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），在发育中的红细胞（RBC）中特异性产生，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被认为是红细胞产生和功能的主要调节因子。因此，预计KLF1突变会产生不良后果，近年来，与KLF1突变相关的RBC疾病患者数量确实迅速增加。其中，KLF1的DNA结合结构域内的杂合E325K突变（氨基酸325处的谷氨酸被赖氨酸取代）与严重形式的RBC疾病有因果关系。然而，这种突变如何影响KLF1在体内的功能，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是严重阻碍了缺乏合适的，足够数量的材料从贫血患者。因此，需要准确模拟RBC疾病的合适的模型细胞系统，但迄今为止尚未获得。我们最近开发了技术，并产生了第一个人永生化的成人红细胞系。这些细胞在体外进行正常的RBC合成过程，并提供可持续的细胞供应。我们还开发了一个平台，使我们能够在基因组的特定位置引入突变，产生一个亚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RBC疾病的模型细胞系统。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中，重新创建疾病基因型和表型。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细胞中异常表达蛋白质的完整谱，以确定紊乱蛋白质组的程度。这些数据也将有助于促进，从而提高诊断的进一步患者的突变，并可能揭示重叠的轮廓RBC疾病的病因不明，促进筛选KLF1突变（ii）描绘的分子机制，E325K突变导致基因调控中断，从而改变蛋白质表达和疾病表型使用全基因组分析技术。KLF1与它所控制的基因的调控区结合，在大多数情况下，诱导它们的表达。因此，我们将确定E325K KLF 1是否干扰正常KLF 1与这些调控区域的结合，反之，E325K KLF 1是否混杂地与红细胞中不正常表达的基因的调控区域结合。然而，基因的表达通常由多个调控区控制，这些调控区在DNA中可能彼此相距一定距离，并且与靶基因相距一定距离，这些调控区必须通过TF结合促进的3D染色质（DNA）结构的改变相互作用以进行基因表达。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调节基因位点处的染色质构型的影响。E325K或正常KLF 1与调节区的异常结合，以及这些区域之间的受阻或不正确的相互作用将用于阻止RBC所需蛋白质的产生或降低RBC所需蛋白质的水平，同时潜在地导致通常不存在于RBC中的蛋白质的产生（iii）确定突变是否干扰KLF 1与其活性所需的辅因子之间的相互作用，如果是这样的话，这些因素的损失对KLF1的基因调控区域的结合的影响，以及揭示的分子机制，E325 K KLF1突变导致观察到的疾病表型，数据也将提供进一步的洞察基因表达的调控，从而红细胞生产KLF1。

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.