scholarly journals Role of the novel endoribonuclease SLFN14 and its disease causing mutations in ribosomal degradation

2018 ◽  
Author(s):  
Sarah J. Fletcher ◽  
Vera P. Pisareva ◽  
Abdullah Khan ◽  
Andrew Tcherepanov ◽  
Neil V. Morgan ◽  
...  
Keyword(s):  
In Vitro Models ◽  
Dominant Negative ◽  
Dominant Negative Effect ◽  
Heterozygous Mutation ◽  
Missense Mutations ◽  
The Novel ◽  
Excessive Bleeding ◽  
Negative Effect ◽  
Inherited Thrombocytopenia

ABSTRACTPlatelets are anucleate and mostly ribosome-free cells within the bloodstream, derived from megakaryocytes within bone marrow and crucial for cessation of bleeding at sites of injury. Inherited thrombocytopenias are a group of disorders characterized by alow platelet count and are frequently associated with excessive bleeding. SLFN14 is one of the most recently discovered genes linked to inherited thrombocytopenia where several heterozygous missense mutations in SLFN14 were identified to cause defective megakaryocyte maturation and platelet dysfunction. Yet, SLFN14 was recently described as a ribosome-associated protein resulting in rRNA and ribosome-bound mRNA degradation in rabbit reticulocytes. To unveil the cellular function of SLFN14 and the link between SLFN14 and thrombocytopenia, we examined SLFN14 (WT/mutants) in in vitro models. Here, we show that all SLFN14 variants co-localize with ribosomes and mediate rRNA endonucleolytic degradation and ribosome clearance. Compare dto SLFN14 WT, expression of mutants is dramatically reduced as a result of post-translational degradation due to partial misfolding of the protein. Moreover, all SLFN14 variants tend to form oligomers. These findings could explain the dominant negative effect of heterozygous mutation on SLFN14 expression in patients’ platelets. Overall we suggest that SLFN14 could be involved in ribosome degradation during platelet formation and maturation.

The Impact of Different DNMT3A Mutations on Outcome in Younger Adults with Acute Myeloid Leukemia

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 67-67
Author(s):  
Rosemary E Gale ◽  
Katarina Lamb ◽  
Christopher Allen ◽  
Dima El-Sharkawi ◽  
Cassandra Stowe ◽  
...  
Keyword(s):  
Conflicts Of Interest ◽  
Multivariable Analysis ◽  
Dominant Negative ◽  
Dominant Negative Effect ◽  
Heterozygous Mutation ◽  
Prognostic Impact ◽  
Missense Mutations ◽  
Limited Information ◽  
Negative Effect ◽  
The Impact

Abstract DNMT3A mutations (DNMT3AMUT) are recurrent in AML. They predominate in patients with intermediate-risk (IR) cytogenetics and are often co-incident with FLT3ITD and NPM1MUT. Their prognostic impact is unclear. Most reports suggest they are associated with a worse outcome, but a large study including 1060 younger adult IR patients found that DNMT3AMUT had no significant impact on survival endpoints. Variable results have also been reported for different FLT3/NPM1 subgroups. Missense mutations at R882 in exon 23 occur in ≈65% of patients, but other missense and truncation mutations occur throughout the gene, mainly in exons 13-23. There is limited information on the prognostic impact of the different mutations, although they may have differing functional consequences. We therefore screened exons 13-23 in DNA samples from 914 younger adult AML patients (median age 43 years) with IR cytogenetics treated on UK MRC trials and evaluated outcome according to type of DNMT3Amutation. Overall, 278 mutations were detected in 272 (30%) patients; 175 (64%) had R882 missense mutations, 59 (22%) other missense mutations, 35 (13%) truncations or in-frame deletions; 3 (1%) had 2 mutations of differing types. Median R882 mutant level in 172 mutated cases was 47% (range 15-85%), consistent with a heterozygous mutation in most cells. Patients with DNMT3AMUT were significantly older than those with DNMT3A wild-type (DNMT3AWT) (P<.0001), more likely to be female (P=.004), have a higher presenting WBC (P<.0001), and a normal rather than abnormal karyotype (P<.0001). The presence of DNMT3AMUT positively correlated with FLT3ITD (P=.0003) and NPM1MUT (P<.0001) and negatively correlated with CEBPAMUT (P<.0001). Patients with R882 mutations had significantly higher WBC (P=.005) and correlation with NPM1MUT (P=.01) than non-R882 mutated patients; non-R882 missense mutated patients had higher WBC (P=.05) and non-significant higher co-incidence with FLT3ITD than those with truncations. Presence of DNMT3AMUT was associated with a poorer prognosis, but this difference was only seen if the results were analyzed separately according to NPM1 genotype, where DNMT3AMUT was associated with higher cumulative incidence of relapse (CIR) than DNMT3AWT in cases with NPM1MUT (49% vs 40%, P=.01) and NPM1WT (61% vs 58%, P=.5) genotype. Similarly, DNMT3AMUT patients had worse overall survival (OS) than DNMT3AWT patients with NPM1MUT (38% vs 50%, P=.008) and NPM1WT (15% vs 25%, P=.09) genotype. This statistical anomaly is an example of Simpson’s paradox. It results from the strong co-incidence of DNMT3A and NPM1 mutations with opposing prognostic associations that mask the effect seen separately when the groups are combined. Although the differences were smaller for NPM1WT cases, tests for heterogeneity showed that the impact of a DNMT3A mutation did not differ between NPM1MUT and NPM1WT for either CIR or OS, nor between the 4 genotypes defined by the combination of NPM1 and FLT3ITD genotypes. In multivariable analysis including age, WBC, NPM1 and FLT3ITD, DNMT3AMUT was a significant adverse risk factor for CIR (HR=1.27, CI=1.01-1.61; P=.04), and showed a trend for being adverse for OS (HR=1.19, CI=.98-1.45; P=.08). When outcome was considered according to the type of mutation (R882, other missense or truncations), for the NPM1MUT genotype cases CIR was highest in R882 and other missense cases (51%, 50%) and truncation cases were similar to DNMT3AWT (35%, 40%). For NPM1WT, CIR was highest in R882 cases (76%), similar in other missense and DNMT3AWT cases (55%, 58%) and lowest in truncation cases (40%). Consistent with this data, for NPM1MUT genotype, OS was lowest in R882 and other missense cases (35%, 38%), better in DNMT3AWT (50%) and highest in truncation cases (57%). For NPM1WT, OS was lowest in R882 cases (11%), and similar in DNMT3AWT, other missense and truncation cases (25%, 21%, 18% respectively). These data suggest that screening cannot be limited to the hotspot R882 mutations and that cases with missense mutations should be treated as poor risk, including those patients currently considered as favorable risk such as NPM1MUTFLT3WT. Conversely, truncation mutations have a different functional impact from missense mutations, more likely to result in haploinsufficiency than a dominant-negative effect, and these cases should be considered as equivalent to DNMT3AWT for prognostication and selection of therapy in 1st remission. Disclosures No relevant conflicts of interest to declare.

scholarly journals Restoration of Silencing in Saccharomyces cerevisiae by Tethering of a Novel Sir2-Interacting Protein, Esc8

Genetics ◽  
2002 ◽  
Vol 162 (2) ◽  
pp. 633-645 ◽  
Author(s):  
Guido Cuperus ◽  
David Shore
Keyword(s):  
Protein A ◽  
Dominant Negative ◽  
Class I ◽  
Dominant Negative Effect ◽  
Rna Pol Ii ◽  
Interacting Protein ◽  
Nucleosome Remodeling ◽  
Negative Effect ◽  
Close Homolog

Abstract We previously described two classes of SIR2 mutations specifically defective in either telomeric/HM silencing (class I) or rDNA silencing (class II) in S. cerevisiae. Here we report the identification of genes whose protein products, when either overexpressed or directly tethered to the locus in question, can establish silencing in SIR2 class I mutants. Elevated dosage of SCS2, previously implicated as a regulator of both inositol biosynthesis and telomeric silencing, suppressed the dominant-negative effect of a SIR2-143 mutation. In a genetic screen for proteins that restore silencing when tethered to a telomere, we isolated ESC2 and an uncharacterized gene, (YOL017w), which we call ESC8. Both Esc2p and Esc8p interact with Sir2p in two-hybrid assays, and the Esc8p-Sir2 interaction is detected in vitro. Interestingly, Esc8p has a single close homolog in yeast, the ISW1-complex factor Ioc3p, and has also been copurified with Isw1p, raising the possibility that Esc8p is a component of an Isw1p-containing nucleosome remodeling complex. Whereas esc2 and esc8 deletion mutants alone have only marginal silencing defects, cells lacking Isw1p show a strong silencing defect at HMR but not at telomeres. Finally, we show that Esc8p interacts with the Gal11 protein, a component of the RNA pol II mediator complex.

scholarly journals Dominant Negative Mutants Implicate STAT5 in Myeloid Cell Proliferation and Neutrophil Differentiation

Blood ◽  
1999 ◽  
Vol 93 (12) ◽  
pp. 4154-4166 ◽  
Author(s):  
Robert L. Ilaria ◽  
Robert G. Hawley ◽  
Richard A. Van Etten
Keyword(s):  
Dna Binding ◽  
Transcriptional Activation ◽  
Dominant Negative ◽  
Dominant Negative Effect ◽  
Cytokine Signaling ◽  
Transactivation Domain ◽  
Negative Effects ◽  
Murine Bone Marrow ◽  
Negative Effect

Abstract STAT5 is a member of the signal transducers and activation of transcription (STAT) family of latent transcription factors activated in a variety of cytokine signaling pathways. We introduced alanine substitution mutations in highly conserved regions of murine STAT5A and studied the mutants for dimerization, DNA binding, transactivation, and dominant negative effects on erythropoietin-induced STAT5-dependent transcriptional activation. The mutations included two near the amino-terminus (W255KR→AAA and R290QQ→AAA), two in the DNA-binding domain (E437E→AA and V466VV→AAA), and a carboxy-terminal truncation of STAT5A (STAT5A/▵53C) analogous to a naturally occurring isoform of rat STAT5B. All of the STAT mutant proteins were tyrosine phosphorylated by JAK2 and heterodimerized with STAT5B except for the WKR mutant, suggesting an important role for this region in STAT5 for stabilizing dimerization. The WKR, EE, and VVV mutants had no detectable DNA-binding activity, and the WKR and VVV mutants, but not EE, were defective in transcriptional induction. The VVV mutant had a moderate dominant negative effect on erythropoietin-induced STAT5 transcriptional activation, which was likely due to the formation of heterodimers that are defective in DNA binding. Interestingly, the WKR mutant had a potent dominant negative effect, comparable to the transactivation domain deletion mutant, ▵53C. Stable expression of either the WKR or ▵53C STAT5 mutants in the murine myeloid cytokine-dependent cell line 32D inhibited both interleukin-3–dependent proliferation and granulocyte colony-stimulating factor (G-CSF)–dependent differentiation, without induction of apoptosis. Expression of these mutants in primary murine bone marrow inhibited G-CSF–dependent granulocyte colony formation in vitro. These results demonstrate that mutations in distinct regions of STAT5 exert dominant negative effects on cytokine signaling, likely through different mechanisms, and suggest a role for STAT5 in proliferation and differentiation of myeloid cells.

scholarly journals The Diagnostic Journey of a Patient with Prader–Willi-Like Syndrome and a Unique Homozygous SNURF-SNRPN Variant; Bio-Molecular Analysis and Review of the Literature

Genes ◽  
2021 ◽  
Vol 12 (6) ◽  
pp. 875
Author(s):  
Karlijn Pellikaan ◽  
Geeske M. van Woerden ◽  
Lotte Kleinendorst ◽  
Anna G. W. Rosenberg ◽  
Bernhard Horsthemke ◽  
...  
Keyword(s):  
Intellectual Disability ◽  
Single Nucleotide Polymorphism Array ◽  
Genetic Defect ◽  
Missense Variant ◽  
Dominant Negative ◽  
Dominant Negative Effect ◽  
Pituitary Hormone ◽  
Negative Effect

Prader–Willi syndrome (PWS) is a rare genetic condition characterized by hypotonia, intellectual disability, and hypothalamic dysfunction, causing pituitary hormone deficiencies and hyperphagia, ultimately leading to obesity. PWS is most often caused by the loss of expression of a cluster of genes on chromosome 15q11.2-13. Patients with Prader–Willi-like syndrome (PWLS) display features of the PWS phenotype without a classical PWS genetic defect. We describe a 46-year-old patient with PWLS, including hypotonia, intellectual disability, hyperphagia, and pituitary hormone deficiencies. Routine genetic tests for PWS were normal, but a homozygous missense variant NM_003097.3(SNRPN):c.193C>T, p.(Arg65Trp) was identified. Single nucleotide polymorphism array showed several large regions of homozygosity, caused by high-grade consanguinity between the parents. Our functional analysis, the ‘Pipeline for Rapid in silico, in vivo, in vitro Screening of Mutations’ (PRiSM) screen, showed that overexpression of SNRPN-p.Arg65Trp had a dominant negative effect, strongly suggesting pathogenicity. However, it could not be confirmed that the variant was responsible for the phenotype of the patient. In conclusion, we present a unique homozygous missense variant in SNURF-SNRPN in a patient with PWLS. We describe the diagnostic trajectory of this patient and the possible contributors to her phenotype in light of the current literature on the genotype–phenotype relationship in PWS.

Novel REST Truncation Mutations Causing Hereditary Gingival Fibromatosis

2021 ◽  
pp. 002203452199662
Author(s):  
J.T. Chen ◽  
C.H. Lin ◽  
H.W. Huang ◽  
Y.P. Wang ◽  
P.C. Kao ◽  
...  
Keyword(s):  
Nuclear Localization ◽  
Genetic Disorder ◽  
Dominant Negative ◽  
Dominant Negative Effect ◽  
Wild Type ◽  
Negative Effect ◽  
Localization Signal ◽  
Gingival Fibromatosis

Hereditary gingival fibromatosis (HGF) is a rare genetic disorder featured by nonsyndromic pathological overgrowth of gingiva. The excessive gingival tissues can cause dental, masticatory, and phonetic problems, which impose severe functional and esthetic burdens on affected individuals. Due to its high recurrent rate, patients with HGF have to undergo repeated surgical procedures of gingival resection, from childhood to adulthood, which significantly compromises their quality of life. Unraveling the genetic etiology and molecular pathogenesis of HGF not only gains insight into gingival physiology and homeostasis but also opens avenues for developing potential therapeutic strategies for this disorder. Recently, mutations in REST (OMIM *600571), encoding a transcription repressor, were reported to cause HGF (GINGF5; OMIM #617626) in 3 Turkish families. However, the functions of REST in gingival homeostasis and pathogenesis of REST-associated HGF remain largely unknown. In this study, we characterized 2 HGF families and identified 2 novel REST mutations, c.2449C>T (p.Arg817*) and c.2771_2793dup (p.Glu932Lysfs*3). All 5 mutations reported to date are nonsenses or frameshifts in the last exon of REST and would presumably truncate the protein. In vitro reporter gene assays demonstrated a partial or complete loss of repressor activity for these truncated RESTs. When coexpressed with the full-length protein, the truncated RESTs impaired the repressive ability of wild-type REST, suggesting a dominant negative effect. Immunofluorescent studies showed nuclear localization of overexpressed wild-type and truncated RESTs in vitro, indicating preservation of the nuclear localization signal in shortened proteins. Immunohistochemistry demonstrated a comparable pattern of ubiquitous REST expression in both epithelium and lamina propria of normal and HGF gingival tissues despite a reduced reactivity in HGF gingiva. Results of this study confirm the pathogenicity of REST truncation mutations occurring in the last exon causing HGF and suggest the pathosis is caused by an antimorphic (dominant negative) disease mechanism.

scholarly journals Crystal structure of MICU2 and comparison with MICU1 reveal insights into the uniporter gating mechanism

2019 ◽  
Vol 116 (9) ◽  
pp. 3546-3555 ◽  
Author(s):  
Kimberli J. Kamer ◽  
Wei Jiang ◽  
Virendar K. Kaushik ◽  
Vamsi K. Mootha ◽  
Zenon Grabarek
Keyword(s):  
Binding Sites ◽  
Mammalian Cells ◽  
Dominant Negative ◽  
Dominant Negative Effect ◽  
Functional Coupling ◽  
Gating Mechanism ◽  
Ef Hand ◽  
Negative Effect ◽  
Oligomeric Complex

The mitochondrial uniporter is a Ca2+-channel complex resident within the organelle’s inner membrane. In mammalian cells the uniporter’s activity is regulated by Ca2+ due to concerted action of MICU1 and MICU2, two paralogous, but functionally distinct, EF-hand Ca2+-binding proteins. Here we present the X-ray structure of the apo form of Mus musculus MICU2 at 2.5-Å resolution. The core structure of MICU2 is very similar to that of MICU1. It consists of two lobes, each containing one canonical Ca2+-binding EF-hand (EF1, EF4) and one structural EF-hand (EF2, EF3). Two molecules of MICU2 form a symmetrical dimer stabilized by highly conserved hydrophobic contacts between exposed residues of EF1 of one monomer and EF3 of another. Similar interactions stabilize MICU1 dimers, allowing exchange between homo- and heterodimers. The tight EF1–EF3 interface likely accounts for the structural and functional coupling between the Ca2+-binding sites in MICU1, MICU2, and their complex that leads to the previously reported Ca2+-binding cooperativity and dominant negative effect of mutation of the Ca2+-binding sites in either protein. The N- and C-terminal segments of the two proteins are distinctly different. In MICU2 the C-terminal helix is significantly longer than in MICU1, and it adopts a more rigid structure. MICU2’s C-terminal helix is dispensable in vitro for its interaction with MICU1 but required for MICU2’s function in cells. We propose that in the MICU1–MICU2 oligomeric complex the C-terminal helices of both proteins form a central semiautonomous assembly which contributes to the gating mechanism of the uniporter.

scholarly journals Dominant Negative Mutants Implicate STAT5 in Myeloid Cell Proliferation and Neutrophil Differentiation

Blood ◽  
1999 ◽  
Vol 93 (12) ◽  
pp. 4154-4166 ◽  
Author(s):  
Robert L. Ilaria ◽  
Robert G. Hawley ◽  
Richard A. Van Etten
Keyword(s):  
Dna Binding ◽  
Transcriptional Activation ◽  
Dominant Negative ◽  
Dominant Negative Effect ◽  
Cytokine Signaling ◽  
Transactivation Domain ◽  
Negative Effects ◽  
Murine Bone Marrow ◽  
Negative Effect

STAT5 is a member of the signal transducers and activation of transcription (STAT) family of latent transcription factors activated in a variety of cytokine signaling pathways. We introduced alanine substitution mutations in highly conserved regions of murine STAT5A and studied the mutants for dimerization, DNA binding, transactivation, and dominant negative effects on erythropoietin-induced STAT5-dependent transcriptional activation. The mutations included two near the amino-terminus (W255KR→AAA and R290QQ→AAA), two in the DNA-binding domain (E437E→AA and V466VV→AAA), and a carboxy-terminal truncation of STAT5A (STAT5A/▵53C) analogous to a naturally occurring isoform of rat STAT5B. All of the STAT mutant proteins were tyrosine phosphorylated by JAK2 and heterodimerized with STAT5B except for the WKR mutant, suggesting an important role for this region in STAT5 for stabilizing dimerization. The WKR, EE, and VVV mutants had no detectable DNA-binding activity, and the WKR and VVV mutants, but not EE, were defective in transcriptional induction. The VVV mutant had a moderate dominant negative effect on erythropoietin-induced STAT5 transcriptional activation, which was likely due to the formation of heterodimers that are defective in DNA binding. Interestingly, the WKR mutant had a potent dominant negative effect, comparable to the transactivation domain deletion mutant, ▵53C. Stable expression of either the WKR or ▵53C STAT5 mutants in the murine myeloid cytokine-dependent cell line 32D inhibited both interleukin-3–dependent proliferation and granulocyte colony-stimulating factor (G-CSF)–dependent differentiation, without induction of apoptosis. Expression of these mutants in primary murine bone marrow inhibited G-CSF–dependent granulocyte colony formation in vitro. These results demonstrate that mutations in distinct regions of STAT5 exert dominant negative effects on cytokine signaling, likely through different mechanisms, and suggest a role for STAT5 in proliferation and differentiation of myeloid cells.

scholarly journals Dominant negative effect of the loss-of-function γ-secretase mutants on the wild-type enzyme through heterooligomerization

2017 ◽  
Vol 114 (48) ◽  
pp. 12731-12736 ◽  
Author(s):  
Rui Zhou ◽  
Guanghui Yang ◽  
Yigong Shi
Keyword(s):  
Alzheimer’S Disease ◽  
Alzheimer's Disease ◽  
Protease Activity ◽  
Underlying Mechanism ◽  
Dominant Negative ◽  
Presenilin 1 ◽  
Dominant Negative Effect ◽  
Loss Of Function ◽  
Negative Effect

γ-secretase is an intramembrane protease complex consisting of nicastrin, presenilin-1/2, APH-1a/b, and Pen-2. Hydrolysis of the 99-residue transmembrane fragment of amyloid precursor protein (APP-C99) by γ-secretase produces β-amyloid (Aβ) peptides. Pathogenic mutations in PSEN1 and PSEN2, which encode the catalytic subunit presenilin-1/2 of γ-secretase, lead to familial Alzheimer’s disease in an autosomal dominant manner. However, the underlying mechanism of how the mutant PSEN gene may affect the function of the WT allele remains to be elucidated. Here we report that each of the loss-of-function γ-secretase variants that carries a PSEN1 mutation suppresses the protease activity of the WT γ-secretase on Aβ production. Each of these γ-secretase variants forms a stable oligomer with the WT γ-secretase in vitro in the presence of the detergent CHAPSO {3-[(3-cholamidopropyl)dimethylammonio]-2-hydroxy-1-propanesulfonate}, but not digitonin. Importantly, robust protease activity of γ-secretase is detectable in the presence of CHAPSO, but not digitonin. These experimental observations suggest a dominant negative effect of the γ-secretase, in which the protease activity of WT γ-secretase is suppressed by the loss-of-function γ-secretase variants through hetero-oligomerization. The relevance of this finding to the genesis of Alzheimer’s disease is critically evaluated.

Cohesin Complex Mutations Impair Differentiation of Human Hematopoietic Stem and Progenitor Cells

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 4785-4785
Author(s):  
Claire Mazumdar ◽  
Rui Li ◽  
Jason Buenrostro ◽  
Howard Y. Chang ◽  
Ravi Majeti
Keyword(s):  
Cord Blood ◽  
Erythroid Differentiation ◽  
Chromatin Accessibility ◽  
Dominant Negative ◽  
Dominant Negative Effect ◽  
Cohesin Complex ◽  
Human Cord Blood ◽  
Hematopoietic Stem ◽  
Negative Effect

Abstract The cohesin complex is a multiprotein complex involved in a number of cellular processes including sister chromatid cohesion in mitosis, replication fork organization, and regulation of chromatin accessibility for gene expression. Mutations in genes encoding the members of the cohesin complex (SMC1A, SMC3, STAG2, and RAD21) occur in about 10-15% of de novo acute myeloid leukemia (AML) patients. Apart from AML, cohesin mutations have been found in many human cancers indicating a central role for this complex in oncogenesis. In AML, our prior studies have demonstrated that cohesin mutations occur in pre-leukemic hematopoietic stem and progenitor cells (HSPC) that retain normal differentiation potential. Thus, these mutations are likely key initiating events in leukemia pathogenesis. Due to their importance in AML evolution, we sought to determine the effect of these mutations on human hematopoiesis. Cohesin mutations typically occur as heterozygous mutations throughout the genes suggesting either a haploinsufficiency or dominant negative effect. Co-immunoprecipitation experiments in primary human AML samples showed marked decrease in binding between RAD21 and SMC1A in RAD21/SMC1A-mutant AML. These results suggest a dominant negative effect of cohesin mutants on complex formation. In an effort to characterize the phenotype of cohesin complex mutations in AML, we generated human AML cell lines engineered to express wildtype (WT) or mutant cohesin components under the control of a doxycycline-inducible promoter. We chose the TF-1 erythroleukemia cell line due to its ability to differentiate down the erythroid lineage in response to erythropoietin (EPO). We found that cohesin mutant cell lines showed a significant decrease in erythroid differentiation upon exposure to EPO as determined by surface expression of glycophorin A (GPA) and RNA expression of fetal hemoglobin and KLF-1, a key erythroid transcription factor, suggesting that cohesin mutations act in a dominant negative manner to impair differentiation. We next investigated the impact of cohesin complex mutations on normal HSPCs from primary human cord blood. We transduced CD34+ cord blood cells with lentivirus encoding constitutive expression of either WT or mutant cohesin components. Transduced cells were isolated and cultured under several conditions. First, cells were cultured with cytokines designed to promote retention of HSPCs, and cord blood cells expressing mutant cohesin showed significant retention of CD34+ expression as compared to WT or control cells. Second, cells were cultured under conditions designed to promote granulocytic/monocytic differentiation, and cohesin mutant-expressing cells showed a significant decrease in CD14+ expression compared to controls. Third, cells were cultured under conditions designed to promote erythroid differentiation, and cohesin mutant cells showed a significant decrease in CD71 and GPA-double positive erythroid cells. Together, this data suggests that cohesin complex mutations impart a differentiation block on primary human HSPCs. Finally, we investigated whether cohesin mutations affected the serial colony replating ability of human HSPCs in vitro. Primary human cord blood HSPCs were transduced with cohesin mutant-encoding lentivirus, sorted, and cultured in methylcellulose for 14 days. No differences were observed in the colony number or type in the primary plating. However, cohesin-mutant cells exhibited increased serial replating potential beyond the 3rd replating, with essentially no control or WT colonies after the 2ndreplating. In summary, our results indicate that cohesin complex mutations impair HSPC differentiation and increase in vitro replating of primary human cells. The mechanisms by which this occurs are currently being investigated, but preliminary data suggests that mutations in cohesin affect global chromatin accessibility. These results are consistent with a model of mutational acquisition in AML that we have proposed, in which pre-leukemic mutations occur in genes involved in global regulation of gene expression through epigenetic mechanisms that impair differentiation and/or affect self-renewal (such as IDH1/2, TET2, DNMT3A, and cohesin), whereas late mutations occur in genes that generally lead to an increase in activated signaling and proliferation (such as FLT3 and RAS). Disclosures No relevant conflicts of interest to declare.

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