triple mutant Arabidopsis seedlings. Gene expression profiling by RNA-seq of wild-type, fpa mutant, bdr1 mutant, bdr2 mutant, bdr3 mutant and bdrs triple mutant Arabidopsis seedlings

We analyzed by RNA-seq the transcriptome of 8-day old Arabidopsis thaliana seedlings for wild-type (Col-0), single mutants for BDR proteins (bdr1/AT5G25520 ; bdr2/AT5G11430 or bdr3/AT2G25640), single mutant for FPA (fpa/AT2G43410, line fpa-7) or a triple mutant of all three BDR proteins (bdrs: cross of bdr1/AT5G25520 mutant SALK_142108C, bdr2/AT5G11430 mutant CS852350 and bdr3/AT2G25640 mutant SALK_059905). We identified hundreds of genes differentially expressed between wild-type and bdrs triple mutant and a significant overlap in DE genes with the fpa mutant. We also analyzed the binding of BDR1 and BDR2 as well as RNA polymerase II and histone marks by ChIP-seq in wild-type, bdrs and fpa-deficient seedlings. Our data support a role of BDRs as negative elongation factors. They bind on the gene body and regulate the expression of genes involved in defense response pathways. Strikingly, by modulating 3' pausing of RNA polymerase II and possibly contributing to gene looping, they also protect a number of genes from transcriptional interferences originating from a highly expressed upstream tandem gene. Thus BDRs proteins are negative elongation factors that act as transcriptional "gatekeepers" in the Arabidopsis thaliana genome. Overall design: mRNA profiles in 8-day old seedlings from wild-type (Col0), bdr single mutants (bdr1/AT5G25520 ; bdr2/AT5G11430 or bdr3/AT2G25640), fpa single mutant (fpa/AT2G43410) or bdr triple mutant were generated by deep sequencing, in triplicates, on Illumina NextSeq500

我们通过RNA测序（RNA-seq）分析了8日龄拟南芥（Arabidopsis thaliana）幼苗的转录组，所涉样本包括野生型（Col-0）、BDR蛋白单突变体（bdr1/AT5G25520；bdr2/AT5G11430或bdr3/AT2G25640）、FPA单突变体（fpa/AT2G43410，品系fpa-7），以及三个BDR蛋白的三重突变体（bdrs：由bdr1/AT5G25520突变体SALK_142108C、bdr2/AT5G11430突变体CS852350与bdr3/AT2G25640突变体SALK_059905杂交构建获得）。我们在野生型与bdrs三重突变体间鉴定到数百个差异表达基因（differentially expressed genes, DE genes），且该差异表达基因集与fpa突变体的差异表达基因存在显著重叠。我们还通过染色质免疫沉淀测序（ChIP-seq），分析了野生型、bdrs突变体及FPA缺陷型幼苗中BDR1、BDR2、RNA聚合酶II与组蛋白修饰的结合分布情况。我们的数据证实BDRs作为负延伸因子发挥功能：它们结合于基因体区域，并调控防御反应通路相关基因的表达。值得关注的是，通过调控RNA聚合酶II的3’暂停并可能参与基因环化过程，BDRs还可保护部分基因免受来自高表达上游串联基因的转录干扰。综上，BDR蛋白作为负延伸因子，在拟南芥基因组中充当转录“守门人”。整体实验设计：采用Illumina NextSeq500平台，对野生型（Col0）、bdr单突变体（bdr1/AT5G25520；bdr2/AT5G11430或bdr3/AT2G25640）、fpa单突变体（fpa/AT2G43410）及bdr三重突变体的8日龄幼苗mRNA进行深度测序，每组设置3次生物学重复。