Regulation of human interferon signaling by transposon exonization.

Innate immune signaling is essential for clearing pathogens and damaged cells, and must be tightly regulated to avoid excessive inflammation or autoimmunity. Here, we found that the alternative splicing of exons derived from transposable elements is a key mechanism controlling immune signaling in human cells. By analyzing long-read transcriptome datasets, we identified numerous transposon exonization events predicted to generate functional protein variants of immune genes, including the type I interferon receptor IFNAR2. We demonstrated that the transposon-derived isoform of IFNAR2 is more highly expressed than the canonical isoform in almost all tissues, and functions as a decoy receptor that potently inhibits interferon signaling including in cells infected with SARS-CoV-2. Our findings uncover a primate-specific axis controlling interferon signaling and show how a transposon exonization event can be co-opted for immune regulation.

Overexpression screen of chromosome 21 genes reveals modulators of Sonic hedgehog signaling relevant to Down syndrome.

Trisomy 21 and mutations in the Sonic hedgehog (SHH) signaling pathway cause overlapping and pleiotropic phenotypes including cerebellar hypoplasia, craniofacial abnormalities, congenital heart defects and Hirschsprung disease. Trisomic cells derived from individuals with Down syndrome possess deficits in SHH signaling, suggesting that overexpression of human chromosome 21 genes may contribute to SHH-associated phenotypes by disrupting normal SHH signaling during development. However, chromosome 21 does not encode any known components of the canonical SHH pathway. Here, we sought to identify chromosome 21 genes that modulate SHH signaling by overexpressing 163 chromosome 21 cDNAs in a series of SHH-responsive mouse cell lines. We confirmed overexpression of trisomic candidate genes using RNA sequencing in the cerebella of Ts65Dn and TcMAC21 mice, model systems for Down syndrome. Our findings indicate that some human chromosome 21 genes, including DYRK1A, upregulate SHH signaling, whereas others, such as HMGN1, inhibit SHH signaling. Individual overexpression of four genes (B3GALT5, ETS2, HMGN1 and MIS18A) inhibits the SHH-dependent proliferation of primary granule cell precursors. Our study prioritizes dosage-sensitive chromosome 21 genes for future mechanistic studies. Identification of the genes that modulate SHH signaling may suggest new therapeutic avenues for ameliorating Down syndrome phenotypes.

Expression of HLA and Autoimmune Pathway Genes in Liver Biopsies of Young Subjects With Autoimmune Hepatitis Type 1.

OBJECTIVES: To test the hypothesis that autoimmune hepatitis (AIH type I) in young subjects is due to genetic differences in proinflammatory genes responding to viral triggers in patients and controls. METHODS: Intrahepatic gene expression was compared between AIH type I (n = 24, age 9-30 years) patients (hereafter referred to as the AIH group) and controls (n = 21, age 4-25 years). RNA sequencing was performed on complementary DNA (cDNA) libraries made from total RNA extracted from formalin-fixed paraffin-embedded (FFPE) liver biopsy samples. Gene expression levels were quantified, and differentially expressed genes were functionally analyzed. Pathway analysis was performed using the databases Kyoto Encyclopedia of Genes and Genomes (KEGG) and PANTHER. The remaining sequences were mapped to the RefSeq complete set of viral genomes. RESULTS: Differential gene analysis identified 181 genes that were significantly differentially expressed (136 upregulated in the AIH group). Autoimmune pathway genes such as CD19 and CD20 which are important in B cell regulation and maturation as well as, CD8 and LY9 , which are T-cell related, were upregulated in our AIH group. Genes implicated in AIH pathogenesis including CXCL10 , which is thought to be associated with AIH severity and progression, complement genes ( C1QA, C1QB , and C1QC ), and human leucocyte antigen ( HLA ) genes ( HLA-DRB1, HLA-DRA, HLA-B , and HLA-C ) were upregulated in samples from the AIH group. Specific viral etiologies were not found. CONCLUSIONS: Unbiased next-generation sequencing and differential gene expression analysis of the AIH group has not only added support for the role of B cells in the pathogenesis and treatment of AIH but also has introduced potential new therapeutic targets: CXCL10 (anti- CXCL10 ) and several complement system-related genes.

CaMKII oxidation is a critical performance/disease trade-off acquired at the dawn of vertebrate evolution.

Antagonistic pleiotropy is a foundational theory that predicts aging-related diseases are the result of evolved genetic traits conferring advantages early in life. Here we examine CaMKII, a pluripotent signaling molecule that contributes to common aging-related diseases, and find that its activation by reactive oxygen species (ROS) was acquired more than half-a-billion years ago along the vertebrate stem lineage. Functional experiments using genetically engineered mice and flies reveal ancestral vertebrates were poised to benefit from the union of ROS and CaMKII, which conferred physiological advantage by allowing ROS to increase intracellular Ca2+ and activate transcriptional programs important for exercise and immunity. Enhanced sensitivity to the adverse effects of ROS in diseases and aging is thus a trade-off for positive traits that facilitated the early and continued evolutionary success of vertebrates.

Development of Methods to Extract RNA From Archived Pediatric Needle Liver Biopsies to Produce Sequencing Data.

ABSTRACT: Genetic susceptibility has been proposed as etiopathogenic in several pediatric liver diseases including autoimmune hepatitis (AIH). High throughput sequencing (HTPS) has been applied to archived needle liver biopsies obtained from adults but rarely to pediatric biopsies. For conclusive diagnosis of AIH, most subjects have an initial formalin-fixed paraffin embedded (FFPE) needle liver biopsy that is eventually archived and may be stored for decades. OBJECTIVE: Our goal was to develop methods to utilize tissue from archived needle liver biopsies for extraction of RNA sufficient to produce HTPS data. METHODS: We extracted total RNA from 45 FFPE needle liver biopsy samples (24 AIH type 1 patients and 21 controls [ages 15_11 and 19_10]; biopsy storage time 0.5-20 years) and constructed cDNA libraries that were then sequenced on an Illumina HiSeq2000 platform. RESULTS: Forty (89%) of the libraries produced high-quality sequences for further analyses. The average number of sequences obtained per library from HTPS was 55,136,519 (range 14,914,291-184,027,499). There was a significant inverse relationship between the number of human reads obtained and the age of the specimen (P < 2_10_7). It was possible to classify more than 90% of the reads as known genes in samples that had been stored for less than 10 years. CONCLUSIONS: Archived needle liver biopsies can be used for sequence based interrogation of the etiologic origins of complex liver diseases of young subjects, such as AIH.

A non-mosaic transchromosomic mouse model of down syndrome carrying the long arm of human chromosome 21.

Animal models of Down syndrome (DS), trisomic for human chromosome 21 (HSA21) genes or orthologs, provide insights into better understanding and treatment options. The only existing transchromosomic (Tc) mouse DS model, Tc1, carries a HSA21 with over 50 protein coding genes (PCGs) disrupted. Tc1 is mosaic, compromising interpretation of results. Here, we "clone" the 34 MB long arm of HSA21 (HSA21q) as a mouse artificial chromosome (MAC). Through multiple steps of microcell-mediated chromosome transfer, we created a new Tc DS mouse model, Tc(HSA21q;MAC)1Yakaz ("TcMAC21"). TcMAC21 is not mosaic and contains 93% of HSA21q PCGs that are expressed and regulatable. TcMAC21 recapitulates many DS phenotypes including anomalies in heart, craniofacial skeleton and brain, molecular/cellular pathologies, and impairments in learning, memory and synaptic plasticity. TcMAC21 is the most complete genetic mouse model of DS extant and has potential for supporting a wide range of basic and preclinical research.

A Prospective Study of the Urinary and Gastrointestinal Microbiome in Prepubertal Males.

OBJECTIVE: To determine if urinary microbial communities similar to those described in adults exist in children and to profile the urinary and gastrointestinal microbiome in children presenting to urology for both routine and complex urologic procedures. METHODS: Prepubertal boys (n = 20, ages 3 months-8 years; median age 15 months) who required elective urologic procedures were eligible. Urine samples were collected via sterile catheterization and fecal samples were obtained by rectal swabs. DNA was extracted from urine pellet and fecal samples and subjected to bacterial profiling via 16S rDNA Illumina sequencing and 16S rDNA quantitative polymerase chain reaction. We assessed within and between sample diversity and differential species abundance between samples. RESULTS: Urine samples had low bacterial biomass that reflected the presence of bacterial populations. The most abundant genera detected in urine samples are not common to skin microbiota and several of the genera have been previously identified in the urinary microbiome of adults. We report presumably atypical compositional differences in both the urinary and gastrointestinal microbiome in children with prior antibiotic exposure and highlight an important case of a child who had undergone lifelong antibiotic treatment as prophylaxis for congenital abnormalities. CONCLUSION: This study provides one of the first characterizations of the urinary microbiome in prepubertal males. Defining the baseline healthy microbiome in children may lay the foundation for understanding the long-term impact of factors such as antibiotic use in the development of a healthy microbiome as well as the development of future urologic and gastrointestinal diseases.

RNA sequencing of cancer reveals novel splicing alterations.

Breast cancer transcriptome acquires a myriad of regulation changes, and splicing is critical for the cell to "tailor-make" specific functional transcripts. We systematically revealed splicing signatures of the three most common types of breast tumors using RNA sequencing: TNBC, non-TNBC and HER2-positive breast cancer. We discovered subtype specific differentially spliced genes and splice isoforms not previously recognized in human transcriptome. Further, we showed that exon skip and intron retention are predominant splice events in breast cancer. In addition, we found that differential expression of primary transcripts and promoter switching are significantly deregulated in breast cancer compared to normal breast. We validated the presence of novel hybrid isoforms of critical molecules like CDK4, LARP1, ADD3, and PHLPP2. Our study provides the first comprehensive portrait of transcriptional and splicing signatures specific to breast cancer sub-types, as well as previously unknown transcripts that prompt the need for complete annotation of tissue and disease specific transcriptome.

Genome-guided transcriptome assembly in the age of next-generation sequencing.

Next-generation sequencing technologies provide unprecedented power to explore the repertoire of genes and their alternative splice variants, collectively defining the transcriptome of a species in great detail. However, assembling the short reads into full-length gene and transcript models presents significant computational challenges. We review current algorithms for assembling transcripts and genes from next-generation sequencing reads aligned to a reference genome, and lay out areas for future improvements.

A method for identifying alternative or cryptic donor splice sites within gene and mRNA sequences. Comparisons among sequences from vertebrates, echinoderms and other groups.

BACKGROUND: As the amount of genome sequencing data grows, so does the problem of computational gene identification, and in particular, the splicing signals that flank exon borders. Traditional methods for identifying splicing signals have been created and optimized using sequences from model organisms, mostly vertebrate and yeast species. However, as genome sequencing extends across the animal kingdom and includes various invertebrate species, the need for mechanisms to recognize splice signals in these organisms increases as well. With that aim in mind, we generated a model for identifying donor and acceptor splice sites that was optimized using sequences from the purple sea urchin, Strongylocentrotus purpuratus. This model was then used to assess the possibility of alternative or cryptic splicing within the highly variable immune response gene family known as 185/333. RESULTS: A donor splice site model was generated from S. purpuratus sequences that incorporates non-adjacent dependences among positions within the 9 nt splice signal and uses position weight matrices to determine the probability that the site is used for splicing. The Purpuratus model was shown to predict splice signals better than a similar model created from vertebrate sequences. Although the Purpuratus model was able to correctly predict the true splice sites within the 185/333 genes, no evidence for alternative or trans-gene splicing was observed. CONCLUSION: The data presented herein describe the first published analyses of echinoderm splice sites and suggest that the previous methods of identifying splice signals that are based largely on vertebrate sequences may be insufficient. Furthermore, alternative or trans-gene splicing does not appear to be acting as a diversification mechanism in the 185/333 gene family.

A non-redundant microarray of genes for two related bacteria.

A microarray with sequences from the annotated open reading frames (ORFs) in Salmonella enterica subspecies 1, serovar Typhimurium was supplemented with annotated chromosomal ORFs from serovar Typhi that are divergent from Typhimurium (>10% DNA sequence divergence). This non- redundant array was used to (i) measure changes in gene copy number in DNA from actively growing versus stationary Typhi and (ii) to reveal the transcriptional response of Typhi to peroxide, a stress similar to that experienced when they are phagocytosed by macrophages. In S.enterica subspecies 1, pairs of genomes differ in the presence or absence of approximately 10% of their genes. An array twice the size of that needed to cover all ORFs for one genome could carry close homologs of all the ORFs for 10 genomes. Non-redundant DNA arrays could be constructed for any group of closely related organisms that differ by the presence and absence of a few genes.