Sullivan C H, O'Farrell S, Grainger R M
Department of Biology, University of Virginia, Charlottesville 22901.
Dev Biol. 1991 May;145(1):40-50. doi: 10.1016/0012-1606(91)90211-k.
In this study we address two questions regarding the control of delta-crystallin gene expression in chick embryos. First we have determined whether delta-crystallin mRNA is found outside of the developing lens, in which it is the predominant mRNA. We find that this mRNA can be detected, although at relatively low levels, in all embryonic tissues we have examined (from the definitive streak stage onward). This low level of transcription may be related to a second function for one or both of the delta-crystallin genes: both genes have a high degree of sequence identity to the enzyme argininosuccinate lyase. This result led us to a second set of experiments in which we reevaluated the possible role of hypomethylation in the expression of the delta-crystallin genes. Previous work showed that particular HhaI and HpaII sites in the crystallin genes undergo hypomethylation early in the process of lens differentiation when there is a burst of delta-crystallin mRNA accumulation. We not find that these sites remain methylated in nonlens tissues, implying that they cannot be required for the delta-crystallin gene activity found in these tissues. Other sites are constitutively hypomethylated, however, and may be functionally linked to this low level of gene activity. From an analysis of the kinetics of the developmentally regulated hypomethylation of HhaI and HpaII sites we also find that complete hypomethylation of these sites is not required for activating high levels of delta-crystallin transcription during lens differentiation. We do find, however, that these sites approach a fully hypomethylated state later in the lens differentiation process. Our analyses of mRNA levels and hypomethylation together lead us to propose that the delta-crystallin genes are regulated by two different mechanisms, one that leads to high levels of expression in the lens and the other which is responsible for low level expression in all other tissues in the chick embryo.
在本研究中，我们探讨了关于鸡胚中δ-晶体蛋白基因表达调控的两个问题。首先，我们确定了δ-晶体蛋白mRNA是否存在于发育中的晶状体之外，在晶状体中它是主要的mRNA。我们发现，在我们检测的所有胚胎组织中（从原条期开始）都能检测到这种mRNA，尽管水平相对较低。这种低水平的转录可能与δ-晶体蛋白基因中的一个或两个基因的第二种功能有关：这两个基因与精氨琥珀酸裂解酶具有高度的序列同一性。这一结果促使我们进行了第二组实验，在这些实验中，我们重新评估了低甲基化在δ-晶体蛋白基因表达中的可能作用。先前的研究表明，在晶状体分化过程早期，当δ-晶体蛋白mRNA大量积累时，晶体蛋白基因中的特定HhaI和HpaII位点会发生低甲基化。我们现在发现，这些位点在非晶状体组织中仍然是甲基化的，这意味着它们对于这些组织中发现的δ-晶体蛋白基因活性并非必需。然而，其他位点是组成性低甲基化的，并且可能在功能上与这种低水平的基因活性相关。通过对HhaI和HpaII位点发育调控的低甲基化动力学分析，我们还发现，在晶状体分化过程中激活高水平的δ-晶体蛋白转录并不需要这些位点完全低甲基化。然而，我们确实发现，在晶状体分化过程后期，这些位点接近完全低甲基化状态。我们对mRNA水平和低甲基化的分析共同使我们提出，δ-晶体蛋白基因受两种不同机制调控，一种机制导致其在晶状体中高水平表达，另一种机制负责其在鸡胚所有其他组织中的低水平表达。
