β-hemoglobinopathies are the most common monogenic disorders worldwide, and are defined based on whether patients have quantitative (β-thalassemia) or qualitative (Sickle Cell Disease (SCD)) defects ...in β-globin synthesis. Unfortunately, there are limited treatment options for β-hemoglobinopathies, and the majority of patients continue to develop life-threatening complications from their diseases. An alternative β-like subunit, γ-globin, has been shown to inhibit pathogenic hemoglobin polymerization in SCD and to functionally replace β-globin in β-thalassemia. The discovery of novel strategies to upregulate γ-globin expression may lead to effective therapies for β-hemoglobinopathies. To identify novel regulators of γ-globin expression, we performed a genome-wide pooled CRISPR activation (CRISPRa) screen, using the Synergistic Activation Mediator (SAM) system. The screen was performed in the HUDEP-2 cell line, which expresses adult β-globin. We generated a clonal HUDEP-2-MPH cell line, which stably expresses the transcriptional activator complex MPH (MS2-P65-HSF1). This cell line was transduced with a viral library that delivers a VP64 activation domain fused to a catalytically dead Cas9 (dCas9-VP64), a blasticidin resistance cassette, and one of 3 unique sgRNAs targeting virtually every coding gene in the human genome (the library consists of 70,290 sgRNAs). At day 8 of erythroid differentiation, the top and bottom 10% γ-expressing cells were sorted, and integrated sgRNAs were sequenced using NextGen sequencing. As expected, sgRNAs targeting BCL11A and ZBTB7A (known negative regulators of γ-globin) were enriched in the bottom 10% γ-expressing cells, while sgRNAs targeting HIF1A (known positive regulator of γ-globin) were enriched in the top 10% γ-expressing cells. Notably, this screen identified several novel candidate positive regulators of γ-globin expression, including the transcriptional repressor Hypermethylated in Cancer 1 (HIC1). In preliminary validation studies, we used 2 independent sgRNAs to activate HIC1 in HUDEP-2-MPH cells and measured the percentage of γ-globin expressing cells (F-cells) by flow cytometry. Compared to cells transduced with control sgRNAs, increased HIC1 transcription (>200-fold) resulted in a profound increase in the proportion of F-cells, from a baseline of ~6% up to ~76%. Similarly, HIC1 overexpression in HUDEP-2 cells resulted in a ~13-fold increase in γ-globin mRNA levels and reduction in BCL11A mRNA level to ~30% of normal. These results suggest that the increased γ-globin expression resulting from HIC1 overexpression may result, at least in part, from reduced BCL11A levels (which we are currently validating). Recently, increased expression of HIC2, a paralogous protein for HIC1, was reported to result in increased γ-globin expression. To exclude the possibility that HIC1 targeting sgRNAs may also target HIC2, we measured the HIC2 mRNA level in HUDEP-2 cells targeted with sgRNAs aimed at increasing HIC1 expression. HIC2 mRNA was not increased in the latter cells. We next overexpressed HIC1 cDNA in erythroid cells differentiated from primary human CD34+ hematopoietic stem and progenitor cells (HSPCs). In early preliminary results, we found that HIC1 overexpression resulted in increased γ-globin expression, validating the HUDEP-2 data. Additional studies are ongoing to define the role of HIC1 in the regulation of γ-globin expression, and to define the impact of HIC1 overexpression on erythroid differentiation. In summary, our screen uncovered HIC1, and other potential novel regulators of γ-globin expression, which we are currently validating. These findings may result in future therapeutic approaches for β-hemoglobinopathies.
Combined deficiency of coagulation factors V and VIII (F5F8D) is a human autosomal recessive bleeding disorder caused by mutations in LMAN1 or MCFD2, which encode the components of the only known ...cargo-receptor complex in the mammalian secretory pathway. The LMAN1/MCFD2 complex facilitates secretion of coagulation factors V (FV) and VIII (FVIII) to the plasma. F5F8D patients exhibit FV and FVIII levels that are ~10-15% of normal. The relationship between Lman1 expression levels and the corresponding secretion of LMAN1-dependent cargos has not been characterized, and additional functions of LMAN1 remain to be determined. We previously described Lman1 mutant mice carrying a gene-trap insertion in intron 10 (Lman1gt1). Lman1gt1/gt1 mice have ~50% of normal FV and FVIII activity levels, and are therefore less severely affected than human F5F8D patients. This difference could be explained by low levels of residual LMAN1 expression, or by differences in FV and FVIII secretion between mice and humans. Lman1gt1/gt1 mice also exhibit a partially penetrant, perinatal lethality that is restricted to certain mouse genetic strains. We generated a second, independent LMAN1-deficient mouse with no Lman1 gene expression (Lman1-/-), as well as hypomorphic LMAN1-deficient mice (Lman1cgt/cgt) that retain ~7% of wildtype Lman1 expression levels on a C57BL/6J genetic background. Plasma FV and FVIII levels in Lman1-/- and Lman1cgt/cgtmice were measured by functional coagulation assays and compared to wildtype (WT) and Lman1gt1/gt1 mice. A dose-response relationship was observed, with Lman1-/- mice exhibiting FV and FVIII levels that are ~50% of normal (compared to ~10-15% in human patients), while hypomorphic Lman1cgt/cgtmice demonstrate an intermediate reduction in FV and FVIII (~70% of WT levels). A similar result was demonstrated for alpha-1 antitrypsin, another known LMAN1-dependent cargo. Taken together with the presence of two null LMAN1 alleles in nearly all reported LMAN1 F5F8D patients, these results suggest that hypomorphic LMAN1 mutations in humans may also result in milder reductions in plasma FV and FVIII that may have been missed clinically, though still contributing to FV and FVIII level variation and to bleeding susceptibility.
The partially penetrant, strain-specific, perinatal lethality previously reported in Lman1gt1/gt1 mice was also confirmed in mice homozygous for the independently generated Lman1- allele on a pure C57BL/6J genetic background (p < 0.007), thereby ruling out a passenger gene effect as the cause of the originally reported Lman1gt1/gt1 lethality. In contrast, Lman1cgt/cgt mice on the same background are viable and observed in the expected Mendelian ratios (p > 0.19). Lman1-/- lethality was not observed on a mixed C57BL/6J and 129S1/SvImJ background (p > 0.79). F5F8D is not associated with fetal loss in humans, nor would fetal loss be expected from moderate deficiency of FV and FVIII. These results thus suggest a critical role for additional, as yet unknown, LMAN1 cargo(s).
Finally, adult Lman1-/- mice exhibit a mild thrombocytopenia compared to WT controls (9.3 x 105 vs. 12.3 x 105 cells/uL, p < 0.004). In contrast, Lman1+/-, Lman1cgt/cgt, and Mcfd2-/- mice all exhibit normal platelet counts indistinguishable from WT controls. No Lman1-/- platelet morphologic abnormalities are evident by standard or transmission electron microscopy, with normal appearance of megakaryocyte number and morphology on bone marrow examination. Although thrombocytopenia has not been noted previously in human F5F8D patients, these results suggest a novel role for an LMAN1-dependent secretory cargo in thrombopoiesis.
No relevant conflicts of interest to declare.
Most proteins secreted into the extracellular space are first recruited from the endoplasmic reticulum into coat protein complex II (COPII)-coated vesicles or tubules that facilitate their transport ...to the Golgi apparatus. Although several secreted proteins have been shown to be actively recruited into COPII vesicles and tubules by the cargo receptors LMAN1 and SURF4, the full cargo repertoire of these receptors is unknown. We now report mass spectrometry analysis of conditioned media and cell lysates from HuH7 cells CRISPR targeted to inactivate the LMAN1 or SURF4 gene. We found that LMAN1 has limited clients in HuH7 cells, whereas SURF4 traffics a broad range of cargoes. Analysis of putative SURF4 cargoes suggests that cargo recognition is governed by complex mechanisms rather than interaction with a universal binding motif..
Proteins carrying a signal peptide and/or a transmembrane domain enter the intracellular secretory pathway at the endoplasmic reticulum (ER) and are transported to the Golgi apparatus via COPII ...vesicles or tubules. SAR1 initiates COPII coat assembly by recruiting other coat proteins to the ER membrane. Mammalian genomes encode two
paralogs,
and
. While these paralogs exhibit ~90% amino acid sequence identity, it is unknown whether they perform distinct or overlapping functions in vivo. We now report that genetic inactivation of
in mice results in lethality during midembryogenesis. We also confirm previous reports that complete deficiency of murine
results in perinatal lethality. In contrast, we demonstrate that deletion of
restricted to hepatocytes is compatible with survival, though resulting in hypocholesterolemia that can be rescued by adenovirus-mediated overexpression of either SAR1A or SAR1B. To further examine the in vivo function of these two paralogs, we genetically engineered mice with the
coding sequence replacing that of
at the endogenous
locus. Mice homozygous for this allele survive to adulthood and are phenotypically normal, demonstrating complete or near-complete overlap in function between the two SAR1 protein paralogs in mice. These data also suggest upregulation of
gene expression as a potential approach for the treatment of SAR1B deficiency (chylomicron retention disease) in humans.
T cell-mediated responses are dependent on their secretion of key effector molecules. However, the critical molecular determinants of the secretion of these proteins are largely undefined. Here, we ...demonstrate that T cell activation increases trafficking via the ER-to-Golgi pathway. To study the functional role of this pathway, we generated mice with a T cell-specific deletion in SEC23B, a core subunit of coat protein complex II (COPII). We found that SEC23B critically regulated the T cell secretome following activation. SEC23B-deficient T cells exhibited a proliferative defect and reduced effector functions in vitro, as well as in experimental models of allogeneic and xenogeneic hematopoietic cell transplantation in vivo. However, T cells derived from 3 patients with congenital dyserythropoietic anemia II (CDAII), which results from Sec23b mutation, did not exhibit a similar phenotype. Mechanistic studies demonstrated that unlike murine KO T cells, T cells from patients with CDAII harbor increased levels of the closely related paralog, SEC23A. In vivo rescue of murine KO by expression of Sec23a from the Sec23b genomic locus restored T cell functions. Together, our data demonstrate a critical role for the COPII pathway, with evidence for functional overlap in vivo between SEC23 paralogs in the regulation of T cell immunity in both mice and humans.
Approximately one-third of the mammalian proteome is transported from the endoplasmic reticulum-to-Golgi via COPII-coated vesicles. SEC23, a core component of coat protein-complex II (COPII), is ...encoded by two paralogous genes in vertebrates (Sec23a and Sec23b). In humans, SEC23B deficiency results in congenital dyserythropoietic anemia type-II (CDAII), while SEC23A deficiency results in a skeletal phenotype (with normal red blood cells). These distinct clinical disorders, together with previous biochemical studies, suggest unique functions for SEC23A and SEC23B. Here we show indistinguishable intracellular protein interactomes for human SEC23A and SEC23B, complementation of yeast Sec23 by both human and murine SEC23A/B, and rescue of the lethality of sec23b deficiency in zebrafish by a sec23a-expressing transgene. We next demonstrate that a Sec23a coding sequence inserted into the murine Sec23b locus completely rescues the lethal SEC23B-deficient pancreatic phenotype. We show that SEC23B is the predominantly expressed paralog in human bone marrow, but not in the mouse, with the reciprocal pattern observed in the pancreas. Taken together, these data demonstrate an equivalent function for SEC23A/B, with evolutionary shifts in the transcription program likely accounting for the distinct phenotypes of SEC23A/B deficiency within and across species, a paradigm potentially applicable to other sets of paralogous genes. These findings also suggest that enhanced erythroid expression of the normal SEC23A gene could offer an effective therapeutic approach for CDAII patients.
Congenital dyserythropoietic anemia type II (CDAII) is an autosomal recessive disease of ineffective erythropoiesis characterized by increased bi/multinucleated erythroid precursors in the bone ...marrow. CDAII results from mutations in SEC23B. The SEC23 protein is a core component of coat protein complex II-coated vesicles, which transport secretory proteins from the endoplasmic reticulum to the Golgi apparatus. Though the genetic defect underlying CDAII has been identified, the pathophysiology of this disease remains unknown. We previously reported that SEC23B-deficient mice die perinatally, exhibiting massive pancreatic degeneration, with this early mortality limiting evaluation of the adult hematopoietic compartment. We now report that mice with SEC23B deficiency restricted to the hematopoietic compartment survive normally and do not exhibit anemia or other CDAII characteristics. We also demonstrate that SEC23B-deficient hematopoietic stem cells (HSC) do not exhibit a disadvantage at reconstituting hematopoiesis when compared directly to wild-type HSC in a competitive repopulation assay. Secondary bone marrow transplants demonstrated continued equivalence of SEC23B-deficient and WT HSC in their hematopoietic reconstitution potential. The surprising discordance in phenotypes between SEC23B-deficient mice and humans may reflect an evolutionary shift in SEC23 paralog function and/or expression, or a change in a specific COPII cargo critical for erythropoiesis.
LMAN1 and MCFD2 encode the components of a mammalian cargo-receptor that facilitates the ER-to-Golgi transport of coagulation factors V (FV) and VIII (FVIII) for secretion to the plasma. Mutations in ...LMAN1 or MCFD2 result in a rare bleeding disorder known as combined deficiency of coagulation factors V and VIII (F5F8D), characterized by FV and FVIII levels that are ~10% of normal. No other clinical phenotypes are known in human patients. Lman1 null mice have ~50% of normal FV and FVIII levels and exhibit a partially penetrant, perinatal lethality, suggesting a critical role for yet unknown LMAN1 secretory cargo(s).
To further characterize the function of the LMAN1/MCFD2 complex and identify new cargos, we generated several murine models of F5F8D, including ubiquitous null Lman1 (Lman1-/-) and Mcfd2 (Mcfd2-/-) mice maintained on a C57BL/6J genetic background. Adult Lman1-/- mice were mildly thrombocytopenic, exhibiting a 25% decrease in platelet count relative to wild-type (WT) mice (9.3 x 105 vs. 12.3 x 105 cells/uL, p < 0.004), but no other CBC abnormalities. In contrast, Lman1 heterozygous and Mcfd2-/- mice exhibited normal platelet counts. To further explore the role of LMAN1 in megakaryocyte/platelet development or survival, bone marrow (BM) histology and platelet transmission electron microscopy were performed. Lman1-/- mice had no platelet morphologic abnormalities by light or transmission electron microscopy, as well as normal number and morphology of BM megakaryocytes. Hematopoietic stem cells and megakaryocyte progenitors were indistinguishable between WT and Lman1-/- mice by flow cytometry.
In order to determine whether the thrombocytopenia phenotype results from LMAN1 deficiency specifically in the hematopoietic compartment, mice with tissue-specific knockout of Lman1 in hematopoietic and endothelial cells (Tie2-Cre) were generated. Platelet counts of mice with LMAN1 deficiency restricted to the hematopoietic compartment were indistinguishable from those in WT controls. In contrast, hepatocyte-specific (Alb-Cre) Lman1 deficiency, resulted in significant thrombocytopenia relative to WT controls (p < 0.017), with platelet counts comparable to those observed in ubiquitous Lman1 null mice. Since thrombopoietin (TPO) is a major hepatocyte-derived regulator of platelet synthesis, plasma TPO levels were measured by ELISA in ubiquitous Lman1 and Mcfd2 null mice. Plasma TPO levels in Lman1-/- mice were ~56% lower than those in WT levels (128.7 x 103 vs. 229.5 x 103 pg/mL, p < 0.0025). However, TPO levels were not reduced in Mcfd2-/- mice (p > 0.17). TPO mRNA expression in the liver of Lman1-/- mice was indistinguishable from livers of WT littermate controls.
In conclusion, global LMAN1-deficient and hepatocyte-specific LMAN1 deficient mice exhibit thrombocytopenia, a phenotype not previously reported in F5F8D patients. Lman1-/- mice, but not Mcfd2-/- mice, exhibit low plasma TPO levels, suggesting that TPO may be a novel LMAN1-dependent secretory cargo. These results raise the possibility that F5F8D patients with LMAN1 mutations may have mild thrombocytopenia, previously unappreciated as a result of the small number of F5F8D patients and the wide range of clinically normal platelet counts.
No relevant conflicts of interest to declare.
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