DISP3 promotes proliferation and delays differentiation of neural progenitor cells
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ABSTRACT: DISP3 (PTCHD2), a sterol-sensing domain-containing protein, is highly expressed in neural tissue but its role in neural differentiation is unknown. In the present study we used a multipotent cerebellar progenitor cell line, C17.2, to investigate the impact of DISP3 on the proliferation and differentiation of neural precursors and its possible involvement in brain malignancies. We found that ectopically expressed DISP3 promotes cell proliferation and alters expression of genes that are involved in tumorigenesis. Finally, the differentiation profile of DISP3-expressing cells were altered, as evidenced by delayed expression of neural specific markers and a reduced capacity to undergo neural differentiation.
Project description:The orphan nuclear receptor Nurr1 has been shown to be critical for the development of ventral midbrain dopaminergic neurons. Consequently, the development of ES cells overexpressing Nurr1 has raised hope for the development of cell replacement therapies for Parkinson's Disease to replace degenerated dopaminergic neurons. However, the molecular consequences of Nurr1 on gene expression in these cells remain unknown. To address this, stable, clonal, c17.2 neural stem cell lines were established that overexpressed the orphan nuclear receptor Nurr1 (clone 42 & clone 48) or parental control cell line (puroB & puroD, respectively). Experiment Overall Design: Stable neural stem cell lines were grown in proliferating conditions and matched for further microarray analysis based on their similar proliferation rates: Experiment Overall Design: clone 42(c42) vs. puroB(pB) Experiment Overall Design: clone 42(c48) vs. puroD(pD)
Project description:We used a next-generation sequencing approach to understand the effects of antioxidant cerium oxide nanoparticles (CeO2) on neuronal stem cell differentiation. As a model we used the murine neuronal progenitor cell line, C17.2, which upon differentiation, is able to generate a mixed culture of neurons and neuroglial cells. As additional controls we used N-acetylcysteine (NAC), a conventional antioxidant and samarium doped cerium oxide nanoparticles (Sm-CeO2), as particle controls (as they bear a reduced antioxidant potential as compared to CeO2 alone). We had a time series approach and we investigates effects after 1, 4 and 7 days during differentiation. We revealed that CeO2 reduce axonal guidance signalling, neuronal differentiation and neuroglial differentiation after 7 days, thus having a negative effect on neuronal development. Overall, these effects are likely due to the antioxidant properties of nanoceria, although some evidence for a particle effect was also provided as indicated by the interference with cytoskeletal as well as integrin signaling genes both by nanoceria and Sm-doped nanoceria, but not by NAC.
Project description:Joint DNA molecules are natural by-products of DNA replication and repair. Persistent joint molecules give rise to ultrafine DNA bridges (UFBs) in mitosis, which compromise sister chromatid separation. The DNA translocase PICH (ERCC6L) plays a central role in UFB resolution. To better understand the genetic context rendering cells dependent on PICH, a genome-wide loss-of-function screen was performed to identify the genetic contexts in which cells become dependent on PICH. In addition to genes involved in DNA cohesion, centromere stability and DNA damage repair, we identified the uncharacterized protein C1orf112. We find that C1orf112 interacts with and stabilizes the AAA+ ATPase FIGNL1. Inactivation of either C1orf112 or FIGNL1 resulted in UFB formation, prolonged retention of RAD51 on chromatin, impaired replication fork dynamics, and consequently impaired genome maintenance. Combined, our data reveal that inactivation of C1orf112 or FIGNL1 dysregulates RAD51 dynamics at replication forks, resulting in DNA replication defects, and a dependency on PICH to preserve cell viability.
Project description:DISP3 (PTCHD2), a sterol-sensing domain-containing protein, is highly expressed in neural tissue but its role in neural differentiation is unknown. To determine the effect of DISP3 on neural stem cells phenotype of NS-5 cells, we impaired Disp3 gene using CRISPR/Cas9 technology. Modification of Disp3 expression levels affects NS-5 capacity to differentiate. In differentiated Disp3 sgRNAs cell cultures we observed higher number of highly differentiated astrocytes and oligodendrocytes and neurons with not only increased βIII-tubulin mRNA expression but also with more developed neurites.
Project description:Tagged versions of thyroid hormone receptors alpha (TRa) and beta (TRb) were stably transfected in two C17.2 cell lines, C17.2a and C17.2b, respectively. Cells were treated with 10-7 M T3 for 6, 12 or 24h or left untreated. We performed DGE by sequencing all polyA RNA according to a SAGE-derived method. Differential gene expression after T3 treatment was computed and the T3 responses induced by the two receptors were compared. We could conclude that, in a similar environment, target genes are only partially shared and that a significant proportion show receptor preference and even selectivity. Examination of thyroid hormone target genes over time in two cell lines (C17.2a, C17.2b), each expressing one of the thyroid hormone receptors (alpha, beta).
Project description:microarray analysis with and without sh-p53 in IMR90 (normal human diploid fiborblasts) Phenotypes were created by overexpression of RasG12V, E1A/RasG12V in IMR90 cells, in growing IMR90, inr quiescent IMR90 cells (3 days confluence) and 24 or 48 hours Etoposide treated IMR90 cells were subjected to lentiviral knockdown of p53 or vector
Project description:Tagged versions of thyroid hormone receptors alpha (TRa) and beta (TRb) were stably transfected in two C17.2 cell lines, C17.2a and C17.2b, respectively. We performed an affinity-based purification of chromatin (ChAP), and high-throughput sequencing was used to assess binding sites of both receptors (ChAP-Seq). Standard ChIP-Seq for RXR was also performed in C17.2a cells. These data allow us to compare binding sites for both receptors and to conclude that they were only partially redundant, with co-existence of receptor-specific sites. Examination of binding sites of the two thyroid hormone receptors (alpha, beta) in two cell lines (C17.2a, C17.2b), each expressing one of the receptors. Examination of RXR binding sites in C17.2a cells.
Project description:p53 ChIP seq and Histone mark ChIP Seq data in IMR90 (normal human diploid fibroblasts) Phenotypes were created by overexpression of RasG12V, E1A/RasG12V in IMR90 cells, growing IMR90 and 24 hour Etoposide treated IMR90 cells were subjected to p53 and histone mark ChIP Seq
Project description:Here we performed DNAse-seq experiments on samples of adherent cultures of mouse neural stem cells (NS5 cell line) under normal growth conditions and upon differentiation by expression of an inducible version of the proneural factor Mash1/Ascl1 (Ascl1-ERT2). This resulted in the generation of genome-wide maps of regions of chromatin accessibility in both conditions.