Role of histone H3 phosphorylation in transcription and chromatin structure

组蛋白氨基末端的共价修饰是调控染色质结构和调控转录的重要机制。特别是，组蛋白H3和H4尾部的赖氨酸残基的乙酰化和甲基化似乎建立了一种密码，用于招募其他蛋白质并改变染色质结构。组蛋白H3在Ser10中的磷酸化作用研究较少，其改变染色质结构的机制也不清楚。这种修饰似乎是有丝分裂过程中染色体凝聚或间期转录激活的信号，但这些明显不同的作用的基础尚不清楚。同样未知的还有这种修饰在多大程度上被用来调节基因表达，H3磷酸化对转录的影响，以及控制Ser10磷酸化H3水平所涉及的酶或调节活性。拟议的实验解决了H3磷酸化是如何调节的问题，以及它在果蝇转录和染色质结构控制中的作用。H3Ser10的磷酸化水平似乎是转录活性的决定因素。H3的磷酸化程度应取决于作用于该组蛋白的蛋白激酶和蛋白磷酸酶的活性。初步结果表明，JIL-1激酶和PP2A磷酸酶是参与这一过程的两个关键酶。实验将试图确认这两种酶的参与和/或确定参与H3磷酸化的其他磷酸酶。SET和pp32蛋白已被证明在体外调节PP2A的活性，并抑制H3乙酰化。因此，这两个蛋白可能是控制组蛋白H3磷酸化和乙酰化之间相互作用的开关的一部分。遗传和分子方法的结合将被用来表征这两种蛋白质，并研究它们在体内控制共价组蛋白修饰的功能。最后，将对JIL-1突变表型的修饰物进行遗传筛选，以确定参与控制H3磷酸化和染色质浓缩的其他因素。该研究项目将作为旨在教育巴尔的摩公立高中生生物医学职业生涯的教学计划的教育基础。将开展的一系列教育活动有两个目标：增加约翰·霍普金斯大学贫困背景本科生的数量，并通过实验室的体验式学习，培训和指导这些学生在高中和霍普金斯大学进行尖端生物学研究。

Covalent modification of histone amino-terminal tails is an important mechanism used to modulate chromatin structure and regulate transcription. In particular, acetylation and methylation of Lys residues in the tails of histones H3 and H4 appear to establish a code that serves to recruit other proteins and alter chromatin structure. The role of histone H3 phosphorylation in Ser10 has been less studied and the mechanisms by which it alters chromatin structure are not understood. This modification appears to be a signal for either chromosome condensation during mitosis or transcription activation during interphase, but the basis for these apparently divergent roles is not known. Also unknown are the extent to which this modification is used to regulate gene expression, the aspects of transcription affected by H3 phosphorylation and the enzymatic or regulatory activities involved in controlling the levels of Ser10-phosphorylated H3. Proposed experiments address the question of how H3 phosphorylation is regulated and what its role is in the control of transcription and chromatin structure in Drosophila. The level of H3 Ser10 phosphorylation appears to be a determinant of transcription activity. The amount of phosphorylated H3 should depend on the activities of protein kinases and protein phosphatases acting on this histone. Preliminary results point to the JIL-1 kinase and PP2A phosphatase as two critical enzymes involved in this process. Experiments will attempt to confirm the involvement of these two enzymes and/or identify additional phosphatases involved in H3 phosphorylation. The proteins SET and pp32 have been shown to regulate PP2A activity in vitro, as well as to inhibit H3 acetylation. Therefore, these two proteins may be part of a switch that controls the interplay between histone H3 phosphorylation and acetylation. A combination of genetic and molecular approaches will be used to characterize these two proteins and study their in vivo function in the control of covalent histone modification. Finally, a genetic screen for modifiers of the JIL-1 mutant phenotype will be carried out in order to identify other factors involved in the control of H3 phosphorylation and chromatin condensation.This research project will serve as the educational basis for an instructional program aimed at educating Baltimore public high school students in biomedical science careers. A series of educational activities will be carried out with two objectives: increasing the number of undergraduate students from underprivileged backgrounds at Johns Hopkins and training and mentoring these students while in high school and at Hopkins in cutting edge biological research through experiential learning in the laboratory.