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Principal Investigator: Merlin Nithya Gnanapragasam
Organization: CLEVELAND STATE UNIVERSITY
Fiscal Year: 2024
Award: $415,031
Funding agency: National Institute of Diabetes and Digestive and Kidney Diseases
Project Summary:
The fetal to adult hemoglobin switching around birth involves an expression shift from γ-globin to β-globin in
erythroid cells. Effective re-expression of fetal γ-globin can ameliorate sickle cell anemia and β-thalassemia.
Hence, identification of inducers of fetal hemoglobin (HbF) is an active area of research and holds immense
therapeutic potential. Unlike the transcriptional and epigenetic regulation, post-transcriptional regulation of β-
globin switching is poorly understood, with few reports on its physiological and clinical relevance. Our preliminary
studies have identified Pumilo-1 (PUM1), an RNA binding protein with no previously reported functions in
erythropoiesis, as a post-transcriptional regulator of β-globin switching. PUM1, whose expression is regulated
by the erythroid master transcription factor, Erythroid Krüppel-like factor (EKLF/KLF1), peaks during erythroid
differentiation, binds fetal γ-globin mRNA, and reduces γ-globin (HBG1) mRNA stability and translational
efficiency, which culminates in reduced γ-globin protein levels. Knockdown of PUM1 leads to a robust increase
in fetal γ-globin (~22% HbF of total hemoglobin), without affecting adult β-globin levels in human erythroid cells.
Importantly, knocking down PUM1 does not limit erythropoiesis progression, providing a potentially safe and
effective treatment strategy in sickle cell anemia and β-thalassemia. In support of this idea, we report elevated
fetal hemoglobin levels in the absence of anemia, in an individual with a novel heterozygous PUM1 mutation in
the RNA binding domain (p.(His1090Profs*16); c.3267_3270delTCAC), suggesting that PUM1 mediated post-
transcriptional regulation is a critical player during human hemoglobin switching. Based on these preliminary
results, we hypothesize that impairing the RNA binding functions of PUM1 in human adult erythroid cells can
induce fetal hemoglobin levels, without deleterious effects on other aspects of erythropoiesis; this induction could
ameliorate the disease phenotypes such as sickling in cultured sickle cell anemia erythroid cells. To test this
hypothesis, we propose the following aims: 1) Decipher the regulatory elements that enable PUM1 mediated
fetal hemoglobin silencing in human erythroid cells, 2) Study the impact of PUM1 knockdown on human erythroid
differentiation, 3) Evaluate the capacity of PUM1 knockdown to relieve sickling in sickle cell anemia erythroid
cells.
Our studies will for the first time describe the post-transcriptional functions of PUM1 in hemoglobin switching and
erythropoiesis and reveal the impact of a novel human PUM1 mutation p.(His1090Profs*16), which we have
identified in a patient with elevated HbF. Further, these studies will advance our mechanistic understanding of
the post-transcriptional silencing of human fetal hemoglobin, which is poorly understood. Finally, since PUM1
functions as a cytoplasmic post-transcriptional regulator, our studies will determine if disrupting PUM1 to induce
fetal hemoglobin could serve as a potential non-gene altering therapeutic target towards ameliorating β-
thalassemia and sickle cell anemia.
Terms: <21+ years old><3' Untranslated Regions><3'UTR><Adult><Adult Human><Affect><Anemia><Area><B-globin><B-thalassemia><Basal Transcription Factor><Basal transcription factor genes><Binding><Binding Sites><Birth><Blood erythrocyte><CRISPR approach><CRISPR based approach><CRISPR method><CRISPR methodology><CRISPR technique><CRISPR technology><CRISPR tools><CRISPR-CAS-9><CRISPR-based method><CRISPR-based technique><CRISPR-based technology><CRISPR-based tool><CRISPR/CAS approach><CRISPR/Cas method><CRISPR/Cas technology><CRISPR/Cas9><CRISPR/Cas9 technology><Cas nuclease technology><Cell Body><Cell Cycle><Cell Division Cycle><Cells><Chromatin><Clinical><Clustered Regularly Interspaced Short Palindromic Repeats approach><Clustered Regularly Interspaced Short Palindromic Repeats method><Clustered Regularly Interspaced Short Palindromic Repeats methodology><Clustered Regularly Interspaced Short Palindromic Repeats technique><Clustered Regularly Interspaced Short Palindromic Repeats technology><Collaborations><Combining Site><Cytoplasm><Data><EKLF><Erythrocytes><Erythrocytic><Erythroid><Erythroid Cells><Erythropoiesis><Fetal Hb><Fetal Hemoglobin><Flow Cytofluorometries><Flow Cytofluorometry><Flow Cytometry><Flow Microfluorimetry><Flow Microfluorometry><Gene Inactivation><Gene Silencing><General Transcription Factor Gene><General Transcription Factors><Genes><Genetic Alteration><Genetic Change><Genetic defect><Genome><Globin><Hb SS disease><HbAS><HbF><HbSS disease><Hemoglobin><Hemoglobin F><Hemoglobin S Disease><Hemoglobin concentration result><Hemoglobin sickle cell disease><Hemoglobin sickle cell disorder><Hereditary Disease><Heterozygote><Human><Impairment><Inborn Genetic Diseases><Individual><Inherited disorder><Marrow erythrocyte><Mediating><Messenger RNA><Modern Man><Molecular><Molecular Interaction><Mutation><New York><Non-Polyadenylated RNA><Nuclear><O element><O2 element><Oxygen><Parturition><Patients><Physiologic><Physiological><Post-Transcriptional Control><Post-Transcriptional Regulation><Process><Proliferating><Proteins><Protocol><Protocols documentation><Putative RNA-Binding Region><RNA><RNA Binding><RNA Binding Domain><RNA Gene Products><RNA Recognition Motif><RNA Seq><RNA bound><RNA sequencing><RNA-Binding Proteins><RNAseq><RNP Domain><RNP Motif><RNP-1 Signature><Reactive Site><Red Blood Cells><Red Cell><Regulatory Element><Reporting><Research><Ribonucleic Acid><Role><Sickle Cell Anemia><Sickle Cell Trait><Site><Symptoms><Testing><Therapeutic><Time><Transcription Factor Proto-Oncogene><Transcription Regulation><Transcription factor genes><Transcriptional Control><Transcriptional Regulation><adulthood><alpha Globin><beta Globin><beta Thalassemia><blood corpuscles><clinical relevance><clinically relevant><disease phenotype><effective therapy><effective treatment><epigenetic regulation><erythroid Kruppel-like factor><erythroid development><erythroid differentiation><fetal><fetal form of hemoglobin><fetal globin><flow cytophotometry><gamma Globin><genome editing><genome mutation><genome wide analysis><genome wide studies><genome-wide analysis><genome-wide identification><genomic editing><hemoglobin level><hereditary disorder><heritable disorder><heterozygosity><inborn error><inherited diseases><inherited genetic disease><inherited genetic disorder><knock-down><knockdown><lentiviral-transduced><lentivirally transduced><lentivirus transduced><mRNA><mRNA Stability><mRNA Translation><novel><p-Globin><p-Thalassemia><post-transcriptional gene regulation><posttranscriptional><posttranscriptional control><posttranscriptional regulation><progenitor><programs><promoter><promotor><sickle cell disease><sickle cell disorder><sickle disease><sicklemia><sickling><social role><therapeutic target><transcription factor><transcriptional silencing><transcriptome sequencing><transcriptomic sequencing><treatment strategy><α-globin><β-globin><β-thalassemia><γ-Globin>