Calcium regulation of the Arabidopsis Na+/K+ transporter HKT1;1 improves seed germination under salt stress.

Calcium is known to improve seed-germination rates under salt stress. We investigated the involvement of calcium ions (Ca2+) in regulating HIGH-AFFINITY K+ TRANSPORTER 1 (HKT1; 1), which encodes a Na+/K+ transporter, and its post-translational regulator TYPE 2C PROTEIN PHOSPHATASE 49 (PP2C49), in germinating Arabidopsis (Arabidopsis thaliana) seedlings. Germination rates of hkt1 mutant seeds under salt stress remained unchanged by CaCl2 treatment in wild-type Arabidopsis, whereas pp2c49 mutant seeds displayed improved salt-stress tolerance in the absence of CaCl2 supplementation. Analysis of HKT1;1 and PP2C49 promoter activity revealed that CaCl2 treatment results in radicle-focused expression of HKT1;1 and reduction of the native radicle-exclusive expression of PP2C49. Ion-content analysis indicated that CaCl2 treatment improves K+ retention in germinating wild-type seedlings under salt stress, but not in hkt1 seedlings. Transgenic seedlings designed to exclusively express HKT1;1 in the radicle during germination displayed higher germination rates under salt stress than the wild type in the absence of CaCl2 treatment. Transcriptome analysis of germinating seedlings treated with CaCl2, NaCl, or both revealed 118 upregulated and 94 downregulated genes as responsive to the combined treatment. Bioinformatics analysis of the upstream sequences of CaCl2-NaCl-treatment-responsive upregulated genes revealed the abscisic acid response element CACGTGTC, a potential CaM-binding transcription activator-binding motif, as most prominent. Our findings suggest a key role for Ca2+ in mediating salt-stress responses during germination by regulating genes that function to maintain Na+ and K+ homeostasis, which is vital for seed germination under salt stress.

CT-FOCS: a novel method for inferring cell type-specific enhancer-promoter maps.

Spatiotemporal gene expression patterns are governed to a large extent by the activity of enhancer elements, which engage in physical contacts with their target genes. Identification of enhancer-promoter (EP) links that are functional only in a specific subset of cell types is a key challenge in understanding gene regulation. We introduce CT-FOCS (cell type FOCS), a statistical inference method that uses linear mixed effect models to infer EP links that show marked activity only in a single or a small subset of cell types out of a large panel of probed cell types. Analyzing 808 samples from FANTOM5, covering 472 cell lines, primary cells and tissues, CT-FOCS inferred such EP links more accurately than recent state-of-the-art methods. Furthermore, we show that strictly cell type-specific EP links are very uncommon in the human genome.

Tauopathy in the young autistic brain: novel biomarker and therapeutic target.

Given our recent discovery of somatic mutations in autism spectrum disorder (ASD)/intellectual disability (ID) genes in postmortem aged Alzheimer's disease brains correlating with increasing tauopathy, it is important to decipher if tauopathy is underlying brain imaging results of atrophy in ASD/ID children. We concentrated on activity-dependent neuroprotective protein (ADNP), a prevalent autism gene. The unique availability of multiple postmortem brain sections of a 7-year-old male, heterozygous for ADNP de novo mutation c.2244Adup/p.His559Glnfs*3 allowed exploration of tauopathy, reflecting on a general unexplored mechanism. The tested subject exhibited autism, fine motor delays, severe intellectual disability and seizures. The patient died after multiple organ failure following liver transplantation. To compare to other ADNP syndrome mutations, immortalized lymphoblastoid cell lines from three different patients (including ADNP p.Arg216*, p.Lys408Valfs*31, and p.Tyr719* heterozygous dominant mutations) and a control were subjected to RNA-seq. Immunohistochemistry, high-throughput gene expression profiles in numerous postmortem tissues followed. Comparisons to a control brain and to extensive datasets were used. Live cell imaging investigated Tau-microtubule interaction, protecting against tauopathy. Extensive child brain tauopathy paralleled by multiple gene expression changes was discovered. Tauopathy was explained by direct mutation effects on Tau-microtubule interaction and correction by the ADNP active snippet NAP. Significant pathway changes (empirical P value < 0.05) included over 100 genes encompassing neuroactive ligand-receptor and cytokine-cytokine receptor interaction, MAPK and calcium signaling, axon guidance and Wnt signaling pathways. Changes were also seen in steroid biosynthesis genes, suggesting sex differences. Selecting the most affected genes by the ADNP mutations for gene expression analysis, in multiple postmortem tissues, identified Tau (MAPT)-gene-related expression changes compared with extensive normal gene expression (RNA-seq) databases. ADNP showed relatively reduced expression in the ADNP syndrome cerebellum, which was also observed for 25 additional genes (representing >50% of the tested genes), including NLGN1, NLGN2, PAX6, SMARCA4, and SNAP25, converging on nervous system development and tauopathy. NAP provided protection against mutated ADNP disrupted Tau-microtubule association. In conclusion, tauopathy may explain brain-imaging findings in ADNP syndrome children and may provide a new direction for the development of tauopathy protecting drug candidates like NAP in ASD/ID.

The EXPANDER Integrated Platform for Transcriptome Analysis.

Genome-wide analysis of cellular transcriptomes using RNA-seq or expression arrays is a major mainstay of current biological and biomedical research. EXPANDER (EXPression ANalyzer and DisplayER) is a comprehensive software package for analysis of expression data, with built-in support for 18 different organisms. It is designed as a "one-stop shop" platform for transcriptomic analysis, allowing for execution of all analysis steps starting with gene expression data matrix. Analyses offered include low-level preprocessing and normalization, differential expression analysis, clustering, bi-clustering, supervised grouping, high-level functional and pathway enrichment tests, and networks and motif analyses. A variety of options is offered for each step, using established algorithms, including many developed and published by our laboratory. EXPANDER has been continuously developed since 2003, having to date over 18,000 downloads and 540 citations. One of the innovations in the recent version is support for combined analysis of gene expression and ChIP-seq data to enhance the inference of transcriptional networks and their functional interpretation. EXPANDER implements cutting-edge algorithms and makes them accessible to users through user-friendly interface and intuitive visualizations. It is freely available to users at http://acgt.cs.tau.ac.il/expander/.

FOCS: a novel method for analyzing enhancer and gene activity patterns infers an extensive enhancer-promoter map.

Recent sequencing technologies enable joint quantification of promoters and their enhancer regions, allowing inference of enhancer-promoter links. We show that current enhancer-promoter inference methods produce a high rate of false positive links. We introduce FOCS, a new inference method, and by benchmarking against ChIA-PET, HiChIP, and eQTL data show that it results in lower false discovery rates and at the same time higher inference power. By applying FOCS to 2630 samples taken from ENCODE, Roadmap Epigenomics, FANTOM5, and a new compendium of GRO-seq samples, we provide extensive enhancer-promotor maps ( http://acgt.cs.tau.ac.il/focs ). We illustrate the usability of our maps for deriving biological hypotheses.