Regulation of DNA hypermethylation in human mammary cells

描述（由申请人提供）：对无病女性的组织活检样本进行培养后，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)。人类乳腺上皮细胞的一个小亚群，变异HMEC (vHMEC)，具有绕过这种增殖屏障的能力，其典型特征是沉默的p16INK4a肿瘤抑制基因，致敏的应激反应程序(2)和获得大量染色体异常(1)。作为沉默p16INK4a的直接结果，HMEC变体获得了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转录因子及其下游靶标（如染色质重塑多梳群（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蛋白被上调并定位到特定的基因组序列，这些基因组序列随后被DNA超甲基化修饰并沉默转录。我们已经证明，在这些条件下，针对DMA超甲基化的位点包括HOXA9，这是一种控制细胞命运和转录程序的转录因子，在乳腺分化中很重要。我们希望利用这个强大的模型系统来研究HOXA9 DNA超甲基化事件的调控方面，以及在这些细胞中p16INK4a沉默时靶向DNA超甲基化的其他基因的身份。我们将通过特定目标来实现这些目标，寻求(1)确定在绝经前和绝经后乳腺组织分离的HMEC中p16INK4a活性丧失后HOXA9调控区域DNA高甲基化的动力学和程度，以及(2)确定HMEC和vHMEC之间DNA高甲基化差异的其他选定基因组位点是否也依赖于p16INK4a活性的丧失。我们将确定该基因的DNA超甲基化动力学是否与在HOXA9位点测量的动力学相似。最后，我们希望确定靶向HOXA9 DNA超甲基化的启动（由p16INK4a活性的丧失引起）是否可以通过pRb途径的其他成员的丧失来表型。我们假设体外HMEC的上述特性与它们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.