The androgen receptor interacts with GATA3 to transcriptionally regulate a luminal epithelial cell phenotype in breast cancer.

BACKGROUND: The androgen receptor (AR) is a tumor suppressor in estrogen receptor (ER) positive breast cancer, a role sustained in some ER negative breast cancers. Key factors dictating AR genomic activity in a breast context are largely unknown. Herein, we employ an unbiased chromatin immunoprecipitation-based proteomic technique to identify endogenous AR interacting co-regulatory proteins in ER positive and negative models of breast cancer to gain new insight into mechanisms of AR signaling in this disease. RESULTS: The DNA-binding factor GATA3 is identified and validated as a novel AR interacting protein in breast cancer cells irrespective of ER status. AR activation by the natural ligand 5α-dihydrotestosterone (DHT) increases nuclear AR-GATA3 interactions, resulting in AR-dependent enrichment of GATA3 chromatin binding at a sub-set of genomic loci. Silencing GATA3 reduces but does not prevent AR DNA binding and transactivation of genes associated with AR/GATA3 co-occupied loci, indicating a co-regulatory role for GATA3 in AR signaling. DHT-induced AR/GATA3 binding coincides with upregulation of luminal differentiation genes, including EHF and KDM4B, established master regulators of a breast epithelial cell lineage. These findings are validated in a patient-derived xenograft model of breast cancer. Interaction between AR and GATA3 is also associated with AR-mediated growth inhibition in ER positive and ER negative breast cancer. CONCLUSIONS: AR and GATA3 interact to transcriptionally regulate luminal epithelial cell differentiation in breast cancer regardless of ER status. This interaction facilitates the tumor suppressor function of AR and mechanistically explains why AR expression is associated with less proliferative, more differentiated breast tumors and better overall survival in breast cancer.

Differential allelic representation (DAR) identifies candidate eQTLs and improves transcriptome analysis.

In comparisons between mutant and wild-type genotypes, transcriptome analysis can reveal the direct impacts of a mutation, together with the homeostatic responses of the biological system. Recent studies have highlighted that, when the effects of homozygosity for recessive mutations are studied in non-isogenic backgrounds, genes located proximal to the mutation on the same chromosome often appear over-represented among those genes identified as differentially expressed (DE). One hypothesis suggests that DE genes chromosomally linked to a mutation may not reflect functional responses to the mutation but, instead, result from an unequal distribution of expression quantitative trait loci (eQTLs) between sample groups of mutant or wild-type genotypes. This is problematic because eQTL expression differences are difficult to distinguish from genes that are DE due to functional responses to a mutation. Here we show that chromosomally co-located differentially expressed genes (CC-DEGs) are also observed in analyses of dominant mutations in heterozygotes. We define a method and a metric to quantify, in RNA-sequencing data, localised differential allelic representation (DAR) between those sample groups subjected to differential expression analysis. We show how the DAR metric can predict regions prone to eQTL-driven differential expression, and how it can improve functional enrichment analyses through gene exclusion or weighting-based approaches. Advantageously, this improved ability to identify probable eQTLs also reveals examples of CC-DEGs that are likely to be functionally related to a mutant phenotype. This supports a long-standing prediction that selection for advantageous linkage disequilibrium influences chromosome evolution. By comparing the genomes of zebrafish (Danio rerio) and medaka (Oryzias latipes), a teleost with a conserved ancestral karyotype, we find possible examples of chromosomal aggregation of CC-DEGs during evolution of the zebrafish lineage. Our method for DAR analysis requires only RNA-sequencing data, facilitating its application across new and existing datasets.

Unbiased gene expression analysis of the delayed fracture healing observed in Zucker diabetic fatty rats.

Aims: Impaired fracture repair in patients with type 2 diabetes mellitus (T2DM) is not fully understood. In this study, we aimed to characterize the local changes in gene expression (GE) associated with diabetic fracture. We used an unbiased approach to compare GE in the fracture callus of Zucker diabetic fatty (ZDF) rats relative to wild-type (WT) littermates at three weeks following femoral osteotomy. Methods: Zucker rats, WT and homozygous for leptin receptor mutation (ZDF), were fed a moderately high-fat diet to induce T2DM only in the ZDF animals. At ten weeks of age, open femoral fractures were simulated using a unilateral osteotomy stabilized with an external fixator. At three weeks post-surgery, the fractured femur from each animal was retrieved for analysis. Callus formation and the extent of healing were assessed by radiograph and histology. Bone tissue was processed for total RNA extraction and messenger RNA (mRNA) sequencing (mRNA-Seq). Results: Radiographs and histology demonstrated impaired fracture healing in ZDF rats with incomplete bony bridge formation and an influx of intramedullary inflammatory tissue. In comparison, near-complete bridging between cortices was observed in Sham WT animals. Of 13,160 genes, mRNA-Seq analysis identified 13 that were differentially expressed in ZDF rat callus, using a false discovery rate (FDR) threshold of 10%. Seven genes were upregulated with high confidence (FDR = 0.05) in ZDF fracture callus, most with known roles in inflammation. Conclusion: These findings suggest that elevated or prolonged inflammation contributes to delayed fracture healing in T2DM. The identified genes may be used as biomarkers to monitor and treat delayed fracture healing in diabetic patients.

The Use of Zebrafish in Transcriptome Analysis of the Early Effects of Mutations Causing Early Onset Familial Alzheimer's Disease and Other Inherited Neurodegenerative Conditions.

The degree to which non-human animals can be used to model Alzheimer's disease is a contentious issue, particularly as there is still widespread disagreement regarding the pathogenesis of this neurodegenerative dementia. The currently popular transgenic models are based on artificial expression of genes mutated in early onset forms of familial Alzheimer's disease (EOfAD). Uncertainty regarding the veracity of these models led us to focus on heterozygous, single mutations of endogenous genes (knock-in models) as these most closely resemble the genetic state of humans with EOfAD, and so incorporate the fewest assumptions regarding pathological mechanism. We have generated a number of lines of zebrafish bearing EOfAD-like and non-EOfAD-like mutations in genes equivalent to human PSEN1, PSEN2, and SORL1. To analyze the young adult brain transcriptomes of these mutants, we exploited the ability of zebrafish to produce very large families of simultaneous siblings composed of a variety of genotypes and raised in a uniform environment. This "intra-family" analysis strategy greatly reduced genetic and environmental "noise" thereby allowing detection of subtle changes in gene sets after bulk RNA sequencing of entire brains. Changes to oxidative phosphorylation were predicted for all EOfAD-like mutations in the three genes studied. Here we describe some of the analytical lessons learned in our program combining zebrafish genome editing with transcriptomics to understand the molecular pathologies of neurodegenerative disease.

Determination of Tr1 cell populations correlating with distinct activation states in acute IAV infection.

Type I regulatory (Tr1) cells are defined as FOXP3-IL-10-secreting clusters of differentiation (CD4+) T cells that contribute to immune suppression and typically express the markers LAG-3 and CD49b and other co-inhibitory receptors. These cells have not been studied in detail in the context of the resolution of acute infection in the lung. Here, we identify FOXP3- interleukin (IL)-10+ CD4+ T cells transiently accumulating in the lung parenchyma during resolution of the response to sublethal influenza A virus (IAV) infection in mice. These cells were dependent on IL-27Rα, which was required for timely recovery from IAV-induced weight loss. LAG-3 and CD49b were not generally co-expressed by FOXP3- IL-10+ CD4+ T cells in this model and four populations of these cells based on LAG-3 and CD49b co-expression were apparent [LAG-3-CD49b- (double negative), LAG-3+CD49b+ (double positive), LAG-3+CD49b- (LAG-3+), LAG-3-CD49b+ (CD49b+)]. However, each population exhibited suppressive potential consistent with the definition of Tr1 cells. Notably, differences between these populations of Tr1 cells were apparent including differential dependence on IL-10 to mediate suppression and expression of markers indicative of different activation states and terminal differentiation. Sort-transfer experiments indicated that LAG-3+ Tr1 cells exhibited the capacity to convert to double negative and double positive Tr1 cells, indicative of plasticity between these populations. Together, these data determine the features and suppressive potential of Tr1 cells in the resolution of IAV infection and identify four populations delineated by LAG-3 and CD49b, which likely correspond to different Tr1 cell activation states.

Parallel recovery of chromatin accessibility and gene expression dynamics from frozen human regulatory T cells.

Epigenetic features such as DNA accessibility dictate transcriptional regulation in a cell type- and cell state- specific manner, and mapping this in health vs. disease in clinically relevant material is opening the door to new mechanistic insights and new targets for therapy. Assay for Transposase Accessible Chromatin Sequencing (ATAC-seq) allows chromatin accessibility profiling from low cell input, making it tractable on rare cell populations, such as regulatory T (Treg) cells. However, little is known about the compatibility of the assay with cryopreserved rare cell populations. Here we demonstrate the robustness of an ATAC-seq protocol comparing primary Treg cells recovered from fresh or cryopreserved PBMC samples, in the steady state and in response to stimulation. We extend this method to explore the feasibility of conducting simultaneous quantitation of chromatin accessibility and transcriptome from a single aliquot of 50,000 cryopreserved Treg cells. Profiling of chromatin accessibility and gene expression in parallel within the same pool of cells controls for cellular heterogeneity and is particularly beneficial when constrained by limited input material. Overall, we observed a high correlation of accessibility patterns and transcription factor dynamics between fresh and cryopreserved samples. Furthermore, highly similar transcriptomic profiles were obtained from whole cells and from the supernatants recovered from ATAC-seq reactions. We highlight the feasibility of applying these techniques to profile the epigenomic landscape of cells recovered from cryopreservation biorepositories.

Differential allelic representation (DAR) identifies candidate eQTLs and improves transcriptome analysis.

In comparisons between mutant and wild-type genotypes, transcriptome analysis can reveal the direct impacts of a mutation, together with the homeostatic responses of the biological system. Recent studies have highlighted that, when homozygous mutations are studied in non-isogenic backgrounds, genes from the same chromosome as a mutation often appear over-represented among differentially expressed (DE) genes. One hypothesis suggests that DE genes chromosomally linked to a mutation may not reflect true biological responses to the mutation but, instead, result from differences in representation of expression quantitative trait loci (eQTLs) between sample groups selected on the basis of mutant or wild-type genotype. This is problematic when inclusion of spurious DE genes in a functional enrichment study results in incorrect inferences of mutation effect. Here we show that chromosomally co-located differentially expressed genes (CC-DEGs) can also be observed in analyses of dominant mutations in heterozygotes. We define a method and a metric to quantify, in RNA-sequencing data, localised differential allelic representation (DAR) between groups of samples subject to differential expression analysis. We show how the DAR metric can predict regions prone to eQTL-driven differential expression, and how it can improve functional enrichment analyses through gene exclusion or weighting of gene-level rankings. Advantageously, this improved ability to identify probable eQTLs also reveals examples of CC-DEGs that are likely to be functionally related to a mutant phenotype. This supports a long-standing prediction that selection for advantageous linkage disequilibrium influences chromosome evolution. By comparing the genomes of zebrafish (Danio rerio) and medaka (Oryzias latipes), a teleost with a conserved ancestral karyotype, we find possible examples of chromosomal aggregation of CC-DEGs during evolution of the zebrafish lineage. The DAR metric provides a solid foundation for addressing the eQTL issue in new and existing datasets because it relies solely on RNA-sequencing data.

Transient Polycomb activity represses developmental genes in growing oocytes.

BACKGROUND: Non-genetic disease inheritance and offspring phenotype are substantially influenced by germline epigenetic programming, including genomic imprinting. Loss of Polycomb Repressive Complex 2 (PRC2) function in oocytes causes non-genetically inherited effects on offspring, including embryonic growth restriction followed by post-natal offspring overgrowth. While PRC2-dependent non-canonical imprinting is likely to contribute, less is known about germline epigenetic programming of non-imprinted genes during oocyte growth. In addition, de novo germline mutations in genes encoding PRC2 lead to overgrowth syndromes in human patients, but the extent to which PRC2 activity is conserved in human oocytes is poorly understood. RESULTS: In this study, we identify a discrete period of early oocyte growth during which PRC2 is expressed in mouse growing oocytes. Deletion of Eed during this window led to the de-repression of 343 genes. A high proportion of these were developmental regulators, and the vast majority were not imprinted genes. Many of the de-repressed genes were also marked by the PRC2-dependent epigenetic modification histone 3 lysine 27 trimethylation (H3K27me3) in primary-secondary mouse oocytes, at a time concurrent with PRC2 expression. In addition, we found H3K27me3 was also enriched on many of these genes by the germinal vesicle (GV) stage in human oocytes, strongly indicating that this PRC2 function is conserved in the human germline. However, while the 343 genes were de-repressed in mouse oocytes lacking EED, they were not de-repressed in pre-implantation embryos and lost H3K27me3 during pre-implantation development. This implies that H3K27me3 is a transient feature that represses a wide range of genes in oocytes. CONCLUSIONS: Together, these data indicate that EED has spatially and temporally distinct functions in the female germline to repress a wide range of developmentally important genes and that this activity is conserved in the mouse and human germlines.

Transcriptional targets of senataxin and E2 promoter binding factors are associated with neuro-degenerative pathways during increased autophagic flux.

Autophagy is an intracellular recycling process that degrades harmful molecules and enables survival during starvation, with implications for diseases including dementia, cancer and atherosclerosis. Previous studies demonstrate how a limited number of transcription factors (TFs) can increase autophagy. However, this knowledge has not resulted in translation into therapy, thus, to gain understanding of more suitable targets, we utilized a systems biology approach. We induced autophagy by amino acid starvation and mTOR inhibition in HeLa, HEK 293 and SH-SY5Y cells and measured temporal gene expression using RNA-seq. We observed 456 differentially expressed genes due to starvation and 285 genes due to mTOR inhibition (PFDR < 0.05 in every cell line). Pathway analyses implicated Alzheimer's and Parkinson's diseases (PFDR ≤ 0.024 in SH-SY5Y and HeLa) and amyotrophic lateral sclerosis (ALS, PFDR < 0.05 in mTOR inhibition experiments). Differential expression of the Senataxin (SETX) target gene set was predicted to activate multiple neurodegenerative pathways (PFDR ≤ 0.04). In the SH-SY5Y cells of neuronal origin, the E2F transcription family was predicted to activate Alzheimer's disease pathway (PFDR ≤ 0.0065). These exploratory analyses suggest that SETX and E2F may mediate transcriptional regulation of autophagy and further investigations into their possible role in neuro-degeneration are warranted.

3DFAACTS-SNP: using regulatory T cell-specific epigenomics data to uncover candidate mechanisms of type 1 diabetes (T1D) risk.

BACKGROUND: Genome-wide association studies (GWAS) have enabled the discovery of single nucleotide polymorphisms (SNPs) that are significantly associated with many autoimmune diseases including type 1 diabetes (T1D). However, many of the identified variants lie in non-coding regions, limiting the identification of mechanisms that contribute to autoimmune disease progression. To address this problem, we developed a variant filtering workflow called 3DFAACTS-SNP to link genetic variants to target genes in a cell-specific manner. Here, we use 3DFAACTS-SNP to identify candidate SNPs and target genes associated with the loss of immune tolerance in regulatory T cells (Treg) in T1D. RESULTS: Using 3DFAACTS-SNP, we identified from a list of 1228 previously fine-mapped variants, 36 SNPs with plausible Treg-specific mechanisms of action. The integration of cell type-specific chromosome conformation capture data in 3DFAACTS-SNP identified 266 regulatory regions and 47 candidate target genes that interact with these variant-containing regions in Treg cells. We further demonstrated the utility of the workflow by applying it to three other SNP autoimmune datasets, identifying 16 Treg-centric candidate variants and 60 interacting genes. Finally, we demonstrate the broad utility of 3DFAACTS-SNP for functional annotation of all known common (> 10% allele frequency) variants from the Genome Aggregation Database (gnomAD). We identified 9376 candidate variants and 4968 candidate target genes, generating a list of potential sites for future T1D or other autoimmune disease research. CONCLUSIONS: We demonstrate that it is possible to further prioritise variants that contribute to T1D based on regulatory function, and illustrate the power of using cell type-specific multi-omics datasets to determine disease mechanisms. Our workflow can be customised to any cell type for which the individual datasets for functional annotation have been generated, giving broad applicability and utility.

Placental Transcription Profiling in 6-23 Weeks' Gestation Reveals Differential Transcript Usage in Early Development.

The human placenta is a rapidly developing transient organ that is key to pregnancy success. Early development of the conceptus occurs in a low oxygen environment before oxygenated maternal blood begins to flow into the placenta at ~10-12 weeks' gestation. This process is likely to substantially affect overall placental gene expression. Transcript variability underlying gene expression has yet to be profiled. In this study, accurate transcript expression profiles were identified for 84 human placental chorionic villus tissue samples collected across 6-23 weeks' gestation. Differential gene expression (DGE), differential transcript expression (DTE) and differential transcript usage (DTU) between 6-10 weeks' and 11-23 weeks' gestation groups were assessed. In total, 229 genes had significant DTE yet no significant DGE. Integration of DGE and DTE analyses found that differential expression patterns of individual transcripts were commonly masked upon aggregation to the gene-level. Of the 611 genes that exhibited DTU, 534 had no significant DGE or DTE. The four most significant DTU genes ADAM10, VMP1, GPR126, and ASAH1, were associated with hypoxia-responsive pathways. Transcript usage is a likely regulatory mechanism in early placentation. Identification of functional roles will facilitate new insight in understanding the origins of pregnancy complications.

Iron Responsive Element-Mediated Responses to Iron Dyshomeostasis in Alzheimer's Disease.

BACKGROUND: Iron trafficking and accumulation is associated with Alzheimer's disease (AD) pathogenesis. However, the role of iron dyshomeostasis in early disease stages is uncertain. Currently, gene expression changes indicative of iron dyshomeostasis are not well characterized, making it difficult to explore these in existing datasets. OBJECTIVE: To identify sets of genes predicted to contain iron responsive elements (IREs) and use these to explore possible iron dyshomeostasis-associated gene expression responses in AD. METHODS: Comprehensive sets of genes containing predicted IRE or IRE-like motifs in their 3' or 5' untranslated regions (UTRs) were identified in human, mouse, and zebrafish reference transcriptomes. Further analyses focusing on these genes were applied to a range of cultured cell, human, mouse, and zebrafish gene expression datasets. RESULTS: IRE gene sets are sufficiently sensitive to distinguish not only between iron overload and deficiency in cultured cells, but also between AD and other pathological brain conditions. Notably, changes in IRE transcript abundance are among the earliest observable changes in zebrafish familial AD (fAD)-like brains, preceding other AD-typical pathologies such as inflammatory changes. Unexpectedly, while some IREs in the 3' untranslated regions of transcripts show significantly increased stability under iron deficiency in line with current assumptions, many such transcripts instead display decreased stability, indicating that this is not a generalizable paradigm. CONCLUSION: Our results reveal IRE gene expression changes as early markers of the pathogenic process in fAD and are consistent with iron dyshomeostasis as an important driver of this disease. Our work demonstrates how differences in the stability of IRE-containing transcripts can be used to explore and compare iron dyshomeostasis-associated gene expression responses across different species, tissues, and conditions.

Genome-Wide CRISPR Screen Identifies RACK1 as a Critical Host Factor for Flavivirus Replication.

Cellular factors have important roles in all facets of the flavivirus replication cycle. Deciphering viral-host protein interactions is essential for understanding the flavivirus life cycle as well as development of effective antiviral strategies. To uncover novel host factors that are co-opted by multiple flaviviruses, a CRISPR/Cas9 genome wide knockout (KO) screen was employed to identify genes required for replication of Zika virus (ZIKV). Receptor for Activated Protein C Kinase 1 (RACK1) was identified as a novel host factor required for ZIKV replication, which was confirmed via complementary experiments. Depletion of RACK1 via siRNA demonstrated that RACK1 is important for replication of a wide range of mosquito- and tick-borne flaviviruses, including West Nile Virus (WNV), Dengue Virus (DENV), Powassan Virus (POWV) and Langat Virus (LGTV) as well as the coronavirus SARS-CoV-2, but not for YFV, EBOV, VSV or HSV. Notably, flavivirus replication was only abrogated when RACK1 expression was dampened prior to infection. Utilising a non-replicative flavivirus model, we show altered morphology of viral replication factories and reduced formation of vesicle packets (VPs) in cells lacking RACK1 expression. In addition, RACK1 interacted with NS1 protein from multiple flaviviruses; a key protein for replication complex formation. Overall, these findings reveal RACK1's crucial role to the biogenesis of pan-flavivirus replication organelles. IMPORTANCE Cellular factors are critical in all facets of viral lifecycles, where overlapping interactions between the virus and host can be exploited as possible avenues for the development of antiviral therapeutics. Using a genome-wide CRISPR knockout screening approach to identify novel cellular factors important for flavivirus replication we identified RACK1 as a pro-viral host factor for both mosquito- and tick-borne flaviviruses in addition to SARS-CoV-2. Using an innovative flavivirus protein expression system, we demonstrate for the first time the impact of the loss of RACK1 on the formation of viral replication factories known as 'vesicle packets' (VPs). In addition, we show that RACK1 can interact with numerous flavivirus NS1 proteins as a potential mechanism by which VP formation can be induced by the former.

Seeing the forest through the trees: prioritising potentially functional interactions from Hi-C.

Eukaryotic genomes are highly organised within the nucleus of a cell, allowing widely dispersed regulatory elements such as enhancers to interact with gene promoters through physical contacts in three-dimensional space. Recent chromosome conformation capture methodologies such as Hi-C have enabled the analysis of interacting regions of the genome providing a valuable insight into the three-dimensional organisation of the chromatin in the nucleus, including chromosome compartmentalisation and gene expression. Complicating the analysis of Hi-C data, however, is the massive amount of identified interactions, many of which do not directly drive gene function, thus hindering the identification of potentially biologically functional 3D interactions. In this review, we collate and examine the downstream analysis of Hi-C data with particular focus on methods that prioritise potentially functional interactions. We classify three groups of approaches: structural-based discovery methods, e.g. A/B compartments and topologically associated domains, detection of statistically significant chromatin interactions, and the use of epigenomic data integration to narrow down useful interaction information. Careful use of these three approaches is crucial to successfully identifying potentially functional interactions within the genome.

In-Frame and Frameshift Mutations in Zebrafish Presenilin 2 Affect Different Cellular Functions in Young Adult Brains.

BACKGROUND: Mutations in PRESENILIN 2 (PSEN2) cause early onset familial Alzheimer's disease (EOfAD) but their mode of action remains elusive. One consistent observation for all PRESENILIN gene mutations causing EOfAD is that a transcript is produced with a reading frame terminated by the normal stop codon-the "reading frame preservation rule". Mutations that do not obey this rule do not cause the disease. The reasons for this are debated. OBJECTIVE: To predict cellular functions affected by heterozygosity for a frameshift, or a reading frame-preserving mutation in zebrafish psen2 using bioinformatic techniques. METHODS: A frameshift mutation (psen2 N140fs ) and a reading frame-preserving (in-frame) mutation (psen2 T141 _ L142delinsMISLISV ) were previously isolated during genome editing directed at the N140 codon of zebrafish psen2 (equivalent to N141 of human PSEN2). We mated a pair of fish heterozygous for each mutation to generate a family of siblings including wild type and heterozygous mutant genotypes. Transcriptomes from young adult (6 months) brains of these genotypes were analyzed. RESULTS: The in-frame mutation uniquely caused subtle, but statistically significant, changes to expression of genes involved in oxidative phosphorylation, long-term potentiation and the cell cycle. The frameshift mutation uniquely affected genes involved in Notch and MAPK signaling, extracellular matrix receptor interactions and focal adhesion. Both mutations affected ribosomal protein gene expression but in opposite directions. CONCLUSION: A frameshift and an in-frame mutation at the same position in zebrafish psen2 cause discrete effects. Changes in oxidative phosphorylation, long-term potentiation and the cell cycle may promote EOfAD pathogenesis in humans.

Tissue and regional expression patterns of dicistronic tRNA-mRNA transcripts in grapevine (Vitis vinifera) and their evolutionary co-appearance with vasculature in land plants.

Transfer RNAs (tRNA) are crucial adaptor molecules between messenger RNA (mRNA) and amino acids. Recent evidence in plants suggests that dicistronic tRNA-like structures also act as mobile signals for mRNA transcripts to move between distant tissues. Co-transcription is not a common feature in the plant nuclear genome and, in the few cases where polycistronic transcripts have been found, they include non-coding RNA species, such as small nucleolar RNAs and microRNAs. It is not known, however, the extent to which dicistronic transcripts of tRNA and mRNAs are expressed in field-grown plants, or the factors contributing to their expression. We analysed tRNA-mRNA dicistronic transcripts in the major horticultural crop grapevine (Vitis vinifera) using a novel pipeline developed to identify dicistronic transcripts from high-throughput RNA-sequencing data. We identified dicistronic tRNA-mRNA in leaf and berry samples from 22 commercial vineyards. Of the 124 tRNA genes that were expressed in both tissues, 18 tRNA were expressed forming part of 19 dicistronic tRNA-mRNAs. The presence and abundance of dicistronic molecules was tissue and geographic sub-region specific. In leaves, the expression patterns of dicistronic tRNA-mRNAs significantly correlated with tRNA expression, suggesting that their transcriptional regulation might be linked. We also found evidence of syntenic genomic arrangements of tRNAs and protein-coding genes between grapevine and Arabidopsis thaliana, and widespread prevalence of dicistronic tRNA-mRNA transcripts among vascular land plants but no evidence of these transcripts in non-vascular lineages. This suggests that the appearance of plant vasculature and tRNA-mRNA occurred concurrently during the evolution of land plants.

Zebrafish Chromosome 14 Gene Differential Expression in the fmr1 h u2787 Model of Fragile X Syndrome.

Zebrafish represent a valuable model for investigating the molecular and cellular basis of Fragile X syndrome (FXS). Reduced expression of the zebrafish FMR1 orthologous gene, fmr1, causes developmental and behavioural phenotypes related to FXS. Zebrafish homozygous for the hu2787 non-sense mutation allele of fmr1 are widely used to model FXS, although FXS-relevant phenotypes seen from morpholino antisense oligonucleotide (morpholino) suppression of fmr1 transcript translation were not observed when hu2787 was first described. The subsequent discovery of transcriptional adaptation (a form of genetic compensation), whereby mutations causing non-sense-mediated decay of transcripts can drive compensatory upregulation of homologous transcripts independent of protein feedback loops, suggested an explanation for the differences reported. We examined the whole-embryo transcriptome effects of homozygosity for fmr1 h u2787 at 2 days post fertilisation. We observed statistically significant changes in expression of a number of gene transcripts, but none from genes showing sequence homology to fmr1. Enrichment testing of differentially expressed genes implied effects on lysosome function and glycosphingolipid biosynthesis. The majority of the differentially expressed genes are located, like fmr1, on Chromosome 14. Quantitative PCR tests did not support that this was artefactual due to changes in relative chromosome abundance. Enrichment testing of the "leading edge" differentially expressed genes from Chromosome 14 revealed that their co-location on this chromosome may be associated with roles in brain development and function. The differential expression of functionally related genes due to mutation of fmr1, and located on the same chromosome as fmr1, is consistent with R.A. Fisher's assertion that the selective advantage of co-segregation of particular combinations of alleles of genes will favour, during evolution, chromosomal rearrangements that place them in linkage disequilibrium on the same chromosome. However, we cannot exclude that the apparent differential expression of genes on Chromosome 14 genes was, (if only in part), caused by differences between the expression of alleles of genes unrelated to the effects of the fmr1 h u2787 mutation and made manifest due to the limited, but non-zero, allelic diversity between the genotypes compared.

Transcriptome analyses of 7-day-old zebrafish larvae possessing a familial Alzheimer's disease-like mutation in psen1 indicate effects on oxidative phosphorylation, ECM and MCM functions, and iron homeostasis.

BACKGROUND: Early-onset familial Alzheimer's disease (EOfAD) is promoted by dominant mutations, enabling the study of Alzheimer's disease (AD) pathogenic mechanisms through generation of EOfAD-like mutations in animal models. In a previous study, we generated an EOfAD-like mutation, psen1Q96_K97del, in zebrafish and performed transcriptome analysis comparing entire brains from 6-month-old wild type and heterozygous mutant fish. We identified predicted effects on mitochondrial function and endolysosomal acidification. Here we aimed to determine whether similar effects occur in 7 day post fertilization (dpf) zebrafish larvae that might be exploited in screening of chemical libraries to find ameliorative drugs. RESULTS: We generated clutches of wild type and heterozygous psen1Q96_K97del 7 dpf larvae using a paired-mating strategy to reduce extraneous genetic variation before performing a comparative transcriptome analysis. We identified 228 differentially expressed genes and performed various bioinformatics analyses to predict cellular functions. CONCLUSIONS: Our analyses predicted a significant effect on oxidative phosphorylation, consistent with our earlier observations of predicted effects on ATP synthesis in adult heterozygous psen1Q96_K97del brains. The dysregulation of minichromosome maintenance protein complex (MCM) genes strongly contributed to predicted effects on DNA replication and the cell cycle and may explain earlier observations of genome instability due to PSEN1 mutation. The upregulation of crystallin gene expression may be a response to defective activity of mutant Psen1 protein in endolysosomal acidification. Genes related to extracellular matrix (ECM) were downregulated, consistent with previous studies of EOfAD mutant iPSC neurons and postmortem late onset AD brains. Also, changes in expression of genes controlling iron ion transport were observed without identifiable changes in the prevalence of transcripts containing iron responsive elements (IREs) in their 3' untranslated regions (UTRs). These changes may, therefore, predispose to the apparent iron dyshomeostasis previously observed in 6-month-old heterozygous psen1Q96_K97del EOfAD-like mutant brains.

Revision of the Litoria watjulumensis (Anura: Pelodryadidae) group from the Australian monsoonal tropics, including the resurrection of L. spaldingi.

We show that the Wotjulum frog, Litoria watjulumensis (Copland, 1957), comprises two deeply divergent mitochondrial DNA lineages that are also reciprocally monophyletic for a nuclear gene locus and have discrete distributions. The taxa are differentiated in multivariate analysis of shape but show no appreciable differences in colour and pattern. The two taxa differ substantially in the degree of female biased sexual size dimorphism, with the western taxon showing considerably more pronounced dimorphism. We subsequently resurrect Litoria (Hyla) spaldingi (Hosmer, 1964) for populations from east of the Daly River system in the Northern Territory through to western Queensland and restrict L. watjulumensis to populations from the Kimberley region of north-western Australia and the Victoria River system of the western Northern Territory. The complex advertisement call of L. spaldingi is described for the first time.

Brain Transcriptome Analysis of a Protein-Truncating Mutation in Sortilin-Related Receptor 1 Associated With Early-Onset Familial Alzheimer's Disease Indicates Early Effects on Mitochondrial and Ribosome Function.

BACKGROUND: The early cellular stresses leading to Alzheimer's disease (AD) remain poorly understood because we cannot access living, asymptomatic human AD brains for detailed molecular analyses. Sortilin-related receptor 1 (SORL1) encodes a multi-domain receptor protein genetically associated with both rare, early-onset familial AD (EOfAD) and common, sporadic, late-onset AD (LOAD). SORL1 protein has been shown to act in the trafficking of the amyloid ß A4 precursor protein (AßPP) that is proteolysed to form one of the pathological hallmarks of AD, amyloid-ß (Aß) peptide. However, other functions of SORL1 in AD are less well understood. OBJECTIVE: To investigate the effects of heterozygosity for an EOfAD-like mutation in SORL1 on the brain transcriptome of young-adult mutation carriers using zebrafish as a model organism. METHODS: We performed targeted mutagenesis to generate an EOfAD-like mutation in the zebrafish orthologue of SORL1 and performed RNA-sequencing on mRNA isolated from the young adult brains of siblings in a family of fish either wild type (non-mutant) or heterozygous for the EOfAD-like mutation. RESULTS: We identified subtle differences in gene expression indicating changes in mitochondrial and ribosomal function in the mutant fish. These changes appear to be independent of changes in mitochondrial content or the expression of AßPP-related proteins in zebrafish. CONCLUSION: These findings provided evidence supporting that EOfAD mutations in SORL1 affect mitochondrial and ribosomal function and provide the basis for future investigation elucidating the nature of these effects.