Identification of LMX1B target genes in a tet-off inducible HeLa cell line and in the mouse kidney

LMX1B is a LIM-homeodomain transcription factor essential for development. Putative LMX1B target genes have been identified through mouse gene targeting studies; however, in the absence of in vivo molecular characterization of their regulation, their identity as direct LMX1B targets remains hypothetical. We describe here the first molecular characterization of LMX1B target gene regulation. A tetracycline-inducible expression system and microarray analysis showed that a subset of NF-kappa B target genes, including IL-6 and IL-8 are upregulated in LMX1B-expressing HeLa cells. Chromatin immunoprecipitation assays revealed that LMX1B binds to the proximal promoter region of IL-6 and IL-8 in vivo, in the vicinity of the characterized kappa B site, and that LMX1B recruitment correlates with an increased NF-kappa B DNA association. Inhibition of NF-kappa B activity by short interfering RNA-mediated knock-down of p65 impairs LMX1B-dependent induction of NF-kappa B target genes, while activation of NF-kappa B activity by TNF-alpha results in a synergistic induction of these genes by LMX1B. IL-6 promoter-driven reporter assays showed that the kappa B site and an adjacent putative LMX1B binding motif are both involved in LMX1B-mediated transcription. Expression of a number of NF-kappa B target genes is affected in the kidney of Lmx1b-/- knock-out mice, thus supporting the biological relevance of the data obtained in the human cell line. Together, these data demonstrate for the first time that LMX1B directly regulates transcription of a subset of NF-kappa B target genes in cooperation with nuclear p50/p65 NF-kappa B. Human subset (GSM303547-GSM303550): Two biological replicates of non-expressing HtTA-LMX1B cells (grown in doxycycline-containing medium) and of LMX1B-expressing HtTA-LMX1B cells (grown for 4 days in doxycycline-free medium) were processed for gene expression array analyses using an Affymetrix platform. Mouse subset (GSM304379-GSM304384): Three kidney samples from newborn wild-type mice and from newborn Lmx1b-/- knock-out mice were processed for gene expression array analyses using an Agilent platform.LMX1B是LIM同源域（LIM-homeodomain）转录因子，对生物体发育至关重要。此前已有研究通过小鼠基因靶向实验鉴定出潜在的LMX1B靶基因，但由于缺乏对其调控过程的体内分子表征，这些基因是否为LMX1B的直接靶标仍属推测。本研究首次针对LMX1B的靶基因调控机制开展分子表征。采用四环素诱导表达系统结合微阵列分析发现，在表达LMX1B的HeLa细胞中，核因子κB（NF-κB）靶基因子集（包含白细胞介素6（IL-6）与白细胞介素8（IL-8））的表达水平显著上调。染色质免疫沉淀实验结果显示，LMX1B可在体内结合IL-6与IL-8的近端启动子区域，位点紧邻已表征的κB结合基序；且LMX1B的招募与NF-κB的DNA结合能力增强呈显著相关。通过小干扰RNA（siRNA）介导的p65基因敲低抑制NF-κB活性，会削弱LMX1B对NF-κB靶基因的诱导作用；而通过肿瘤坏死因子α（TNF-α）激活NF-κB活性，则可与LMX1B协同增强这些靶基因的诱导表达。以IL-6启动子驱动的报告基因实验证实，κB结合位点与邻近的推定LMX1B结合基序均参与了LMX1B介导的转录调控过程。Lmx1b基因敲除（Lmx1b-/-）小鼠的肾脏组织中，多个NF-κB靶基因的表达水平发生改变，这验证了人类细胞系实验结果的生物学相关性。综上，本研究首次证实LMX1B可与核p50/p65 NF-κB协同，直接调控部分NF-κB靶基因的转录。人类样本子集（GSM303547-GSM303550）：分别收集两批生物学重复的未表达LMX1B的HtTA-LMX1B细胞（培养于含多西环素的培养基中）与表达LMX1B的HtTA-LMX1B细胞（于不含多西环素的培养基中培养4天），采用Affymetrix平台开展基因表达微阵列分析。小鼠样本子集（GSM304379-GSM304384）：分别收集新生野生型小鼠与新生Lmx1b基因敲除小鼠的3份肾脏组织样本，采用Agilent平台开展基因表达微阵列分析。