Molecular mechanism by which the E325K mutation of human KLF1 causes a severe dyserythropoietic anemia, utilising a novel model system of RBC disease

KLF1是一种转录因子（TF），专门在发育中的红细胞（rbc）中产生，它对调节许多基因的表达以及这些细胞中合成的蛋白质至关重要。事实上，KLF1被认为是红细胞生成和功能的主要调节因子。因此，可以预期KLF1突变会产生不良后果，近年来，与KLF1突变相关的RBC疾病个体数量迅速增加，确实是这种情况。其中，KLF1 DNA结合区域内的杂合子E325K突变（氨基酸325处的谷氨酸被赖氨酸取代）与一种严重的红细胞疾病有因果关系。然而，这种突变如何影响体内KLF1的功能从而导致疾病表型目前尚不清楚。研究许多红细胞疾病背后的缺陷严重阻碍了缺乏合适的，足够数量的贫血患者的材料。因此，需要合适的模型细胞系统来准确地模拟红细胞疾病，但迄今为止还没有可用的。我们最近开发了技术，并产生了第一个人类不朽的成人红细胞系。这些细胞在体外进行正常的红细胞合成过程，并提供持续的细胞供应。我们还开发了一个平台，允许我们在基因组的特定位置引入突变，产生一个子系并提供具有突变的细胞用于研究。因此，我们有独特的机会来创建模型红细胞疾病的细胞系统。我们建议从E325K突变的患者中创建一个品系，并将E325K突变引入我们现有的一个品系，重新创建该疾病的基因型和表型。我们将使用这些系统(i)使用比较蛋白质组学技术获得E325K KLF1细胞中异常表达的完整蛋白质库的全面图谱，以确定紊乱蛋白质组的程度。这些数据也将有助于促进，从而提高对更多的突变患者的诊断，并可能揭示与未知病因的红细胞疾病的重叠，促进KLF1突变的筛选（ii）描述E325K突变导致基因调控中断的分子机制，从而使用全基因组分析技术改变蛋白质表达和疾病表型。KLF1结合到它控制的基因的调控区域，在大多数情况下，诱导它们的表达。因此，我们将确定E325K KLF1是否干扰正常KLF1与这些调控区域的结合，反之，E325K KLF1是否混杂地结合在红细胞中不正常表达的基因的调控区域。然而，基因的表达通常受到多个调控区域的控制，这些区域可能位于DNA中彼此之间的距离，以及与靶基因之间的距离，它们必须通过3D染色质（DNA）结构的改变相互作用，由TF结合促进，以实现基因表达。因此，为了确定E325K KLF1如何扭曲细胞的遗传读数，我们将分析其对所选KLF1调节基因位点上的这种染色质配置的影响。E325K或正常KLF1与调控区域的异常结合，以及这些区域之间的阻碍或不正确的相互作用将有助于阻止红细胞所需蛋白质的产生或降低其水平，同时可能导致红细胞中不正常存在的蛋白质的产生（iii）确定突变是否扰乱了KLF1与其活性所需的辅助因子之间的相互作用，如果是这样，这些因子的丢失对KLF1与基因调控区域结合的影响。除了揭示E325K KLF1突变导致所观察到的疾病表型的分子机制外，该数据还将进一步深入了解KLF1对基因表达的调控，从而促进红细胞的产生。

KLF1 is a transcription factor (TF) specifically produced in developing red blood cells (RBCs) where it is essential for regulating the expression of many genes, and thus the proteins synthesized in these cells. Indeed, KLF1 is considered a master regulator of red blood cell production and function. It would therefore be anticipated that mutations in KLF1 have adverse outcomes, and this is indeed the case with the number of individuals identified with RBC disorders associated with mutations in KLF1 increasing rapidly over recent years. Of these the heterozygous E325K mutation (substitution of glutamic acid with lysine at amino acid 325) within the DNA binding domain of KLF1, is causally linked to a severe form of RBC disease. However, how this mutation effects the function of KLF1 in vivo to cause the disease phenotype is currently not known. Studying the defects behind many RBC diseases is severely impeded by paucity of suitable, and adequate quantities of material from anaemic patients. Hence suitable model cell systems are required that accurately mimic RBC diseases, but to date have not been available. We have recently developed technology and generated the first human immortalized adult RBC lines. The cells undergo the normal process of RBC synthesis in vitro, and provide a sustainable supply of cells. We have also developed a platform, allowing us to introduce mutations in specific positions in the genome, producing a sub-line and supply of cells with the mutation for study. We thus have the unique opportunity to create model cellular systems of RBC disease. We propose to create a line from a patient with the E325K mutation, and introduce the E325K mutation into one of our existing lines, recreating the disease genotype and phenotype. We will use these systems to (i) obtain a comprehensive map of the complete repertoire of proteins aberrantly expressed in cells with E325K KLF1 using comparative proteomic techniques, to determine the extent of the disordered proteome. These data will also serve to facilitate, and thus improve diagnosis of further patients with the mutation, and may reveal overlap with profiles of RBC disorders of unknown etiology, prompting screening for KLF1 mutations (ii) delineate the molecular mechanisms by which the E325K mutation results in disrupted gene regulation, and thus altered protein expression and the disease phenotype using genome-wide analysis techniques. KLF1 binds to the regulatory regions of the genes it controls to, in most cases, induce their expression. We will therefore determine if E325K KLF1 interferes with the binding of normal KLF1 to such regulatory regions, and conversely if E325K KLF1 binds promiscuously to the regulatory regions of genes not normally expressed in RBCs. However, expression of a gene is often controlled by multiple regulatory regions that may lie at a distance from each other in the DNA, and from the target gene, which must interact via alterations in the 3D chromatin (DNA) structure, facilitated by TF binding, for gene expression. Therefore, to determine how E325K KLF1 may distort the genetic readout of cells we will analyse its effect on such chromatin configuration at the loci of selected KLF1 regulated genes. Aberrant binding of E325K or normal KLF1 to regulatory regions, and impeded or incorrect interaction between such regions would serve to prevent production or reduce the level of proteins required by RBCs, whilst potentially cause proteins not normally present in RBCs to be produced (iii) determine if the mutation perturbs the interaction between KLF1 and co-factors required for its activity, and if so the effect of the loss of such factors on the binding of KLF1 to gene regulatory regions.As well as revealing the molecular mechanisms by which the E325K KLF1 mutation results in the observed disease phenotype, the data will also provide further insight into the regulation of gene expression and thus red blood cell production by KLF1.