Elevating fetal hemoglobin - recently discovered regulators and mechanisms.

It has been known for over half a century that humans produce different forms of hemoglobin, a tetramer of α- and ß-like hemoglobin chains, throughout ontogeny. The switch from fetal to adult hemoglobin occurs around the time of birth when erythropoiesis shifts from the fetal liver to the bone marrow. Naturally, diseases caused by defective adult ß-globin genes, such as sickle cell disease and ß-thalassemia manifest themselves as production of fetal hemoglobin fades. Reversal of this developmental switch has been a major goal to treat these diseases and has been a driving force to understand its underlying molecular biology. Several review articles have illustrated the long and at times arduous paths that led to the discovery of the first transcriptional regulators involved in this process. Here we survey recent developments, spurred by the discovery of CRISPR tools that enabled for the first time high throughput genetic screens for new molecules that impact the fetal to adult hemoglobin switch. Numerous opportunities for therapeutic intervention thus came to light, offering hope for effective pharmacologic intervention for patients for whom gene therapy is out of reach.

let-7 miRNAs repress HIC2 to regulate BCL11A transcription and hemoglobin switching.

ABSTRACT: The switch from fetal hemoglobin (γ-globin, HBG) to adult hemoglobin (ß-globin, HBB) gene transcription in erythroid cells serves as a paradigm for a complex and clinically relevant developmental gene regulatory program. We previously identified HIC2 as a regulator of the switch by inhibiting the transcription of BCL11A, a key repressor of HBG production. HIC2 is highly expressed in fetal cells, but the mechanism of its regulation is unclear. Here we report that HIC2 developmental expression is controlled by microRNAs (miRNAs), as loss of global miRNA biogenesis through DICER1 depletion leads to upregulation of HIC2 and HBG messenger RNA. We identified the adult-expressed let-7 miRNA family as a direct posttranscriptional regulator of HIC2. Ectopic expression of let-7 in fetal cells lowered HIC2 levels, whereas inhibition of let-7 in adult erythroblasts increased HIC2 production, culminating in decommissioning of a BCL11A erythroid enhancer and reduced BCL11A transcription. HIC2 depletion in let-7-inhibited cells restored BCL11A-mediated repression of HBG. Together, these data establish that fetal hemoglobin silencing in adult erythroid cells is under the control of a miRNA-mediated inhibitory pathway (let-7 ⊣ HIC2 ⊣ BCL11A ⊣ HBG).

Gene silencing dynamics are modulated by transiently active regulatory elements.

Transcriptional enhancers have been extensively characterized, but cis-regulatory elements involved in acute gene repression have received less attention. Transcription factor GATA1 promotes erythroid differentiation by activating and repressing distinct gene sets. Here, we study the mechanism by which GATA1 silences the proliferative gene Kit during murine erythroid cell maturation and define stages from initial loss of activation to heterochromatinization. We find that GATA1 inactivates a potent upstream enhancer but concomitantly creates a discrete intronic regulatory region marked by H3K27ac, short noncoding RNAs, and de novo chromatin looping. This enhancer-like element forms transiently and serves to delay Kit silencing. The element is ultimately erased via the FOG1/NuRD deacetylase complex, as revealed by the study of a disease-associated GATA1 variant. Hence, regulatory sites can be self-limiting by dynamic co-factor usage. Genome-wide analyses across cell types and species uncover transiently active elements at numerous genes during repression, suggesting that modulation of silencing kinetics is widespread.

Molecular basis of polycomb group protein-mediated fetal hemoglobin repression.

The switch from fetal hemoglobin (HbF) to adult hemoglobin (HbA) is a paradigm for developmental gene expression control with relevance to sickle cell disease and ß-thalassemia. Polycomb repressive complex (PRC) proteins regulate this switch, and an inhibitor of PRC2 has entered a clinical trial for HbF activation. Yet, how PRC complexes function in this process, their target genes, and relevant subunit composition are unknown. Here, we identified the PRC1 subunit BMI1 as a novel HbF repressor. We uncovered the RNA binding proteins LIN28B, IGF2BP1, and IGF2BP3 genes as direct BMI1 targets, and demonstrate that they account for the entirety of BMI1's effect on HbF regulation. BMI1 functions as part of the canonical PRC1 (cPRC1) subcomplex as revealed by the physical and functional dissection of BMI1 protein partners. Lastly, we demonstrate that BMI1/cPRC1 acts in concert with PRC2 to repress HbF through the same target genes. Our study illuminates how PRC silences HbF, highlighting an epigenetic mechanism involved in hemoglobin switching.

HIC2 controls developmental hemoglobin switching by repressing BCL11A transcription.

The fetal-to-adult switch in hemoglobin production is a model of developmental gene control with relevance to the treatment of hemoglobinopathies. The expression of transcription factor BCL11A, which represses fetal ß-type globin (HBG) genes in adult erythroid cells, is predominantly controlled at the transcriptional level but the underlying mechanism is unclear. We identify HIC2 as a repressor of BCL11A transcription. HIC2 and BCL11A are reciprocally expressed during development. Forced expression of HIC2 in adult erythroid cells inhibits BCL11A transcription and induces HBG expression. HIC2 binds to erythroid BCL11A enhancers to reduce chromatin accessibility and binding of transcription factor GATA1, diminishing enhancer activity and enhancer-promoter contacts. DNA-binding and crystallography studies reveal direct steric hindrance as one mechanism by which HIC2 inhibits GATA1 binding at a critical BCL11A enhancer. Conversely, loss of HIC2 in fetal erythroblasts increases enhancer accessibility, GATA1 binding and BCL11A transcription. HIC2 emerges as an evolutionarily conserved regulator of hemoglobin switching via developmental control of BCL11A.

Dual function NFI factors control fetal hemoglobin silencing in adult erythroid cells.

The mechanisms by which the fetal-type ß-globin-like genes HBG1 and HBG2 are silenced in adult erythroid precursor cells remain a fundamental question in human biology and have therapeutic relevance to sickle cell disease and ß-thalassemia. Here, we identify via a CRISPR-Cas9 genetic screen two members of the NFI transcription factor family-NFIA and NFIX-as HBG1/2 repressors. NFIA and NFIX are expressed at elevated levels in adult erythroid cells compared with fetal cells, and function cooperatively to repress HBG1/2 in cultured cells and in human-to-mouse xenotransplants. Genomic profiling, genome editing and DNA binding assays demonstrate that the potent concerted activity of NFIA and NFIX is explained in part by their ability to stimulate the expression of BCL11A, a known silencer of the HBG1/2 genes, and in part by directly repressing the HBG1/2 genes. Thus, NFI factors emerge as versatile regulators of the fetal-to-adult switch in ß-globin production.

Identification and characterization of RBM12 as a novel regulator of fetal hemoglobin expression.

The fetal-to-adult hemoglobin transition is clinically relevant because reactivation of fetal hemoglobin (HbF) significantly reduces morbidity and mortality associated with sickle cell disease (SCD) and ß-thalassemia. Most studies on the developmental regulation of the globin genes, including genome-wide genetics screens, have focused on DNA binding proteins, including BCL11A and ZBTB7A/LRF and their cofactors. Our understanding of RNA binding proteins (RBPs) in this process is much more limited. Two RBPs, LIN28B and IGF2BP1, are known posttranscriptional regulators of HbF production, but a global view of RBPs is still lacking. Here, we carried out a CRISPR/Cas9-based screen targeting RBPs harboring RNA methyltransferase and/or RNA recognition motif (RRM) domains and identified RNA binding motif 12 (RBM12) as a novel HbF suppressor. Depletion of RBM12 induced HbF expression and attenuated cell sickling in erythroid cells derived from patients with SCD with minimal detrimental effects on cell maturation. Transcriptome and proteome profiling revealed that RBM12 functions independently of major known HbF regulators. Enhanced cross-linking and immunoprecipitation followed by high-throughput sequencing revealed strong preferential binding of RBM12 to 5' untranslated regions of transcripts, narrowing down the mechanism of RBM12 action. Notably, we pinpointed the first of 5 RRM domains as essential, and, in conjunction with a linker domain, sufficient for RBM12-mediated HbF regulation. Our characterization of RBM12 as a negative regulator of HbF points to an additional regulatory layer of the fetal-to-adult hemoglobin switch and broadens the pool of potential therapeutic targets for SCD and ß-thalassemia.

FACS and immunomagnetic isolation of early erythroid progenitor cells from mouse fetal liver.

Early erythroid progenitors are transit-amplifying cells with high proliferative capacity committed to undergoing red cell differentiation. CD71/CD24low progenitors are less mature and have greater proliferative capacity than CD71/CD24high. We present protocols for isolation of CD71/CD24low progenitors from mouse fetal liver using both fluorescence-activated cell sorting (FACS) and immunomagnetic enrichment. CD71/CD24low progenitors isolated with both approaches show similar transcriptomes at single-cell resolution and exhibit characteristic proliferative responses to glucocorticoids. For complete details on the use and execution of this protocol, please refer to Li et al. (2019).

ZMYND8-regulated IRF8 transcription axis is an acute myeloid leukemia dependency.

The transformed state in acute leukemia requires gene regulatory programs involving transcription factors and chromatin modulators. Here, we uncover an IRF8-MEF2D transcriptional circuit as an acute myeloid leukemia (AML)-biased dependency. We discover and characterize the mechanism by which the chromatin "reader" ZMYND8 directly activates IRF8 in parallel with the MYC proto-oncogene through their lineage-specific enhancers. ZMYND8 is essential for AML proliferation in vitro and in vivo and associates with MYC and IRF8 enhancer elements that we define in cell lines and in patient samples. ZMYND8 occupancy at IRF8 and MYC enhancers requires BRD4, a transcription coactivator also necessary for AML proliferation. We show that ZMYND8 binds to the ET domain of BRD4 via its chromatin reader cassette, which in turn is required for proper chromatin occupancy and maintenance of leukemic growth in vivo. Our results rationalize ZMYND8 as a potential therapeutic target for modulating essential transcriptional programs in AML.

Heterogeneity of fetal hemoglobin production in adult red blood cells.

PURPOSE OF REVIEW: Small amounts of fetal hemoglobin can be expressed in a subset of adult red blood cells called F-cells. This review examines the potential mechanisms and clinical implications of the heterogeneity of fetal hemoglobin expression. RECENT FINDINGS: Although the heterocellular nature of fetal hemoglobin expression in adult red blood cells has been noted for over 70 years, the molecular basis of this phenomenon has been unclear. Recent discoveries of novel regulators of fetal hemoglobin as well as technological advances have shed new light on these cells. SUMMARY: Fetal hemoglobin reactivation in adult red blood cells through genetic or pharmacological approaches can involve both increasing the number of F-cells and cellular fetal hemoglobin content. New technologies enable the study and eventually the improvement of these parameters in patients with sickle cell disease and ß-thalassemia.

HRI depletion cooperates with pharmacologic inducers to elevate fetal hemoglobin and reduce sickle cell formation.

Increasing fetal hemoglobin (HbF) provides clinical benefit in patients with sickle cell disease (SCD). We recently identified heme-regulated inhibitor (HRI, EIF2AK1), as a novel HbF regulator. Because HRI is an erythroid-specific protein kinase, it presents a potential target for pharmacologic intervention. We found that maximal HbF induction required >80% to 85% HRI depletion. Because it remains unclear whether this degree of HRI inhibition can be achieved pharmacologically, we explored whether HRI knockdown can be combined with pharmacologic HbF inducers to achieve greater HbF production and minimize potential adverse effects associated with treatments. Strongly cooperative HbF induction was observed when HRI depletion was combined with exposure to pomalidomide or the EHMT1/2 inhibitor UNC0638, but not to hydroxyurea. Mechanistically, reduction in the levels of the HbF repressor BCL11A reflected the cooperativity of HRI loss and pomalidomide treatment, whereas UNC0638 did not modulate BCL11A levels. In conjunction with HRI loss, pomalidomide maintained its HbF-inducing activity at 10-fold lower concentrations, in which condition there were minimal observed detrimental effects on erythroid cell maturation and viability, as well as fewer alterations in the erythroid transcriptome. When tested in cells from patients with SCD, combining HRI depletion with pomalidomide or UNC0638 achieved up to 50% to 60% HbF and 45% to 50% HbF, respectively, as measured by high-performance liquid chromatography, and markedly counteracted cell sickling. In summary, this study provides a foundation for the exploration of combining future small-molecule HRI inhibitors with additional pharmacologic HbF inducers to maximize HbF production and preserve erythroid cell functionality for the treatment of SCD and other hemoglobinopathies.

The HRI-regulated transcription factor ATF4 activates BCL11A transcription to silence fetal hemoglobin expression.

Reactivation of fetal hemoglobin remains a critical goal in the treatment of patients with sickle cell disease and ß-thalassemia. Previously, we discovered that silencing of the fetal γ-globin gene requires the erythroid-specific eIF2α kinase heme-regulated inhibitor (HRI), suggesting that HRI might present a pharmacologic target for raising fetal hemoglobin levels. Here, via a CRISPR-Cas9-guided loss-of-function screen in human erythroblasts, we identify transcription factor ATF4, a known HRI-regulated protein, as a novel γ-globin regulator. ATF4 directly stimulates transcription of BCL11A, a repressor of γ-globin transcription, by binding to its enhancer and fostering enhancer-promoter contacts. Notably, HRI-deficient mice display normal Bcl11a levels, suggesting species-selective regulation, which we explain here by demonstrating that the analogous ATF4 motif at the murine Bcl11a enhancer is largely dispensable. Our studies uncover a linear signaling pathway from HRI to ATF4 to BCL11A to γ-globin and illustrate potential limits of murine models of globin gene regulation.

Understanding heterogeneity of fetal hemoglobin induction through comparative analysis of F and A erythroblasts.

Reversing the developmental switch from fetal hemoglobin (HbF, α2γ2) to adult hemoglobin (HbA, α2ß2) is an important therapeutic approach in sickle cell disease (SCD) and ß-thalassemia. In healthy individuals, SCD patients, and patients treated with pharmacologic HbF inducers, HbF is present only in a subset of red blood cells known as F cells. Despite more than 50 years of observations, the cause for this heterocellular HbF expression pattern, even among genetically identical cells, remains unknown. Adult F cells might represent a reversion of a given cell to a fetal-like epigenetic and transcriptional state. Alternatively, isolated transcriptional or posttranscriptional events at the γ-globin genes might underlie heterocellularity. Here, we set out to understand the heterogeneity of HbF activation by developing techniques to purify and profile differentiation stage-matched late erythroblast F cells and non-F cells (A cells) from the human HUDEP2 erythroid cell line and primary human erythroid cultures. Transcriptional and proteomic profiling of these cells demonstrated very few differences between F and A cells at the RNA level either under baseline conditions or after treatment with HbF inducers hydroxyurea or pomalidomide. Surprisingly, we did not find differences in expression of any known HbF regulators, including BCL11A or LRF, that would account for HbF activation. Our analysis shows that F erythroblasts are not significantly different from non-HbF-expressing cells and that the primary differences likely occur at the transcriptional level at the ß-globin locus.

Corrigendum: The Spectrum of SPTA1-Associated Hereditary Spherocytosis.

[This corrects the article DOI: 10.3389/fphys.2019.00815.].

The autophagy-activating kinase ULK1 mediates clearance of free α-globin in ß-thalassemia.

In ß-thalassemia, accumulated free α-globin forms intracellular precipitates that impair erythroid cell maturation and viability. Protein quality control systems mitigate ß-thalassemia pathophysiology by degrading toxic free α-globin, although the associated mechanisms are poorly understood. We show that loss of the autophagy-activating Unc-51-like kinase 1 (Ulk1) gene in ß-thalassemic mice reduces autophagic clearance of α-globin in red blood cell precursors and exacerbates disease phenotypes, whereas inactivation of the canonical autophagy-related 5 (Atg5) gene has relatively minor effects. Systemic treatment with the mTORC1 inhibitor rapamycin reduces α-globin precipitates and lessens pathologies in ß-thalassemic mice via an ULK1-dependent pathway. Similarly, rapamycin reduces free α-globin accumulation in erythroblasts derived from CD34+ cells of ß-thalassemic individuals. Our findings define a drug-regulatable pathway for ameliorating ß-thalassemia.

The Spectrum of SPTA1-Associated Hereditary Spherocytosis.

Hereditary spherocytosis (HS) is the most common red blood cell (RBC) membrane disorder causing hereditary hemolytic anemia. Patients with HS have defects in the genes coding for ankyrin (ANK1), band 3 (SLC4A1), protein 4.2 (EPB42), and α (SPTA1) or ß-spectrin (SPTB). Severe recessive HS is most commonly due to biallelic SPTA1 mutations. α-spectrin is produced in excess in normal erythroid cells, therefore SPTA1-associated HS ensues with mutations causing significant decrease of normal protein expression from both alleles. In this study, we systematically compared genetic, rheological, and protein expression data to the varying clinical presentation in eleven patients with SPTA1-associated HS. The phenotype of HS in this group of patients ranged from moderately severe to severe transfusion-dependent anemia and up to hydrops fetalis which is typically fatal if transfusions are not initiated before term delivery. The pathogenicity of the mutations could be corroborated by reduced SPTA1 mRNA expression in the patients' reticulocytes. The disease severity correlated to the level of α-spectrin protein in their RBC cytoskeleton but was also affected by other factors. Patients carrying the low expression αLEPRA allele in trans to a null SPTA1 mutation were not all transfusion dependent and their anemia improved or resolved with partial or total splenectomy, respectively. In contrast, patients with near-complete or complete α-spectrin deficiency have a history of having been salvaged from fatal hydrops fetalis, either because they were born prematurely and started transfusions early or because they had intrauterine transfusions. They have suboptimal reticulocytosis or reticulocytopenia and remain transfusion dependent even after splenectomy; these patients require either lifetime transfusions and iron chelation or stem cell transplant. Comprehensive genetic and phenotypic evaluation is critical to provide accurate diagnosis in patients with SPTA1-associated HS and guide toward appropriate management.

The E3 ligase adaptor molecule SPOP regulates fetal hemoglobin levels in adult erythroid cells.

Reactivation of fetal hemoglobin (HbF) production benefits patients with sickle cell disease and ß-thalassemia. To identify new HbF regulators that might be amenable to pharmacologic control, we screened a protein domain-focused CRISPR-Cas9 library targeting chromatin regulators, including BTB domain-containing proteins. Speckle-type POZ protein (SPOP), a substrate adaptor of the CUL3 ubiquitin ligase complex, emerged as a novel HbF repressor. Depletion of SPOP or overexpression of a dominant negative version significantly raised fetal globin messenger RNA and protein levels with minimal detrimental effects on normal erythroid maturation, as determined by transcriptome and proteome analyses. SPOP controls HbF expression independently of the major transcriptional HbF repressors BCL11A and LRF. Finally, pharmacologic HbF inducers cooperate with SPOP depletion during HbF upregulation. Our study implicates SPOP and the CUL3 ubiquitin ligase system in controlling HbF production in human erythroid cells and may offer new therapeutic strategies for the treatment of ß-hemoglobinopathies.