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

点击翻译按钮获取中文摘要

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.