NFI Genes Research Articles

HGG-01. DURABLE RESPONSE TO IMMUNOTHERAPY IN AN ADOLESCENT PATEINT WITH CONSTITUTIONAL MISMATCH REPAIR DEFICIENCY CMMRD AND SYNCHRONOUS THREE PRIMARY MALIGNANCIES

Abstract BACKGROUND Familial Cancer Syndrome FCS is of particular concern in Saudi Arabia due to the high rates of consanguinity. Constitutional mismatch repair deficiency Syndrome (CMMRD) is an autosomal recessive FCS with a wide spectrum of malignancies Caused by Biallelic germline mutations in one of 4 mismatch repair genes (MLH1, PMS2, MSH2, and MSH6). METHODS we report 13 yr. Saudi boy, previously healthy with multiple café-au-lait (CAL) macules, and Consanguineous parents with a positive family history of Malignancy. presented with progressive headache, vomiting, abdominal pain, weight loss, and microcytic anemia. MRI Brain showed a Large left hemispheric necrotic mass, MRI abdomen: Circumferential gastric body wall thickening Distal transverse colon intraluminal polypoid mass, and liver hypodense lesion. He had a Resection of the brain tumor and Upper GI endoscopy + colonoscopy with polypectomy: which showed an Ugly gastric ulcer, and multiple colonic polyps. RESULTS Brain tumor molecular pathology: Gliosarcoma WHO grade 4 with negative IHC expression in PMS2 protein, hypermutant tumor with somatic PMS2 ATRX, TP53, NFI, and PIK3R1 genes mutation. stomach biopsy confirmed invasive gastric adenocarcinoma and Colonic polyp biopsy: Tubulovillous adenoma. Genetic testing Detect pathogenic Biallelic germline PMS2 mutation. He received adjuvant focal Brain radiation therapy 59.4Gy/33 F concurrent with immunotherapy (PD-1) inhibitor Nivolumab. Follow-up Abdominal imaging showed excellent response to immunotherapy with no significant residual lesion. MRI of the brain showed surgical bed multi-cystic lesions with thick nodular enhancement suggestive either of radiation necrosis vs. progressions/pseudoprogression. repeated MRI confirmed further progression of the enhancing surgical cavity lesion which was unresectable. CTLA-4 Inhibitor ipilimumab was added to Nivolumab to optimize therapy. UpToDate our patient is clinically stable on Maintenance Nivolumab with no side effects. CONCLUSION Our case confirms immunotherapy’s efficacy for treating CMMRD-related cancers, which can improve survival while reducing toxicity from less effective conventional therapies.

Genomic Landscape of Primary and Recurrent Anal Squamous Cell Carcinomas in Relation to HPV Integration, Copy-Number Variation, and DNA Damage Response Genes.

The incidence of anal squamous cell carcinoma (ASCC) has been increasing, particularly in populations with HIV. Human papillomavirus (HPV) is the causal factor in 85% to 90% of ASCCs, but few studies evaluated HPV genotypes and integrations in relation to genomic alterations in ASCC. Using whole-exome sequence data for primary (n = 56) and recurrent (n = 31) ASCC from 72 patients, we detected HPV DNA in 87.5% of ASCC, of which HPV-16, HPV-18, and HPV-6 were detected in 56%, 22%, and 33% of HIV-positive (n = 9) compared with 83%, 3.2%, and 1.6% of HIV-negative cases (n = 63), respectively. Recurrent copy-number variations (CNV) involving genes with documented roles in cancer included amplification of PI3KCA and deletion of APC in primary and recurrent tumors; amplifications of CCND1, MYC, and NOTCH1 and deletions of BRCA2 and RB1 in primary tumors; and deletions of ATR, FANCD2, and FHIT in recurrent tumors. DNA damage response genes were enriched among recurrently deleted genes in recurrent ASCCs (P = 0.001). HPV integrations were detected in 29 of 76 (38%) ASCCs and were more frequent in stage III-IV versus stage I-II tumors. HPV integrations were detected near MYC and CCND1 amplifications and recurrent targets included NFI and MUC genes. These results suggest HPV genotypes in ASCC differ by HIV status, HPV integration is associated with ASCC progression, and DNA damage response genes are commonly disrupted in recurrent ASCCs. IMPLICATIONS: These data provide the largest whole-exome sequencing study of the ASCC genomic landscape to date and identify HPV genotypes, integrations, and recurrent CNVs in primary or recurrent ASCCs.

NFIA and NFIB function as tumour suppressors in high-grade glioma in mice.

Nuclear factor one (NFI) transcription factors are implicated in both brain development and cancer in mice and humans and play an essential role in glial differentiation. NFI expression is reduced in human astrocytoma samples, particularly those of higher grade, whereas over-expression of NFI protein can induce the differentiation of glioblastoma cells within human tumour xenografts and in glioblastoma cell lines in vitro. These data indicate that NFI proteins may act as tumour suppressors in glioma. To test this hypothesis, we generated complex mouse genetic crosses involving six alleles to target gene deletion of known tumour suppressor genes that induce endogenous high-grade glioma in mice, and overlaid this with loss of function Nfi mutant alleles, Nfia and Nfib, a reporter transgene and an inducible Cre allele. Deletion of Nfi resulted in reduced survival time of the mice, increased tumour load and a more aggressive tumour phenotype than observed in glioma mice with normal expression of NFI. Together, these data indicate that NFI genes represent a credible target for both diagnostic analyses and therapeutic strategies to combat high-grade glioma.

ETMR-13. NFI GENES IN ETMR TUMORIGENESIS AND NEURODEVELOPMENT

Embryonal tumors with multilayered rosettes (ETMRs) are aggressive pediatric embryonal brain tumors with a universally poor prognosis. These tumors are commonly characterized by amplification of C19MC, but other miRNA-related aberrations, such as DICER mutations or MIR17HG amplifications, are also observed. Nevertheless, it remains unknown how these aberrations are driving the tumorigenesis. We applied miRNA target prediction to investigate the downstream targets shared by these aberrations affecting normal brain development and tumorigenesis. The nuclear factor one (NFI) family of transcription factors were found to be top candidates shared by both miRNA clusters. These genes are expressed at very low levels in ETMRs, in contrast to other brain tumors. During normal brain development these genes are expressed in radial glial progenitors and are required for the transition of proliferation to differentiation. Since radial glial progenitors are the potential cell-of-origin of ETMRs, we hypothesize that downregulation of NFI is required for the proliferative, undifferentiated state of ETMRs. Indeed, mouse models with deletion of an Nfi family member display sustained proliferation and delayed differentiation of radial glial progenitors during development. This leads into brain overgrowth, which has also been observed in humans with intellectual disabilities caused by NFI haploinsufficiency. When multiple Nfi family members are simultaneously targeted in mice, the progenitors are retained and both neurogenesis and gliogenesis are inhibited, resulting in a neuropathology similar to that of human ETMR tumors. Hence, downregulation of NFI genes resulting from miRNA aberrations could contribute to the developmental state and possibly tumorigenesis of ETMRs.

Common Regulatory Targets of NFIA, NFIX and NFIB during Postnatal Cerebellar Development.

Transcriptional regulation plays a central role in controlling neural stem and progenitor cell proliferation and differentiation during neurogenesis. For instance, transcription factors from the nuclear factor I (NFI) family have been shown to co-ordinate neural stem and progenitor cell differentiation within multiple regions of the embryonic nervous system, including the neocortex, hippocampus, spinal cord and cerebellum. Knockout of individual Nfi genes culminates in similar phenotypes, suggestive of common target genes for these transcription factors. However, whether or not the NFI family regulates common suites of genes remains poorly defined. Here, we use granule neuron precursors (GNPs) of the postnatal murine cerebellum as a model system to analyse regulatory targets of three members of the NFI family: NFIA, NFIB and NFIX. By integrating transcriptomic profiling (RNA-seq) of Nfia- and Nfix-deficient GNPs with epigenomic profiling (ChIP-seq against NFIA, NFIB and NFIX, and DNase I hypersensitivity assays), we reveal that these transcription factors share a large set of potential transcriptional targets, suggestive of complementary roles for these NFI family members in promoting neural development.

Variants in nuclear factor I genes influence growth and development.

The nuclear factor one (NFI) site-specific DNA-binding proteins represent a family of transcription factors that are important for the development of multiple organ systems, including the brain. During brain development in mice, the expression patterns of Nfia, Nfib, and Nfix overlap, and knockout mice for each of these exhibit overlapping brain defects, including megalencephaly, dysgenesis of the corpus callosum, and enlarged ventricles, which implies a common but not redundant function in brain development. In line with these models, human phenotypes caused by haploinsufficiency of NFIA, NFIB, and NFIX display significant overlap, sharing neurodevelopmental deficits, macrocephaly, brain anomalies, and variable somatic overgrowth. Other anomalies may be present depending on the NFI gene involved. The possibility of variants in NFI genes should therefore be considered in individuals with intellectual disability and brain overgrowth, with individual NFI-related conditions being differentiated from one another by additional signs and symptoms. The exception is provided by specific NFIX variants that act in a dominant negative manner, as these cause a recognizable entity with more severe cognitive impairment and marked bone dysplasia, Marshall-Smith syndrome. NFIX duplications are associated with a phenotype opposite to that of haploinsufficiency, characterized by short stature, small head circumference, and delayed bone age. The spectrum of NFI-related disorders will likely be further expanded, as larger cohorts are assessed.

DNA polymerase I proofreading exonuclease activity is required for endonuclease V repair pathway both in vitro and in vivo

Deamination of adenine can occur spontaneously under physiological conditions to generate the highly mutagenic lesion, deoxyinosine (hypoxanthine deoxyribonucleotide, dI). In DNA, dI preferably pairs with cytosine rather than thymine and results in A:T to G:C transition mutations after DNA replication. The deamination of adenine is enhanced by ROS from exposure of DNA to ionizing radiation, UV light, nitrous acid, or heat. In Escherichia coli, dI repair is initiated by endonuclease V (endo V; nfi gene product) nicking but a complete repair mechanism has yet to be elucidated. Using in vitro minimum component reconstitution assays, we previously showed that endo V, DNA polymerase I (pol I), and E. coli DNA ligase were sufficient to repair this dI lesions efficiently and that the 3′-5′ exonuclease of pol I is essential. Here we employed a phagemid-based T-I substrate mimicking adenine deamination product to demonstrate pol I proofreading exonuclease is required by the endo V repair pathway both in vitro and in vivo. In vivo we found that the repair level of an nfi mutant (11%) was almost 8-fold lower than the wild type (87%). while the polA-D424A strain, a pol I mutant defective in 3′-5′ exonuclease, showed a high repair level similar to wild type (both more than 80%). Using additional C–C mismatch as strand discrimination marker we found that the high level of dI removal in polA-D424A was due to strand loss (more than 60%) associated with incomplete repair. Thus, pol I proofreading exonuclease is the major function responsible for dI lesion removal after endoV nicking both in vitro and in vivo. Finally, using MALDI-TOF to analyze single-nucleotide extension product we show that the pol I proofreading exonuclease excises only 2-nt 5′ upstream of endo V incision site further honing the role of pol I in the endoV dI dependent repair pathway.

Nfib hemizygous mice are protected from hyperoxic lung injury and death.

Nuclear Factor I (Nfi) genes encode transcription factors essential for the development of organ systems including the lung. Nfib null mice die at birth with immature lungs. Nfib hemizygous mice have reduced lung maturation with decreased survival. We therefore hypothesized that these mice would be more sensitive to lung injury and would have lower survival to hyperoxia. Adult Nfib hemizygous mice and their wild‐type (Wt) littermates were exposed to 100% O2 for 89, 80, 72 and 66 h for survival studies with lung outcome measurements at 66 h. Nfib hemizygous and Wt controls were also studied in RA at 66 h. Cell counts and cytokines were measured in bronchoalveolar lavage (BAL); lung sections examined by histopathology; lung angiogenic and oxidative stress gene expression assessed by real‐time PCR. Unexpectedly, Nfib hemizygous mice (0/14–0%) had significantly lower mortality compared to Wt mice (10/22–45%) at 80 h of hyperoxia (P < 0.003). LD 50 was 80 h in the Wt group versus 89 h in the hemizygous group. There were no differences in BAL cell counts between the groups. Among the cytokines studied, MIP‐2 was significantly lower in hemizygous mice exposed to hyperoxia. New vessel formation, edema, congestion, and alveolar hemorrhage were noted on histopathology at 72 and 80 h in wild‐type mice. Nfib hemizygous lungs had significant downregulation of genes involved in redox signaling and inflammatory pathways. Adult Nfib hemizygous mice are relatively resistant to hyperoxia compared to wild‐type littermates. Mechanisms contributing to this resistance are not clear; however, transcription factors such as Nfib may regulate cell survival and play a role in modulating postnatal lung development.

Abstract 3536: The role of Nuclear factor I transcription factors in glioma

Abstract Gliomas are the most common brain tumors in adults. Among the malignant gliomas, grade IV glioma, or glioblastoma (GBM), is the most aggressive form. Despite harsh treatments such as surgery, radiotherapy and chemotherapy, the median survival of GBM patients remains at just 12-15 months. GBM tumors could originate from cells of the glial lineage that have escaped the normal glial differentiation mechanisms. Therefore, by applying knowledge about normal glial development, we might understand how pathways that normally drive differentiation are affected in these tumors. If reactivating these pathways induces the differentiation of proliferative tumor cells, we could use this as a novel target for therapy. Our research is focused on the Nuclear factor I (NFI) transcription factors, one of the key factors to induce glial differentiation during normal development and is implicated in glioma. In insertional mutagenesis glioma mouse models, Nfi genes are consistently disrupted, suggesting a role for NFI in glioma initiation. Furthermore, loss of NFIB is common in human astrocytoma, while loss of NFIA is associated with oligodendroglioma. To determine whether loss of Nfi alters glioma initiation, progression or tumor histology, we have crossed inducible glioma mice with conditional Nfi deletion mice to follow tumor initiation and progression using fluorescence imaging and immunohistochemistry. Based on analyses of glioma mRNA expression data sets, NFIB expression correlates inversely with tumor grade and survival. Using immunofluorescence, we determined that both NFIA and NFIB are mainly expressed within the non-proliferative cells in GBM tissue, suggesting that NFI may play a direct role in tumor cell differentiation. To establish whether NFI expression can indeed inhibit tumor proliferation and induce differentiation, U251 GBM cells were transfected with NFI. While overexpression of NFIA and NFIB reduced cell proliferation, an NFI dominant-negative protein enhanced proliferation. We are currently validating these finding in vivo by electroporating NFI expression constructs into patient-derived GBM xenografts in mice. Our preliminary data show NFIB electroporated cells are indeed differentiated and non-proliferative. Our data suggests that NFI-mediated glial differentiation can be induced in glioma to inhibit tumor growth and may prevent recurrence. Hence, activation of NFI could be a potential target for a glial differentiation-based approach to treat glioma. Citation Format: Kok Siong Chen, Linda J. Richards, Jens Bunt. The role of Nuclear factor I transcription factors in glioma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3536. doi:10.1158/1538-7445.AM2017-3536

Cervical Dumbbell: Ganglioneuromas Causing Myelopathy in a Case of Neurofibromatosis Type-I: Case Report and Literature Review

Background Data: Ganglioneuromas are rare benign tumors arising from the sympathetic nervous system. They occasionally grow in a dumbbell fashion extending into the spinal canal extradurally. However, ganglioneuromas of the cervical spine with intradural extension or multiple locations or in association with neurofibromatosis type-I are rare.Purpose: Our aim is to describe a man with cervical compressive myelopathy at C2 level caused by dumbbell shaped intradural extramedullary ganglioneuroma and multilevel spinal lesions (ganglioneuromas) associated with neurofibromatosis type-I with genetic predisposition.Study Design: Case report and review of the literature.Patient and Method: Twenty eight year-old man with NF-I presented with acute history of progressive quadriparesis. Magnetic resonance imaging showed bilateral dumbbell masses at the C2 level compressing the spinal cord. A systemic imaging survey of the patient showed numerous asymptomatic foraminal and extraforaminal tumors at all neuroforamina of the spine.Results: Surgical decompression of the spinal cord by resections of bilateral tumors at the C2 level alleviated patient symptoms. Histopathological diagnosis was ganglioneuroma. The patient was heterozygous in the NF-I gene for a sequence variant defined as c.970T>C, and predicted to result in the amino acid substitution p.Cys324Arg.Conclusion: The case reported here is very unusual because of its type, its location as dumbbell spinal ganglioneuroma at level of 2nd cervical spine, being associated with other ganglioneuromas at other spinal levels and being associated with NF-I molecular mutation. (2017ESJ143)

PNR-12GENOME-WIDE PROFILING OF EMBRYONAL TUMORS WITH MULTILAYERED ROSETTES (ETMR)

PNR-12. GENOME-WIDE PROFILING OF EMBRYONAL TUMORS WITH MULTILAYERED ROSETTES (ETMR) Sander Lambo1, Jens Bunt2, Christine Schmidt1, Stefan M. Pfister1, Linda J. Richards2, Lukas Chavez1, Andrey Korshunov3, and Marcel Kool1; Division of Pediatric Neurooncology, DKFZ, Heidelberg, Germany; Cortical Development and Axon Guidance, Queensland Brain Institute, Brisbane, Australia; Clinical Cooperation Unit Neuropathology, DKFZ, Heidelberg, Germany ETMRs are rare pediatric brain tumors with a dismal prognosis and largely unknown mechanisms driving the tumors. Characteristic to ETMR tumors are high expression of LIN28A/B and fusion of a Chr19 microRNA cluster (C19MC) to TTYH1 coupled with amplification of the cluster. Our goal is to understand the mechanisms that play a role in tumorigenesis and possibly identify new treatment strategies. We generated genomic data from 23 ETMRs, including three that lack the C19MC amplification, to understand their genome, transcriptome and epigenome. Whole genome sequencing identified no other recurrent genetic events than the fusion and C19MC amplification, frequentlyaccompanied byother complex rearrangements. Interestingly, gene and miRNA expression profiles of ETMRs w/o C19MC amplification wereverysimilar.Analysisof whole genome bisulfite sequencing data supported this similarity, demonstrating large overlapping regions of strong hypomethylation between tumors. Additionally, we generated genome-wide chromatin maps for the major (ENCODE) histone marks. The resulting enhancer landscape, combined with gene and miRNA expression, revealed high similarity with neural tube cells in the forebrain lineage. miRNA target prediction algorithms predicted that NFI transcription factors, highly downregulated in ETMRs, are targeted by multiple miRNAs in C19MC, suggesting a role for NFI genes in ETMR tumorigenesis. Inhibition of NFI protein function in the mouse brain indeed resulted in formation of ectopic rosette-like structures in the forebrain and an increased size of the ventricular zone, but the mice died before tumors could eventually develop. Together our data provides many novel insights into (epi)genetic aberrations and transcriptional networks driving ETMRs. Neuro-Oncology 18:iii7–iii15, 2016. doi:10.1093/neuonc/now067.10 #The Author(s) 2016. Published by Oxford University Press on behalf of the Society for Neuro-Oncology. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

Adherence to abiotic surface induces SOS response in Escherichia coli K-12 strains under aerobic and anaerobic conditions

During the colonization of surfaces, Escherichia coli bacteria often encounter DNA-damaging agents and these agents can induce several defence mechanisms. Base excision repair (BER) is dedicated to the repair of oxidative DNA damage caused by reactive oxygen species (ROS) generated by chemical and physical agents or by metabolism. In this work, we have evaluated whether the interaction with an abiotic surface by mutants derived from E. coli K-12 deficient in some enzymes that are part of BER causes DNA damage and associated filamentation. Moreover, we studied the role of endonuclease V (nfi gene; 1506 mutant strain) in biofilm formation. Endonuclease V is an enzyme that is involved in DNA repair of nitrosative lesions. We verified that endonuclease V is involved in biofilm formation. Our results showed more filamentation in the xthA mutant (BW9091) and triple xthA nfo nth mutant (BW535) than in the wild-type strain (AB1157). By contrast, the mutant nfi did not present filamentation in biofilm, although its wild-type strain (1466) showed rare filaments in biofilm. The filamentation of bacterial cells attaching to a surface was a consequence of SOS induction measured by the SOS chromotest. However, biofilm formation depended on the ability of the bacteria to induce the SOS response since the mutant lexA Ind(-) did not induce the SOS response and did not form any biofilm. Oxygen tension was an important factor for the interaction of the BER mutants, since these mutants exhibited decreased quantitative adherence under anaerobic conditions. However, our results showed that the presence or absence of oxygen did not affect the viability of BW9091 and BW535 strains. The nfi mutant and its wild-type did not exhibit decreased biofilm formation under anaerobic conditions. Scanning electron microscopy was also performed on the E. coli K-12 strains that had adhered to the glass, and we observed the presence of a structure similar to an extracellular matrix that depended on the oxygen tension. In conclusion, it was proven that bacterial interaction with abiotic surfaces can lead to SOS induction and associated filamentation. Moreover, we verified that endonuclease V is involved in biofilm formation.

Nfi Genes Are Novel Regulators Of Murine Hematopoietic Stem- and Progenitor Cell Survival

Hematopoietic stem cells (HSCs) are responsible for life-long maintenance of hematopoiesis.HSC transplantation represents one of the most heavily exploited cell based therapies, routinely used to treat a myriad of life threating disorders, such as leukemia and bone marrow failure. Identifying the molecular pathways that regulate HSC engraftment is crucial to further improving outcomes in patients that rely on HSC transplantation as a curative therapy.By examining the global gene expression profiles of highly purified HSC (Lineage-Sca-1+c-Kit+CD150+CD48-), we recently identified the following members of the Nfi gene family of transcription factors as highly expressed by HSC (McKinney-Freeman et al., Cell Stem Cell, 2012): Nfix, Nfia, and Nfic. These data suggest that Nfi genes may play a novel role in regulating HSC function. To test this hypothesis, HSCs were enriched from adult bone marrow (Lineage-, c-kit+, Sca-1+ (LSK) cells) and then transduced, individually, with lentiviruses carrying shRNAs targeting each Nfi gene. Twenty-four hours post-transduction, cells were injected into lethally irradiated mice along with untransduced bone marrow LSK competitor cells congenic at the CD45 allele. The peripheral blood of recipient mice was then analyzed periodically over 16 weeks for engraftment of the Nfi-depleted cells. Although shRNA mediated knockdown of Nfi gene expression had no effect on the in vitro cell growth or viability of LSK cells, Nfi-depleted HSCs displayed a significant loss of short- and long-term in vivo hematopoietic repopulating activity. This was true for Nfia-, Nfic-, and Nfix-deficient HSC. While Nfia and Nfic are only expressed by bone marrow HSC, Nfix is highly expressed by both bone marrow and fetal liver HSC. When Nfix was depleted by shRNAs from LSK cells purified from E14.5 fetal liver, a similar loss in competitive repopulating potential was seen. Lineage analysis of peripheral blood of recipients showed no significant differences in the distribution of the major blood lineages derived from LSK cells transduced with Nfi-specific shRNAs compared to controls. When the bone marrow of recipients transplanted with Nfix- depleted cells was examined 4 and 16 weeks post-transplant, a general loss of all hematopoietic stem- and progenitor compartments examined was seen relative to control. Thus, the observed decrease in repopulating activity occurs at the level of HSCs and multipotent progenitors. To confirm an essential role for an Nfi gene family member in the regulation of HSC engraftment post-transplant, LSK cells were purified from Nfix fl/fl mice, transduced with lentiviral Cre recombinase and subsequently introduced into lethally irradiated recipients alongside congenic competitor cells. Like LSK transduced with Nfix-specific shRNAs, Nfix-/- LSK cells failed to repopulate the peripheral blood of recipient mice as efficiently as control and similar trends were detected in all stem- and progenitor cell populations examined.Time-course experiments immediately following transplantation revealed that Nfix-depleted LSK cells establish themselves in the marrow of recipient mice as efficiently as control at 5 days post-transplant, but thereafter exhausted rapidly. Examination 10 days post-transplant revealed a 5-fold increase in apoptosis specifically in the LSK compartment, but not in its differentiated progeny, in recipients transplanted with Nfix-depleted LSK cells compared to control. The increase in apoptosis was not associated with any apparent change in the cell cycle status of the LSK cells. These data suggest that Nfi genes are necessary for the survival of HSC post-transplantation.In an effort to identify the molecular pathways regulated by Nfi genes in HSC, we acquired the global gene expression profiles of Nfix-depleted HSC. In agreement with our observation that Nfix-deficient HSC displays elevated levels of apoptosis following transplantation in vivo, we observed a significant decrease in multiple genes known to be important for HSC survival, such as Erg, Mecom and Mpl, in Nfix-depleted HSC.In summary, we have for the first time established a role for the Nfi gene family in HSC biology, as evident by a decrease in bone marrow repopulating activity in Nfi-depleted HSCs. By dissecting the precise role of Nfi genes in HSC biology, we will glean insights that could improve our understanding of graft failure in clinical bone marrow transplantations. Disclosures:No relevant conflicts of interest to declare.

Nfix is a novel regulator of murine hematopoietic stem and progenitor cell survival

AbstractHematopoietic stem cells are both necessary and sufficient to sustain the complete blood system of vertebrates. Here we show that Nfix, a member of the nuclear factor I (Nfi) family of transcription factors, is highly expressed by hematopoietic stem and progenitor cells (HSPCs) of murine adult bone marrow. Although short hairpin RNA–mediated knockdown of Nfix expression in Lineage−Sca-1+c-Kit+ HSPCs had no effect on in vitro cell growth or viability, Nfix-depleted HSPCs displayed a significant loss of colony-forming potential, as well as short- and long-term in vivo hematopoietic repopulating activity. Analysis of recipient mice at 4 to 20 days posttransplant revealed that Nfix-depleted HSPCs are established in the bone marrow, but fail to persist due to increased apoptotic cell death. Gene expression profiling of Nfix-depleted HSPCs reveals that loss of Nfix expression in HSPCs is concomitant with a decrease in the expression of multiple genes known to be important for HSPCs survival, such as Erg, Mecom, and Mpl. These data reveal that Nfix is a novel regulator of HSPCs survival posttransplantation and establish a role for Nfi genes in the regulation of this cellular compartment.

The Human Homolog of Escherichia coli Endonuclease V Is a Nucleolar Protein with Affinity for Branched DNA Structures

Loss of amino groups from adenines in DNA results in the formation of hypoxanthine (Hx) bases with miscoding properties. The primary enzyme in Escherichia coli for DNA repair initiation at deaminated adenine is endonuclease V (endoV), encoded by the nfi gene, which cleaves the second phosphodiester bond 3′ of an Hx lesion. Endonuclease V orthologs are widespread in nature and belong to a family of highly conserved proteins. Whereas prokaryotic endoV enzymes are well characterized, the function of the eukaryotic homologs remains obscure. Here we describe the human endoV ortholog and show with bioinformatics and experimental analysis that a large number of transcript variants exist for the human endonuclease V gene (ENDOV), many of which are unlikely to be translated into functional protein. Full-length ENDOV is encoded by 8 evolutionary conserved exons covering the core region of the enzyme, in addition to one or more 3′-exons encoding an unstructured and poorly conserved C-terminus. In contrast to the E. coli enzyme, we find recombinant ENDOV neither to incise nor bind Hx-containing DNA. While both enzymes have strong affinity for several branched DNA substrates, cleavage is observed only with E. coli endoV. We find that ENDOV is localized in the cytoplasm and nucleoli of human cells. As nucleoli harbor the rRNA genes, this may suggest a role for the protein in rRNA gene transactions such as DNA replication or RNA transcription.

Expression of nuclear factor one A and -B in the olfactory bulb

The nuclear factor one (NFI) family of transcription factors consists of four members in vertebrates, NFIA, NFIB, NFIC, and NFIX, which share a highly conserved N-terminal DNA-binding domain. NFI genes are widely expressed in the developing mouse brain, and mouse mutants lacking NFIA, NFIB, or NFIX exhibit developmental deficits in several areas, including the cortex, hippocampus, pons, and cerebellum. Here we analyzed the expression of NFIA and NFIB in the developing and adult olfactory bulb (OB), rostral migratory stream (RMS), and subventricular zone (SVZ). We found that NFIA and NFIB are expressed within these regions during embryonic and postnatal development and in the adult. Immunohistochemical analysis using cell-type-specific markers revealed that migrating neuroblasts in the adult brain express NFI transcription factors, as do astrocytes within the RMS and progenitor cells within the SVZ. Moreover, astrocytes within the OB express NFIA, whereas mitral cells within the OB express NFIB. Taken together these data show that NFIA and NFIB are expressed in both the developing and the adult OB and in the RMS and SVZ, indicative of a regulatory role for these transcription factors in the development of this facet of the olfactory system.

Human endonuclease V as a repair enzyme for DNA deamination

The human endonuclease V gene is located in chromosome 17q25.3 and encodes a 282 amino acid protein that shares about 30% sequence identity with bacterial endonuclease V. This study reports biochemical properties of human endonuclease V with respect to repair of deaminated base lesions. Using soluble proteins fused to thioredoxin at the N-terminus, we determined repair activities of human endonuclease V on deoxyinosine (I)-, deoxyxanthosine (X)-, deoxyoxanosine (O)- and deoxyuridine (U)-containing DNA. Human endonuclease V is most active with deoxyinosine-containing DNA but with minor activity on deoxyxanthosine-containing DNA. Endonuclease activities on deoxyuridine and deoxyoxanosine were not detected. The endonuclease activity on deoxyinosine-containing DNA follows the order of single-stranded I>G/I>T/I>A/I>C/I. The preference of the catalytic activity correlates with the binding affinity of these deoxyinosine-containing DNAs. Mg2+ and to a much less extent, Mn2+, Ni2+, Co2+ can support the endonuclease activity. Introduction of human endonuclease V into Escherichia coli cells deficient in nfi, mug and ung genes caused three-fold reduction in mutation frequency. This is the first report of deaminated base repair activity for human endonuclease V. The relationship between the endonuclease activity and deaminated deoxyadenosine (deoxyinosine) repair is discussed.

Endonuclease V-mediated deoxyinosine excision repair in vitro

Deoxyinosine (dI) in DNA can arise from hydrolytic or nitrosative deamination of deoxyadenosine. It is excised in a repair pathway that is initiated by endonuclease V, the nfi gene product, in Escherichia coli. Repair was studied in vitro using M13mp18 derived heteroduplexes containing a site-specific deoxyinosine. Unpaired dI/G mismatch resides within the recognition site for XhoI restriction endonucleases, permitting evaluation of repair occurring on deoxyinosine-containing DNA strand. Our results show that dI lesions were efficiently repaired in nfi+E. coli extracts but the repair level was much reduced in nfi mutant extracts. We subjected the deoxyinosine-containing heteroduplex to a purified system consisting of soluble endonuclease V fusion protein, DNA polymerase I, and DNA ligase, along with the four deoxynucleoside triphosphates. Interestingly we found these three proteins alone are sufficient to process the dI lesion efficiently. We also found that the 3′-exonuclease activity of DNA polymerase I is sufficient to remove the dI lesion in this minimum reconstituted assay.

Tissue- and Cell-Specific Alternative Splicing of NFIC

Alternative splicing is an important mechanism for increasing genetic complexity leading to multiple transcripts from single genes and gene regulation through alternative promoters. Splicing often leads to unique tissue-specific patterns of mRNAs with specific biological functions. Nuclear factor I-C (NFI-C), a member of the NFI gene family, is expressed in numerous tissues including brain, liver, spleen and heart. However, the unique dental phenotype of Nfic^–/– mice lacking molar roots demonstrates a critical role for this transcription factor in root formation. In humans, the NFI-C gene is alternatively spliced producing 4 isoforms. However, different spliced variants have not been studied in association with tissue specificity. The main objective of this study is to identify the NFI-C isoforms expressed in dental cells/tissues, comparing them to the spliced variants in nondental cells/tissues and to analyze their relative expression levels in various cell types. Using bioinformatics, we analyzed the NFI-C gene structure, identifying 2 potential alternative promoters driving expression of selective mRNA transcripts. Our studies show the expression of 3 NFI-C transcripts with the overall splicing pattern conserved between dental and nondental cells tested. Furthermore, by quantitative real-time PCR analysis, we found that although the relative levels of these transcripts were similar in dental and nondental cells, significant differences were observed within the dental cells tested. These are the first studies to analyze the expression of NFI-C isoforms in dental versus nondental cells/tissues, finding subtle cell-/tissue-specific expression patterns that could explain the dental phenotype of Nfic^–/– mice.

SURGICAL CORRECTION OF SPINAL DEFORMITY IN NEUROFIBROMATOSIS: EXPERIENCE IN CDI APPLICATION

Objective. To analyze immediate and long-term results of surgical correction of spinal deformity in patients with neurofibromatosis type I. Material and Methods. A total of 24 patients (14 boys, 10 girls), mean age 14.2 years, were operated on during the period of 1996–2007. Out of them 16 patients had a rightsided curve, and 8 patients — left-sided one. Spinal deformity correction was performed with Cotrel — Dubousset instrumentation. In 14 cases main intervention was preceded by discectomy and interbody fusion with bone autograft at the apex of the primary scoliotic curve. In 10 cases both procedures were conducted under a single anesthetic event. Mean postoperative follow-up term was 1.4 years. Results. Mean initial value of the primary scoliotic curve was 102.6°, after surgery — 61.3°, and at final follow-up — 67.9°. Thoracic kyphosis before operation was 82.5°, after operation — 51.2°, and at final follow-up — 64.0°. The tilt of the lowest instrumented vertebra before operation was 16.7°, after operation — 9.5° and at final follow-up — 10.9°. Only mechanical complications were noted: rod fracture in 3 patients, and cranial hook dislocation in 1 patient. Histological analysis of surgical material showed that NF-I gene mutation led to the disturbance of organospecific differentiation of chondroblasts and to staging process of chondroblast — chondrocyte — osteoblast — osteocyte differentiation, which explains postoperative progression of the spinal deformity and formation of pseudoarthroses in the fusion mass. Conclusion. Spinal deformities associated with neurofibromatosis are rare but due to specificity of their pre- and postoperative course present significant challenge.