Nearby transposable elements impact plant stress gene regulatory networks: a meta-analysis in A. thaliana and S. lycopersicum

Background Transposable elements (TE) make up a large portion of many plant genomes and are playing innovative roles in genome evolution. Several TEs can contribute to gene regulation by influencing expression of nearby genes as stress-responsive regulatory motifs. To delineate TE-mediated plant stress regulatory networks, we took a 2-step computational approach consisting of identifying TEs in the proximity of stress-responsive genes, followed by searching for cis-regulatory motifs in these TE sequences and linking them to known regulatory factors. Through a systematic meta-analysis of RNA-seq expression profiles and genome annotations, we investigated the relation between the presence of TE superfamilies upstream, downstream or within introns of nearby genes and the differential expression of these genes in various stress conditions in the TE-poor Arabidopsis thaliana and the TE-rich Solanum lycopersicum. Results We found that stress conditions frequently expressed genes having members of various TE superfamilies in their genomic proximity, such as SINE upon proteotoxic stress and Copia and Gypsy upon heat stress in A. thaliana, and EPRV and hAT upon infection, and Harbinger, LINE and Retrotransposon upon light stress in S. lycopersicum. These stress-specific gene-proximal TEs were mostly located within introns and more detected near upregulated than downregulated genes. Similar stress conditions were often related to the same TE superfamily. Additionally, we detected both novel and known motifs in the sequences of those TEs pointing to regulatory cooption of these TEs upon stress. Next, we constructed the regulatory network of TFs that act through binding these TEs to their target genes upon stress and discovered TE-mediated regulons targeted by TFs such as BRB/BPC, HD, HSF, GATA, NAC, DREB/CBF and MYB factors in Arabidopsis and AP2/ERF/B3, NAC, NF-Y, MYB, CXC and HD factors in tomato. Conclusions Overall, we map TE-mediated plant stress regulatory networks using numerous stress expression profile studies for two contrasting plant species to study the regulatory role TEs play in the response to stress. As TE-mediated gene regulation allows plants to adapt more rapidly to new environmental conditions, this study contributes to the future development of climate-resilient plants.

New genomic signals underlying the emergence of human proto-genes

De novo genes are novel genes which emerge from non-coding DNA. Until now, little is known about de novo genes properties, correlated to their age and mechanisms of emergence. In this study, we investigate four properties: introns, upstream regulatory motifs, 5 prime UTRs and protein domains, in 23135 human proto-genes. We found that proto-genes contain introns, whose number and position correlates with the genomic position of proto-gene emergence. The origin of these introns is debated, as our result suggest that 41% proto-genes might have captured existing introns, as well as the fact that 13.7% of them do not splice the ORF. We show that proto-genes which emerged via overprinting tend to be more enriched in core promotor motifs, while intergenic and intronic ones are more enriched in enhancers, even if the motif TATA is most expressed upstream these genes. Intergenic and intronic 5 prime UTRs of proto-genes have a lower potential to stabilise mRNA structures than exonic proto-genes and established human genes. Finally, we confirm that proto-genes gain new putative domains with age. Overall, we find that regulatory motifs inducing transcription and translation of previously non-coding sequences may facilitate proto-gene emergence. Our paper demonstrates that introns, 5 prime UTRs, and domains have specific properties in proto-genes. We also show the importance of studying proto-genes in relation to their genomic position, as it strongly impacts these properties.

Bistability in macrophage polarization and metabolic implications

Macrophages are essential innate immune cells characterized by a high diversity and plasticity. In vitro, their full dynamic range of activation profiles include the classical pro-inflammatory (M1) and the alternative anti-inflammatory (M2) program. Bistability usually arises in biological systems that contain a positive-feedback loop or a mutually inhibitory, double-negative-feedback loop, which are common regulatory motifs reported for macrophage transitions from one activation state to the other one. This switch-like behavior of macrophage is observed at four different levels. First, a decision-making module in signal transduction includes mutual inhibitory interactions between M1 (STAT1 and NF-KB/p50-p65) and M2 (STAT3 and NF-KB/p50-p50) signaling pathways. Second, a switch-like behavior at the gene expression level includes complex network motifs of transcription factors and miRNAs. Third, those changes impact metabolic gene expression leading to several switches in energy production, NADPH and ROS production, TCA cycle functionality, biosynthesis and nitrogen metabolism. Fourth, metabolic changes are monitored by specialized metabolic sensors coupled to AMPK and mTOR activity to provide stability by maintaining the signals to promote either M1 or M2 activation. The targeting of robust molecular switches has the potential to treat a broad range of widespread diseases such as sepsis, cancer or chronic inflammatory diseases.

monaLisa: an R/Bioconductor package for identifying regulatory motifs

Proteins binding to specific nucleotide sequences, such as transcription factors, play key roles in the regulation of gene expression. Their binding can be indirectly observed via associated changes in transcription, chromatin accessibility, DNA methylation and histone modifications. Identifying candidate factors that are responsible for these observed experimental changes is critical to understand the underlying biological processes. Here we present monaLisa, an R/Bioconductor package that implements approaches to identify relevant transcription factors from experimental data. The package can be easily integrated with other Bioconductor packages and enables seamless motif analyses without any software dependencies outside of R.AvailabilitymonaLisa is implemented in R and available on Bioconductor at https://bioconductor.org/packages/monaLisa with the development version hosted on GitHub at https://github.com/fmicompbio/[email protected]

Inference of Rift Valley Fever pathogenesis in Bos taurus using a gene co-expression network

Background: Rift Valley Fever (RVF) is a viral disease caused by the Rift Valley Fever virus and spread mainly by the Aedes and Culex mosquito species. The disease primarily infects domestic animals such as sheep, goats, and cattle, resulting in a spectrum of clinical outcomes including morbidity, massive storm abortions, and neonatal fatalities. RVF outbreaks are closely linked to above-average rainfall and flooding, which create an ideal environment for mosquitos to breed, multiply, and transmit the virus to animals. The outcomes of human RVF infection range from self-limiting febrile illness to potentially fatal hemorrhagic diatheses and miscarriage in pregnant women. Collectively, the economic losses due to the zoonotic RVF disease is immense. Methods: Using the Weighted Gene Co-expression Network Analysis (WGCNA) package, RNA-Seq data generated from five healthy Bos taurus steer calves aged 4-6 months was obtained from the Gene Expression Omnibus (GEO) database (Accession number GSE71417). The data was utilized to construct a gene co-expression network. Enriched modules containing genes potentially involved in RVF infection progression were identified. Moreover, using the Multiple Expectation Maximizations for Motif Elicitation (MEME) suite, consensus regulatory motifs of enriched gene clusters were deciphered and the most abundant putative regulatory motif in each enriched module unveiled by comparative analysis with publicly available motifs using the TOMTOM motif comparison tool. The potential roles of the identified regulatory motifs were inferred by literature mining. Results: The constructed gene co-expression network revealed thirty-three (33) modules, nine of which were enriched for Gene Ontology terms linked to RVF pathogenesis. Functional enrichment in two (red and turquoise) of the nine modules was significant. ASH1-like histone lysine methyltransferase and Astrotactin were the hub genes for the red and turquoise modules respectively. ASH1-like histone lysine methyltransferase gene is involved in chromatin epigenetic modification while Astrotactin is a vertebrate-specific gene that plays an important role in neurodevelopment. Additionally, consensus regulatory motifs located on the 3' end of genes in each enriched module was identified. Conclusions: In this study, we have developed a gene co-expression network that has aided in the unveiling of functionally related genes, intramodular hub genes, and immunity genes potentially involved in RVF pathogenesis. The discovery of functional genes with putative critical roles in the establishment of RVF infection establishment will contribute to the understanding of the molecular mechanism of RVF pathogenesis. Importantly, the putative regulatory motifs identified are plausible targets for RVF drug and vaccine development. Keywords: Rift Valley Fever, Bos taurus, Gene co-expression network, modules, hub genes, Regulatory motifs.

Pharmacogenomics to Predict Mucositis in Acute Myeloid Leukemia

Introduction Acute myeloid leukemia (AML) is a heterogenous hematologic malignancy that primarily affects older adults, with a median age of diagnosis at 68 years and five-year relative survival of 29.5%. The incidence of AML diagnosis is expected to increase with an aging population in the United States, encouraging the exploration for treatment risk stratification to inform care. One factor impacting survivorship is treatment-related toxicity. Cytarabine (active metabolite, Ara-C) continues as a mainstay agent for initial induction regimens in AML, but efforts to minimize its toxicity remain a challenge. Interpatient variability presents another intricacy, where response to cytarabine can manifest as a subset of patients who have an inadequate response or those who are overly sensitive to its effects. Pharmacogenes in the cytarabine pathway may be responsible for this inconsistent response and could influence toxicity susceptibility. We conducted a retrospective analysis of AML patients treated with cytarabine to evaluate whether single nucleotide polymorphisms (SNPs) in its pharmacogenes are associated with treatment-related mucositis. Specifically, mucositis can negatively impact quality of life with pain and difficulty eating and swallowing, Thus, identifying patients likely to develop mucositis can assist in timely supportive care. Methods Patients from the University of Florida Shand's Hospital who received cytarabine during their first induction therapy from 2009 to 2019 for de novo AML were screened for mucositis within the first thirty days. We obtained FFPE tissues of these patients and extracted genomic DNA. Using TaqMan real-time PCR assays (QuantStudio 3) in-house, we genotyped for ten SNPs from genes involved in the cytarabine metabolic pathway. Logistic regression models were used to test for association between these SNPs and the incidence of mucositis. All analyses were performed using Rstudio v4.1.0. SNPs with significant results were then tested for other SNPs that occur in linkage disequilibrium (LD) using HaploReg v4.1, in addition to testing for association with gene expression using the Genotype-Tissue Expression Portal (GTEx). Results In total, 184 patients were included in this study, with 92 in each group - mucositis and no mucositis (control). The median age was 64 years, 58% were male, 83% were white, 12% African American or black, and 0.2% were Asian. No difference in the incidence of mucositis was observed by sex or race. Logistic regression models identified two SNPs significantly associated (p<0.05) with incidence of mucositis within the first thirty days of cytarabine exposure. For rs5841, a synonymous coding SNP in NME4, presence of the variant T allele (CT/TT genotype) was associated with significantly increased incidence of mucositis (OR=1.51; 95% CI [1.09-2.11]) (Figure 1A and B). rs5841 occurs in LD with seven other SNPs impacting multiple regulatory motifs. Consistent with these results, presence of the T allele is associated with higher NME4 expression (Figure 1C), implying that higher ara-C activation in the variant T group is contributing to greater incidence of mucositis. For rs17103168, an intronic SNP in CMPK, patients homozygous for the variant allele (GG genotype) experienced significantly higher incidence of mucositis compared to those with AG or AA genotypes (OR=1.56; 95% CI [0.95-2.58]) (Figure 2A and B). This SNP occurs in LD with sixty other SNPs impacting numerous regulatory motifs as well. Presence of the GG genotype is associated with high gene expression (Figure 2C), suggesting that this increased gene expression in GG genotypes may result in higher ara-C levels, and subsequently increasing risk of toxicities. Conclusion Overall, we report a significant association between the risk of mucositis after initial cytarabine exposure and two SNPs in cytarabine metabolic pathway genes. Identifying those who may experience adverse toxicity using such SNP-based prediction models will allow for clinically meaningful interventions. Consideration of interpatient variability with cytarabine may lend valuable insight in shared-decision making between patient and clinician when weighing risks vs. benefit of treatment. Ongoing and future studies are focused on expanding this cohort to include evaluation of cytarabine pharmacogenomics with respect to disease progression and survival outcome in AML. Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare.

Activation of the essential kinase PDK1 by phosphoinositide-driven autophosphorylation

3-phosphoinositide-dependent kinase 1 (PDK1) is an essential serine/threonine protein kinase, which plays a crucial role in cell growth and proliferation. It is often referred to as a master kinase due to its ability to activate at least 23 downstream protein kinases implicated in various signaling pathways. In this study, we have elucidated the mechanism of phosphoinositide-driven PDK1 auto-activation. We show that PDK1 trans-autophosphorylation is mediated by a PIP3-mediated face-to-face dimer. We report regulatory motifs in the kinase-PH interdomain linker that allosterically activate PDK1 autophosphorylation via a linker-swapped dimer mechanism. Finally, we show that PDK1 is autoinhibited by its PH domain and that positive cooperativity of PIP3 binding drives switch-like activation of PDK1. Our work implies that the PDK1-mediated activation of effector kinases, including Akt, PKC, Sgk, S6K and RSK, many of whom are not directly regulated by phosphoinositides, is also likely to be dependent on PIP3 or PI(3,4)P2.

MicroSalmon: A Comprehensive, Searchable Resource of Predicted MicroRNA Targets and 3′UTR Cis-Regulatory Elements in the Full-Length Sequenced Atlantic Salmon Transcriptome

Complete 3′UTRs unambiguously assigned to specific mRNA isoforms from the Atlantic salmon full-length (FL) transcriptome were collected into a 3′UTRome. miRNA response elements (MREs) and other cis-regulatory motifs were subsequently predicted and assigned to 3′UTRs of all FL-transcripts. The MicroSalmon GitHub repository provides all results. RNAHybrid and sRNAtoolbox tools predicted the MREs. UTRscan and the Teiresias algorithm predicted other 3′UTR cis-acting motifs, both known vertebrate motifs and putative novel motifs. MicroSalmon provides search programs to retrieve all FL-transcripts targeted by a miRNA (median number 1487), all miRNAs targeting an FL-transcript (median number 27), and other cis-acting motifs. As thousands of FL-transcripts may be targets of each miRNA, additional experimental strategies are necessary to reduce the likely true and relevant targets to a number that may be functionally validated. Low-complexity motifs known to affect mRNA decay in vertebrates were over-represented. Many of these were enriched in the terminal end, while purine- or pyrimidine-rich motifs with unknown functions were enriched immediately downstream of the stop codon. Furthermore, several novel complex motifs were over-represented, indicating conservation and putative function. In conclusion, MicroSalmon is an extensive and useful, searchable resource for study of Atlantic salmon transcript regulation by miRNAs and cis-acting 3′UTR motifs.

Probing 3′UTRs as modular regulators of gene expression

Genes are commonly abstracted into a coding sequence and cis-regulatory elements (CREs), such as promoter and terminator regions, and short sequence motifs within these regions. Modern cloning techniques allow easy assembly of synthetic genetic constructs from discrete cis-regulatory modules. However, it is unclear how much the contributions of CREs to gene expression depend on other CREs in the host gene. Using budding yeast, we probe the extent of composability, or independent effects, of distinct CREs. We confirm that the quantitative effect of a terminator on gene expression depends on both promoter and coding sequence. We then explore whether individual cis-regulatory motifs within terminator regions display similar context dependence, focusing on putative regulatory motifs inferred using transcriptome-wide datasets of mRNA decay. We construct a library of diverse reporter genes, consisting of different combinations of motifs within various terminator contexts, paired with different promoters, to test the extent of composability. Our results show that the effect of a motif on RNA abundance depends both on its host terminator, and also on the associated promoter sequence. Consequently, this emphasises the need for improved motif inference algorithms that include both local and global context effects, which in turn could aid researchers in the accurate use of diverse CREs for the engineering of synthetic genetic constructs.

Gene co-expression reveals the modularity and integration of C4 and CAM in Portulaca

C4 and Crassulacean acid metabolism (CAM) have been considered as largely independent photosynthetic adaptations in spite of sharing key biochemical modules. Portulaca is a geographically widespread clade of over 100 annual and perennial angiosperm species that primarily use C4 photosynthesis, but facultatively exhibit CAM when drought stressed, a photosynthetic system known as C4+CAM. It has been hypothesized that C4+CAM is rare because of pleiotropic constraints, but these have not been deeply explored. We generated a chromosome-level genome assembly of P. amilis and sampled mRNA from P. amilis and P. oleracea during CAM induction. Gene co-expression network analyses identified C4 and CAM gene modules shared and unique to both Portulaca species. A conserved CAM module linked phosphoenolpyruvate carboxylase (PEPC) to starch turnover during the day-night transition and was enriched in circadian clock regulatory motifs in the P. amilis genome. Preservation of this co-expression module regardless of water status suggests that Portulaca constitutively operate a weak CAM cycle that is transcriptionally and post-transcriptionally upregulated during drought. C4 and CAM mostly used mutually exclusive paralogs for primary carbon fixation and, although it is likely that nocturnal CAM malate stores are shuttled into diurnal C4 decarboxylation pathways, we find some evidence that metabolite cycling may occur at low levels. C4 likely evolved in Portulaca through co-option of redundant paralogs and integration of the diurnal portion of CAM. Thus, the ancestral CAM system did not strongly constrain C4 evolution because photosynthetic gene networks are not co-regulated for both daytime and nighttime functions.