Department of Cell Biology and Cancer Center, University of Massachusetts Medical School, Worcester, Massachusetts 01655, USA.
J Cell Physiol. 2011 May;226(5):1383-9. doi: 10.1002/jcp.22465.
The nuclear matrix bound transcription factor RUNX2 is a lineage-specific developmental regulator that is linked to cancer. We have previously shown that RUNX2 controls transcription of both RNA polymerase II genes and RNA polymerase I-dependent ribosomal RNA genes. RUNX2 is epigenetically retained through mitosis on both classes of target genes in condensed chromosomes. We have used fluorescence recovery after photobleaching to measure the relative binding kinetics of enhanced green fluorescent protein (EGFP)-RUNX2 at transcription sites in the nucleus and nucleoli during interphase, as well as on mitotic chromosomes. RUNX2 becomes more strongly bound as cells go from interphase through prophase, with a doubling of the most tightly bound "immobile fraction." RUNX2 exchange then becomes much more facile during metaphase to telophase. During interphase the less tightly bound pool of RUNX2 exchanges more slowly at nucleoli than at subnuclear foci, and the non-exchanging immobile fraction is greater in nucleoli. These results are consistent with a model in which the molecular mechanism of RUNX2 binding is different at protein-coding and ribosomal RNA genes. The binding interactions of RUNX2 change as cells go through mitosis, with binding affinity increasing as chromosomes condense and then decreasing through subsequent mitotic phases. The increased binding affinity of RUNX2 at mitotic chromosomes may reflect its epigenetic function in "bookmarking" of target genes in cancer cells.
核基质结合转录因子 RUNX2 是一种与癌症相关的谱系特异性发育调节剂。我们之前已经表明，RUNX2 控制 RNA 聚合酶 II 基因和 RNA 聚合酶 I 依赖性核糖体 RNA 基因的转录。在浓缩染色体上，RUNX2 通过有丝分裂在两类靶基因上保持表观遗传。我们使用光漂白后荧光恢复来测量间期核和核仁中增强型绿色荧光蛋白（EGFP）-RUNX2 在转录位点的相对结合动力学，以及有丝分裂染色体上的结合动力学。随着细胞从间期进入前期，RUNX2 的结合变得更加紧密，最紧密结合的“固定分数”增加了一倍。随后，在中期到末期，RUNX2 的交换变得更加容易。在间期，RUNX2 的结合较弱，在核仁中的交换速度比在亚核焦点中慢，并且核仁中非交换的固定分数更大。这些结果与 RUNX2 结合的分子机制在蛋白质编码基因和核糖体 RNA 基因中不同的模型一致。随着细胞通过有丝分裂，RUNX2 的结合相互作用发生变化，随着染色体浓缩，结合亲和力增加，然后在随后的有丝分裂阶段降低。在有丝分裂染色体上 RUNX2 增加的结合亲和力可能反映了其在癌细胞中靶基因“标记”的表观遗传功能。
