Project description:The induction of pluripotency or trans-differentiation of one cell type to another can be accomplished with cell lineage-specific transcription factors. Here we report that repression of a single RNA binding protein PTB, which occurs during normal brain development via the action of miR-124, is sufficient to induce trans-differentiation of fibroblasts into functional neurons. Besides its traditional role in regulated splicing, we show that PTB has a previously undocumented function in the regulation of microRNA functions, suppressing or enhancing microRNA targeting by competitive binding on target mRNA or altering local RNA secondary structure. A key event during neuronal induction is the relief of PTB-mediated blockage of microRNA action on multiple components of the REST complex, thereby de-repressing a large array of neuronal genes, including miR-124 and multiple neuronal-specific transcription factors, in non-neuronal cells. This converts a negative feedback loop to a positive one to elicit cellular reprogramming to the neuronal lineage. Examination of PTB regulated AGO2/microRNA targeting in Hela cells by CLIP-seq (two biological replicates) , paired-end RNA-seq (control and PTB knockdown) and 3’end stability RNA-seq (control and PTB knockdown)
Project description:Ectopic expression of neuronal microRNAs (miRNAs), miR-9/9* and miR-124 (miR-9/9*-124) in adult human fibroblasts has been found to evoke extensive reconfigurations of the chromatin and direct the fate conversion to neurons. We found that miR-9/9* and miR-124 led to the repression of REST, a transcriptional repressor of neuronal genes, during microRNA-mediated neuronal conversion and knockdown of REST enhanced the activation of BAF53b, a mature neuronal marker. Furthermore, time series analysis of the transcriptome of cells undergoing the miR-9/9*-124-induced conversion indicated upregulated genetic pathways that were predicted to be targeted by REST although the transcript level of REST remained unchanged (Abernathy et al., 2017). Therefore, we reasoned that knocking down REST in addition to miR-9/9*-124 at an early time point (day 7), in which REST repression is minimally evident during neuronal conversion, would speed up the adoption of neuronal identity. We performed the RNA-seq analysis to compared differentially expressed genes (DEGs) between human adult fibroblasts expressing control shRNA (shCTL) and reprogramming cells expressing shCTL or shREST at day 7. Finally, we found that the repression of REST constitutes an important component of microRNA-mediated neuronal reprogramming of human fibroblasts.
Project description:This dataset includes microRNA profiling of 61 early-passage metastatic melanoma cell lines. The data are provided as single-end small RNA seq fastq files.
Project description:AIM: To find molecular signatures associated to the siRNA-mediated knockdowns in order to be able to identify similarities among different knockdowns. DESCRIPTION: Each sample includes biological triplicates for 35 siRNA-mediated knockdowns targeting 30 chromatin-associated proteins during in early reprogramming to iPS at day 6. A daily timecourse from reprogramming cells, without treatment from MEFs until day 6 is also included in triplicate.
