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Expressed Genes (Degs) and Annotated Under GO, COG, KEGG and TF bioRxiv preprint doi: https://doi.org/10.1101/563916; this version posted March 1, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license. 1 Transcriptome Profiling of Mouse Brain and Lung under 2 Dip2A Regulation Using RNA-Sequencing 3 4 Rajiv kumar sah1, Anlan Yang1, Fatoumata Binta Bah1, Salah Adlat1, Ameer Ali 5 Bohio2, Zin Mar Oo1, Chenhao Wang1, May zun Zaw Myint1, Noor Bahadar1, 6 Luqing Zhang1,2*, Xuechao Feng1,2*, Yaowu Zheng1,2* 7 1 Transgenic Research Center, School of Life Sciences, Northeast Normal University, 8 Changchun, China, 9 2 Key Laboratory of Molecular Epigenetics of Ministry of Education, Northeast 10 Normal University, Changchun, China, 11 12 * [email protected] (LQZ), *[email protected] (XCF); [email protected] (YWZ) 13 Abstract: 14 Disconnected interacting 2 homolog A (DIP2A) gene is highly 15 expressed in nervous system and respiratory system of developing 16 embryos. However, genes regulated by Dip2a in developing brain and 17 lung have not been systemically studied. Transcriptome of brain and lung 18 in embryonic 19.5 day (E19.5) were compared between wild type and 19 Dip2a-/- mice. Total RNAs were extracted from brain and lung of E19.5 20 embryos for RNA-Seq. Clean reads were mapped to mouse reference 21 sequence (mm9) using Tophat and assembled into transcripts by 22 Cufflinks. Edge R and DESeq were applied to identify differentially 1 bioRxiv preprint doi: https://doi.org/10.1101/563916; this version posted March 1, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license. 23 expressed genes (DEGs) and annotated under GO, COG, KEGG and TF. 24 An average of 50 million reads per sample was mapped to the reference 25 sequence. A total of 214 DEGs were detected in brain (82 up and 132 26 down) and 1900 DEGs in lung (1259 up and 641 down). GO enrichment 27 analysis indicated that DEGs in both Brain and Lung were mainly 28 enriched in biological processes ‘DNA-templated transcription and 29 Transcription from RNA polymerase II promoter’, ‘multicellular 30 organism development’, ‘cell differentiation’ and ‘apoptotic process’. In 31 addition, COG classification showed that both were mostly involved in 32 ‘Replication, Recombination and Repair’, ‘Signal transduction and 33 mechanism’, ‘Translation, Ribosomal structure and Biogenesis’ and 34 ‘Transcription’. KEGG enrichment analysis showed that brain was 35 mainly enriched in ‘Thryoid cancer’ pathway whereas lung in 36 ‘Complement and Coagulation Cascades’ pathway. Transcription factor 37 (TF) annotation analysis identified Zinc finger domain containing (ZF) 38 proteins were mostly regulated in lung and brain. Interestingly, study 39 identified genes Skor2, Gpr3711, Runx1, Erbb3, Frmd7, Fut10, Sox11, 40 Hapln1, Tfap2c and Plxnb3 from brain that play important roles in 41 neuronal cell maturation, differentiation and survival; genes Hoxa5, 42 Eya1, Ctsh, Erff1, Lama1, Lama2, Rspo2, Sox11, Spry4, Shh, Igf1 and 43 Wnt7a from lung are important in lung development and morphogenesis. 44 Expression levels of the candidate genes were validated by qRT-PCR. 2 bioRxiv preprint doi: https://doi.org/10.1101/563916; this version posted March 1, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license. 45 Genome wide transcriptional analysis using wild type and Dip2a 46 knockout mice in brain and lung at embryonic day 19.5 (E19.5) provided 47 a genetic basis of molecular function of these genes. 48 49 Introduction 50 DIP2A is a member of Disconnected (disco)-interacting 2 (DIP2) 51 protein family whose molecular anatomical function remains to be 52 clarified. DIP2A was firstly identified in Drosophila as a novel 53 transcription factor that interacts with Disconnected (disco) gene needed 54 for proper neural connection during visual system development in 55 Drosophila [1-3]. Previous studies have shown that Dip2A is highly 56 expressed in human brain and may play a role in axon patterning in 57 Central Nervous System (CNS) [4]. Bioinformatics analysis using 58 Homolog Gene suggests that DIP2A is a receptor molecule with DMAP, 59 AMP and CaiC binding domains [5]. At DNA replication site, DIP2A, in 60 a complex with DMAP1-DNMT1-HDAC, regulates neurite outgrowth 61 and synaptic plasticity [6]. Moreover, Dip2a has been previously 62 identified as a risk gene associated with neurodevelopment diseases like 63 autism spectrum disorder, Development dyslexia and Alzheimer diseases 64 [7-9]. These all evidence strongly supports the role of Dip2a gene in both 65 vertebrate and invertebrate nervous system development. However, 66 which biological process or molecular function is regulated by Dip2a 3 bioRxiv preprint doi: https://doi.org/10.1101/563916; this version posted March 1, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license. 67 gene during embryonic brain development is not known. 68 In order to systematically understand expression pattern and 69 physiological role of Dip2a gene, Dip2a-LacZ knockin and Dip2a 65kb 70 knockout (Dip2a-/-) mouse models were generated using CRISPR/Cas9 71 system [10]. Dip2a expression via endogenous expression of 72 β-Galactosidase gene (LacZ) have shown that Dip2a is highly expressed 73 in brain neurons, retinal ganglia cell, reproductive, vascular and Lung 74 tissue in adult and ectodermal tissue in developing embryos [11]. RNA 75 sequencing (RNA-Seq) has rapidly emerged as a favorite approach for 76 high throughput gene expression and function studies. Through 77 RNA-Seq, gene expression and gene interactions at any time point or in a 78 particular tissue can be investigated [12]. In present study, Transcriptome 79 (RNA-seq) analysis of E19.5 brain and lung of WT and Dip2a-/- embryo 80 was performed. 81 Dip2a role in brain and lung development has not been studied before. 82 A global Transcriptome analysis of brain and lung will help us in 83 understanding of Dip2a function in regulating brain and lung 84 development. A total of 214 genes in brain and 1900 genes in lung were 85 identified differentially expressed under Dip2A, suggesting that these 86 genes are potentially relevant to brain and lung development and function. 87 Those genes are further explicated and discussed in this study. 88 4 bioRxiv preprint doi: https://doi.org/10.1101/563916; this version posted March 1, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license. 89 Materials and methods 90 Animals 91 All procedures were conducted following guidelines recommended in 92 the guide for Care and Use of Laboratory Animals of National Institutes 93 of Health with approval of Institutional Animal Care and Use Committee 94 of Northeast Normal University (NENU/IACUC, AP2013011). Mice 95 were housed in clean facility in individual IVC cages under a normal 96 12:12h light:dark cycles in a temperature of 20oC and humidity 50 ± 20% 97 in Northeast Normal University. All mice were anesthetized before 98 euthanasia with 1% pentobarbital at a dose of 10mg/kg and all effort was 99 made to minimize suffering. 100 RNA isolation and library preparation for RNA-Seq 101 Dip2a heterozygous mice (Dip2a+/-) were intercrossed, female mice 102 checked for presence of copulation plug (Vaginal plug) and designated as 103 E0.5 day. At age of E19.5, pregnant dams were euthanized and embryos 104 were collected on ice-cold 1X PBS. Brains and lungs were dissected out 105 from each embryo and frozen immediately in liquid nitrogen. Total RNA 106 from brain and lung was isolated using RNAiso plus reagent (Takara, 107 Dalian) in accordance with the manufacturer’s instruction and followed 108 by additional step of DNase I digestion to eliminate genomic DNA 109 contamination. The quality and purity of RNA was checked by Nano drop 110 ND-1000 spectrophotometer (Thermo Fisher Scientific, USA) and 5 bioRxiv preprint doi: https://doi.org/10.1101/563916; this version posted March 1, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license. 111 Agilent 2100 Bio analyzer (Santa Clara, CA, USA). Library was 112 constructed and sequenced using Illumina HiseqTM 2500 (Biomarker, 113 Beijing, China). 114 Sequence Mapping, assembly and gene annotation 115 Clean data (clean reads) were obtained by filtering out adapter only 116 reads, ambiguous “N” base containing reads (N>5%) and reads with low 117 Q-value (< 10) from raw data. The clean reads were then mapped to 118 mouse reference genome (mm9) using Tophat v2.2.0 that allows up to 119 two mismatches [13]. The mapped reads were then assembled into 120 transcripts using Cufflinks v2.2.1 [13]. The transcripts were defined as 121 unigenes and analyzed via BLASTX alignment (E-value < 1.0E-5) with 122 NR (NCBI non-redundant protein) and Swiss-Prot databases under 123 BLAST2GO platform [14-15].
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