BAP1 regulation of the key adaptor protein NCoR1 is critical for γ-globin gene repression.

Human globin gene production transcriptionally "switches" from fetal to adult synthesis shortly after birth and is controlled by macromolecular complexes that enhance or suppress transcription by cis elements scattered throughout the locus. The DRED (direct repeat erythroid-definitive) repressor is recruited to the ε-globin and γ-globin promoters by the orphan nuclear receptors TR2 (NR2C1) and TR4 (NR2C2) to engender their silencing in adult erythroid cells. Here we found that nuclear receptor corepressor-1 (NCoR1) is a critical component of DRED that acts as a scaffold to unite the DNA-binding and epigenetic enzyme components (e.g., DNA methyltransferase 1 [DNMT1] and lysine-specific demethylase 1 [LSD1]) that elicit DRED function. We also describe a potent new regulator of γ-globin repression: The deubiquitinase BRCA1-associated protein-1 (BAP1) is a component of the repressor complex whose activity maintains NCoR1 at sites in the ß-globin locus, and BAP1 inhibition in erythroid cells massively induces γ-globin synthesis. These data provide new mechanistic insights through the discovery of novel epigenetic enzymes that mediate γ-globin gene repression.

An erythroid-to-myeloid cell fate conversion is elicited by LSD1 inactivation.

Histone H3 lysine 4 methylation (H3K4Me) is most often associated with chromatin activation, and removing H3K4 methyl groups has been shown to be coincident with gene repression. H3K4Me demethylase KDM1a/LSD1 is a therapeutic target for multiple diseases, including for the potential treatment of ß-globinopathies (sickle cell disease and ß-thalassemia), because it is a component of γ-globin repressor complexes, and LSD1 inactivation leads to robust induction of the fetal globin genes. The effects of LSD1 inhibition in definitive erythropoiesis are not well characterized, so we examined the consequences of conditional inactivation of Lsd1 in adult red blood cells using a new Gata1creERT2 bacterial artificial chromosome transgene. Erythroid-specific loss of Lsd1 activity in mice led to a block in erythroid progenitor differentiation and to the expansion of granulocyte-monocyte progenitor-like cells, converting hematopoietic differentiation potential from an erythroid fate to a myeloid fate. The analogous phenotype was also observed in human hematopoietic stem and progenitor cells, coincident with the induction of myeloid transcription factors (eg, PU.1 and CEBPα). Finally, blocking the activity of the transcription factor PU.1 or RUNX1 at the same time as LSD1 inhibition rescued myeloid lineage conversion to an erythroid phenotype. These data show that LSD1 promotes erythropoiesis by repressing myeloid cell fate in adult erythroid progenitors and that inhibition of the myeloid-differentiation pathway reverses the lineage switch induced by LSD1 inactivation.

GATA2 controls lymphatic endothelial cell junctional integrity and lymphovenous valve morphogenesis through miR-126.

Mutations in the transcription factor GATA2 cause lymphedema. GATA2 is necessary for the development of lymphatic valves and lymphovenous valves, and for the patterning of lymphatic vessels. Here, we report that GATA2 is not necessary for valvular endothelial cell (VEC) differentiation. Instead, GATA2 is required for VEC maintenance and morphogenesis. GATA2 is also necessary for the expression of the cell junction molecules VE-cadherin and claudin 5 in lymphatic vessels. We identified miR-126 as a target of GATA2, and miR-126-/- embryos recapitulate the phenotypes of mice lacking GATA2. Primary human lymphatic endothelial cells (HLECs) lacking GATA2 (HLECΔGATA2) have altered expression of claudin 5 and VE-cadherin, and blocking miR-126 activity in HLECs phenocopies these changes in expression. Importantly, overexpression of miR-126 in HLECΔGATA2 significantly rescues the cell junction defects. Thus, our work defines a new mechanism of GATA2 activity and uncovers miR-126 as a novel regulator of mammalian lymphatic vascular development.

Small molecule therapeutics to treat the ß-globinopathies.

PURPOSE OF REVIEW: The current review focuses on recent insights into the development of small molecule therapeutics to treat the ß-globinopathies. RECENT FINDINGS: Recent studies of fetal γ-globin gene regulation reveal multiple insights into how γ-globin gene reactivation may lead to novel treatment for ß-globinopathies. SUMMARY: We summarize current information regarding the binding of transcription factors that appear to be impeded or augmented by different hereditary persistence of fetal hemoglobin (HPFH) mutations. As transcription factors have historically proven to be difficult to target for therapeutic purposes, we next address the contributions of protein complexes associated with these HPFH mutation-affected transcription factors with the aim of defining proteins that might provide additional targets for chemical molecules to inactivate the corepressors. Among the enzymes associated with the transcription factor complexes, a group of corepressors with currently available inhibitors were initially thought to be good candidates for potential therapeutic purposes. We discuss possibilities for pharmacological inhibition of these corepressor enzymes that might significantly reactivate fetal γ-globin gene expression. Finally, we summarize the current clinical trial data regarding the inhibition of select corepressor proteins for the treatment of sickle cell disease and ß-thalassemia.

The LSD1 inhibitor RN-1 induces fetal hemoglobin synthesis and reduces disease pathology in sickle cell mice.

Inhibition of lysine-specific demethylase 1 (LSD1) has been shown to induce fetal hemoglobin (HbF) levels in cultured human erythroid cells in vitro. Here we report the in vivo effects of LSD1 inactivation by a selective and more potent inhibitor, RN-1, in a sickle cell disease (SCD) mouse model. Compared with untreated animals, RN-1 administration leads to induced HbF synthesis and to increased frequencies of HbF-positive cells and mature erythrocytes, as well as fewer reticulocytes and sickle cells, in the peripheral blood of treated SCD mice. In keeping with these observations, histologic analyses of the liver and spleen of treated SCD mice verified that they do not exhibit the necrotic lesions that are usually associated with SCD. These data indicate that RN-1 can effectively induce HbF levels in red blood cells and reduce disease pathology in SCD mice, and may therefore offer new therapeutic possibilities for treating SCD.

Roles of Nuclear Orphan Receptors TR2 and TR4 during Hematopoiesis.

TR2 and TR4 (NR2C1 and NR2C2, respectively) are evolutionarily conserved nuclear orphan receptors capable of binding direct repeat sequences in a stage-specific manner. Like other nuclear receptors, TR2 and TR4 possess important roles in transcriptional activation or repression with developmental stage and tissue specificity. TR2 and TR4 bind DNA and possess the ability to complex with available cofactors mediating developmental stage-specific actions in primitive and definitive erythrocytes. In erythropoiesis, TR2 and TR4 are required for erythroid development, maturation, and key erythroid transcription factor regulation. TR2 and TR4 recruit and interact with transcriptional corepressors or coactivators to elicit developmental stage-specific gene regulation during hematopoiesis.

Murine erythroid differentiation kinetics in vivo under normal and anemic stress conditions.

Our current understanding of the kinetics and dynamics of erythroid differentiation is based almost entirely on the ex vivo expansion of cultured hematopoietic progenitor cells. In this study, we used an erythroid-specific, inducible transgenic mouse line to investigate for the first time, the in vivo erythroid differentiation kinetics under steady-state conditions. We demonstrated that bipotent premegakaroycyte/erythroid (PreMegE) progenitor cells differentiate into erythroid-committed proerythroblast/basophilic erythroblasts (ProBasoE) after 6.6 days under steady-state conditions. During this process, each differentiation phase (from PreMegE to precolony forming unit-erythroid [PreCFU-E], PreCFU-E to CFU-E, and CFU-E to ProBasoE) took ∼2 days in vivo. Upon challenge with 5-flurouracil (5-FU), which leads to the induction of stress erythropoiesis, erythroid maturation time was reduced from 6.6 to 4.7 days. Furthermore, anemia induced in 5-FU-treated mice was shown to be due not only to depleted bone marrow erythroid progenitor stores but also to a block in reticulocyte exit from the bone marrow into the circulation, which differed from the mechanism induced by acute blood loss.

Identification of novel γ-globin inducers among all potential erythroid druggable targets.

Human γ-globin is predominantly expressed in fetal liver erythroid cells during gestation from 2 nearly identical genes, HBG1 and HBG2, that are both perinatally silenced. Reactivation of these fetal genes in adult red blood cells can ameliorate many symptoms associated with the inherited ß-globinopathies, sickle cell disease, and Cooley anemia. Although promising genetic strategies to reactivate the γ-globin genes to treat these diseases have been explored, there are significant barriers to their effective implementation worldwide; alternatively, pharmacological induction of γ-globin synthesis could readily reach the majority of affected individuals. In this study, we generated a CRISPR knockout library that targeted all erythroid genes for which prospective or actual therapeutic compounds already exist. By probing this library for genes that repress fetal hemoglobin (HbF), we identified several novel, potentially druggable, γ-globin repressors, including VHL and PTEN. We demonstrate that deletion of VHL induces HbF through activation of the HIF1α pathway and that deletion of PTEN induces HbF through AKT pathway stimulation. Finally, we show that small-molecule inhibitors of PTEN and EZH induce HbF in both healthy and ß-thalassemic human primary erythroid cells.

A new murine Rpl5 (uL18) mutation provides a unique model of variably penetrant Diamond-Blackfan anemia.

Ribosome dysfunction is implicated in multiple abnormal developmental and disease states in humans. Heterozygous germline mutations in genes encoding ribosomal proteins are found in most individuals with Diamond-Blackfan anemia (DBA), whereas somatic mutations have been implicated in a variety of cancers and other disorders. Ribosomal protein-deficient animal models show variable phenotypes and penetrance, similar to human patients with DBA. In this study, we characterized a novel ENU mouse mutant (Skax23m1Jus) with growth and skeletal defects, cardiac malformations, and increased mortality. After genetic mapping and whole-exome sequencing, we identified an intronic Rpl5 mutation, which segregated with all affected mice. This mutation was associated with decreased ribosome generation, consistent with Rpl5 haploinsufficiency. Rpl5Skax23-Jus/+ animals had a profound delay in erythroid maturation and increased mortality at embryonic day (E) 12.5, which improved by E14.5. Surviving mutant animals had macrocytic anemia at birth, as well as evidence of ventricular septal defect (VSD). Surviving adult and aged mice exhibited no hematopoietic defect or VSD. We propose that this novel Rpl5Skax23-Jus/+ mutant mouse will be useful in studying the factors influencing the variable penetrance that is observed in DBA.

The Endoplasmic Reticulum Cargo Receptor SURF4 Facilitates Efficient Erythropoietin Secretion.

Erythropoietin (EPO) stimulates erythroid differentiation and maturation. Though the transcriptional regulation of EPO has been well studied, the molecular determinants of EPO secretion remain unknown. Here, we generated a HEK293T reporter cell line that provides a quantifiable and selectable readout of intracellular EPO levels and performed a genome-scale CRISPR screen that identified SURF4 as an important mediator of EPO secretion. Targeting SURF4 with multiple independent single guide RNAs (sgRNAs) resulted in intracellular accumulation and extracellular depletion of EPO. Both of these phenotypes were rescued by expression of SURF4 cDNA. Additionally, we found that disruption of SURF4 resulted in accumulation of EPO in the endoplasmic reticulum (ER) compartment and that SURF4 and EPO physically interact. Furthermore, SURF4 disruption in Hep3B cells also caused a defect in the secretion of endogenous EPO under conditions mimicking hypoxia, ruling out an artifact of heterologous overexpression. This work demonstrates that SURF4 functions as an ER cargo receptor that mediates the efficient secretion of EPO. Our findings also suggest that modulating SURF4 may be an effective treatment for disorders of erythropoiesis that are driven by aberrant EPO levels. Finally, we show that SURF4 overexpression results in increased secretion of EPO, suggesting a new strategy for more efficient production of recombinant EPO.

Global dynamics of stage-specific transcription factor binding during thymocyte development.

In vertebrates, multiple transcription factors (TFs) bind to gene regulatory elements (promoters, enhancers, and silencers) to execute developmental expression changes. ChIP experiments are often used to identify where TFs bind to regulatory elements in the genome, but the requirement of TF-specific antibodies hampers analyses of tens of TFs at multiple loci. Here we tested whether TF binding predictions using ATAC-seq can be used to infer the identity of TFs that bind to functionally validated enhancers of the Cd4, Cd8, and Gata3 genes in thymocytes. We performed ATAC-seq at four distinct stages of development in mouse thymus, probing the chromatin accessibility landscape in double negative (DN), double positive (DP), CD4 single positive (SP4) and CD8 SP (SP8) thymocytes. Integration of chromatin accessibility with TF motifs genome-wide allowed us to infer stage-specific occupied TF binding sites within known and potentially novel regulatory elements. Our results provide genome-wide stage-specific T cell open chromatin profiles, and allow the identification of candidate TFs that drive thymocyte differentiation at each developmental stage.