Heterochromatin biology

我们正在研究将构成异色区域保持在浓缩形式的机制。为此，我们使用了位于鸡β珠蛋白基因上游的16kb异染色质片段。在其他已发表的研究中，我们测量了这种片段的流体力学性质，这是很大一部分脊椎动物基因组的典型特征，如果不受调控，它可能会以一种有害于细胞功能的方式沉默相邻的基因。我们发现，在该区域内，组蛋白乙酰化水平的低水平与凝集结构的维持是耦合的，而使用组蛋白去乙酰化酶抑制剂提高组蛋白乙酰化水平显著增加转录。现在我们发现，当组蛋白乙酰化增加时，凝聚结构也会部分被破坏。我们还发现，当DICER或Argacete2酶耗尽时，也可以观察到类似的变化：整个结构域的转录水平上升，组蛋白乙酰化增加，组蛋白H3 K9甲基化减少，所有这些都与染色质结构域的打开一致，整个区域变得更容易受到限制酶MspI的攻击。DICER基因敲除也会导致异染色质区域产生的小干扰RNA分子消失。我们的结果表明，脊椎动物结构性异染色质的维持机制可能使用一些与分裂酵母相同的机制，并显示了如何在脊椎动物中建立和维持这种结构域。 我们现在已经将我们的研究扩展到全基因组范围内，调查Ago2蛋白在重复序列中的分布。在重复序列中发现高浓度的Ago2。我们现在正在完成对这些地区的分析。

We are studying the mechanisms that maintain constitutive heterochromatic regions in a condensed form. For this purpose we are using a well characterized 16 kb heterochromatin segment located upstream of the chicken beta globin locus. In other published studies we have measured the hydrodynamic properties of this fragment, which is typical of a large proportion of vertebrate genomes, and which has the potential if unregulated to silence adjacent genes in a manner deleterious to cellular function. We found that maintenance of the condensed structure is coupled to low level transcription in the region, and that elevation of levels of histone acetylation by use of histone deacetylase inhibitors markedly increases transcription. Now we have found that the condensed structure is also disrupted in part when histone acetylation increases. We have also found that similar changes can be observed when the enzymes dicer or argonaute2 are depleted: Levels of transcription rise across the domain, histone acetylation increases, and histone H3 K9 methylation decreases, all consistent with opening of the chromatin domain, and the entire region becomes more accessible to attack by the restriction enzyme MspI. Dicer knockdown also results in the disappearance of small interfering RNA molecules derived from the hetercohromatic region. Our results show that mechanisms of maintenance of constitutive heterochromatin in vertebrates may use some of the same mechanisms used by fission yeast, and they show how such domains may be established and maintained in vertebrates. We have now extended our studies to a genome-wide survey of the distribution of the protein Ago2 at repetitive sequences. High concentrations of Ago2 are found at repeat sequences. We are now completing our analysis of these regions.