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作为一个模型系统，可以研究在红系发育过程中该家族的集群和个体成员的调控机制。我们将重点放在鸡β -珠蛋白位点5'端的1.2 kb绝缘子DNA序列上。这种绝缘体既能阻断外部增强子的影响，又能防止可能关闭整个区域表达的浓缩染色质的侵入。我们之前已经将增强子阻断活性缩小到1.2 kb序列内的一个小DNA片段，并表明该活性与普遍存在的脊椎动物核蛋白CTCF的结合有关。我们发现CTCF结合位点在小鼠和人的Igf2/H19位点的印迹中也起着重要作用。该绝缘体还具有保护被稳定地转染到细胞系或动物中的报告基因免受位置效应的能力。我们发现这种保护能力存在于？在1.2 kb绝缘子内删除250 bp长的core'元件，并且删除core中包含CTCF位点的子区域不会影响活性。然而，与核蛋白结合位点相对应的其他四个亚区对位置效应保护很重要。我们已经确定了至少一个相关因子，与其中两个位点结合，作为我们实验室先前描述的蛋白质，并且对寡核苷酸G序列具有强烈的偏好。珠蛋白绝缘体似乎作为一个屏障，防止上游浓缩染色质区域的侵犯，该区域的长度约为16kb。我们已经开发了高精度分析这一段水动力特性的方法，现在正在使用这些方法来确定这一重要元素的压实状态。针对位置效应的保护试验利用了显示在细胞表面的蛋白质的报告基因，从而可以确定单个细胞的活性以及总mRNA丰度。在未绝缘的细胞系中，该基因的表达通常在培养10-80天后消失。由于这种灭绝通常以DNA甲基化增加为标志，我们利用亚硫酸盐作图来确定启动子和部分编码区域中每个CpG甲基化位点的甲基化状态。DNA甲基化的时间过程与mRNA的消失速度相比是缓慢的。然而，信使RNA （mRNA）的丢失与组蛋白乙酰化的丢失相关。随着灭绝的进行，H3赖氨酸9的甲基化水平（我们已经证明这是珠蛋白染色质失活的标志）也在缓慢增加。数据支持一种模型，其中组蛋白去乙酰化是该报告基因失活的控制步骤。我们发现，被隔离的记者的行为完全不同，他们受到保护，不会被噤声。在这里，启动子和编码区都维持乙酰化（即使在具有突变增强子的基因中），但DNA甲基化仅在启动子上受到抑制。这似乎足以防止灭绝。我们提出了一个模型，其中组蛋白乙酰化是维持活性的主要步骤。这反过来使启动子上的核小体更具移动性，并允许转录因子结合。这些结合蛋白占据潜在的DNA甲基化位点，进而防止失活。这与早期已知的调控环完全不同，其中DNA甲基化是导致组蛋白去乙酰化酶募集的主要事件。这证实了我们之前提出的针对位置效应的绝缘体保护机制。

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. 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 had earlier narrowed down enhancer blocking activity to a small DNA fragment within the 1.2 kb sequence, and shown that this activity involves binding of the ubiquitous vertebrate nuclear protein CTCF. We showed that the CTCF binding site also plays an important role in imprinting at the Igf2/H19 locus in mouse and humans. The insulator also has the ability to protect against position effects reporter genes that are stably transfected into cell lines or animals. We showed 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. We have identified at least one of the involved factors, binding to two of these sites, as a protein previously described in our laboratory, and which has a strong preference for oligo G sequences. 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 segment with high precision, and are now using these methods to determine the state of compaction of this important element. Assays for protection against position effects make use of a reporter gene for a protein that is displayed on the cell surface, making possible determination of individual cell activities as well as total mRNA abundance. In uninsulated lines expression of this gene is typically extinguished over 10-80 days in culture. Because such extinction is usually marked by increased DNA methylation we made use of bisulfite mapping to determine the methylation state of each CpG methylation site in the promoter and part of the coding region. The time course of DNA methylation is slow compared to the rate of disappearance of mRNA. However the loss of messenger RNA (mRNA) correlates with the loss of histone acetylation. As extinction proceeds, levels of H3 lysine 9 methylation, which we have shown is a mark of inactive globin chromatin, also increase slowly. The data support a model in which histone deacetylation is the controlling step in inactivation of this reporter. We find that insulated reporters, which are protected from silencing, behave quite differently. Here acetylation is maintained over both promoter and coding region (even in a gene with a mutated enhancer), but DNA methylation is only inhibited over the promoter. This appears to be sufficient to protect against extinction. We propose a model in which once again histone acetylation is the primary step in maintaining activity. This in turn makes nucleosomes over the promoter more mobile, and allows binding of transcription factors. Occupancy of potential DNA methylation sites by these bound proteins in turn protects against inactivation. This is completely different from the earlier known regulatory loop in which DNA methylation is the primary event leading to recruitment of histone deacetylases. It serves to confirm the mechanism we have previously proposed for insulator protection against position effects.