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SULT4A1 Deletion in Zebrafish Alters Expression of Genes Implicated in Neurological Disorders

Sulfotransferase 4A1 (SULT4A1), a member of the cytosolic sulfotransferases (SULTs) superfamily, was first identified and molecularly cloned by our lab in 2000, from the brains of humans and rats. Considered an orphan enzyme, no known substrate or function has been ascribed to SULT4A1; however, linkage disequilibrium studies have suggested SULT4A1 may play a role in schizophrenia susceptibility. Of all the SULTs, SULT4A1 is the most conserved family member and is expressed in a wide variety of vertebrates. For example, zebrafish (Danio rerio) and humans share approximately 87% sequence identity and 92% amino acid similarity, making zebrafish a candidate model to study SULT4A1. To that end, our lab has published several studies on the potential function of SULT4A1 in zebrafish. First, we reported an upregulation of genes associated with phototransduction, as well as, genes associated with circadian rhythm after SULT4A1 knockdown using specific morpholino antisense oligonucleotides and deep RNA sequencing (RNA‐Seq) on zebrafish embryos. To further probe the role of SULT4A1 in zebrafish, we generated SULT4A1 knockout mutants using transcription activator‐like effector nucleases (TALEN) technology. Indeed, we identified an 8‐nucleotide (Δ8) deletion in exon 2 of SULT4A1, causing a premature stop codon. Casual observations by our lab noted SULT4A1Δ8/Δ8 fish displayed extended periods of inactivity during daylight hours compared to wild type (WT) SULT4A fish, like humans, zebrafish are a diurnal species. EthoVision tracking software was used to analyze behavior in WT and mutant fish over a 48 hour period. Statistical analysis confirmed SULT4A1Δ8/Δ8 showed increased inactivity levels, inactivity bout length, and frequency when compared to SULT4A1WT/WT zebrafish. To identify potential biological pathways altered in SULT4A1Δ8/Δ8 fish, RNA‐Seq was performed on both WT and SULT4A1 mutant neuronal and eye tissues. Here we report, statistical analysis of the sequencing data showed a total of 82 genes/transcripts (48 upregulated; 34 downregulated) significantly altered between SULT4A1Δ8/Δ8 and SULT4A1WT/WT zebrafish. Pathway analysis of these genes/transcripts were associated with neurological disease (p=1.8E‐6), developmental disorders (p=1.8E‐6), and auditory disease (p=1.80E‐6). Among the top genes that were altered several have been linked to neurological disorders including LRFN5 (schizophrenia), FOLH1 (circadian entrainment), while SPINK4 and STK31 are linked with epilepsy. The 82 altered genes/transcripts expressed were localized to 11 of the 25 zebrafish chromosomes. Moreover, 56% (47/82) of the genes/transcripts were localized to chromosomes 1, 12, 14, and 16 indicating a selective genomic effect of SULT4A1 deletion, although the significance remains unclear. Sequencing analysis suggests that SULT4A1 is important in normal brain biochemistry and homeostasis in zebrafish. Currently, we are in the process of validating gene expression data by quantitative PCR (qPCR) and confirming the behavioral phenotype in the progeny of SULT4A1Δ8/Δ8 zebrafish. Investigation into possible SULT4A1 interaction partners and potential SULT4A1 substrates is ongoing.