Active Allele Research Articles

Comparative genetic characteristics of microsatellite profile of holstein and purebred kholmogorsky bulls

Relevance. Due to widespread metisation the Kholmogorskaya breed is on the verge of complete extinction, which requires the development of measures aimed at preserving not only the animals themselves, but also their genetic diversity. Microsatellites can be considered the most convenient for analyzing the genetic state of a population, due to the high level of their polymorphism.Methods. The material for genetic examination was cryocon-served sperm of bulls-producers belonging to the RSUP «Komi» for breeding work (Syktyvkar). Samples from 64 pure-bred Kholmogorsky and 36 with different levels of Holstein breeding bulls were selected for analysis. The allelic structure of STR markers was determined in the laboratory of DNA technologies of FGBNU «VNIIplem». The genetic and statistical analysis was carried out according to the methods generally accepted in biology and animal science.Results. Among purebred bulls 9 privat-alleles were identified, and in crossbreed producers — 13. The most polymorphic among Holstein animals were loci TGLA122 (Na = 10) and TGLA227 (Na = 11), in purebred animals — TGLA227 (Na = 9) and TGLA53 (Na = 10). The maximum correspondence between the number of actual and effective alleles was observed at the BM1824 locus, and the minimum at TGLA227. The Shannon index was the highest for the locus TGLA122 (2,046) in crossbreeds and for TGLA53 (2,011) in purebred bulls, and the lowest for BM1818 (1,050 and 1,174). The diversity of the allelofund of the analyzed loci in the metalized animals was slightly higher (by 0.26). The average fixation index in crossbreeds deviated towards a slight deficit of heterozygosity (0.017), and in purebred bulls an excess (-0.025). The greatest genetic relationship between the two samples was observed at the SPS115 locus (0.996), and the smallest at CSSM66 (0.827).

Co-administration of angiotensin II and simvastatin triggers kidney injury upon heme oxygenase-1 deficiency

Kidneys are pivotal organ in iron redistribution and can be severely damaged in the course of hemolysis. In our previous studies, we observed that induction of hypertension with angiotensin II (Ang II) combined with simvastatin administration results in a high mortality rate or the appearance of signs of kidney failure in heme oxygenase-1 knockout (HO-1 KO) mice. Here, we aimed to address the mechanisms underlying this effect, focusing on heme and iron metabolism.We show that HO-1 deficiency leads to iron accumulation in the renal cortex. Higher mortality of Ang II and simvastatin-treated HO-1 KO mice coincides with increased iron accumulation and the upregulation of mucin-1 in the proximal convoluted tubules. In vitro studies showed that mucin-1 hampers heme- and iron-related oxidative stress through the sialic acid residues. In parallel, knock-down of HO-1 induces the glutathione pathway in an NRF2-depedent manner, which likely protects against heme-induced toxicity.To sum up, we showed that heme degradation during heme overload is not solely dependent on HO-1 enzymatic activity, but can be modulated by the glutathione pathway. We also identified mucin-1 as a novel redox regulator. The results suggest that hypertensive patients with less active HMOX1 alleles may be at higher risk of kidney injury after statin treatment.

TP53 Alterations in Myelodysplastic Syndromes and Acute Myeloid Leukemia.

TP53 mutations are less frequent in myelodysplastic syndromes (MDS) and acute myeloid leukemia (AML) than in solid tumors, except in secondary and therapy-related MDS/AMLs, and in cases with complex monosomal karyotype. As in solid tumors, missense mutations predominate, with the same hotspot mutated codons (particularly codons 175, 248, 273). As TP53-mutated MDS/AMLs are generally associated with complex chromosomal abnormalities, it is not always clear when TP53 mutations occur in the pathophysiological process. It is also uncertain in these MDS/AML cases, which often have inactivation of both TP53 alleles, if the missense mutation is only deleterious through the absence of a functional p53 protein, or through a potential dominant-negative effect, or finally a gain-of-function effect of mutant p53, as demonstrated in some solid tumors. Understanding when TP53 mutations occur in the disease course and how they are deleterious would help to design new treatments for those patients who generally show poor response to all therapeutic approaches.

CRISPR/Cas9-mediated Cxcr4 disease allele inactivation for gene therapy in a mouse model of WHIM syndrome

AbstractWHIM syndrome is an autosomal dominant immunodeficiency disorder caused by gain-of-function mutations in chemokine receptor CXCR4 that promote severe panleukopenia because of retention of mature leukocytes in the bone marrow (BM). We previously reported that Cxcr4-haploinsufficient (Cxcr4+/o) hematopoietic stem cells (HSCs) have a strong selective advantage for durable hematopoietic reconstitution over wild-type (Cxcr4+/+) and WHIM (Cxcr4+/w) HSCs and that a patient with WHIM was spontaneously cured by chromothriptic deletion of the disease allele in an HSC, suggesting that WHIM allele inactivation through gene editing may be a safe genetic cure strategy for the disease. We have developed a 2-step preclinical protocol of autologous hematopoietic stem and progenitor cell (HSPC) transplantation to achieve this goal. First, 1 copy of Cxcr4 in HSPCs was inactivated in vitro by CRISPR/Cas9 editing with a single guide RNA (sgRNA) that does not discriminate between Cxcr4+/w and Cxcr4+/+ alleles. Then, through in vivo natural selection, WHIM allele–inactivated cells were enriched over wild-type allele–inactivated cells. The WHIM allele–inactivated HSCs retained long-term pluripotency and selective hematopoietic reconstitution advantages. To our knowledge, this is the first example of gene therapy for an autosomal dominant gain-of-function disease using a disease allele inactivation strategy in place of the less efficient disease allele repair approach.

Bright side of the MAOA-uVNTR on trait and situational forgiveness

The stress-and-coping theory of forgiveness posits that forgiveness and aggression are alternative ways of coping with stress of interpersonal offences. Inspired by the link between aggression and MAOA-uVNTR (a genetic variant involving in catabolism of monoamines), we investigated the relationship between this variant and forgiveness with two studies. Study 1 examined the relationship between the MAOA-uVNTR and trait forgiveness in students, and study 2 examined the effect of this variant on third-party forgiveness in response to situational offences in male inmates. The results showed that the MAOA-H (a high activity allele) was associated with higher trait forgiveness in male students and greater third-party forgiveness to accidentally committed harm and attempted but failed harm in male inmates than the MAOA-L. These findings highlight the bright side of MAOA-uVNTR on trait and situational forgiveness.

Two independent loss‐of‐function mutations in anthocyanidin synthase homeologous genes are responsible for the all‐green phenotype of sweet basil

Sweet basil, Ocimum basilicum L., is an important culinary herb grown worldwide. Although basil is green, many landraces, breeding lines, and exotic cultivars have purple stems and flowers. This anthocyanin pigmentation is unacceptable in traditional Italian basil used for Pesto sauce production. In the current study, we aimed to resolve the genetics that underlines the different colors. We used the recently published sweet basil genome to map quantitative trait loci (QTL) for flower and stem color in a bi-parental F2 population. It was found that the pigmentation is governed by a single QTL, harboring an anthocyanidin synthase (ANS) gene (EC 1.14.20.4). Further analysis revealed that the basil genome harbors two homeologous ANS genes, each carrying a loss-of-function mutation. ObANS1 carries a single base pair insertion resulting in a frameshift, and ObANS2 carries a missense mutation within the active site. In the purple-flower parent, ANS1 is functional, and ANS2 carries a nonsense mutation. The functionality of the ObANS1 active allele was validated by complementation assay in an Arabidopsis ANS mutant. Moreover, we have restored the functionality of the missense-mutated ObANS2 using site-directed activation. We found that the non-functional alleles were expressed to similar levels as the functional allele, suggesting polyploids invest futile effort in expressing non-functional genes, offsetting their advantageous redundancy. This work demonstrated the usefulness of the genomics and genetics of basil to understand the basic mechanism of metabolic traits and raise fundamental questions in polyploid plant biology.

Mutation and Phenotype Analysis in Patients with Osteopetrosis Caused by Loss-of-Function Mutation of <I>CLCN7 </I>Single Allele

Objective: To investigate the relationship between gene mutation and clinical phenotype in patients with autosomal dominant osteopetrosis type 2 (ADO2) caused by single allele mutation of <I>CLCN7</I>. Methods: The two ADO2 cases were clinically described in detail, <I>CLCN7</I> gene were screened by the next generation sequence assay, and validated by Sanger method. The hazard of mutations was evaluated on PolyPhen2 website. Literature review helped to browse and gather ADO2 cases with <I>CLCN7</I> data, accordingly all found <I>CLCN7</I> mutant loci were mapped on the peptide chain, and clinical presentations in each case were tentatively summarized corresponding gene mutation. Phenotype was classified as mild type, intermediate type and severe type according to the degree of clinical presentation. Mild type presents mainly with bone and/or joint pain and occasional bone fracture, intermediate type with additional multiple and recurrent bone fracture, sever type with hematopoietic dysfunction in addition to recurrent bone fractures. Results: The first case was a 30 year old man presenting as mild ADO2 with shorter stature than familial hereditary height, as well as back pain and knee joint pain for up-to nine years. The second case was a 13 year old boy who suffered from recurrent bone fractures since four years ago in addition to short stature and more severe bone pain, but had normal peripheral blood picture. His prominent bone change showed by X-Ray indicated a more typical and severe osteopetrosis than the former. Gene analysis indicated a mutation of c.2284C>T (E24), p.R762W in the first case and c.856C>T (E10), p.R286W in the second case. Literature review gathered 42 pathogenic mutations, mild, intermediate and severe clinical type account for 33.3%, 46.7% and 20.0%, respectively. Among family members of probands, 58.8% had unaffected mutation-carrier, and 44.7% had family history of the disease. Conclusions: In spite of single allele inactivation, <I>CLCN7</I> related ADO2 may cause highly various clinical phenotype of osteopetrosis, covering normal unaffected, mild, intermediate and severe conditions. The relationship between genotype and phenotype remains obscure in the disease.

ZmCCT10-relayed photoperiod sensitivity regulates natural variation in the arithmetical formation of male germinal cells in maize.

Extensive mutational screening studies have documented genes regulating anther and pollen development. Knowledge concerning how formation of male germinal cell is arithmetically controlled in natural populations, under different environmental conditions, is lacking. We counted pollen number within a single anther and a maize-teosinte BC2 S3 recombinant inbred line population to identify ZmCCT10 as a major determinant of pollen number variation. ZmCCT10 was originally identified as a photoperiod-sensitive negative regulator of flowering. ZmCCT10 inactivation, after transposon insertion within its promoter, is proposed to have accelerated maize spread toward higher latitudes, thus allowing temperate maize to flower under long-day conditions. We showed that the active ZmCCT10 allele decreased pollen formation. As different active and inactive ZmCCT10 alleles have been found in natural maize populations, this represents the first report of a gene controlling pollen number in a crop natural population. These findings suggest that higher pollen number, which provides a competitive advantage in open-pollinated populations, may have been one of the major driving forces for the selection of an inactive ZmCCT10 allele during tropical maize domestication. We provide evidence that ZmCCT10 has opposite effects on cell proliferation of archesporial and tapetum cells and it modulates expression of key regulators during early anther development.

Genetic Strategies to Study T Cell Development.

Genetics approaches have been instrumental to deciphering T cell development in the thymus, including gene disruption by homologous recombination and more recently Crispr-based gene editing and transgenic gene expression, especially of specific T cell antigen receptors (TCR). This brief chapter describes commonly used tools and strategies to modify the genome of thymocytes, including mouse strains with lineage- and stage-specific expression of the Cre recombinase used for conditional allele inactivation or expressing unique antigen receptor specificities.

OR04-4 Loss of KDM1A in Bilateral Macronodular Adrenal Hyperplasia With GIP-Dependent Cushing's Syndrome and in Acromegaly With Paradoxical GH Response to Oral Glucose

Abstract Context Primary bilateral macronodular adrenal hyperplasia (PBMAH) with glucose-dependent insulinotropic polypeptide (GIP)-dependent Cushing's syndrome is caused by ectopic expression of GIP receptor (GIPR) in the adrenal lesions. Such ectopic expression of GIPR was also reported in other endocrine neoplasm, notably in somatotroph pituitary adenomas from acromegalic patients with paradoxical increase of GH after oral glucose load, suggesting a common molecular pathogenesis. We aimed to identify the driver event responsible for GIP-dependent PBMAH with Cushing's syndrome and ectopic GIPR expression in somatotropinomas. Methods We conducted an international, multicenter, cohort study. We collected blood and adrenal samples from patients who had undergone unilateral or bilateral adrenalectomy for GIP-dependent PBMAH with Cushing's syndrome. Adrenal samples from patients with PBMAH and Cushing's syndrome without food-dependent cortisol production were used as controls. We further collected somatotropinoma specimens from acromegaly patients followed at two expert endocrine centers in France. Results 17 patients with familial or sporadic GIP-dependent PBMAH with Cushing's syndrome were studied. We identified germline heterozygous mutations in the lysine demethylase 1A (KDM1A) gene in all 17 patients. We further identified a recurrent deletion of the short arm of chromosome 1 harboring the KDM1A locus in the adrenal lesions of affected patients. None of the 25 patients in the control group had KDM1A germline or somatic alterations. Concomitant genetic inactivation of both KDM1A alleles resulted in loss of KDM1A expression in the adrenal lesions. RNA-sequencing revealed the global impact of KDM1A loss in adrenal tissue on gene transcription and identified differentially regulated genes including those encoding for GIPR and other G-Protein-Coupled Receptors that may be involved in adrenal tumorigenesis and regulation of steroidogenesis. In vitro pharmacologic inhibition, silencing and knock-out by CRISPR-Cas9 genome editing of KDM1A led to an increase in GIPR transcripts and protein in human adrenocortical H295R cells. Somatotropinoma samples from 78 patients with acromegaly were studied. 24% of these patients presented with a paradoxical rise of GH after oral glucose load and expressed ectopically GIP-receptor in their somatotropinoma. None of the somatotropinomas harbored KDM1A pathogenic variants, but those from patients with paradoxical GH response displayed a recurrent chromosome 1p loss. Discussion We identified germline inactivating KDM1A mutations and loss of heterozygosity as a genetic predisposition to GIP-dependent PBMAH with Cushing's syndrome following a tumor suppressor gene model of tumorigenesis. We currently perform genetic screening in first-degree relatives of patients with GIP-dependent PBMAH with Cushing's syndrome and clinical examination with biochemical testing in asymptomatic KDM1A variant carriers. We did not identify somatic KDM1A mutations in somatotropinomas expressing GIPR ectopically, however their recurrent 1p chromosome loss suggests that KDM1A haploinsufficiency may contribute to GIPR expression in those tumors. Presentation: Saturday, June 11, 2022 12:15 p.m. - 12:30 p.m.

Nitrogen repression of deoxynivalenol biosynthesis is mediated by Mep2 ammonium permease in Fusarium graminearum.

Fusarium graminearum is an important wheat pathogen and a producer of deoxynivalenol (DON). Biosynthesis of DON is suppressed by ammonium and induced by arginine and polyamines. To better understand ammonium repression of DON biosynthesis, in this study, we functionally characterized three ammonium permease (MEP) genes in F. graminearum. All the mep deletion mutants were normal in growth on V8 agar. Whereas deletion of MEP1 had no detectable phenotypes, the mep2 and mep3 mutants had defects in hyphal growth under ammonium limiting conditions and infection of wheat heads, with the latter having less severe defects. Deletion of MEP2 but not MEP3 affected nitrogen repression of DON biosynthesis and genes involved in nitrate metabolism. The mep2 mep3 double mutant had more severe defects in nitrogen repression than the mep2 mutant and was defective in ascospore releasing. Mutant alleles of MEP2 with truncated C-terminal cytoplasmic tail (CT) failed to complement the mep2 mutant. Expression of a dominant active allele of RAS2 partially rescued the defects of mep2 in nitrogen repression. Taken together, these results suggest that Mep2 acts as the major sensor of ammonium availability in F. graminearum and its CT region functions in nitrogen repression via RAS2 and downstream signalling pathways.

A HST1-like gene controls tiller angle through regulating endogenous auxin in common wheat.

Tiller angle is one of the most important agronomic traits and one key factor for wheat ideal plant architecture, which can both increase photosynthetic efficiency and greatly enhance grain yield. Here, a deacetylase HST1-like (TaHST1L) gene controlling wheat tiller angle was identified by the combination of a genome-wide association study (GWAS) and bulked segregant analysis (BSA). Ethyl methane sulfonate (EMS)-mutagenized tetraploid wheat lines with the premature stop codon of TaHST1L exhibited significantly smaller tiller angles than the wild type. TaHST1L-overexpressing (OE) plants exhibited significantly larger tiller angles and increased tiller numbers in both winter and spring wheat, while TaHST1L-silenced RNAi plants displayed significantly smaller tiller angles and decreased tiller numbers. Moreover, TaHST1L strongly interacted with TaIAA17 and inhibited its expression at the protein level, and thus possibly improved the content of endogenous auxin in the basal tissue of tillers. The transcriptomics and metabolomics results indicated that TaHST1L might change plant architecture by mediating auxin signal transduction and regulating endogenous auxin levels. In addition, a 242-bp insertion/deletion (InDel) in the TaHST1L-A1 promoter altered transcriptional activity and TaHST1L-A1b allele with the 242-bp insertion widened the tiller angle of TaHST1L-OE transgenic rice plants. Wheat varieties with TaHST1L-A1b allele possessed the increased tiller angle and grain yield. Further analysis in wheat and its progenitors indicated that the 242-bp InDel possibly originated from wild emmer and was strongly domesticated in the current varieties. Therefore, TaHST1L involved in the auxin signalling pathway showed the big potential to improve wheat yield by controlling plant architecture.

Maternal Education Potentially Moderates the MAOA uVNTR Effects on Externalizing Behavior in Black South African Children

Interactions between the MAOA uVNTR and rearing environment are suggested to influence the developmental manifestations of childhood internalizing and externalizing behavior. However, few studies in the MAOA literature have included continental African children, or focused on non-clinical samples. We explored the main and interactive effects of the MAOA uVNTR (high and low activity alleles) in Black South African male (n = 478) and female (n = 540) children who were part of the longitudinal Birth to Twenty Plus cohort. Historical data on birth weight, gestational age at delivery, socioeconomic status, and maternal education were combined with genotypic information and analyzed using regression modeling. We found no significant main effects for the MAOA uVNTR on childhood behavior in either sex. A significant interaction (p = .04) was identified between MAOA and maternal education, suggesting that externalizing behavior in boys carrying a low activity MAOA allele varied in direct proportion to the education levels of their mothers. However, the model fit failed to reach significance, possibly due to our inclusion of only non-clinical pre-pubertal males. No significant interactions were detected for female children. Our findings lend tentative credibility to the Environmental Sensitivity metaframework, which suggests that MAOA is an important plasticity factor in childhood development.

CBP/p300 and HDAC activities regulate H3K27 acetylation dynamics and zygotic genome activation in mouse preimplantation embryos

Epigenome reprogramming after fertilization enables transcriptionally quiescent maternal and paternal chromatin to acquire a permissive state for subsequent zygotic genome activation (ZGA). H3K27 acetylation (H3K27ac) is a well‐established chromatin marker of active enhancers and promoters. However, reprogramming dynamics of H3K27ac during maternal‐to‐zygotic transition (MZT) in mammalian embryos are not well‐studied. By profiling the allelic landscape of H3K27ac during mouse MZT, we show that H3K27ac undergoes three waves of rapid global transitions between oocyte stage and 2‐cell stage. Notably, germinal vesicle oocyte and zygote chromatin are globally hyperacetylated, with noncanonical, broad H3K27ac domains that correlate with broad H3K4 trimethylation (H3K4me3) and open chromatin. H3K27ac marks genomic regions primed for activation including ZGA genes, retrotransposons, and active alleles of imprinted genes. We show that CBP/p300 and HDAC activities play important roles in regulating H3K27ac dynamics and are essential for preimplantation development. Specifically, CBP/p300 acetyltransferase broadly deposits H3K27ac in zygotes to induce the opening of condensed chromatin at putative enhancers and ensure proper ZGA. On the contrary, HDACs revert broad H3K27ac domains to canonical domains and safeguard ZGA by preventing premature expression of developmental genes. In conclusion, coordinated activities of CBP/p300 and HDACs during mouse MZT are essential for ZGA and preimplantation development.

Abstract IA013: Rhabdomyosarcoma: visions through the looking glass

Abstract Rhabdomyosarcoma (RMS) is the most common soft tissue sarcoma in children. Despite aggressive treatment clinical outcomes for RMS have not improved for three decades, emphasizing the need to uncover the molecular underpinnings of the disease. RMS has been presumed to originate from derailed muscle progenitors based on the histologic appearance and gene expression pattern of the tumors resembling embryonic developing skeletal muscle. However, an origin restricted to skeletal muscle does not explain RMS occurring in tissues devoid of skeletal muscle such as the prostate, bladder, and salivary gland. Previously, we described that activation of Sonic Hedgehog signaling through expression of a conditional, constitutively active Smoothened allele, SmoM2, under control of a presumed adipocyte-restricted adipose protein 2 (aP2)-Cre recombinase transgene in mice gives rise to aggressive skeletal muscle tumors. These tumors display the histologic and molecular characteristics of human embryonal fusion-negative RMS (FN-RMS). This model suggested a potential non-myogenic origin of FN-RMS and an avenue to explain FN-RMS development in anatomic sites devoid of skeletal muscle. Lineage tracing showed that RMS can originate from cell reprogramming and transdifferentiation of endothelial progenitor cells. Hedgehog pathway activation in committed endothelial progenitors results in Tbx1 expression and subsequent Myod1 expression driving a partially myogenic program characteristic of FN-RMS. Our work identifies reprogramming cell fate as a mechanism of transformation in pediatric sarcoma and illustrates that it is dangerous to assume the cell of origin from the characteristics of the tumor cell. The cell-reprogramming mechanism that shifts endothelial progenitors to muscle-like cells provides a unique system to define the core regulatory circuitry controlling RMS cell fate and to determine in vivo if targeting this network is a therapeutic vulnerability. Genomic profiling of human FN-RMS failed to uncover a unique mutation that drives oncogenesis. However, the PTEN cis-regulatory region is hypermethylated in more than 90% of all human FN-ERMS tumors, resulting in decreased expression. However, inhibiting the PI3K/AKT/mTOR pathway has had varied efficacy in RMS. In our RMS mouse model, PTEN localizes to the cytoplasm and nucleus, suggesting that PTEN could have functions other than regulating the PI3K/AKT/mTOR pathway. We demonstrate that Pten loss cooperates in RMS tumorigenesis and results in tumors more reflective of human FN-RMS. We show that Pten loss drives expression of the transcription factor PAX7 and identified PAX7 as a dependency in human FN-RMS. Furthermore, Pax7 deletion completely rescues the deleterious effects of Pten loss but also alters tumor cell fate, giving rise to a smooth muscle tumor. Thus, PTEN loss drives the expression of PAX7, a key member of the RMS core regulatory circuitry dictating tumor cell fate. This highlights a synthetic essential relationship between PTEN and PAX7 in FN-RMS tumor maintenance and tumor-fate decisions. Citation Format: Mark E. Hatley, Casey G. Langdon, Katherine E. Gadek, Matthew R. Garcia, Catherine J. Drummond, Jason A. Hanna, Hongjian E. Jin, Jerold E. Rehg. Rhabdomyosarcoma: visions through the looking glass [abstract]. In: Proceedings of the AACR Special Conference: Sarcomas; 2022 May 9-12; Montreal, QC, Canada. Philadelphia (PA): AACR; Clin Cancer Res 2022;28(18_Suppl):Abstract nr IA013.

Azathioprine-induced severe pancytopenia: A serious complication in a patient with normal TPMT activity

Azathioprine is an immunomodulator commonly used in renal transplant recipients and to treat lupus erythematous, and inflammatory bowel disease, and as second-line therapy in cases of rheumatoid arthritis. Here, we present a case of non-segmental vitiligo treated with low-dose azathioprine developing life-threatening pancytopenia with febrile neutropenia and trephine biopsy-proven drug-induced myelosuppression. The patient needed broad-spectrum antibiotics and granulocyte colony-stimulating factor (G-CSF) support with intermittent transfusion of blood products to completely recover from myelosuppression. It took almost a month for the patient to completely recover from the cytotoxicity caused by azathioprine. Interestingly, the patient had normal thiopurine methyl transferase (TPMT) activity and wild-type TPMT allele. We tried to outline how these types of patients can be managed and to emphasize the need for continuous monitoring of blood counts in patients receiving azathioprine to prevent life-threatening cytopenia even with normal TPMT activity and wild-type TPMT allele.

Social and maternal behavior in mesoderm specific transcript (Mest)-deficient mice.

Mesoderm specific transcript (Mest)/paternally expressed gene-1 (Peg1) is an imprinted gene expressed predominantly from the paternal allele. Aberrations in maternal behavior were previously reported in a Mest global knockout mouse (Mesttm1Masu). In this study, we performed in-depth social and maternal behavioral testing in a mouse model of Mest inactivation developed in our laboratory (Mesttm1.2Rkz). Mice with paternal allele inactivation (MestpKO) did not show anxiety after testing in the elevated plus maze, open field trial, and marble burying; nor depression-like behaviors in the tail suspension test. MestpKO showed normal social behaviors and memory/cognition in the three-chamber box test and the novel object recognition test, respectively. Primiparous MestpKO and MestgKO (biallelic Mest inactivation) female mice exhibited normal nest building and maternal behavior; and, virgin MestpKO and MestgKO female mice showed normal maternal instinct. Analyses of gene expression in adult hypothalamus, embryonic day 14.5 whole brain and adult whole brain demonstrated full abrogation of Mest mRNA in MestpKO and MestgKO mice with no effect on miR-335 expression. Our data indicates no discernible impairments in object recognition memory, social behavior or maternal behavior resulting from loss of Mest. The basis for the differences in maternal phenotypic behaviors between Mesttm1Masu and Mesttm1.2Rkz is not known.

Gene replacement therapy in a schwannoma mouse model of neurofibromatosis type 2

Loss of function of the neurofibromatosis type 2 (NF2) tumor suppressor gene leads to the formation of schwannomas, meningiomas, and ependymomas, comprising ∼50% of all sporadic cases of primary nervous system tumors. NF2 syndrome is an autosomal dominant condition, with bi-allelic inactivation of germline and somatic alleles resulting in loss of function of the encoded protein merlin and activation of mammalian target of rapamycin (mTOR) pathway signaling in NF2-deficient cells. Here we describe a gene replacement approach through direct intratumoral injection of an adeno-associated virus vector expressing merlin in a novel human schwannoma model in nude mice. In culture, the introduction of an AAV1 vector encoding merlin into CRISPR-modified human NF2-null arachnoidal cells (ACs) or Schwann cells (SCs) was associated with decreased size and mTORC1 pathway activation consistent with restored merlin activity. In vivo, a single injection of AAV1-merlin directly into human NF2-null SC-derived tumors growing in the sciatic nerve of nude mice led to regression of tumors over a 10-week period, associated with a decrease in dividing cells and an increase in apoptosis, in comparison with vehicle. These studies establish that merlin re-expression via gene replacement in NF2-null schwannomas is sufficient to cause tumor regression, thereby potentially providing an effective treatment for NF2.

Abstract 3767: Identification of regulators of epigenetic silencing of the p16INK4a gene

Abstract Epigenetic silencing of tumor suppressor genes plays critical roles in oncogenesis. The CDKN2A/p16INK4a gene is one of the most frequently silenced tumor suppressor genes. To elucidate molecular mechanisms of maintenance of the silenced status of the p16INK4a gene, we applied the insertional chromatin immunoprecipitation (iChIP) method. The promoter region of the transcriptionally active and silenced alleles of the p16INK4a gene were tagged with a bacterial DNA-binding protein LexA and its binding elements. Subsequently, the promoter regions were affinity purified with antibody against a tag fused with LexA. Proteins more or less abundantly associated with the silenced promoter were identified by stable isotope labeling using amino acids in cell culture (SILAC), one of quantitative mass spectrometry methods. Knock-down experiments of the identified enzymes revealed essential components of maintenance of epigenetic silencing. The essential enzymes can be good targets of development of drugs reactivating expression of the silenced tumor suppressor genes in cancers. We are currently performing drug discovery research to identify small molecules that bind and inhibit those candidate drug target enzymes. Our locus-specific ChIP technology consisting of iChIP and the engineered DNA-binding molecule-mediated ChIP (enChIP) method is a powerful tool to identify novel drug targets for development of drugs regulating gene expression in intractable diseases. Citation Format: Toshitsugu Fujita, Hodaka Fujii. Identification of regulators of epigenetic silencing of the p16INK4a gene [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3767.

Abstract LB559: The X-linked gene for the helicase DDX3X is required for lymphoid differentiation and MYC-driven lymphomagenesis

Abstract The X-linked gene DDX3X encodes an RNA helicase and is mutated at high frequencies in several types of human B-cell lymphoma. Females have two active DDX3X alleles and males carry a DDX3Y homolog on the Y chromosome. We show here that pan-hematopoietic, homozygous deletion of Ddx3x in female mice perturbs erythropoiesis causing early developmental arrest. However, both hemizygous male and heterozygous female embryos develop normally, suggesting that one Ddx3x allele is sufficient for fetal hematopoietic development in females and that the Ddx3y allele can compensate for the loss of Ddx3x in males. In adult mice, loss of DDX3X affects hematopoietic progenitors, early lymphoid development, marginal zone, and germinal center B-cells as well as lymphomagenesis driven by an Eμ-Myc or λ-Myc transgene in a sex-dependent manner. A B-cell-specific depletion of both Ddx3x alleles induces a delay in tumorigenesis in female mice while Ddx3x deletion in male mice does not impact tumorigenesis. However, male Eμ-Myc mice lacking Ddx3x on a pan-hematopoietic way remained for the majority lymphoma-free. The few tumors that appeared in these male mice showed upregulated expression of DDX3Y indicating a requirement of DDX3 activity for lymphomagenesis. Our data reveal sex-specific roles of DDX3X in erythro- and lymphopoiesis as well as in MYC-driven lymphomagenesis, which are important when considering inhibition of DDX3 as a treatment of Myc dependent B-cell lymphoma. Citation Format: Marion Lacroix, Hugues Beauchemin, Julie Ross, Jennifer Fraszczak, Peiman Shooshtarizadeh, Riyan Chen, Tarik Moroy. The X-linked gene for the helicase DDX3X is required for lymphoid differentiation and MYC-driven lymphomagenesis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr LB559.