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

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.

KLF1 is an erythroid specific transcription factor essential for erythropoiesis. In recent years the number of individuals identified with disease phenotypes mapped to KLF1 mutations has increased rapidly. Of these the most severe, a form of dyserythropoietic anemia, is causally linked with the monoallelic mutation E325K; the effect of this mutation more severe than monoallelic loss of function KLF1 mutations. However, how the mutation affects the function of KLF1 in vivo resulting in a dominant phenotype is not known. Studying the molecular defects behind many RBC diseases is severely impeded by paucity of suitable and adequate quantities of material from anemic patients. We have recently developed technology and generated the first human immortalized adult erythroid lines, which recapitulate erythropoiesis, and provide a sustainable supply of cells. We have also developed a platform for CRISPR-Cas9 genome editing of these lines. We will use these technologies to create model disease cellular systems, generating a line from a patient with the E325K mutation, and introducing the mutation into an existing line. These systems will be used to (i) obtain a comprehensive map of the complete repertoire of proteins aberrantly expressed in cells expressing E325K KLF1 by comparative proteomics (ii) delineate molecular mechanisms by which the E325K mutation results in disrupted gene regulation, specifically determining if E325K KLF1 exhibits altered DNA binding specificity in vivo and if it interferes with the occupancy rate of wild type KLF1 at gene regulatory regions by ChIP-seq, and how E325K KLF1 then distorts the genetic readout of cells via analysis of chromatin configuration at gene loci required for interaction of regulatory elements and thus regulated gene expression using Capture-C 3C (iii) whether the mutation perturbs KLF1-co-factor binding required for such interactions. The data will also provide further insight into KLF1-directed transcriptional control.

The immediate impact of our studies will be in scientific advancement stemming from increased understanding of the molecular functions of KLF1, a transcription factor (TF) critical for erythropoiesis and how the E325K mutation effects KLF1 function and results in a severe disease phenotype. Findings will therefore be important to researchers in the UK and international academic communities and education, working in related fields. The number of individuals with phenotypes mapped to KLF1 mutations have increased rapidly in recent years, and it is likely that KLF1 mutations are causative in a range of further hematologic disorders. Hence comprehensive delineation of the altered proteome and disrupted genetic readout associated with the E325K KLF1 mutation may enable other RBC disorders with phenotypic similarities but of unknown etiology to be screened for KLF1 mutations, thus aiding diagnosis and opening-up new avenues for treatment strategies and potentially novel therapeutic approaches. More widely, for synthetic biology approaches, understanding of how substitution of residues in TFs effect function will facilitate the design of engineered TFs with enhanced or altered functions. Furthermore, identification of the molecular defect(s) resulting in severely impaired enucleation of erythroid cells in patients with the E325K KLF1 mutation may inform the enucleation defect of iPSC derived erythroid cells, and hence facilitate their use as a progenitor source for in vitro erythropoietic systems for novel therapeutics.
Also of impact to academic researchers, health service professionals and commercially will be our technology to create model cellular systems of RBC disease, and the disease lines created. Presently, studying the molecular defects behind many RBC diseases is severely impeded by paucity of suitable, and adequate quantities of material from anemic patients. Hence suitable model cellular systems are required that accurately mimic the disease state, but to date have not been available. Our original immortalised adult erythroid cell line (BEL-A), that forms the basis of these approaches has had significant impact with interest commercially and from academic researchers globally, and we anticipate similar interest for our disease systems. Such impact was also recently exemplified by the extensive press interest globally, following a press release on the BEL-A publication by the University Press Office.
Impact will also stem from major publications in high-profile peer-reviewed journals; invitations to disseminate data at conferences both home and abroad; from our long standing collaboration with National Health Service Blood & Transplant (NHSBT) in Bristol, Oxford and Cambridge, presenting data at research seminars and at the annual NHSBT R&D conference; the influence of research findings on the academic and industrial sector for understanding of the aetiology of human disease; formation of new national and international academic and health institute collaborations in order to exploit the acquired information; further funding applications.
Impact for the appointed post-doctoral researchers and academic lead will be in skills enhancement and career progression. The former will receive high-class training in multi-disciplinary research, essential for modern medically orientated research in academia, healthcare or industry along with experience gained in running a successful research lab, perform collaborative research and disseminating research findings in written and oral formats.
Impact on the wider community of young researchers will incur by our continued commitment to the training of international and national postgraduate students, undergraduate and sixth form pupils. We also anticipate considerable impact generated through collaborative links already established nationally and internationally, and with project partners Prof Hughes and Bieker who have distinct areas of expertise, their groups and institutes.