Chromatin Structure And Function

我们继续对表达基因附近的染色质结构进行研究。鸡红系细胞中的珠蛋白基因家族是一个模型系统，在该系统中可以研究红系发育过程中家族和个体成员的调控机制。我们将注意力集中在鸡β-珠蛋白基因5‘端1.2kb的绝缘子DNA序列及其上游元件上。这种绝缘体既能阻止外部增强剂的影响，又能防止可能关闭整个区域表达的浓缩染色质的侵蚀。我们发现，增强子阻断活性与单一蛋白CTCF与增强子内的一个位点结合有关。绝缘体还具有单独的能力，以防止位置效应报告基因稳定地转染到细胞系或动物中，作为防止浓缩染色质侵蚀的边界。我们发现，这种保护能力存在于1.2kb绝缘子内的250个碱基的核心元件中，并且核心中包含CTCF位点的亚区的缺失不会影响活性。然而，对应于核蛋白结合位点的另外四个亚区对于位置效应保护(边界功能)是重要的。我们现在已经确定了与这些位点结合的几个相关因素。一种是我们实验室之前描述的一种蛋白质，它对寡聚G序列有强烈的偏好。它绑定到四个站点中的两个。其他因素与另外两个网站有关。此外，我们还能够在体内检测到已知组蛋白修饰酶的辅助因子在绝缘体上的结合。这些结果与我们提出的一个模型是一致的，在该模型中，屏障功能与绝缘子附近的多个组蛋白修饰有关。珠蛋白绝缘体似乎是一种屏障，可以防止长度约16kb的浓缩染色质上游区域的侵蚀。我们开发了高精度分析这一上游段的水动力特性的方法。除了非常精确地测量其沉降系数外，我们还测量了这一特定碎片的浮力密度。这使我们能够计算其摩擦系数，并得出结论，即颗粒形状是一个延伸的杆，与许多实验室早期的结构建议一致。这一点很重要，因为脊椎动物基因组中有大量这种散布的浓缩染色质片段，而且几乎对它们的结构一无所知。我们还继续研究转基因沉默的动力学，使用我们的测试构建表达IL2受体的片段。虽然在一些基因系统中，DNA甲基化是基因沉默的主要事件，但我们在这里发现，组蛋白H3和H4的去乙酰化首先发生，或多或少与mRNA的丢失同时发生。然而，DNA甲基化发生的时间要晚得多，这不是沉默的原因，而是其他更早变化的结果。

We have continued our studies of chromatin structure in the neighborhood of expressed genes. The globin gene family in chicken erythroid cells serves as a model system in which it is possible to study the mechanisms associated with regulation of the cluster and individual members of the family during erythroid development. We have focused attention on the 1.2 kb insulator DNA sequence at the 5' end of the chicken beta-globin locus, and elements upstream of it. This insulator is capable both of blocking the influence of outside enhancers and of preventing the encroachment of condensed chromatin that might shut down expression of the entire region. We showed that enhancer blocking activity is associated with binding of a single protein, CTCF, to a site within the enhancer. The insulator also has the separate ability to protect against position effects reporter genes that are stably transfected into cell lines or animals, serving as a boundary against encroachment of condensed chromatin. We found that this protective ability is present in a ?core' element, 250 bp long, from within the 1.2 kb insulator, and that deletion of subregions within the core that contain the CTCF site do not affect activity. However four other subregions corresponding to binding sites for nuclear proteins are important for position effect protection (boundary function). We have now identified several of the involved factors binding to these sites. One is a protein previously described in our laboratory, and which has a strong preference for oligo G sequences. It binds to two of the four sites. Other factors are associated with the other two sites. We have furthermore been able to detect binding at the insulator in vivo of co-factors that are known histone modification enzymes. These results are consistent with a model we have proposed in which barrier function is connected with multiple histone modifications in the neighborhood of the insulator. The globin insulator appears to serve as a barrier against encroachment of an upstream region of condensed chromatin about 16 kb in length. We have developed methods for analyzing the hydrodynamic properties of this upstream segment with high precision. In addition to measuring its sedimentation coefficient very accurately, we have also measured the buoyant density of this specific fragment. This allows us to calculate its frictional coefficient and leads to the conclusion that the particle shape is an extended rod, consistent with earlier structural proposals from a number of laboratories. This is important because vertebrate genomes have large numbers of interspersed condensed chromatin segments of this kind, and virtually nothing is known of their structure. We have also continued studies of the kinetics of transgene silencing, using our test construct which expresses a fragment of the IL2 receptor. Although in some gene systems DNA methylation is the primary event in gene silencing, we find here that histone H3 and H4 deacetylation occurs first, more or less simultaneously with loss of mRNA. However DNA methylation occurs much later and cannot be the cause of the silencing, but rather a consequence of other, earlier changes.