Regulation of DNA hypermethylation in human mammary cells

描述（由申请人提供）：在培养来自无疾病女性的组织活检样本时，大多数人乳腺上皮细胞（HMEC）进入增殖屏障，该屏障在几次群体倍增后被激活（1）。人乳腺上皮细胞的一个小亚群，变体HMEC（vHMEC），具有绕过这种增殖屏障的能力，并且以沉默的p16INK4a肿瘤抑制基因、致敏应激反应程序（2）和获得大量染色体异常（1）为代表。作为沉默的p16INK4a的直接结果，变体HMEC获得对恶性转化至关重要的几种表型。例如，在最近的工作中，我们已经表明vHMEC含有E2F1转录因子及其几个下游靶点（如染色质重塑多梳组（PcG）蛋白、EZH2和SUZ12）的表达增加。当亲本HMEC细胞失去p16INK4a活性时，这些PcG蛋白被上调并定位于特定的基因组序列，随后通过DNA超甲基化修饰并沉默转录。我们已经证明，在这些条件下，靶向DMA超甲基化的位点包括HOXA 9，一种控制细胞命运和乳腺分化中重要的转录程序的转录因子。我们希望使用这个强大的模型系统来研究HOXA 9 DNA超甲基化事件的调控方面，以及在这些细胞中p16INK4a沉默后靶向DNA超甲基化的其他基因的身份。我们将通过特定目标实现这些目标，这些目标旨在（1）确定从绝经前和绝经后乳腺组织分离的HMEC中p16 INK4a活性丧失后HOXA 9调控区DNA超甲基化的动力学和程度，以及（2）确定HMEC和vHMEC之间DNA超甲基化不同的其他选择的基因组位点是否也依赖于p16INK4a的缺失活动我们将确定该基因中DNA超甲基化的动力学是否与HOXA 9基因座处测量的动力学相似。最后，我们希望确定靶向HOXA 9 DNA超甲基化（由p16 INK4a活性丧失引起）的起始是否可以通过pRb途径其他成员的丧失而表型复制。我们假设HMEC在体外的上述性质与其在体内的转化过程密切相关，并可能提供临床相关的生物标志物。

DESCRIPTION (provided by applicant): Upon culture of tissue biopsy samples from disease-free women, the majority of human mammary epithelial cells (HMEC) enter a proliferation barrier that is activated after several population doublings (1). A small subpopulation of human mammary epithelial cells, variant HMEC (vHMEC), have the ability to bypass this proliferation barrier and are typified by a silenced p16INK4a tumor suppressor gene, a sensitized stress response program (2) and the acquisition of a tremendous number of chromosomal abnormalities (1). Variant HMEC acquire several phenotypes critical to malignant transformation as a direct result of silenced p16INK4a. For example, in recent work we have shown that vHMEC contain an increased expression of the E2F1 transcription factor and several of its downstream targets such as the chromatin remodeling polycomb group (PcG) proteins, EZH2 and SUZ12. Upon loss of p16INK4a activity from parental HMEC cells, these PcG proteins are upregulated and localize to specific genomic sequences that are subsequently modified by DNA hypermethylation and silenced for transcription. We have demonstrated that loci targeted for DMA hypermethylation under these conditions include HOXA9, a transcription factor which controls cell fate and transcriptional programs important in mammary gland differentiation. We wish to use this powerful model system to investigate regulatory aspects of the HOXA9 DNA hypermethylation event as well as the identity of other genes that are targeted for DNA hypermethylation upon the silencing of p16INK4a in these cells. We will accomplish these goals through Specific Aims that seek to (1) determine the kinetics and extent of DNA hypermethylation at the HOXA9 regulatory region that takes place after the loss of p16INK4a activity in HMEC isolated from pre- and postmenopausal breast tissue and (2) determine if additional selected genomic sites that differ in DNA hypermethylation between HMEC and vHMEC are also dependent on the loss of p16INK4a activity. We will determine if the kinetics of DNA hypermethylation in this gene is similar to that measured at the HOXA9 locus. Finally, we wish to determine if the initiation of targeted HOXA9 DNA hypermethylation (brought about by loss of p16INK4a activity) can be phenocopied by the loss of other members of the pRb pathway. We hypothesize that the above-described properties of HMEC in vitro are critically relevant to their transformation processes in vivo and may provide clinically relevant biomarkers.