Lack of Stim2 Affects Vision-Dependent Behavior and Sensitivity to Hypoxia.

Stromal interaction molecules (STIMs) are endoplasmic reticulum-resident proteins that regulate Ca2+ homeostasis and signaling by store-operated calcium entry (SOCE). The different properties and functions of STIM1 and STIM2 have been described mostly based on work in vitro. STIM2 knockout mice do not survive until adulthood. Therefore, we generated and characterized stim2a and stim2b double-knockout zebrafish. The (stim2a;stim2b)-/- zebrafish did not have any apparent morphological phenotype. However, RNA sequencing revealed 1424 differentially expressed genes. One of the most upregulated genes was annexin A3a, which is a marker of activated microglia. This corresponded well to an increase in Neutral Red staining in the in vivo imaging of the (stim2a;stim2b)-/- zebrafish brain. The lack of Stim2 decreased zebrafish survival under low oxygen conditions. Behavioral tests, such as the visual-motor response test and dark-light preference test, indicated that (stim2a;stim2b)-/- larvae might have problems with vision. This was consistent with the downregulation of many genes that are related to light perception. The periodic acid-Schiff staining of retina sections from adult zebrafish revealed alterations of the stratum pigmentosum, suggesting the involvement of a Stim2-dependent process in visual perception. Altogether, these data reveal new functions for Stim2 in the nervous system.

Targeting integrated stress response with ISRIB combined with imatinib treatment attenuates RAS/RAF/MAPK and STAT5 signaling and eradicates chronic myeloid leukemia cells.

The integrated stress response (ISR) facilitates cellular adaptation to unfavorable conditions by reprogramming the cellular response. ISR activation was reported in neurological disorders and solid tumors; however, the function of ISR and its role as a possible therapeutic target in hematological malignancies still remain largely unexplored. Previously, we showed that the ISR is activated in chronic myeloid leukemia (CML) cells and correlates with blastic transformation and tyrosine kinase inhibitor (TKI) resistance. Moreover, the ISR was additionally activated in response to imatinib as a type of protective internal signaling. Here, we show that ISR inhibition combined with imatinib treatment sensitized and more effectively eradicated leukemic cells both in vitro and in vivo compared to treatment with single agents. The combined treatment specifically inhibited the STAT5 and RAS/RAF/MEK/ERK pathways, which are recognized as drivers of resistance. Mechanistically, this drug combination attenuated both interacting signaling networks, leading to BCR-ABL1- and ISR-dependent STAT5 activation. Consequently, leukemia engraftment in patient-derived xenograft mice bearing CD34+ TKI-resistant CML blasts carrying PTPN11 mutation responsible for hyperactivation of the RAS/RAF/MAPK and JAK/STAT5 pathways was decreased upon double treatment. This correlated with the downregulation of genes related to the RAS/RAF/MAPK, JAK/STAT5 and stress response pathways and was associated with lower expression of STAT5-target genes regulating proliferation, viability and the stress response. Collectively, these findings highlight the effect of imatinib plus ISRIB in the eradication of leukemic cells resistant to TKIs and suggest potential clinical benefits for leukemia patients with TKI resistance related to RAS/RAF/MAPK or STAT5 signaling. We propose that personalized treatment based on the genetic selection of patients carrying mutations that cause overactivation of the targeted pathways and therefore make their sensitivity to such treatment probable should be considered as a possible future direction in leukemia treatment.

Genomic and physiological analyses of the zebrafish atrioventricular canal reveal molecular building blocks of the secondary pacemaker region.

The atrioventricular canal (AVC) is the site where key structures responsible for functional division between heart regions are established, most importantly, the atrioventricular (AV) conduction system and cardiac valves. To elucidate the mechanism underlying AVC development and function, we utilized transgenic zebrafish line sqet31Et expressing EGFP in the AVC to isolate this cell population and profile its transcriptome at 48 and 72 hpf. The zebrafish AVC transcriptome exhibits hallmarks of mammalian AV node, including the expression of genes implicated in its development and those encoding connexins forming low conductance gap junctions. Transcriptome analysis uncovered protein-coding and noncoding transcripts enriched in AVC, which have not been previously associated with this structure, as well as dynamic expression of epithelial-to-mesenchymal transition markers and components of TGF-ß, Notch, and Wnt signaling pathways likely reflecting ongoing AVC and valve development. Using transgenic line Tg(myl7:mermaid) encoding voltage-sensitive fluorescent protein, we show that abolishing the pacemaker-containing sinoatrial ring (SAR) through Isl1 loss of function resulted in spontaneous activation in the AVC region, suggesting that it possesses inherent automaticity although insufficient to replace the SAR. The SAR and AVC transcriptomes express partially overlapping species of ion channels and gap junction proteins, reflecting their distinct roles. Besides identifying conserved aspects between zebrafish and mammalian conduction systems, our results established molecular hallmarks of the developing AVC which underlies its role in structural and electrophysiological separation between heart chambers. This data constitutes a valuable resource for studying AVC development and function, and identification of novel candidate genes implicated in these processes.

The Hunt for Ancient Prions: Archaeal Prion-Like Domains Form Amyloid-Based Epigenetic Elements.

Prions, proteins that can convert between structurally and functionally distinct states and serve as non-Mendelian mechanisms of inheritance, were initially discovered and only known in eukaryotes, and consequently considered to likely be a relatively late evolutionary acquisition. However, the recent discovery of prions in bacteria and viruses has intimated a potentially more ancient evolutionary origin. Here, we provide evidence that prion-forming domains exist in the domain archaea, the last domain of life left unexplored with regard to prions. We searched for archaeal candidate prion-forming protein sequences computationally, described their taxonomic distribution and phylogeny, and analyzed their associated functional annotations. Using biophysical in vitro assays, cell-based and microscopic approaches, and dye-binding analyses, we tested select candidate prion-forming domains for prionogenic characteristics. Out of the 16 tested, eight formed amyloids, and six acted as protein-based elements of information transfer driving non-Mendelian patterns of inheritance. We also identified short peptides from our archaeal prion candidates that can form amyloid fibrils independently. Lastly, candidates that tested positively in our assays had significantly higher tyrosine and phenylalanine content than candidates that tested negatively, an observation that may help future archaeal prion predictions. Taken together, our discovery of functional prion-forming domains in archaea provides evidence that multiple archaeal proteins are capable of acting as prions-thus expanding our knowledge of this epigenetic phenomenon to the third and final domain of life and bolstering the possibility that they were present at the time of the last universal common ancestor.

Open chromatin landscape of rat microglia upon proinvasive or inflammatory polarization.

Microglia are brain-resident, myeloid cells that play important roles in health and brain pathologies. Herein, we report a comprehensive, replicated, false discovery rate-controlled dataset of DNase-hypersensitive (DHS) open chromatin regions for rat microglia. We compared the open chromatin landscapes in untreated primary microglial cultures and cultures stimulated for 6 hr with either glioma-conditioned medium (GCM) or lipopolysaccharide (LPS). Glioma-secreted factors induce proinvasive and immunosuppressive activation of microglia, and these cells then promote tumor growth. The open chromatin landscape of the rat microglia consisted of 126,640 reproducible DHS regions, among which 2,303 and 12,357 showed a significant change in openness following stimulation with GCM or LPS, respectively. Active genes exhibited constitutively open promoters, but there was no direct dependence between the aggregated openness of DHS regions near a gene and its expression. Individual regions mapped to the same gene often presented different patterns of openness changes. GCM-regulated DHS regions were more frequent in areas away from gene bodies, while LPS-regulated regions were more frequent in introns. GCM and LPS differentially affected the openness of regions mapped to immune checkpoint genes. The two treatments differentially affected the aggregated openness of regions mapped to genes in the Toll-like receptor signaling and axon guidance pathways, suggesting that the molecular machinery used by migrating microglia is similar to that of growing axons and that modulation of these pathways is instrumental in the induction of proinvasive polarization of microglia by glioma. Our dataset of open chromatin regions paves the way for studies of gene regulation in rat microglia.

Gliosarcoma Is Driven by Alterations in PI3K/Akt, RAS/MAPK Pathways and Characterized by Collagen Gene Expression Signature.

Gliosarcoma is a very rare brain tumor reported to be a variant of glioblastoma (GBM), IDH-wildtype. While differences in molecular and histological features between gliosarcoma and GBM were reported, detailed information on the genetic background of this tumor is lacking. We intend to fill in this knowledge gap by the complex analysis of somatic mutations, indels, copy number variations, translocations and gene expression patterns in gliosarcomas. Using next generation sequencing, we determined somatic mutations, copy number variations (CNVs) and translocations in 10 gliosarcomas. Six tumors have been further subjected to RNA sequencing analysis and gene expression patterns have been compared to those of GBMs. We demonstrate that gliosarcoma bears somatic alterations in gene coding for PI3K/Akt (PTEN, PI3K) and RAS/MAPK (NF1, BRAF) signaling pathways that are crucial for tumor growth. Interestingly, the frequency of PTEN alterations in gliosarcomas was much higher than in GBMs. Aberrations of PTEN were the most frequent and occurred in 70% of samples. We identified genes differentially expressed in gliosarcoma compared to GBM (including collagen signature) and confirmed a difference in the protein level by immunohistochemistry. We found several novel translocations (including translocations in the RABGEF1 gene) creating potentially unfavorable combinations. Collected results on genetic alterations and transcriptomic profiles offer new insights into gliosarcoma pathobiology, highlight differences in gliosarcoma and GBM genetic backgrounds and point out to distinct molecular cues for targeted treatment.

Genome-Wide Analysis of Drosophila RBf2 Protein Highlights the Diversity of RB Family Targets and Possible Role in Regulation of Ribosome Biosynthesis.

RBf2 is a recently evolved retinoblastoma family member in Drosophila that differs from RBf1, especially in the C-terminus. To investigate whether the unique features of RBf2 contribute to diverse roles in gene regulation, we performed chromatin immunoprecipitation sequencing for both RBf2 and RBf1 in embryos. A previous model for RB-E2F interactions suggested that RBf1 binds dE2F1 or dE2F2, whereas RBf2 is restricted to binding to dE2F2; however, we found that RBf2 targets approximately twice as many genes as RBf1. Highly enriched among the RBf2 targets were ribosomal protein genes. We tested the functional significance of this finding by assessing RBf activity on ribosomal protein promoters and the endogenous genes. RBf1 and RBf2 significantly repressed expression of some ribosomal protein genes, although not all bound genes showed transcriptional effects. Interestingly, many ribosomal protein genes are similarly targeted in human cells, indicating that these interactions may be relevant for control of ribosome biosynthesis and growth. We carried out bioinformatic analysis to investigate the basis for differential targeting by these two proteins and found that RBf2-specific promoters have distinct sequence motifs, suggesting unique targeting mechanisms. Association of RBf2 with these promoters appears to be independent of dE2F2/dDP, although promoters bound by both RBf1 and RBf2 require dE2F2/dDP. The presence of unique RBf2 targets suggest that evolutionary appearance of this corepressor represents the acquisition of potentially novel roles in gene regulation for the RB family.