RUNX1 Deficiency Cooperates with SRSF2 Mutation to Further Disrupt RNA Splicing and Exacerbate Myelodysplastic Syndromes in Mouse Models

Abstract Myelodysplastic syndromes (MDS) and leukemias require the acquisition of multiple mutations during disease development resulting in clonal diversity and different responses. Splicing factors, transcription factors, epigenetic regulators, and cell signaling proteins are the common molecular events mutated during disease evolution and those events rarely occur alone. However, it remains unclear how the combinations of mutations in different categories may have cooperative effects in gene regulation and disease etiology. Mutations in the splicing factor SRSF2 and the transcription factor RUNX1 are closely associated in MDS patients, and their co-existence is linked to poor prognosis. To understand the functional contribution of the coexistence in vivo, we utilized Mx1-Cre based conditional knock-in Srsf2-P95H mutation (P95H/+) mice, and Mx1-Cre based Runx1 conditional knockout mice (Runx1 f/f). We crossed these two strains to establish a new mouse model with inducible double mutations (Srsf2 P95H/+ Runx1Δ/Δ). Double mutant mice showed pancytopenia with MDS features including severe leukopenia in multiple lineages, macrocytic anemia, thrombocytopenia, and dysplastic morphology in peripheral blood. Double mutant mice also displayed more dramatic skewing toward the myeloid lineage at the expense of the B cell lineage when compared to single mutant mice. In competitive bone marrow transplantation assays, SRSF2 P95H cooperated with RUNX1 deficiency to confer a competitive disadvantage in vivo. To investigate the mechanistic basis of this cooperation, differential splicing and gene expression were assessed by RNA sequencing of Lineage- c-kit+ cells isolated from WT, SRSF2 P95H, RUNX1 KO, and Double mutant bone marrow cells. Interestingly, deletion of the Runx1 gene alone resulted in significant changes to RNA splicing in 1120 genes, while the SRSF2 P95H mutation itself induced splicing changes in 935 genes. Furthermore, 2468 splice junctions in 1677 genes showed splicing changes in double mutant samples compared to wildtype controls. Among these altered splicing events, intriguingly, exon skipping was the major alteration in single and double mutants. Furthermore, the double mutant demonstrated increased aberrant splicing events when compared to the single mutants alone. We performed pathway analysis using the differentially spliced genes identified in double mutant cells. Pathways in cancer, DNA replication/repair, cell death and survival, hematological disease and inflammatory response were enriched. Splicing changes were detected in genes recurrently mutated in blood malignancies, including Fanca, Fance, Fancl, Ezh2, Atm, Gnas, Braf, Bcor, Fyn, and Wsb1 as well as in genes critical for splicing regulation, such as Srsf6, Fus, Hnrnpa2b1, and Srrm2. Gene expression analysis revealed 869 significantly differentially expressed genes in double mutant cells. Within the events in the double mutant population, 60% of the differentially expressed genes were also observed in RUNX1 single mutant cells, while only 2% of the differentially expressed genes were observed in SRSF2 single mutant cells, and 38% of the differentially expressed genes were uniquely presented in the double mutant cells. These results suggest that the gene expression program is heavily affected by loss of RUNX1 and the coexistence of an SRSF2 mutation contributes to certain synergistic effects in transcriptional regulation. Furthermore, we identified 101 genes that showed both differential splicing and expression, including Jak3, Jag2, Csf3r, Fcer1g, CD244 which are important in hematologic disorders. Together, these results suggest that the deficiency of compound RUNX1 and SRSF2 P95H mutations impairs multi-lineage hematopoiesis and exacerbates the disease phenotypes caused by single mutations alone. At the genome-wide level, loss of the transcription factor RUNX1 itself dysregulates splicing outcomes and cooperates with the splicing factor SRSF2 P95H mutation to further perturb the expression and splicing of key regulators involved in hematopoietic stem/progenitor cell development, inflammatory responses, DNA damage, and RNA splicing. Disclosures No relevant conflicts of interest to declare.

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Hyperactive Nras and Dose Reduction of Tet2 Collaborate to Dys-Regulate Hematopoietic Stem Cells and Promote Leukemogenesis

Blood ◽

10.1182/blood.v128.22.1204.1204 ◽

2016 ◽

Vol 128 (22) ◽

pp. 1204-1204

Author(s):

Xi Jin ◽

Tingting Qin ◽

Nathanael G Bailey ◽

Meiling Zhao ◽

Kevin B Yang ◽

...

Keyword(s):

Gene Expression ◽

Stem Cell ◽

Double Mutant ◽

Mutant Mice ◽

Cytokine Signaling ◽

Self Renewal ◽

Hematopoietic Stem ◽

Single Mutant ◽

Tet2 Mutation

Abstract Activating mutations in RAS and somatic loss-of-function mutations in the ten-eleven translocation 2 (TET2) are frequently detected in hematologic malignancies. Global genomic sequencing revealed the co-occurrence of RAS and TET2 mutations in chronic myelomonocytic leukemias (CMMLs) and acute myeloid leukemias (AMLs), suggesting that the two mutations collaborate to induce malignant transformation. However, how the two mutations interact with each other, and the effects of co-existing RAS and TET2 mutations on hematopoietic stem cell (HSC) function and leukemogenesis, remains unknown. In this study, we generated conditional Mx1-Cre+;NrasLSL-G12D/+;Tet2fl/+mice (double mutant) and activated the expression of mutant Nras and Tet2 in hematopoietic tissues with poly(I:C) injections. Double mutant mice had significantly reduced survival compared to mice expressing only NrasG12D/+ or Tet2+/-(single mutants). Hematopathology and flow-cytometry analyses showed that these mice developed accelerated CMML-like phenotypes with higher myeloid cell infiltrations in the bone marrow and spleen as compared to single mutants. However, no cases of AML occurred. Given that CMML is driven by dys-regulated HSC function, we examined stem cell competitiveness, self-renewal and proliferation in double mutant mice at the pre-leukemic stage. The absolute numbers of HSCs in 10-week old double mutant mice were comparable to that observed in wild type (WT) and single mutant mice. However, double mutant HSCsdisplayed significantly enhanced self-renewal potential in colony forming (CFU) replating assays. In vivo competitive serial transplantation assays using either whole bone marrow cells or 15 purified SLAM (CD150+CD48-Lin-Sca1+cKit+) HSCs showed that while single mutant HSCs have increased competitiveness and self-renewal compared to WT HSCs, double mutants have further enhanced HSC competitiveness and self-renewal in primary and secondary transplant recipients. Furthermore, in vivo BrdU incorporation demonstrated that while Nras mutant HSCs had increased proliferation rate, Tet2 mutation significantly reduced the level of HSC proliferation in double mutants. Consistent with this, in vivo H2B-GFP label-retention assays (Liet. al. Nature 2013) in the Col1A1-H2B-GFP;Rosa26-M2-rtTA transgenic mice revealed significantly higher levels of H2B-GFP in Tet2 mutant HSCs, suggesting that Tet2 haploinsufficiency reduced overall HSC cycling. Overall, these findings suggest that hyperactive Nras signaling and Tet2 haploinsufficiency collaborate to enhance HSC competitiveness through distinct functions: N-RasG12D increases HSC self-renewal, proliferation and differentiation, while Tet2 haploinsufficiency reduces HSC proliferation to maintain HSCs in a more quiescent state. Consistent with this, gene expression profiling with RNA sequencing on purified SLAM HSCs indicated thatN-RasG12D and Tet2haploinsufficiencyinduce different yet complementary cellular programs to collaborate in HSC dys-regulation. To fully understand how N-RasG12D and Tet2dose reduction synergistically modulate HSC properties, we examined HSC response to cytokines important for HSC functions. We found that when HSCs were cultured in the presence of low dose stem cell factor (SCF) and thrombopoietin (TPO), only Nras single mutant and Nras/Tet2 double mutant HSCs expanded, but not WT or Tet2 single mutant HSCs. In the presence of TPO and absence of SCF, HSC expansion was only detected in the double mutants. These results suggest that HSCs harboring single mutation of Nras are hypersensitive to cytokine signaling, yet the addition of Tet2 mutation allows for further cytokine independency. Thus, N-RasG12D and Tet2 dose reduction collaborate to promote cytokine signaling. Together, our data demonstrate that hyperactive Nras and Tet2 haploinsufficiency collaborate to alter global HSC gene expression and sensitivity to stem cell cytokines. These events lead to enhanced HSC competitiveness and self-renewal, thus promoting transition toward advanced myeloid malignancy. This model provides a novel platform to delineate how mutations of signaling molecules and epigenetic modifiers collaborate in leukemogenesis, and may identify opportunities for new therapeutic interventions. Disclosures No relevant conflicts of interest to declare.

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Mutations in the RNA Splicing Factor SF3B1 Promote Transformation through MYC Stabilization

Blood ◽

10.1182/blood-2018-99-110328 ◽

2018 ◽

Vol 132 (Supplement 1) ◽

pp. 882-882

Author(s):

Akihide Yoshimi ◽

Zhaoqi Liu ◽

Wang Jiguang ◽

Hana Cho ◽

Stanley C Lee ◽

...

Keyword(s):

Rna Splicing ◽

Research Funding ◽

Double Mutant ◽

Splicing Factor ◽

Rna Seq ◽

Aberrant Splicing ◽

Fty 720 ◽

The Impact ◽

Mutant Cells

Abstract Mutations in the RNA splicing factor SF3B1 are recurrent in CLL and myeloid neoplasms but their functional role in promoting tumorigenesis remain poorly understood. While SF3B1 mutations have been identified as promoting use of aberrant 3' splice sites (3'ss), consistent identification of mis-spliced transcripts and pathways that functionally link mutant SF3B1 to transformation remains elusive. Moreover, large-scale analyses of the impact of mutant SF3B1 on gene expression and gene regulatory networks, which may be distinct from aberrant splicing changes, remain to be performed. We therefore sought to elucidate the effects of SF3B1 mutations across hematopoietic malignancies and cancer lineages at the level of both mRNA splicing and expression. To this end, we collected RNA-seq data from 79 tumors and 12 isogenic cell lines harboring SF3B1 hotspot mutations. The most frequent hotspot, K700E, was the most common mutation in CLL and breast cancers while mutations at position R625 were restricted to melanomas (Figure A, B). Regulatory network analysis of differentially expressed genes in SF3B1 mutated CLL identified MYC as the top master regulator (Figure C). MYC activation in SF3B1 mutated CLL was also verified by differential expression analyses (Figure D) and was common to SF3B1K700E mutant cancers while absent in cancers with mutations affecting R625. Taken together, these observations suggested that tumors harboring SF3B1K700E mutations activate the MYC transcriptional program. We next sought to verify the effects of c-Myc activation by mutant Sf3b1 in the B-cell lineage in vivo. We crossed Cd19-cre Sf3b1K700E/+ mice with Eμ-Myc transgenic mice to generate Cd19-cre+ control, Sf3b1K700E/+, Eμ-MycTg/+, and Sf3b1K700E/+Eμ-MycTg/+ double-mutant mice. While control or single mutant primary mice did not develop disease over one year, double-mutant mice developed a lethal B-cell malignancy. This effect was consistent in serial transplantation, where mice transplanted with double-mutant cells had shorter survival compared to single-mutant controls (Figure E). These data provide the first evidence that SF3B1 mutations contribute to tumorigenesis in vivo. To understand the molecular mechanism for MYC activation across SF3B1 mutant human and mouse cells, we analyzed RNA-seq data from CLL patients, isogenic Nalm-6 cells, and splenic B-cells from the mouse models. This revealed a significant overlap in aberrant (3'ss) events across SF3B1 mutant samples. Interestingly, mis-spliced events across mouse and human SF3B1K700E mutant samples identified aberrant 3'ss usage and decay of PPP2R5A (Figure F), a gene whose product has previously been shown to regulate c-MYC protein stability and the only gene whose aberrant splicing was most prominent in K700E compared with R625 mutant SF3B1. PPP2R5A is a subunit of the PP2A phosphatase complex that dephosphorylates Serine 62 (S62) of c-MYC, resulting in an unstable form of c-MYC that is a substrate for proteasomal degradation. Consistent with this, SF3B1K700E mutant cells exhibited dramatic increase in S62-phosphorylated c-MYC and increased stability of c-MYC protein. MYC expression, stability, and S62 phosphorylation could be abrogated in SF3B1 mutant cells by restoring PPP25RA expression. In addition to c-MYC S62 phosphorylation, PPP2R5A-containing PP2A reduced S70 phosphorylation of BCL2 (a modification important for apoptosis induction) in SF3B1 mutant cells. To functionally evaluate the importance of impaired PP2A enzymatic activity in SF3B1 mutant cells further, we assessed the therapeutic potential of the FDA-approved oral PP2A activator, FTY-720. SF3B1 mutant cells were more sensitive to FTY-720 treatment than SF3B1 WT counterparts, experiencing growth arrest at lower concentration (Figure G). Moreover, both S62-phosphorylated c-MYC and S70-phosphorylated BCL2 decreased in a dose-dependent manner upon treatment with FTY-720 (Figure H). Here through combined evaluation of the effects of the SF3B1 mutation on splicing, gene expression, and transcriptional networks across cancer types, we identify a novel mechanism by which mutant SF3B1-mediated alterations in RNA splicing contribute to activation of oncogenic MYC through effects on MYC proteolysis. Moreover, these data highlight a novel therapeutic approach targeting the impact of mutant SF3B1 on post-translational modification of MYC. Figure. Figure. Disclosures Mato: Janssen: Consultancy, Honoraria; Celgene: Consultancy; Prime Oncology: Speakers Bureau; TG Therapeutics: Research Funding; Regeneron: Research Funding; Abbvie: Consultancy; Sunesis: Honoraria, Research Funding; Acerta: Research Funding; AstraZeneca: Consultancy; Pharmacyclics: Consultancy, Honoraria, Research Funding.

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Genome-Wide Analysis of a Novel Murine Model of Chronic Lymphocytic Leukemia

Blood ◽

10.1182/blood.v128.22.967.967 ◽

2016 ◽

Vol 128 (22) ◽

pp. 967-967

Author(s):

Lili Wang ◽

Rutendo Gambe ◽

Jing Sun ◽

Sachet Shukla ◽

Jaegil Kim ◽

...

Keyword(s):

B Cells ◽

Murine Model ◽

Rna Splicing ◽

Double Mutant ◽

Splice Variants ◽

Genetic Alterations ◽

Lymphocytic Leukemia ◽

Mutant Mice ◽

Genome Wide

Abstract Large-scale cancer sequencing efforts worldwide have yielded numerous novel cancer drivers; however, how these genetic alterations functionally lead to cancer remains largely unknown. An indispensible approach for establishing the causal features of disease is through in vivo animal models. In chronic lymphocytic leukemia (CLL), only limited mouse models are currently available and most do not reflect the genetics of human CLL. Studies of whole-exome sequencing (WES) using CLL samples have consistently pointed to the common co-occurrence of mutations in the RNA splicing factor gene SF3B1 and mutations in the DNA damage response gene ATM or deletion of chromosome 11q (del(11q), whose minimally deleted region encompasses ATM). We therefore asked whether this combination of traits would be productive of CLL in mice. To this end, we modeled the effects of these combined alterations by crossing mice with conditional knockout of Atm and mice with a conditional knock-in allele of SF3B1 mutation (Sf3b1-K700E). We achieved B cell-restricted expression of heterozygous Sf3b1 mutation and Atm deletion by breeding these mice with CD19-Cre homozygous transgenic mice. We found that in vivo co-expression of these two mutations in B cells, but not of either single lesion alone, led to clonal expansion of CD19+CD5+ B cells in blood, marrow and spleen (at low penetrance) in aged (18 to 24-month old) but not young mice. These malignant cells could be propagated by in vivo passaging, with detectable disease within 4 weeks following transfer, thus making this mouse line amenable to further drug discovery and biologic investigations. To better understand how Sf3b1 mutation and Atm deletion synergistically contribute to CLL, we asked if RNA level changes are present in the double mutant mice. We performed transcriptome sequencing of splenic B cell RNA collected from age-matched mice that either express wild-type, or singly mutant alleles of Sf3b1 or Atm, or doubly mutant alleles with or without CLL-like disease (n=2-6 samples, per group). Using the tool JuncBASE, we classified and quantified splice variants associated with the different genetic alterations. Consistent with prior findings in human CLL, we observed that the splice variants in micewith mutated Sf3b1 alone (without CLL) were highly enriched at 3' splice sites (27 of 77 splice variants, t-test q<0.05, absolute ΔPSI >10%). On the other hand, mice with Atm single deletion displayed an RNA splicing pattern with enrichment of alternative first and last exons (11 and 12 of 52, chi-squared test, p=4.5 x 10-4). B cells with the combined Sf3b1 and Atm mutations displayed a combination of splicing patterns that comprised of both alternative 3' splice variants, as well as alternative first and last exons. Moreover, we identified unique CLL splice variants in genes (Setdb2, Phf11c) previously demonstrated to be associated with CLL. We further investigated the differential gene expression between B cells from double mutant mice with and without CLL-like disease. We identified 1,875 CLL-specific genes (DESeq2, q<0.01). Gene set enrichment analysis (GSEA) of these genes indicated their involvement in cellular processes such as IL2-STAT5 signaling and the interferon gamma response, both pathways implicated in human CLL. In parallel, we asked if there are DNA level changes in the doubly mutant mice. We examined the mutation rate in DNA derived from splenic B cells collected from mice with a singly mutated allele of Sf3b1 or Atm, or with doubly mutated alleles with and without CLL-like disease through comparison against matched germline DNA from kidney by whole-genome sequencing. Preliminarily, we have observed that co-expression of Sf3b1 mutation and deletion of Atm results in a higher mutation rate compared to cells with only single mutation. In summary, we have generated a genetically-engineered murine model that faithfully recapitulates human CLL genetics. This is the first demonstration that expression of putative CLL driver events identified from unbiased genome-wide sequencing indeed initiates CLL-like disease. Genome-wide DNA and RNA analysis using this model has revealed that altered RNA splicing, dysregulation of gene expression, and genomic instability all contribute to CLL leukemogenesis. We anticipate that further dissection of this murine model will shed light on mechanistic understanding of cooperation between Atm deletion and SF3B1 mutation in CLL. Disclosures No relevant conflicts of interest to declare.

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6 EXPRESSION PROFILING OF SINGLE BOVINE EMBRYOS REVEALS SIGNIFICANT EFFECTS OF IN VITRO MATURATION, FERTILIZATION AND CULTURE

Reproduction Fertility and Development ◽

10.1071/rdv18n2ab6 ◽

2006 ◽

Vol 18 (2) ◽

pp. 111

Author(s):

S. L. Smith ◽

L.-Y. Sung ◽

R. Page ◽

B. Henderson ◽

F. Du ◽

...

Keyword(s):

Gene Expression ◽

Differentially Expressed Genes ◽

Oocyte Maturation ◽

Expression Profiling ◽

Bovine Serum ◽

In Vitro Maturation ◽

Differentially Expressed ◽

In Vitro Oocyte Maturation

Cattle and sheep embryos transferred after in vitro production are often afflicted by large offspring syndrome (LOS), which has been correlated with the presence of serum and/or cell co-culture. Previous research indicates that post-fertilization culture affects blastocyst quality and gene expression, and in vitro oocyte maturation and fertilization impact developmental competence. To dissect the effects of in vitro maturation, fertilization, and culture, we compared the expression profiles of single bovine blastocysts generated by: (1) in vitro maturation, fertilization and culture (IVF, n = 15); (2) in vivo maturation, in vivo fertilization, and in vitro culture (IVD, n = 14); and (3) in vivo maturation, fertilization, and development (AI, n = 14). For in vitro culture, the embryos were cultured for 2 days in CR1aa medium with bovine serum albumin (BSA) and then transferred to CR1aa with 10% fetal bovine serum (FBS) with cumulus cells until Day 7, at which time the embryos were vitrified. IVD zygotes were surgically collected from two superovulated Holstein donor cows 24 h post-insemination and cultured in the same system. To conduct expression profiling, total RNA was isolated from individual thawed embryos. The RNA was subjected to three rounds of amplification utilizing a previously adapted and validated T7 linear amplification protocol. Amplified RNA from each embryo and from a standard reference was indirectly labeled with Cy3 or Cy5 by dye swap and hybridized to a custom bovine cDNA microarray containing ~6300 unique genes. After Loess normalization, an ANOVA model (GeneSpring 6.1 and SAS 9.0) was used to identify differentially expressed genes. The P-values were adjusted for multiple comparisons using the false discovery rate approach, and a e2-fold differential criterion was applied. A subset of the differentially expressed genes was verified by real-time RT-PCR. The blastocyst rates for IVF and IVD embryos were 37% and 75%, respectively. There were 305, 365, and 200 genes differentially expressed between the AI and IVD, the IVF and IVD, and the AI and IVF comparisons, respectively. Interestingly, 44 differentially expressed genes were identified between the AI embryos and both the IVF and the IVD embryos, making these potential candidates for LOS. There were 61 genes differentially expressed between the IVF embryos and the AI and IVD embryos. The Gene Ontology categories 'RNA processing' and 'RNA binding' were over-represented among the genes that were down-regulated in the IVF embryos, indicating an effect of in vitro oocyte maturation/fertilization on embryonic gene expression. This work was supported by USDA grants to X.Y., H.A.L., and X.C.T.

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A Combined Survival Model Integrating Gene Expression and Alternative Splicing Events Provides Higher Predicative Power for Risk Stratification

Blood ◽

10.1182/blood.v116.21.1929.1929 ◽

2010 ◽

Vol 116 (21) ◽

pp. 1929-1929

Author(s):

Parantu K Shah ◽

Hervé Avet-Loiseau ◽

Stephane Minvielle ◽

Samir B. Amin ◽

Florence Magrangeas ◽

...

Keyword(s):

Gene Expression ◽

Alternative Splicing ◽

Risk Stratification ◽

Cell Lines ◽

Differentially Expressed Genes ◽

Differentially Expressed ◽

Differential Splicing ◽

Combined Model ◽

Healthy Donors ◽

Alternative Splicing Events

Abstract Abstract 1929 Considerable efforts have been spent evaluating impact of global gene expression profile on clinical outcome. Although significant correlation has been described between outcome and expressed gene signature, the overall predictability of such models have reached a plateau. Biologically this is expected as gene function is modulated at multiple levels. Besides the change in level of expression, post-transcriptional changes such as alternate splicing alter specificity of gene function and may affect the eventual outcome. Although various genes have normal alternate spliced form, dysregulation of alternative splicing that alters protein function has been implicated in number of disease processes including cancer. We have observed significant level of dysregulated splicing events in multiple myeloma (MM). We hypothesize that a combined model that includes dysregulated splicing events, besides level of expressed genes, may provide superior survival model in MM. To develop a combined model we have hybridized RNA isolated from CD138+ purified MM cells collected at the time of diagnosis from 170 newly-diagnosed patients treated homogeneously in tandem transplantation IFM trials, 23 MM cell lines and 6 Healthy donors on Affymetrix Exon 1.0 ST GeneChip arrays. Exon array not only provides an accurate measure of expression levels for genes, but also allows simultaneous identification of alternative splicing events. Pre-processing and normalization methods in aroma, affymetrix and robust multichip analysis model in FIRMA, followed by t-tests with Benjamini-Hochberg multiple hypothesis corrections were used respectively to identify differential expression and alternative splicing. We identified 1454 differentially expressed genes and 759 differential splicing events between healthy donors and MM patients, and 5476 differentially expressed genes and 4012 differential splicing events between healthy donors and MM cell lines. There are 1071 differentially expressed genes and 286 alternative spliced exons shared between MM samples and MM cell lines. Univariate survival analysis using FIRMA scores of exons identified a total of 89 genes with more than 10 alternative splicing events between healthy donors and MM patients associated with survival with Cox proportional hazard model and log-rank tests. We have now built 3 different survival models considering: 1) gene expression only, 2) alternative splicing only, and 3) a combined model integrating gene expression and alternative splicing events. We utilized refined regularized variable selection methods to handle these high-dimensional feature space. Our analysis suggests that composite model using gene expression and alternative splicing information performs significantly better than the gene expression only model in identifying high-risk patients, when the data were divided in median or quartiles. Specifically, the difference in overall survival is 32.6 months to 38.5 months using the median survival, and 18 months vs 23 months for median event free survival. We are currently in the process of validating the combined model. Our data suggests the need for inclusion of modifiers of transcriptome to develop a comprehensive model that will have higher predicative power for risk stratification as well as for selection of therapeutic intervention. Disclosures: Anderson: Millennium Pharmaceuticals: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau. Munshi:Millennium Pharmaceuticals: Honoraria, Speakers Bureau.

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Transcriptome Sequencing of the Spleen Reveals Antiviral Response Genes in Chickens Infected with CAstV

Viruses ◽

10.3390/v13122374 ◽

2021 ◽

Vol 13 (12) ◽

pp. 2374

Author(s):

Joanna Sajewicz-Krukowska ◽

Jan Paweł Jastrzębski ◽

Maciej Grzybek ◽

Katarzyna Domańska-Blicharz ◽

Karolina Tarasiuk ◽

...

Keyword(s):

Gene Expression ◽

Differentially Expressed Genes ◽

Egg Production ◽

Global Analysis ◽

Antiviral Response ◽

Differentially Expressed ◽

P Value ◽

Rna Seq ◽

Qrt Pcr

Astrovirus infections pose a significant problem in the poultry industry, leading to multiple adverse effects such as a decreased egg production, breeding disorders, poor weight gain, and even increased mortality. The commonly observed chicken astrovirus (CAstV) was recently reported to be responsible for the “white chicks syndrome” associated with an increased embryo/chick mortality. CAstV-mediated pathogenesis in chickens occurs due to complex interactions between the infectious pathogen and the immune system. Many aspects of CAstV–chicken interactions remain unclear, and there is no information available regarding possible changes in gene expression in the chicken spleen in response to CAstV infection. We aim to investigate changes in gene expression triggered by CAstV infection. Ten 21-day-old SPF White Leghorn chickens were divided into two groups of five birds each. One group was inoculated with CAstV, and the other used as the negative control. At 4 days post infection, spleen samples were collected and immediately frozen at −70 °C for RNA isolation. We analyzed the isolated RNA, using RNA-seq to generate transcriptional profiles of the chickens’ spleens and identify differentially expressed genes (DEGs). The RNA-seq findings were verified by quantitative reverse-transcription PCR (qRT-PCR). A total of 31,959 genes was identified in response to CAstV infection. Eventually, 45 DEGs (p-value < 0.05; log2 fold change > 1) were recognized in the spleen after CAstV infection (26 upregulated DEGs and 19 downregulated DEGs). qRT-PCR performed on four genes (IFIT5, OASL, RASD1, and DDX60) confirmed the RNA-seq results. The most differentially expressed genes encode putative IFN-induced CAstV restriction factors. Most DEGs were associated with the RIG-I-like signaling pathway or more generally with an innate antiviral response (upregulated: BLEC3, CMPK2, IFIT5, OASL, DDX60, and IFI6; downregulated: SPIK5, SELENOP, HSPA2, TMEM158, RASD1, and YWHAB). The study provides a global analysis of host transcriptional changes that occur during CAstV infection in vivo and proves that, in the spleen, CAstV infection in chickens predominantly affects the cell cycle and immune signaling.

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Genome-Wide Expression Analysis of Burkholderia pseudomallei Infection ina Hamster Model of Acute Melioidosis

Infection and Immunity ◽

10.1128/iai.00737-06 ◽

2006 ◽

Vol 74 (10) ◽

pp. 5465-5476 ◽

Cited By ~ 46

Author(s):

Apichai Tuanyok ◽

Marina Tom ◽

John Dunbar ◽

Donald E. Woods

Keyword(s):

Gene Expression ◽

Differentially Expressed Genes ◽

Energy Production ◽

Burkholderia Pseudomallei ◽

Expression Profiles ◽

Differentially Expressed ◽

Bacterial Growth Rate ◽

Hamster Model

ABSTRACT Burkholderia pseudomallei is the causative agent of melioidosis and represents a potential bioterrorism threat. In the current studies we have examined gene expression in B. pseudomallei in an animal model of acute melioidosis using whole-genome microarrays. Gene expression profiles were generated by comparing transcriptional levels of B. pseudomallei-expressed genes in infected hamster organs including liver, lung, and spleen following intraperitoneal and intranasal routes of infection to those from bacteria grown in vitro. Differentially expressed genes were similar in infected livers irrespective of the route of infection. Reduced expression of a number of housekeeping genes suggested a lower bacterial growth rate during infection. Energy production during growth in vivo involved specific biochemical pathways such as isomerization of 3-phosphoglycerate, catabolism of d-glucosamine and inositol, and biosynthesis of particular amino acids. In addition, the induction of genes known to be involved in oxidative phosphorylation including ubiquinol oxidase, ferredoxin oxidoreductase, and formate dehydrogenase enzymes suggested the use of alternative pathways for energy production, while the expression of genes coding for ATP-synthase and NADH-dehydrogenase enzymes was reduced. Our studies have identified differentially expressed genes which include potential virulence genes such as those for a putative phospholipase C and a putative two-component regulatory system, and they have also provided a better understanding of bacterial metabolism in response to the host environment during acute melioidosis.

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CircFAM13B promotes the proliferation of hepatocellular carcinoma by sponging miR-212, upregulating E2F5 expression and activating the P53 pathway

Cancer Cell International ◽

10.1186/s12935-021-02120-6 ◽

2021 ◽

Vol 21 (1) ◽

Author(s):

Ying Xie ◽

Xiaofeng Hang ◽

Wensheng Xu ◽

Jing Gu ◽

Yuanjing Zhang ◽

...

Keyword(s):

Gene Expression ◽

Hepatocellular Carcinoma ◽

Gene Expression Omnibus ◽

Differentially Expressed ◽

In Vivo Studies ◽

Circular Rnas ◽

Novel Target ◽

Underlying Mechanisms

Abstract Background Most of the biological functions of circular RNAs (circRNAs) and the potential underlying mechanisms in hepatocellular carcinoma (HCC) have not yet been discovered. Methods In this study, using circRNA expression data from HCC tumor tissues and adjacent tissues from the Gene Expression Omnibus database, we identified out differentially expressed circRNAs and verified them by qRT-PCT. Functional experiments were performed to evaluate the effects of circFAM13B in HCC in vitro and in vivo. Results We found that circFAM13B was the most significantly differentially expressed circRNA in HCC tissue. Subsequently, in vitro and in vivo studies also demonstrated that circFAM13B promoted the proliferation of HCC. Further studies revealed that circFAM13B, a sponge of miR-212, is involved in the regulation of E2F5 gene expression by competitively binding to miR-212, inhibits the activation of the P53 signalling pathway, and promotes the proliferation of HCC cells. Conclusions Our findings revealed the mechanism underlying the regulatory role played by circFAM13B, miR-212 and E2F5 in HCC. This study provides a new theoretical basis and novel target for the clinical prevention and treatment of HCC.

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Differential gene expression analysis of ankylosing spondylitis shows deregulation of the HLA-DRB, HLA-DQB, ITM2A, and CTLA4 genes

Egyptian Journal of Medical Human Genetics ◽

10.1186/s43042-021-00161-0 ◽

2021 ◽

Vol 22 (1) ◽

Author(s):

Rowan AlEjielat ◽

Anas Khaleel ◽

Amneh H. Tarkhan

Keyword(s):

Gene Expression ◽

Ankylosing Spondylitis ◽

Differentially Expressed Genes ◽

Gene Network ◽

Disease Diagnosis ◽

Receptor Activation ◽

Functional Enrichment ◽

Differentially Expressed ◽

Inflammatory Disorder ◽

Data Set

Abstract Background Ankylosing spondylitis (AS) is a rare inflammatory disorder affecting the spinal joints. Although we know some of the genetic factors that are associated with the disease, the molecular basis of this illness has not yet been fully elucidated, and the genes involved in AS pathogenesis have not been entirely identified. The current study aimed at constructing a gene network that may serve as an AS gene signature and biomarker, both of which will help in disease diagnosis and the identification of therapeutic targets. Previously published gene expression profiles of 16 AS patients and 16 gender- and age-matched controls that were profiled on the Illumina HumanHT-12 V3.0 Expression BeadChip platform were mined. Patients were Portuguese, 21 to 64 years old, were diagnosed based on the modified New York criteria, and had Bath Ankylosing Spondylitis Disease Activity Index scores > 4 and Bath Ankylosing Spondylitis Functional Index scores > 4. All patients were receiving only NSAIDs and/or sulphasalazine. Functional enrichment and pathway analysis were performed to create an interaction network of differentially expressed genes. Results ITM2A, ICOS, VSIG10L, CD59, TRAC, and CTLA-4 were among the significantly differentially expressed genes in AS, but the most significantly downregulated genes were the HLA-DRB6, HLA-DRB5, HLA-DRB4, HLA-DRB3, HLA-DRB1, HLA-DQB1, ITM2A, and CTLA-4 genes. The genes in this study were mostly associated with the regulation of the immune system processes, parts of cell membrane, and signaling related to T cell receptor and antigen receptor, in addition to some overlaps related to the IL2 STAT signaling, as well as the androgen response. The most significantly over-represented pathways in the data set were associated with the “RUNX1 and FOXP3 which control the development of regulatory T lymphocytes (Tregs)” and the “GABA receptor activation” pathways. Conclusions Comprehensive gene analysis of differentially expressed genes in AS reveals a significant gene network that is involved in a multitude of important immune and inflammatory pathways. These pathways and networks might serve as biomarkers for AS and can potentially help in diagnosing the disease and identifying future targets for treatment.

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Behavioral sensitization induced by methamphetamine causes differential alterations in gene expression and histone acetylation of the prefrontal cortex in rats

BMC Neuroscience ◽

10.1186/s12868-021-00616-5 ◽

2021 ◽

Vol 22 (1) ◽

Author(s):

Hui Li ◽

Jing-An Chen ◽

Qian-Zhi Ding ◽

Guan-Yi Lu ◽

Ning Wu ◽

...

Keyword(s):

Gene Expression ◽

Prefrontal Cortex ◽

Histone Acetylation ◽

Differentially Expressed Genes ◽

Behavioral Sensitization ◽

Functional Enrichment ◽

Differentially Expressed ◽

Adult Rats ◽

Mrna Microarray ◽

H3 Acetylation

Abstract Background Methamphetamine (METH) is one of the most widely abused illicit substances worldwide; unfortunately, its addiction mechanism remains unclear. Based on accumulating evidence, changes in gene expression and chromatin modifications might be related to the persistent effects of METH on the brain. In the present study, we took advantage of METH-induced behavioral sensitization as an animal model that reflects some aspects of drug addiction and examined the changes in gene expression and histone acetylation in the prefrontal cortex (PFC) of adult rats. Methods We conducted mRNA microarray and chromatin immunoprecipitation (ChIP) coupled to DNA microarray (ChIP-chip) analyses to screen and identify changes in transcript levels and histone acetylation patterns. Functional enrichment analyses, including Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses, were performed to analyze the differentially expressed genes. We then further identified alterations in ANP32A (acidic leucine-rich nuclear phosphoprotein-32A) and POU3F2 (POU domain, class 3, transcription factor 2) using qPCR and ChIP-PCR assays. Results In the rat model of METH-induced behavioral sensitization, METH challenge caused 275 differentially expressed genes and a number of hyperacetylated genes (821 genes with H3 acetylation and 10 genes with H4 acetylation). Based on mRNA microarray and GO and KEGG enrichment analyses, 24 genes may be involved in METH-induced behavioral sensitization, and 7 genes were confirmed using qPCR. We further examined the alterations in the levels of the ANP32A and POU3F2 transcripts and histone acetylation at different periods of METH-induced behavioral sensitization. H4 hyperacetylation contributed to the increased levels of ANP32A mRNA and H3/H4 hyperacetylation contributed to the increased levels of POU3F2 mRNA induced by METH challenge-induced behavioral sensitization, but not by acute METH exposure. Conclusions The present results revealed alterations in transcription and histone acetylation in the rat PFC by METH exposure and provided evidence that modifications of histone acetylation contributed to the alterations in gene expression caused by METH-induced behavioral sensitization.

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