The Role of Methyl CpG Binding Domain Protein 2 (MBD2) in the Regulation of Embryonic and Fetal β-type Globin Genes

The reexpression of the fetal γ-globin gene in adult erythrocytes is of therapeutic interest due to its ameliorating effects in β-hemoglobinopathies. We recently showed that Methyl CpG Binding Domain Protein2 (MBD2) contributes to the silencing of the chicken embryonic ρ-globin and human fetal γ-globin genes. We further biochemically characterized an erythroid MeCP1 complex that is recruited by MBD2 to mediate the silencing of these genes. These observations suggest that the disruption of the MeCP1 complex could augment the expression of the fetal/embryonic globin genes. In the studies presented in chapter 2, we have pursued a structural and biophysical analysis of the interaction between two of the six components of the MeCP1 complex: MBD2 and p66α. These studies show that the coiled coil regions from MBD2 and p66α form a highly stable heterodimeric complex. Further, overexpressing the p66α coiled coil domain in adult erythroid cells can augment the expression of the chicken ρ-globin and human γ-globin genes, by disrupting the assembly of a functional MeCP1 complex. This indicates that the exogenously expressed p66α coiled coil peptide competes with the endogenous p66α for the interaction with the coiled coil domain of MBD2. These studies show that the coiled coil interaction between MBD2 and p66α could serve as a potential targets for the therapeutic induction of fetal hemoglobin. The laboratory showed that knockout of MBD2 in transgenic mice carrying the human β-globin gene cluster, results in an elevated expression of γ-globin in adult erythrocytes. However, MBD2 does not directly bind to the γ-globin gene to mediate its silencing. In the work presented in chapter 3, we have tested the hypothesis that MBD2 may suppress γ-globin gene transcription in adult erythrocytes indirectly, by binding to and repressing transcription of intermediary gene/s which may be involved in γ-globin gene regulation. Employing microarray technology, we have identified Gab1 and ZBTB32 as candidate genes that may be involved in the MBD2 mediated silencing of γ-globin

PUM1 Mediates the Posttranscriptional Regulation of Human Fetal Hemoglobin

The fetal-to-adult hemoglobin switching at about the time of birth involves a shift in expression from γ-globin to β-globin in erythroid cells. Effective re-expression of fetal γ-globin can ameliorate sickle cell anemia and β-thalassemia. Despite the physiological and clinical relevance of this switch, its posttranscriptional regulation is poorly understood. Here, we identify Pumilo 1 (PUM1), an RNA-binding protein with no previously reported functions in erythropoiesis, as a direct posttranscriptional 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 γ-globin messenger RNA (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 hemoglobin (∼22% HbF) without affecting β-globin levels in human erythroid cells. Importantly, targeting PUM1 does not limit the progression of erythropoiesis, which provides a potentially safe and effective treatment strategy for sickle cell anemia and β-thalassemia. In support of this idea, we report elevated levels of HbF 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), which suggests that PUM1-mediated posttranscriptional regulation is a critical player during human hemoglobin switching

A Systems Approach Identifies Essential FOXO3 Functions at Key Steps of Terminal Erythropoiesis

<div><p>Circulating red blood cells (RBCs) are essential for tissue oxygenation and homeostasis. Defective terminal erythropoiesis contributes to decreased generation of RBCs in many disorders. Specifically, ineffective nuclear expulsion (enucleation) during terminal maturation is an obstacle to therapeutic RBC production <i>in vitro</i>. To obtain mechanistic insights into terminal erythropoiesis we focused on FOXO3, a transcription factor implicated in erythroid disorders. Using an integrated computational and experimental systems biology approach, we show that FOXO3 is essential for the correct temporal gene expression during terminal erythropoiesis. We demonstrate that the FOXO3-dependent genetic network has critical physiological functions at key steps of terminal erythropoiesis including enucleation and mitochondrial clearance processes. FOXO3 loss deregulated transcription of genes implicated in cell polarity, nucleosome assembly and DNA packaging-related processes and compromised erythroid enucleation. Using high-resolution confocal microscopy and imaging flow cytometry we show that cell polarization is impaired leading to multilobulated <i>Foxo3</i>^<i>-/-</i> erythroblasts defective in nuclear expulsion. Ectopic FOXO3 expression rescued <i>Foxo3</i>^<i>-/-</i> erythroblast enucleation-related gene transcription, enucleation defects and terminal maturation. Remarkably, FOXO3 ectopic expression increased wild type erythroblast maturation and enucleation suggesting that enhancing FOXO3 activity may improve RBCs production. Altogether these studies uncover FOXO3 as a novel regulator of erythroblast enucleation and terminal maturation suggesting FOXO3 modulation might be therapeutic in disorders with defective erythroid maturation.</p></div

Model.

<p><b>(A)</b> Depiction of expression of clusters Q and R genes in <i>Foxo3</i>^<i>-/-</i> versus wild type erythroblasts. Cluster Q is enriched for nucleosome assembly, heme biosynthesis, and DNA packaging-related processes while cluster R is enriched for autophagy and catabolic processes. <b>(B)</b> Model for gene expression in terminally maturing erythroblasts. Complexes of core erythroid transcription factors regulate the genetic programs required for maturation of the initial erythroblast stages. These transcription factor complexes may also induce <i>Foxo3</i> expression in immature erythroblasts. In turn, FOXO3 cooperates with these factors to sustain and/or enhance the erythroid transcriptional program during the later stages of terminal maturation.</p

Defective enucleation in <i>Foxo3</i>^<i>-/-</i> bone marrow erythroblasts.

<p><b>(A)</b> Enucleation was analyzed by immunofluorescence of freshly isolated bone marrow cells from WT (n = 5) and <i>Foxo3</i>^<i>-/-</i> (n = 3) mice using anti-TER119 antibody (green), Rhodamine Phalloidin (red) and Hoechst (blue). Images were obtained by confocal microscopy and abnormal enucleating cells counted. Representative images of enucleating cells are shown, with white asterisks denoting abnormally enucleating cells. At least 10 enucleating cells were counted per bone marrow and the results indicate the percentage of abnormal enucleating cells in each bone marrow as mean ± SEM. <b>(B)</b> Quantification of abnormal nuclei within the orthochromatic faction of WT and <i>Foxo3</i>^<i>-/-</i> erythroblasts by imaging flow cytometry. Abnormal nuclei were defined as having high 3-fold symmetry of the nucleus [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005526#pgen.1005526.ref064" target="_blank">64</a>]. Representative images of normal and abnormal nuclei from <i>Foxo3</i> mutants are shown are shown. Results are mean ± SEM of n = 4. *<i>P</i> < 0.05 **<i>P <</i> 0.01 ***<i>P</i> < 0.001, Student’s <i>t</i> test. <b>(C)</b> Model for the impact of loss of FOXO3 on the enucleation process. <b>(D)</b> Heatmap of RNA-Seq data of CDC42-related gene cluster (both upstream and downstream of CDC42) implicated in polarity and actin polymerization in Gates I-II and III of <i>Foxo3</i> wild type and mutant erythroblasts.</p

Deregulated gene expression in maturing <i>Foxo3</i>^<i>-/-</i> erythroblasts.

<p><b>(A)</b> Flow cytometry strategy used to FACS sort pro-, basophilic and polychromatic erythroblasts (Gates I to III respectively, in red) from wild type and <i>Foxo3</i>^<i>-/-</i> bone marrow according to their TER119 and CD44 cell surface expression and forward scatter properties for RNA-Seq. Gate IV cells (depicted in black) are purified in subsequent experiments for experimental validation purposes. <b>(B)</b> Heatmap of differentially expressed genes in WT erythroblasts (low in green to high in red). Clustering of genes was performed according to their expression level in WT pro-, basophilic and polychromatic erythroblasts. Only the 5514 genes that varied at least 2 fold from pro- to polychromatic erythroblasts were used for clustering. <b>(C)</b> Heatmap of differentially expressed genes in WT versus <i>Foxo3</i>^<i>-/-</i> erythroblasts at each gate (low in green to high in red). Clustering of the 3904 differentially expressed genes between WT and <i>Foxo3</i>^<i>-/-</i> samples (amplitude ≥ 2) is shown. Amplitude was calculated as the difference between the same gates of WT and <i>Foxo3</i>^<i>-/-</i> erythroblasts.</p