Gene-Expression Profiling Suggests Impaired Signaling via the Interferon Pathway in Cstb(-/-) Microglia

Progressive myoclonus epilepsy of Unverricht-Lundborg type (EPM1, OMIM254800) is an autosomal recessive neurodegenerative disorder characterized by stimulus-sensitive and action-activated myoclonus, tonic-clonic epileptic seizures, and ataxia. Loss-of-function mutations in the gene encoding the cysteine protease inhibitor cystatin B (CSTB) underlie EPM1. The deficiency of CSTB in mice (Cstb(-/-) mice) generates a phenotype resembling the symptoms of EPM1 patients and is accompanied by microglial activation at two weeks of age and an upregulation of immune system-associated genes in the cerebellum at one month of age. To shed light on molecular pathways and processes linked to CSTB deficiency in microglia we characterized the transcriptome of cultured Cstb(-/-) mouse microglia using microarray hybridization and RNA sequencing (RNA-seq). The gene expression profiles obtained with these two techniques were in good accordance and not polarized to either pro- or anti-inflammatory status. In Cstb(-/-) microglia, altogether 184 genes were differentially expressed. Of these, 33 genes were identified by both methods. Several interferon-regulated genes were weaker expressed in Cstb(-/-) microglia compared to control. This was confirmed by quantitative real-time PCR of the transcripts Irf7 and Stat1. Subsequently, we explored the biological context of CSTB deficiency in microglia more deeply by functional enrichment and canonical pathway analysis. This uncovered a potential role for CSTB in chemotaxis, antigen-presentation, and in immune-and defense response-associated processes by altering JAK-STAT pathway signaling. These data support and expand the previously suggested involvement of inflammatory processes to the disease pathogenesis of EPM1 and connect CSTB deficiency in microglia to altered expression of interferon-regulated genes.Peer reviewe

NANOG priming before full reprogramming may generate germ cell tumours

Reprogramming somatic cells into a pluripotent state brings patient-tailored, ethical controversy-free cellular therapy closer to reality. However, stem cells and cancer cells share many common characteristics; therefore, it is crucial to be able to discriminate between them. We generated two induced pluripotent stem cell (iPSC) lines, with NANOG pre-transduction followed by OCT3/4, SOX2, and LIN28 overexpression. One of the cell lines, CHiPS W, showed normal pluripotent stem cell characteristics, while the other, CHiPS A, though expressing pluripotency markers, failed to differentiate and gave rise to germ cell-like tumours in vivo. Comparative genomic hybridisation analysis of the generated iPS lines revealed that they were genetically more stable than human embryonic stem cell counterparts. This analysis proved to be predictive for the differentiation potential of analysed cells. Moreover, the CHiPS A line expressed a lower ratio of p53/p21 when compared to CHiPS W. NANOG pre-induction followed by OCT3/4, SOX2, MYC, and KLF4 induction resulted in the same tumour-inducing phenotype. These results underline the importance of a re-examination of the role of NANOG during reprogramming. Moreover, this reprogramming method may provide insights into primordial cell tumour formation and cancer stem cell transformation

Evolutionary origin and functional divergence of totipotent cell homeobox genes in eutherian mammals

BACKGROUND: A central goal of evolutionary biology is to link genomic change to phenotypic evolution. The origin of new transcription factors is a special case of genomic evolution since it brings opportunities for novel regulatory interactions and potentially the emergence of new biological properties. RESULTS: We demonstrate that a group of four homeobox gene families (Argfx, Leutx, Dprx, Tprx), plus a gene newly described here (Pargfx), arose by tandem gene duplication from the retinal-expressed Crx gene, followed by asymmetric sequence evolution. We show these genes arose as part of repeated gene gain and loss events on a dynamic chromosomal region in the stem lineage of placental mammals, on the forerunner of human chromosome 19. The human orthologues of these genes are expressed specifically in early embryo totipotent cells, peaking from 8-cell to morula, prior to cell fate restrictions; cow orthologues have similar expression. To examine biological roles, we used ectopic gene expression in cultured human cells followed by high-throughput RNA-seq and uncovered extensive transcriptional remodelling driven by three of the genes. Comparison to transcriptional profiles of early human embryos suggest roles in activating and repressing a set of developmentally-important genes that spike at 8-cell to morula, rather than a general role in genome activation. CONCLUSIONS: We conclude that a dynamic chromosome region spawned a set of evolutionarily new homeobox genes, the ETCHbox genes, specifically in eutherian mammals. After these genes diverged from the parental Crx gene, we argue they were recruited for roles in the preimplantation embryo including activation of genes at the 8-cell stage and repression after morula. We propose these new homeobox gene roles permitted fine-tuning of cell fate decisions necessary for specification and function of embryonic and extra-embryonic tissues utilised in mammalian development and pregnancy.This work was supported primarily by the European Research Council under the European Union’s Seventh Framework Programme (FP7/2007-2013 ERC grant 268513) to PWHH. In addition, support was provided by the Spanish Ministry of Economy and Competitiveness (BFU2014-55076-P) to MI and a fellowship from ‘la Caixa’-Severo Ochoa to CDR