BACKGROUND CCAAT/Enhancer Binding Proteindelta (C/EBPdelta) is a member of the highly conserved C/EBP family of leucine zipper (bZIP) proteins. C/EBPdelta is highly expressed in G0 growth arrested mammary epithelial cells (MECs) and "loss of function" alterations in C/EBPdelta have been associated with impaired contact inhibition, increased genomic instability and increased cell migration. Reduced C/EBPdelta expression has also been reported in breast cancer and acute myeloid leukemia (AML). C/EBPdelta functions as a transcriptional activator, however, only a limited number of C/EBPdelta target genes have been reported. As a result, the role of C/EBPdelta in growth control and the potential mechanisms by which "loss of function" alterations in C/EBPdelta contribute to tumorigenesis are poorly understood. The goals of the present study were to identify C/EBPdelta target genes using Chromatin Immunoprecipitation coupled with a CpG Island (HCG12K) Array gene chip ("ChIP-chip") assay and to assess the expression and potential functional roles of C/EBPdelta target genes in growth control. RESULTS ChIP-chip assays identified approximately 100 C/EBPdelta target gene loci which were classified by gene ontology (GO) into cell adhesion, cell cycle regulation, apoptosis, signal transduction, intermediary metabolism, gene transcription, DNA repair and solute transport categories. Conventional ChIP assays validated the ChIP-chip results and demonstrated that 14/14 C/EBPdelta target loci were bound by C/EBPdelta in G0 growth arrested MCF-12A MECs. Gene-specific RT-PCR analysis also demonstrated C/EBPdelta-inducible expression of 14/14 C/EBPdelta target genes in G0 growth arrested MCF-12A MECs. Finally, expression of endogenous C/EBPdelta and selected C/EBPdelta target genes was also demonstrated in contact-inhibited G0 growth arrested nontransformed human MCF-10A MECs and in mouse HC11 MECs. The results demonstrate consistent activation and downstream function of C/EBPdelta in growth arrested human and murine MECs. CONCLUSION C/EBPdelta target genes were identified by a global gene array approach and classified into functional categories that are consistent with biological contexts in which C/EBPdelta is induced, such as contact-mediated G0 growth arrest, apoptosis, metabolism and inflammation. The identification and validation of C/EBPdelta target genes provides new insights into the mechanistic role of C/EBPdelta in mammary epithelial cell biology and sheds new light on the potential impact of "loss of function" alterations in C/EBPdelta in tumorigenesis.

中文翻译：

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G0 生长停滞乳腺上皮细胞中 CCAAT/增强子结合蛋白δ (C/EBPδ) 靶基因的鉴定和表征。


背景CCAAT/增强子结合蛋白δ(C/EBPδ)是高度保守的亮氨酸拉链(bZIP)蛋白C/EBP家族的成员。 C/EBPδ 在 G0 生长停滞的乳腺上皮细胞 (MEC) 中高度表达，C/EBPδ 中的“功能丧失”改变与接触抑制受损、基因组不稳定性增加和细胞迁移增加有关。据报道，乳腺癌和急性髓系白血病 (AML) 中 C/EBPδ 表达降低。 C/EBPdelta 作为转录激活剂发挥作用，然而，仅报道了有限数量的 C/EBPdelta 靶基因。因此，人们对 C/EBPδ 在生长控制中的作用以及 C/EBPδ 中“功能丧失”改变导致肿瘤发生的潜在机制知之甚少。本研究的目标是使用染色质免疫沉淀结合 CpG 岛 (HCG12K) 阵列基因芯片（“ChIP 芯片”）测定来鉴定 C/EBPdelta 靶基因，并评估 C/EBPdelta 靶标的表达和潜在功能作用控制生长的基因。结果 ChIP 芯片检测鉴定出大约 100 个 C/EBPdelta 靶基因位点，根据基因本体论 (GO) 将其分为细胞粘附、细胞周期调节、细胞凋亡、信号转导、中间代谢、基因转录、DNA 修复和溶质转运类别。常规 ChIP 测定验证了 ChIP 芯片结果，并证明 14/14 C/EBPdelta 靶位点与 G0 生长停滞的 MCF-12A MEC 中的 C/EBPdelta 结合。基因特异性 RT-PCR 分析还证明了 G0 生长停滞的 MCF-12A MEC 中 14/14 C/EBPdelta 靶基因的 C/EBPdelta 诱导表达。 最后，在接触抑制的 G0 生长停滞的非转化人 MCF-10A MEC 和小鼠 HC11 MEC 中也证实了内源 C/EBPdelta 和选定的 C/EBPdelta 靶基因的表达。结果表明，在生长停滞的人和小鼠 MEC 中，C/EBPdelta 的激活和下游功能是一致的。结论 通过全局基因阵列方法鉴定了 C/EBPdelta 靶基因，并将其分类为与诱导 C/EBPdelta 的生物学背景一致的功能类别，例如接触介导的 G0 生长停滞、细胞凋亡、代谢和炎症。 C/EBPdelta靶基因的鉴定和验证为C/EBPdelta在乳腺上皮细胞生物学中的机制作用提供了新的见解，并为C/EBPdelta“功能丧失”改变在肿瘤发生中的潜在影响提供了新的线索。