Intrinsically disordered regions in nucleosome positioning barriers

核小体缺失启动子区（NDRs）后接规则的核小体阵列（刻板的NDRs阵列模式）是真核生物中活性基因的普遍染色质模式，并且对于转录调控是必不可少的，但仍然无法解释。一般调节因子（GRF），如酵母Abf 1/Reb 1或哺乳动物CTCF，不是经典的转录反式激活因子，但被认为可以产生NDRs并作为阵列形成的核小体屏障。事实上，我们发现基本的NDR阵列模式在Abf 1/Reb 1和ATP依赖性染色质重塑物（如ISW 2）的最小系统中重建。现在，我们要问是什么使GRF成为核小体屏障。除了DNA结合结构域，GRF高度富集在固有无序区（IDR）。我们假设特定的IDR特性是GRF核小体屏障功能的关键。为了研究这一点，我们与Rohit Pappu教授（圣路易斯华盛顿大学）的团队合作，他们在IDR的生物物理学和合理的IDR设计方面具有丰富的经验。Pappu组的第一个序列分析已经显示了GRF的特征性和保守的IDR组织，即，DNA结合域侧翼的长酸性IDR，其不同于经典反式激活因子中的IDR组织。这提供了一个概念框架，以研究哪些IDR特性通过a）随机诱变（遗传筛选）; B）结构域交换; c）比较基因组学;和d）根据IDR生物物理学的从头设计赋予核小体屏障功能。我们将在体内以及通过我们独特的重建系统中的直接生化评估来测试屏障功能。通过这种方式，我们将定义核小体屏障功能的特定分子要求。

Nucleosome depleted promoter regions (NDRs) followed by regular nucleosomal arrays (stereotypic NDR-array patterns) are the universal chromatin pattern of active genes in eukaryotes and are essential for transcription regulation, but remain unexplained. General regulatory factors (GRFs), like yeast Abf1/Reb1 or maybe mammalian CTCF, are not classical transactivators of transcription but suggested to generate NDRs and function as nucleosome barriers for array formation. Indeed, we showed that basic NDR-array patterns are reconstituted in a minimal system of Abf1/Reb1 and ATP dependent chromatin remodelers, like ISW2. Now, we ask what makes a GRF a nucleosome barrier. Besides DNA binding domains, GRFs are highly enriched in intrinsically disordered regions (IDRs). We hypothesize that specific IDR properties are key to the GRF nucleosome barrier function. To study this, we collaborate with the group of Prof. Rohit Pappu (Washington University, St. Louis) that has extensive experience in the biophysics of IDRs and in rational IDR design to interrogate function. First sequence analyses in the Pappu group already showed a characteristic and conserved IDR organization of GRFs, i.e., long acidic IDRs flanking the DNA binding domain, that are distinct from IDR organizations in classical transactivators. This provides a conceptional framework to study which IDR properties confer nucleosome barrier function by a) random mutagenesis (genetic screens); b) domain swaps; c) comparative genomics; and d) de novo design according to IDR biophysics. We will test for barrier function both in vivo as well as by direct biochemical assessment in our unique reconstitution system. This way we will define the specific molecular requirements for nucleosome barrier function.