Mutations In Zinc Finger Research Articles

Morc2a variants cause hydroxyl radical-mediated neuropathy and are rescued by restoring GHKL ATPase.

Mutations in the Microrchidia CW-type zinc finger 2 (MORC2) GHKL ATPase module cause a broad range of neuropathies, such as Charcot-Marie-Tooth disease type 2Z; however, the aetiology and therapeutic strategy are not fully understood. Previously, we reported that the Morc2a p.S87L mouse model exhibited neuropathy and muscular dysfunction through DNA damage accumulation. In the present study, we analysed the gene expression of Morc2a p.S87L mice and designated the primary causing factor. We investigated the pathological pathway using Morc2a p.S87L mouse embryonic fibroblasts and human fibroblasts harbouring MORC2 p.R252W. We subsequently assessed the therapeutic effect of gene therapy administered to Morc2a p.S87L mice. This study revealed that Morc2a p.S87L causes a protein synthesis defect, resulting in the loss of function of Morc2a and high cellular apoptosis induced by high hydroxyl radical levels. We considered the Morc2a GHKL ATPase domain as a therapeutic target because it simultaneously complements hydroxyl radical scavenging and ATPase activity. We used the adeno-associated virus (AAV)-PHP.eB serotype, which has a high CNS transduction efficiency, to express Morc2a or Morc2a GHKL ATPase domain protein in vivo. Notably, AAV gene therapy ameliorated neuropathy and muscular dysfunction with a single treatment. Loss-of-function characteristics due to protein synthesis defects in Morc2a p.S87L were also noted in human MORC2 p.S87L or p.R252W variants, indicating the correlation between mouse and human pathogenesis. In summary, CMT2Z is known as an incurable genetic disorder, but the present study demonstrated its mechanisms and treatments based on established animal models. This study demonstrates that the Morc2a p.S87L variant causes hydroxyl radical-mediated neuropathy, which can be rescued through AAV-based gene therapy.

Genomic, clinical characteristics, and treatment outcomes of patients with metastatic castration-sensitive prostate cancer with SPOP mutations: Analysis from the Kaiser Permanente Northern California Healthcare System.

261 Background: Androgen deprivation therapy (ADT) plus either Abiraterone, Docetaxel or Enzalutamide (ADE) improves overall survival (OS) of men with metastatic M1 castrate-sensitive prostate cancer (mCSPC), compared to ADT alone and is currently standard of care. Men with mCSPC with a point mutation of the speckle-type pox virus and zinc finger mutation (mutSPOP) derive superior response to ADT compared to wild type SPOP. The additive benefit of ADE to ADT in this subclass of CSPC is unclear. We sought to evaluate the benefit of therapy in patients with mutSPOP in a real-world setting. Methods: Between November 2017 and July 2022, 1002, patients in the Kaiser Permanente Northern California (KPNC) health system with advanced prostate cancer had tumor specimens examined by next generation sequencing (NGS). Of these patients, we retrospectively identified 70 mCSPC patients with mutSPOP. Patient records were reviewed to determine baseline characteristics, therapy for CSPC, subsequent therapy for Castrate Resistant Prostate Cancer (CRPC), and clinical outcome. The primary outcome was progression-free survival (PFS) for patients who received ADT alone or ADT plus ADE. PFS was defined as time to either PSA progression by PCWG2 criteria, clinical progression, or time to next line of therapy. Results: Median age for the 70 patients with mutSPOP was 75 years (range 50-91). Thirty-six (51%) were non-Hispanic white; 11 (16%) were African American; and 13 (19%) were Asian. F133 was the most frequently mutated allele (64%), TP53 was the most common co-mutated gene (27%), and mutations in MSH2, MSH6 and BRCA1 were identified in one patient each. Two patients had concurrent ERG fusion. Thirty-six patients (51%) had a Gleason score 8-10 and 48 (69%) were diagnosed with de-novo metastatic disease. Twenty-seven patients (39%) received ADT alone, 41 (59%) received ADT + ADE, 2 patients elected observation only. PFS was 28.1 months (mos) for ADT alone vs 35 for ADT+ADE (p=0.08). Twenty-nine patients (41%) received 2nd line therapy at CRPC; among them 24 received ADT plus Abiraterone or Enzalutamide. Median PFS for all the CRPC patients who received 2nd line treatment was 15mos. Median PFS for CRPC patients who received 2nd line treatment with Abiraterone, or Enzalutamide was 15.3 mos. Median overall survival for the entire group was 173 mos (95% CI 135-NR). Conclusions: In this real-world study within KPNC, we confirmed the durable benefit of ADT based therapy in patients with mutSPOP. Patients with mutSPOP derived similar benefit regardless of 1st line therapy (ADT vs ADT + ADE). We also noted durable responses to abiraterone or enzalutamide for CRPC after initial treatment with ADE for CSPC. Confirmation of these findings from an another large NGS database is ongoing and will be reported later.

Regulation of human ZNF687, a gene associated with Paget's disease of bone.

Mutations in Zinc finger 687 (ZNF687) were associated with Paget's disease of bone (PDB), a disease characterized by increased bone resorption and excessive bone formation. It was suggested that ZNF687 plays a role in bone differentiation and development. However, the mechanisms involved in ZNF687 regulation remain unknown. This study aimed to obtain novel knowledge regarding ZNF687 transcriptional and epigenetic regulation. Through in silico analysis, we hypothesized three ZNF687 promoter regions located upstream exon 1A, 1B, and 1C and denominated promoter regions 1, 2, and 3, respectively. Their functionality was confirmed by luciferase activity assays and positive/negative regulatory regions were identified using promoter deletions constructs. In silico analysis revealed a high density of CpG islands in these promoter regions and in vitro methylation suppressed promoters' activity. Using bioinformatic approaches, bone-associated transcription factor binding sites containing CpG dinucleotides were identified, including those for NFκB, PU.1, DLX5, and SOX9. By co-transfection in HEK293 and hFOB cells, we found that DLX5 specifically activated ZNF687 promoter region 1, and its methylation impaired DLX5-driven promoter stimulation. NFκB repressed and activated promoter regions 1 and 2, respectively, and these activities were affected by methylation. PU.1 induced ZNF687 promoter region 1 which was affected by methylation. SOX9 differentially regulated ZNF687 promoters in HEK293 and hFOB cells that were impaired after methylation. In conclusion, this study provides novel insights into ZNF687 regulation by demonstrating that NFκB, PU.1, DLX5, and SOX9 are regulators of ZNF687 promoters, and DNA methylation influences their activity. The contribution of the dysregulation of these mechanisms in PDB should be further elucidated.

Novel compound heterozygous mutations in UHRF1 are associated with atypical immunodeficiency, centromeric instability and facial anomalies syndrome with distinctive genome-wide DNA hypomethylation.

Immunodeficiency, centromeric instability and facial anomalies (ICF) syndrome is in most cases caused by mutations in either DNA methyltransferase (DNMT)3B, zinc finger and BTB domain containing 24, cell division cycle associated 7 or helicase lymphoid-specific. However, the causative genes of a few ICF patients remain unknown. We, herein, identified ubiquitin-like with plant homeodomain and really interesting new gene finger domains 1 (UHRF1) as a novel causative gene of one such patient with atypical symptoms. This patient is a compound heterozygote for two previously unreported mutations in UHRF1: c.886C > T (p.R296W) and c.1852C > T (p.R618X). The R618X mutation plausibly caused nonsense-mediated decay, while the R296W mutation changed the higher order structure of UHRF1, which is indispensable for the maintenance of CG methylation along with DNMT1. Genome-wide methylation analysis revealed that the patient had a centromeric/pericentromeric hypomethylation, which is the main ICF signature, but also had a distinctive hypomethylation pattern compared to patients with the other ICF syndrome subtypes. Structural and biochemical analyses revealed that the R296W mutation disrupted the protein conformation and strengthened the binding affinity of UHRF1 with its partner LIG1 and reduced ubiquitylation activity of UHRF1 towards its ubiquitylation substrates, histone H3 and proliferating cell nuclear antigen -associated factor 15 (PAF15). We confirmed that the R296W mutation causes hypomethylation at pericentromeric repeats by generating the HEK293 cell lines that mimic the patient's UHRF1 molecular context. Since proper interactions of the UHRF1 with LIG1, PAF15 and histone H3 are essential for the maintenance of CG methylation, the mutation could disturb the maintenance process. Evidence for the importance of the UHRF1 conformation for CG methylation in humans is, herein, provided for the first time and deepens our understanding of its role in regulation of CG methylation.

A Missense Mutation in Zinc Finger 4 of WT1 Might Lead to Focal Segmental Glomerular Sclerosis due to Its Mislocalization and Downstream Dysregulation

A Missense Mutation in Zinc Finger 4 of WT1 Might Lead to Focal Segmental Glomerular Sclerosis due to Its Mislocalization and Downstream Dysregulation

P.220 Neuropsychological functioning in CMT type 2Z: A case report of language deficits masquerading as intellectual disability

CMT type 2Z is an increasingly studied form of CMT caused by mutation in the microrchidia family CW-type zinc finger 2 gene (<i>MORC2</i>). Intellectual disability is commonly reported in the small available literature in individuals with CMT2Z; however, neuropsychological data is lacking in this population, and especially in children. Here we present a case report of an 8-year-old boy with genetically confirmed CMT2Z due to a <i>de novo</i> pathogenic variant in the <i>MORC2</i> gene. This boy was referred for comprehensive neuropsychological evaluation by his neuromuscular multidisciplinary team. Results revealed a pattern of strengths and weaknesses consistent with parent report of developmental, language, and academic difficulties. However, overall thinking skills were highly variable, with Average visual and visuospatial skills in contrast to impaired verbal, working memory, and processing speed skills (WISC-V FSIQ=70, GAI=81; VCI=65, VSI=100, FRI=97, WMI=69, PSI=49). Level of independence day-to-day as rated by his mother fell in the impaired range (Vineland-3 ABC=67). Academic achievement was also impaired across skills (WIAT-III Early Reading Skills SS=40, Single Word Reading SS=52, Alphabet Writing Fluency SS=72, Spelling SS=51, Math Problem Solving SS=51, Numerical Operations SS=68). No clinically significant concerns were endorsed by his mother in terms of social-emotional functioning (i.e., externalizing, internalizing, or behavioral symptoms). Diagnostically, this child presents with a complex picture. Impairments in adaptive functioning raise concern for intellectual disability; however, given this child's uneven intellectual profile, with Average visual/visuospatial skills compared to impaired verbal skills, and impairments in virtually all other receptive and expressive language skills measured on exam, observed qualitatively, and reported by his mother, the most appropriate diagnosis is a language disorder. Children with language disorders have persistent difficulties in use of language due to deficits in production including reduced vocabulary, limited sentence structure, and impairments in discourse. Children with language disorders also have difficulties learning adaptive skills and with academic learning, which is also consistent with this child's profile. More research is needed, but the neurocognitive profile of children with CMT2Z is likely more complex than currently reported.

Specific effects of somatic GATA2 zinc finger mutations on erythroid differentiation

GATA2 zinc-finger (ZF) mutations are associated with distinct entities of myeloid malignancies. The specific distribution of these mutations points toward different mechanisms of leukemogenesis depending on the ZF domain affected. In this study, we compared recurring somatic mutations in ZF1 and ZF2. All tested ZF mutants disrupted DNA binding in vitro. In transcription assays, co-expression of FOG1 counteracted GATA2-dependent transcriptional activation, while a variable response to FOG1-mediated repression was observed for individual GATA2 mutants. In primary murine bone marrow cells, GATA2 wild-type (WT) expression inhibited colony formation, while this effect was reduced for both mutants A318T (ZF1) and L359V (ZF2) with a shift toward granulopoiesis. In primary human CD34+ bone marrow cells and in the myeloid cell line K562, ectopic expression of GATA2 L359V, but not A318T or G320D, caused a block of erythroid differentiation accompanied by downregulation of GATA1, STAT5B, and PLCG1. Our findings may explain the role of GATA2 L359V during the progression of chronic myeloid leukemia and the collaboration of GATA2 ZF1 alterations with CEBPA double mutations in erythroleukemia.

Structure\u2013function relationships explain CTCF zinc finger mutation phenotypes in cancer

CCCTC-binding factor (CTCF) plays fundamental roles in transcriptional regulation and chromatin architecture maintenance. CTCF is also a tumour suppressor frequently mutated in cancer, however, the structural and functional impact of mutations have not been examined. We performed molecular and structural characterisation of five cancer-specific CTCF missense zinc finger (ZF) mutations occurring within key intra- and inter-ZF residues. Functional characterisation of CTCF ZF mutations revealed a complete (L309P, R339W, R377H) or intermediate (R339Q) abrogation as well as an enhancement (G420D) of the anti-proliferative effects of CTCF. DNA binding at select sites was disrupted and transcriptional regulatory activities abrogated. Molecular docking and molecular dynamics confirmed that mutations in residues specifically contacting DNA bases or backbone exhibited loss of DNA binding. However, R339Q and G420D were stabilised by the formation of new primary DNA bonds, contributing to gain-of-function. Our data confirm that a spectrum of loss-, change- and gain-of-function impacts on CTCF zinc fingers are observed in cell growth regulation and gene regulatory activities. Hence, diverse cellular phenotypes of mutant CTCF are clearly explained by examining structure–function relationships.

Effect of the Forth and Fifth Zinc Finger Deletions of MTF-1 on the Expression of Metal Ion Metabolism Related Gene.

Metal response element binding transcription factor 1 (MTF-1) is one of the important regulatory proteins involved in the mediation of intracellular metal ion balance, which is zinc dependent. The changes of zinc finger effected its function. MTF-1 mutant 293T cell line was obtained by transferring the vector of MTF-1 4th or 5th mutant zinc finger into 293T cell line that knocked out MTF-1 gene. The results showed that the mutant of 4th zinc finger in MTF-1 protein showed a significant difference on target gene expression compared with 5th zinc finger. Further RNA-seq assay showed that 4th and 5th zinc finger of MTF-1 have a different effect on molecular biological functions, cellular components, and biological process. The mutant of 4th and 5th zinc finger in MTF-1 protein changed different signaling pathways and metal ion metabolism related to genes. The present study evaluated that 4th or 5th mutant zinc finger in MTF-1 gene were associated with the function of MTF-1 protein.

PARP7 mono-ADP-ribosylates the agonist conformation of the androgen receptor in the nucleus.

We recently described a signal transduction pathway that contributes to androgen receptor (AR) regulation based on site-specific ADP-ribosylation by PARP7, a mono-ADP-ribosyltransferase implicated in several human cancers. ADP-ribosylated AR is recognized by PARP9/DTX3L, a heterodimeric complex that contains an ADP-ribose reader (PARP9) and a ubiquitin E3 ligase (DTX3L). Here, we have characterized the cellular and biochemical requirements for AR ADP-ribosylation by PARP7. We found that the reaction requires nuclear localization of PARP7 and an agonist-induced conformation of AR. PARP7 contains a Cys3His1-type zinc finger (ZF), which also is critical for AR ADP-ribosylation. The Parp7 ZF is required for efficient nuclear import by a nuclear localization signal encoded in PARP7, but rescue experiments indicate the ZF makes a contribution to AR ADP-ribosylation that is separable from the effect on nuclear transport. ZF mutations do not detectably reduce PARP7 catalytic activity and binding to AR, but they do result in the loss of PARP7 enhancement of AR-dependent transcription of the MYBPC1 gene. Our data reveals critical roles for AR conformation and the PARP7 ZF in AR ADP-ribosylation and AR-dependent transcription.

Eukaryotic initiation factor EIF-3.G augments mRNA translation efficiency to regulate neuronal activity.

The translation initiation complex eIF3 imparts specialized functions to regulate protein expression. However, understanding of eIF3 activities in neurons remains limited despite widespread dysregulation of eIF3 subunits in neurological disorders. Here, we report a selective role of the C. elegans RNA-binding subunit EIF-3.G in shaping the neuronal protein landscape. We identify a missense mutation in the conserved Zinc-Finger (ZF) of EIF-3.G that acts in a gain-of-function manner to dampen neuronal hyperexcitation. Using neuron-type-specific seCLIP, we systematically mapped EIF-3.G-mRNA interactions and identified EIF-3.G occupancy on GC-rich 5'UTRs of a select set of mRNAs enriched in activity-dependent functions. We demonstrate that the ZF mutation in EIF-3.G alters translation in a 5'UTR-dependent manner. Our study reveals an in vivo mechanism for eIF3 in governing neuronal protein levels to control neuronal activity states and offers insights into how eIF3 dysregulation contributes to neurological disorders.

Severe Congenital Neutropenia-Associated Mutations Reveal Stage-Specific Gfi1-Dependent Checkpoints in Myeloid Development

Growth factor independent-1 (GFI1) is a zinc-finger transcription factor that is critically required for both murine and human granulopoiesis. Mutations in GFI1 zinc fingers (e.g. N382S in zinc finger 5) have previously been found in humans with severe congenital neutropenia (SCN). We noted that clinical resequencing of neutropenic patients identified a number of novel GFI1 sequence changes of unknown significance. To clarify their impact, we used viral vectors to force expression of 13 GFI1 polymorphisms/mutations in primary-murine progenitors followed by colony-forming-unit assays for differentiation. While these assays infer new potential clinical significance to some genetic alterations, we focused on the sixth zinc finger of GFI1. While Gfi1 zinc-finger 6 was previously found to be dispensable for DNA binding and repression of a high affinity GFI1 DNA-binding-site reporter, the biological impact of expressing GFI1 zinc finger 6 mutants suggests a functional role for this domain in granulopoiesis. Therefore, we generated mice with patient-derived SCN-associated mutations (zinc finger 5; N382S, zinc finger 6; K403R, and R412X) in the murine Gfi1 locus. The resulting defects in murine neutrophil production are proportional to the severity of SCN in patients with the same GFI1 mutation (ranging from mild to profound neutropenia); with neonates displaying more profound defects. Neonatal and adult Gfi1N382S/- and Gfi1R412X/- mice are neutropenic, but Gfi1K403R/- mice have normal steady-state neutrophil levels. While Gfi1-/- HSC are profoundly abnormal, SCN-associated mutant HSC are phenotypically normal; dissociating Gfi1 control of hematopoietic stem cell biology from granulopoiesis. Similar to Gfi1-/-, the Gfi1N382S/- and Gfi1R412X/- mice accumulate cells within the “granulocyte monocyte progenitor” (GMP) gate as well as those that express Sca1 (known as emergency GMP; eGMP); however, unlike Gfi1-/- mice, the serum levels of known cytokine inducers of eGMP are normal in SCN-associated mutants. Moreover, isolated progenitors from all three mutants form significantly more monocytic colonies at the expense of granulocytic colonies in methylcellulose assays. Together, these data suggest that the mutations induce a cell-intrinsic defect.We recently utilized single-cell RNA-Seq (scRNA-Seq) to identify a gene regulatory network (GRN) centered on the Gfi1 and Irf8 transcription factors that underlies the granulocytic versus monocytic lineage decision of myeloid progenitors at steady state. To pinpoint the molecular deregulation underlying Gfi1-mutant progenitors, we performed scRNA-Seq. Notably, Gfi1R412X/- induced deregulation of gene expression which increases in amplitude and complexity as myeloid differentiation proceeds; coincident with induction of Gfi1 expression. ChIP-Seq analysis reveals that most of the deregulated genes are normally bound by Gfi1. Using murine fluorescent-protein knock-in reporter alleles for Gfi1 and Irf8, we demonstrate the stage-specific regulation of the granulocyte-monocyte lineage decision, and how SCN mutations deregulate the Gfi1-Irf8 network. Finally, we developed an alternative flow-cytometric strategy that highlights individual Gfi1-dependent checkpoints in myeloid differentiation (identified by comparative analysis of Gfi1-/- versus knock-in mutants). We find that the accumulated Gfi1N382S/- GMP are unspecified (similar to Gfi1-/-); however, Gfi1R412X/- GMP exhibit only a modest reduction in the granulocytic specified population and instead display a profound block at granulocytic commitment. The latter effects can be partially explained by the protein-destabilizing effect of mutations in zinc finger 6; suggesting that there are dose-dependent roles for Gfi1 in granulopoietic specification and commitment. In summary, Gfi1 knock-in mice provide a mouse model of human SCN, and both flow-cytometric and scRNA-Seq dissection of hematopoiesis reveal myelopoietic-stage-specific deregulation to both block granulopoiesis and deregulate monocytic subsets. DisclosuresNo relevant conflicts of interest to declare.

A Point Mutation in IKAROS ZF1 Causes a B Cell Deficiency in Mice.

IKZF1 (IKAROS) is essential for normal lymphopoiesis in both humans and mice. Previous Ikzf1 mouse models have demonstrated the dual role for IKZF1 in both B and T cell development and have indicated differential requirements of each zinc finger. Furthermore, mutations in IKZF1 are known to cause common variable immunodeficiency in patients characterized by a loss of B cells and reduced Ab production. Through N-ethyl-N-nitrosourea mutagenesis, we have discovered a novel Ikzf1 mutant mouse with a missense mutation (L132P) in zinc finger 1 (ZF1) located in the DNA binding domain. Unlike other previously reported murine Ikzf1 mutations, this L132P point mutation (Ikzf1L132P ) conserves overall protein expression and has a B cell-specific phenotype with no effect on T cell development, indicating that ZF1 is not required for T cells. Mice have reduced Ab responses to immunization and show a progressive loss of serum Igs compared with wild-type littermates. IKZF1L132P overexpressed in NIH3T3 or HEK293T cells failed to localize to pericentromeric heterochromatin and bind target DNA sequences. Coexpression of wild-type and mutant IKZF1, however, allows for localization to pericentromeric heterochromatin and binding to DNA indicating a haploinsufficient mechanism of action for IKZF1L132P Furthermore, Ikzf1+/L132P mice have late onset defective Ig production, similar to what is observed in common variable immunodeficiency patients. RNA sequencing revealed a total loss of Hsf1 expression in follicular B cells, suggesting a possible functional link for the humoral immune response defects observed in Ikzf1L132P/L132P mice.

Effects of &lt;i&gt;Zrsr2&lt;/i&gt; Mutations in Mice Oogenesis, Peripheral Blood Cells and Muscle Strength

ZRSR2 is a splicing factor essential for 3’ss recognition of U12-type introns and is frequently mutated in myeloid malignancies and in several tumors. To explore the biological role of ZRSR2 we generated three Zrsr2 mutant mice with deletions localized immediately after the AUG initiation and in two splicing sensitive elements present in the two zinc finger domains. The three Zrsr2 mutant mice exhibited muscle weakness, blood cell anomalies, and in the 2 lines with zinc finger mutations, oogenesis was blocked at the secondary follicle stage. RNA-seq of Zrsr2mu follicles showed aberrations in gene expression and biological pathways, and showed altered alternative splicing (AS) events involving enrichment of U2 and U12-type intron retention (IR), suggesting a functional crosstalk between the major and minor spliceosomes. These results suggest that ZRSR2 is essential for efficient U12 intron splicing and reveals that AS regulation of U12 introns constitute a potential underlying mechanism governing oogenesis.

The zinc finger domains in U2AF26 and U2AF35 have diverse functionalities including a role in controlling translation

ABSTRACT Recent work has associated point mutations in both zinc fingers (ZnF) of the spliceosome component U2AF35 with malignant transformation. However, surprisingly little is known about the functionality of the U2AF35 ZnF domains in general. Here we have analysed key functionalities of the ZnF domains of mammalian U2AF35 and its paralog U2AF26. Both ZnFs are required for splicing regulation, whereas only ZnF2 controls protein stability and contributes to the interaction with U2AF65. These features are confirmed in a naturally occurring splice variant of U2AF26 lacking ZnF2, that is strongly induced upon activation of primary mouse T cells and localized in the cytoplasm. Using Ribo-Seq in a model T cell line we provide evidence for a role of U2AF26 in activating cytoplasmic steps in gene expression, notably translation. Consistently, an MS2 tethering assay shows that cytoplasmic U2AF26/35 increase translation when localized to the 5ʹUTR of a model mRNA. This regulation is partially dependent on ZnF1 thus providing a connection between a core splicing factor, the ZnF domains and the regulation of translation. Altogether, our work reveals unexpected functions of U2AF26/35 and their ZnF domains, thereby contributing to a better understanding of their role and regulation in mammalian cells.

GATA2 Zinc Finger Mutations Influence Erythroid Differentiation and Chemotherapy Response

The transcription factor GATA2 plays an important role in cell lineage decisions during hematopoiesis. GATA2 Zinc-Finger (ZF) mutations are associated with distinct entities of myeloid malignancies. Alterations of the N-terminal ZF1 were identified in AML patients with biallelic CEBPA mutations, whereas the C-terminal ZF2 is typically affected by germline mutations predisposing to MDS and AML, or by somatic lesions in CML blast crisis. Nevertheless, the context-dependent mechanisms underlying GATA2 ZF mutations remain mostly unclear. Here, we set out to study the functional consequences of GATA2 ZF mutations. To test the effect on differentiation, we expressed GATA2 wild-type (WT) or GATA2 ZF mutants in human CD34+ hematopoietic stem and progenitor cells, stimulated with appropriate cytokines. Differentiation was evaluated by FACS-measurements of surface marker expression (erythroid markers: CD71, CD235a; granulocytic/monocytic markers: CD15, CD14). GATA2 WT caused a block of erythroid differentiation that is overcome by the ZF1 mutants (A318T and G320D), whereas the ZF2 mutant L359V may aggravate this block. For granulocytic/monocytic differentiation an overall block was observed for GATA2 WT and all the ZF mutants tested (Figure 1 A, B). Recently, we and others observed GATA2 mutation gain in AML relapse (Greif et al., 2018 Clin Cancer Res; Christopher et al., 2018, NEJM), pointing towards a potential role in therapy resistance. Therefore, we treated K562 cells stably expressing GATA2 WT or mutants with Daunorubicin (one of the two drugs commonly used in AML chemotherapy). Expression of GATA2 A318T in K562 cells correlated with higher sensitivity to Daunorubicin and lower expression levels of IDH2. (Figure 1 C, D). Interestingly, this particular ZF1 mutation was the only one that got lost at relapse in the study by Christopher and colleagues, consistent with therapy sensitivity, whereas most of the GATA2 mutations gained at relapse were localized in the ZF2 domain. In summary, GATA2 ZF mutations influence hematopoietic differentiation and chemotherapy response in a position-dependent manner. In the present study, we report distinct roles for individual GATA2 mutations depending on the affected ZF domain and altered amino acid positions. Understanding the oncogenic collaboration of GATA2 mutations with other driver genes in distinct patient subgroups is a challenge ahead. Figure.1 Disclosures Hiddemann: Roche: Consultancy, Honoraria, Research Funding; Gilead: Consultancy, Honoraria; Bayer: Research Funding; Vector Therapeutics: Consultancy, Honoraria; Celgene: Consultancy, Honoraria; Janssen: Consultancy, Honoraria, Research Funding.

Novel GLI3 Mutations in Chinese Patients with Non-syndromic Post-axial Polydactyly.

Polydactyly, characterized by supernumerary digits in the upper or lower extremities, is the most common congenital digital abnormalities. It derives from the defective patterning of anteroposterior axis of the developing limb, with various etiology and clinical heterogeneity. The patients with post-axial polydactyly type A (PAPA) have the typical symptom of a well-formed supernumerary digit outside the fifth digit. The aim of present study was to identify the causative mutations of two unrelated Han Chinese patients with non-syndromic PAPA. Two unrelated Han Chinese patients and 100 ethnicity-matched, unrelated normal controls were recruited for this study. BGISEQ-500 exome sequencing was performed in the two patients, followed by validation in the patients and 100 controls by using Sanger sequencing. Two mutations in the GLI family zinc finger 3 gene (GLI3), including a frameshift mutation c.3437_3453delTCGAGCAGCCCTGCCCC (p.L1146RfsX95) and a nonsense mutation c.3997C>T (p.Q1333X), were identified in two patients but were absent in the 100 healthy controls. The two GLI3 mutations, p.L1146RfsX95 and p.Q1333X, may account for non-syndromic PAPA in the two patients, respectively. The findings of this study may expand the mutational spectrum of GLI3-PAPA and provide novel insights into the genetic basis of polydactyly.

Breast cancer mutation in GATA3 zinc finger 1 induces conformational changes leading to the closer binding of ZnFn2 with a wrapping architecture

GATA3 is a transcription factor, known to regulate the transcriptional network and several pathways using two zinc fingers. Its mutation is associated with a higher risk of breast cancer. The molecular mechanisms of these mutations are poorly understood. It recognizes -GATA- sites on the DNA, using its two zinc fingers ZnFn1 and ZnFn2. Mutations in ZnFn2 have been studied in the past and well known but recently ZnFn1 mutations are also being reported very frequently in breast cancer patients and there is very less knowledge available regarding the binding modes and mechanism. Here, we have investigated the structural and functional impact of GATA3 mutation M294K on the DNA-binding property. The structure was obtained and mutation was induced before subjecting it to the molecular docking followed by MD simulation. Our findings indicate that the somatic mutation M294K, reported in the GATA3 gene destabilizes the unbound structure but, when it forms the DNA-complex, its overall structural stability is restored by the wrapping architecture of ZnFn2 around the DNA in the palindromic region, leading to the enhanced kinetic stability. The mutation not only affects the ZnFn1 region alone but also influences the whole complex by inducing the conformational changes in the linker region between the two zinc fingers, bringing the two zinc fingers to closer proximity representing the flexibility in binding sites. Our findings provide a better understanding of ZnFn1 mutations and the possibility of a different strategy to target these genes for their clinical relevance.Communicated by Ramaswamy H. Sarma

GATA2 Zinc Finger Mutations Affect DNA-Binding and Promote Granulopoietic Differentiation

GATA2 Zinc-Finger (ZF) mutations are associated with specific subgroups of myeloid malignancies. Alterations of the N-terminal ZF1 were identified in AML patients with biallelic CEBPA mutations, whereas the C-terminal ZF2 is typically affected by germline mutations, predisposing to MDS and AML, or by somatic lesions in CML blast crisis. Nevertheless, the context-dependent mechanism underlying GATA2 ZF2 mutations remains mostly unclear. Here, we set out to study the functional consequences of GATA2 ZF mutations in myeloid malignancies.In particular, we performed DNA-pulldown experiments, using FLAG-tagged full length GATA2 ZF WT and mutant proteins after expression in HEK293T cells and incubation of the cell lysates with biotinylated oligonucleotides encoding the binding-motif GATC. These experiments showed disruption of DNA binding for all GATA2 ZF mutants tested in vitro - regardless of the mutant positions within the ZF domains (Figure 1 A).Moreover, we studied the impact of GATA2 ZF mutations on the protein-interaction with Friend of GATA Protein 1 (FOG1; HGNC symbol ZFPM1). The influence of FOG1 on GATA2-dependent transcriptional activation was evaluated using a GATA-specific luciferase reporter. All GATA2 mutants tested were able to activate this reporter, although to variable extent. While co-expression of FOG1 overall counteracted GATA2-dependent transcriptional activation, this effect was significantly reduced for the GATA2 mutants L321F (located in ZF1) and T354M (located in ZF2). This suggested that both ZF domains are involved in the FOG1-interaction.To gain insights into the influence of GATA2 ZF1 mutation on hematopoiesis we performed colony forming cell (CFC)-assays. Lin- primary murine bone marrow cells expressing GATA2 WT or mutants were plated in methylcellulose supplemented with cytokines. Consistent with previous reports, GATA2 WT expression led to a reduced colony number, while this effect was decreased for both mutants A318T (ZF1) and L359V (ZF2). In particular, a higher number of CFU-G colonies were observed for the GATA2 mutant-expressing cells indicating a lineage shift towards granulopoiesis (Figure 1 B).In summary, we have shown that GATA2 mutations influence DNA-binding, protein-interactions and myeloid differentiation. Our findings further suggest that GATA2 ZF1 mutations may contribute to myeloid leukemogenesis through increased proliferation of granulocytic progenitors. Understanding the oncogenic collaboration of GATA2 mutations with other driver genes in distinct patient subgroups is a challenge ahead. [Display omitted] DisclosuresHiddemann:Celgene: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; F. Hoffman-La Roche: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Janssen: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Bayer: Consultancy, Research Funding.

Neutropenia-Associated Mutations Differentially Impact Developmental Cell-States

Efforts to understand the genomic impact of human-disease-relevant genetic lesions and how they disrupt the normal sequence of cell-state transitions is hampered by a lack of defined hierarchical cellular states and corresponding networks of regulatory genes (transcription factors). Severe congenital neutropenia (SCN) patients display inherited and de novo mutations in Growth factor independent-1 (GFI1), which encodes a zinc-finger transcription factor. We identified known (e.g. N382S in zinc finger 5) and novel GFI1 sequence changes in SCN patients, then used lentiviral mediated expression to functionally evaluate them. GFI1-N382S, GFI1-K403R and GFI1-R412X mutations (in zinc finger 6) significantly elevated the expression of the Gfi1 target gene, Irf8. We generated mice with these patient-derived SCN-associated mutations in the murine Gfi1 locus. Neonatal and adult Gfi1N382S/- and Gfi1R412X/- mice are neutropenic, but Gfi1K403R/- mice have normal steady-state neutrophil levels. The resulting steady-state dysgranulopoiesis in adult mice was further pronounced in neonates. We noted that Gfi1R412X/- mice accumulate less Gfi1 protein than Gfi1+/+, while Gfi1R412X/R412X homozygous alleles genetically rescued both the hypomorphic protein defect and substantially restored neutrophil numbers (though not to normal). In contrast, functional challenge with neutrophil-dependent pathogens in vivo revealed a broad susceptibility for all Gfi1-mutant mice.To determine the underlying mechanism of neutropenia and immune defects, we first used novel flow cytometry analyses and Fluidigm C1 single cell RNA-Seq to establish the successive genomic states encompassing normal granulocyte specification and commitment. Independent CITE-Seq/10x sequencing analysis provided direct correlation between flow cytometry populations and genomic information, while also establishing the trajectory through genomic states traversed during terminal granulopoiesis. Next, using a novel bioinformatics algorithm (cellHarmony) we assigned Gfi1-mutant cells to their respective wild-type cell states and then determined differential gene expression. We find few genes deregulated across granulopoiesis, and that the bulk of transcriptional impact on Gfi1 target genes is specific to successive granulopoietic cell states. These insights facilitated Gfi1R412X/- Irf8+/- genetic rescue of granulocytic specification, but not post-commitment defects. We noted that a portion of Gfi1R412X/- gene deregulation unrepaired by genetic rescue was enriched for chromosome organization, proteolysis, and innate immune effectors. Electron microscopy revealed uncondensed chromatin in mature Gfi1R412X/- neutrophils while SWATH proteomics identified a loss of neutrophil granule proteins and members of the NADPH oxidase complex (potentially linking SCN with chronic granulomatous disease genes). To this end, we functionally validated impaired NADPH oxidase complex function in neutrophils from Gfi1-mutant mice. We noted Gfi1 mutant mice have consistently elevated levels of granulocyte colony stimulating factor (but not other cytokines), and so we extended our analysis of oxidative burst to GCSF-rescued human SCN patients to find profound defects; underscoring the inability of genetic or cytokine rescued specification to resolve post-commitment defects. We illustrate a work flow that can be broadly applied to molecularly dissect translationally relevant mouse models of disease, and underscores the necessity of evaluating mutations within the context of relevant cell states. DisclosuresDwivedi:Abbvie: Employment. Myers:Bellicum Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees; Novartis: Membership on an entity's Board of Directors or advisory committees. Nazor:BioLegend: Employment.