Rational Targeting of a NuRD Sub-Complex for Fetal Hemoglobin Induction Following Comprehensive in Situ Mutagenesis

Sickle cell disease and β-thalassemia are major hemoglobin disorders for which induction of fetal hemoglobin (HbF) can mitigate disease severity. However, the molecular mechanisms underlying the developmental repression of HbF remain incompletely understood. The nucleosome remodeling and deacetylase (NuRD) complex is a major negative regulator of HbF level. In this study, we sought to identify possible rational therapeutic strategies targeting critical NuRD determinants. We employed comprehensive dense mutagenesis using pooled CRISPR screening in HUDEP-2 human erythroid precursors to disrupt protein coding sequences of all 13 genes of the NuRD complex, including CHD, MTA, GATAD2, HDAC, MBD, and RBBP family members. The custom sgRNA library included 5,038 sgRNAs. We found that only 5 genes, CHD4, MTA2, GATAD2A, HDAC2, and MBD2, were required for HbF repression, suggesting that a non-redundant NuRD sub-complex contributes to HbF silencing. We validated the existence of this NuRD sub-complex by mass spectrometry analysis after immunoprecipitation of CHD4 and MTA2 as well as MTA2-BioID2 mediated proximity labeling. Remarkably, 5 of the 6 NuRD subunit proteins commonly detected by these three methods were identified as functional by CRISPR screening (MTA2, RBBP4, CHD4, GATAD2A, HDAC2). Disruption of CHD4 resulted in the highest HbF induction of any of the NuRD subunits. However, unlike the other NuRD genes, CHD4 disruption also led to cellular toxicity. We observed a small group of sgRNAs within the CHDCT2 domain of CHD4 associated with high HbF induction yet relatively modest negative fitness. We validated by electroporation of Cas9:sgRNA to CD34+ HSPC primary erythroid precursors that in-frame mutations of CHD4 CHDCT2 escape cellular toxicity while inducing HbF. Similarly, we targeted homologous amino acid residues within mouse Chd4 CHDCT2 domain by Cas9 mutagenesis in mouse oocytes. While loss of Chd4 is lethal at the blastocyst stage, homozygous in-frame deletions within the Chd4 CHDCT2 domain are tolerated in mouse embryos and result in increased γ-globin expression in mid-gestation embryos bearing transgenic human β-globin gene clusters. To investigate the mechanism whereby in-frame deletions at CHD4 CHDCT2 impact NuRD, we performed glycerol gradient density sedimentation, which revealed that these in-frame mutations impair the recruitment of CHD4 to the NuRD complex. A recent study demonstrated that the previously poorly characterized CHD4 CHDCT2 domain directly binds to GATAD2 factors (Torrado et al, FEBS J, 2017). We observed a cluster of sgRNAs associated with heightened HbF enrichment scores at the C-terminal region of GATAD2A encompassing a C2C2-type GATA zinc finger. We hypothesized that ectopic expression of this GATAD2A zinc finger might competitively bind to CHD4 and displace CHD4 from NuRD. Overexpression of the GATAD2A zinc finger in both HUDEP-2 and CD34+ HSPC derived primary erythroid precursors led to robust induction of HbF without negatively impacting cellular fitness. Immunoprecipitation of the GATAD2A zinc finger enriched CHD4 but not other endogenous NuRD components, such as GATAD2A or MBD2. Moreover, glycerol gradient density sedimentation showed that the GATAD2A zinc finger co-sedimented with sub-NuRD fractions of CHD4. Together these data suggest that expression of the GATAD2A zinc finger sequesters CHD4 from NuRD, yet spares cytotoxicity. In summary, we show that biochemical disruption of the CHD4-GATAD2A interaction could serve as a rational therapeutic strategy to potently induce HbF for the β-hemoglobin disorders while preventing cellular toxicity associated with complete CHD4 inhibition. Disclosures No relevant conflicts of interest to declare.

A method for the quantitative extraction of gold nanoparticles from human bronchoalveolar lavage fluids through a glycerol gradient

We report a simple method for the clinically-oriented extraction of sub-ppm gold nanoparticles from human bronchoalveolar lavage fluids.

The Distinctive Morphogenesis of Infantile Megakaryopoiesis Is Associated with Altered P-TEFb Regulation By the Oncofetal Factor IGF2BP3

Infantile (fetal/neonatal) megakaryocytes (Mk) differ from "adult" Mk in several important aspects. They are smaller, more proliferative, less polyploid and show leaky expression of erythroid genes. Their distinctive properties contribute to multiple disease states including neonatal thrombocytopenia, poor platelet recovery post umbilical cord blood (CB) transplantation, and acute Mk leukemias (AMkL). Their leukemic propensity is highlighted by the capacity of the AMkL oncoprotein GATA1s to transform infantile but not adult Mk. Despite their altered morphogenesis, infantile megakaryocytes retain most features of the adult differentiation and signaling program. Their principal signaling perturbation has been characterized as excessive responsiveness to thrombopoietin (Tpo) particularly with regard to the mTOR pathway. Thus Tpo agonists used to treat adult thrombocytopenia may not offer appropriate therapy for neonatal thrombocytopenia. We previously identified a signaling pathway that drives megakaryocyte morphogenesis and is disrupted by the GATA1s oncoprotein in AMkL (Elagib et al., Dev. Cell, 2013). A central feature of this pathway is the irreversible activation of the P-TEFb kinase (Cdk9/Cyclin T). This cascade is initiated by downregulation of core components of the repressive 7SK snRNP complex (MePCE, LARP7, 7SK snRNA). The resulting constitutive P-TEFb activation drives multiple features of Mk differentiation: induction of cytoskeletal morpho-genetic factors, silencing of erythroid genes, and promotion of histone H2B K120 monoubiquitiniation (H2BUb1). A critical, rate-limiting step triggering this pathway comprises MePCE proteolysis by calpain 2. GATA1s disrupts this pathway by preventing induction of calpain 2 by wild type GATA1. We now report that infantile Mk display intrinsic defects in the Mk P-TEFb activation pathway. In repeated experiments, human CB Mk failed to upregulate P-TEFb-dependent cytoskeletal factors, exhibited global deficiency in H2BUb1, and incompletely silenced erythroid antigen expression. Their defective P-TEFb activation resulted from an inability to downregulate 7SK snRNA, despite downregulation of the key 7SK stabilizers MePCE and LARP7. The inexplicable stabilization of 7SK in CB Mk argues for the existence of an alternative, infantile 7SK snRNP complex refractory to activation by calpain. Accordingly, screening studies identified candidate 7SK binding factors preferentially expressed in CB as opposed to adult progenitors. Among these factors, the RNA binding protein IGF2BP3 showed high abundance in CB Mk but complete absence from adult peripheral blood-derived (PB) Mk. Furthermore, immunoprecipitation studies consistently showed interaction of endogenous IGF2BP3 and HEXIM1 in K562 cells. HEXIM1 is the 7SK snRNP component that mediates repression of P-TEFb. Immunoprecipitation of epitope-tagged IGF2BP3 from HEK293 cells consistently identified an association with endogenous 7SK snRNA. In addition, enforced expression of IGF2BP3 in HEK293 cells, to levels seen in CB Mk, shifted the fractionation pattern of HEXIM1 on glycerol gradient sedimentation. Notably, a similar difference in HEXIM1 fractionation was seen when comparing CB and adult Mk by glycerol gradient sedimentation. Thus, IGF2BP3 represents a fetal/neonatal factor that reconfigures the composition and/or conformation of the 7SK snRNP, potentially altering regulation of P-TEFb. Contribution of IGF2BP3 to the infantile Mk phenotype was supported by experiments in which shRNA-mediated knockdown in CB Mk consistently enhanced enlargement, polyploidization, growth arrest, and erythroid silencing. Conversely, enforced expression of IGF2BP3 in adult Mk inhibited their enlargement, polyploidization, and growth arrest. Our results thus implicate IGF2BP3 as a key contributor to the infantile Mk phenotype, interfering with morphogenesis by stabilizing 7SK and thwarting irreversible P-TEFb activation. In light of our prior published results on the inhibitory effects of GATA1s in Mk morphogenesis (Elagib et al., Dev. Cell, 2013), our current findings highlight P-TEFb regulation as a convergence point for oncogenic stimuli during megakaryopoiesis. Disclosures No relevant conflicts of interest to declare.

Functional Association of the Microprocessor Complex with the Spliceosome

ABSTRACT The majority of human microRNAs (miRNAs) are located in the introns of other genes (A. Rodriguez, S. Griffiths-Jones, J. L. Ashurst, and A. Bradley, Genome Res. 14:1902-1910, 2004). Based on the discovery that artificial insertion of pre-miRNAs in introns did not hamper mRNA production and that the miRNA-harboring introns were spliced more slowly than the adjacent introns, a model was previously proposed in which Drosha crops the pre-miRNA and the two cropped fragments from the pre-mRNA are subsequently trans spliced (Y. K. Kim and V. N. Kim, EMBO J. 26:775-783, 2007). However, the molecular basis for this model was not elucidated. To analyze the molecular mechanism of intronic miRNA processing, we developed an in vitro system in which both pre-miRNA processing and mRNA splicing are detected simultaneously. Our analysis using this system showed that pre-miRNA cropping from the pre-mRNA could occur kinetically faster than splicing. Glycerol gradient sedimentation experiments revealed that part of the pre-miRNA was cofractionated with the spliceosome. Furthermore, coimmunoprecipitation experiments with an anti-Drosha antibody demonstrated that Drosha was associated not only with the cropping products but also with a Y-shaped branch intron and a Y-shaped splicing intermediate. These results provide a molecular basis for the postulated existence of a pathway in which the Microprocessor complex becomes associated with the spliceosome, pre-miRNA cropping occurs prior to splicing, and trans splicing takes place between the cropped products.

Oligomeric forms of the 90-kDa heat shock protein

Two isoforms of the 90-kDa heat shock protein, HSP90α and HSP90β, are present in the cytosol of mammalian cells. Analysis by polyacrylamide gel electrophoresis under nondenaturing conditions (native PAGE) revealed that HSP90α predominantly exists as a homodimer and that HSP90β is present mainly as a monomer [Minami, Kawasaki, Miyata, Suzuki and Yahara (1991) J. Biol. Chem. 266, 10099-10103]. However, only the dimeric form has been observed under other analytical conditions such as gradient centrifugation. In this study, therefore, we investigated native forms of HSP90 by use of immunochemical techniques with isoform-specific monoclonal antibodies recently developed in our laboratory. Glycerol gradient centrifugation at the physiological salt concentration as well as native PAGE analysis of rat liver cytosol revealed oligomeric forms of HSP90α sedimenting at 8-10S as predominant ones. On the other hand, the glycerol gradient centrifugation revealed multiple forms of HSP90β oligomers sedimenting at 6-12S. All of the HSP90β oligomers, however, migrated at 100-kDa monomer and 190-kDa dimer positions on native PAGE. A novel two-dimensional double native PAGE revealed that the entity was converted from the HSP90β dimer to monomers during the electrophoresis. The same PAGE further revealed that the HSP90α oligomer also dissociated into dimers during the electrophoresis. Full-length form of bacterially-expressed human HSP90α migrated as dimers, but a considerable amount did not penetrate into the gel under native PAGE conditions, indicating the existence of oligomeric forms. Electrophoretic studies of deletion mutants of HSP90 demonstrated that the C-terminal 200 amino acids were capable of forming oligomers. Taken together, we conclude that both of the HSP90 isoforms predominantly exist as oligomeric forms in the cytosol even under unstressed conditions but that they artificially dissociate into smaller forms when subjected to native PAGE.

Prp31p promotes the association of the U4/U6 x U5 tri-snRNP with prespliceosomes to form spliceosomes in Saccharomyces cerevisiae.

The PRP31 gene encodes a factor essential for the splicing of pre-mRNA in Saccharomyces cerevisiae. Cell extracts derived from a prp31-1 strain fail to form mature spliceosomes upon heat inactivation, although commitment complexes and prespliceosome complexes are detected under these conditions. Coimmunoprecipitation experiments indicate that Prp31p is associated both with the U4/U6 x U5 tri-snRNP and, independently, with the prespliceosome prior to assembly of the tri-snRNP into the splicing complex. Nondenaturing gel electrophoresis and glycerol gradient analyses demonstrate that while Prp31p may play a role in maintaining the assembly or stability of tri-snRNPs, functional protein is not essential for the formation of U4/U6 or U4/U6 x U5 snRNPs. These results suggest that Prp31p is involved in recruiting the U4/U6 x U5 tri-snRNP to prespliceosome complexes or in stabilizing these interactions.