Unknown,Transcriptomics,Genomics,Proteomics

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RNA-Seq analysis of mouse dopamine neurons identifies transcriptional repression of PlexinC1 by Lmx1a and Lmx1b

ABSTRACT: In order to identify axon guidance molecules regulated by Lmx1a and Lmx1b RNA sequencing was performed on three biological replicates for the control and four biological replicates for Lmx1a/b cKO mice. For each sample, two technical replicates were performed. For each E15.5 embryo used, VTA and SNpc were dissected from 8 antero- posterior levels across the entire dopaminergic domain as revealed by TH immnostaining. Following RNA extraction, RNAseq libraries were constructed using the Illumina TruSeq Stranded RNA protocol with oligo dT pulldown and sequenced on Illumina HiSeq2500 by 150-bp paired-end sequencing. One sample was excluded before the sequencing because the amount of RNA was too low to measure the RNA quality.

INSTRUMENT(S): Illumina HiSeq 2500

ORGANISM(S): Mus musculus

SUBMITTER: Emmanouil Metzakopian

PROVIDER: E-MTAB-5986 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:LMX1B is a LIM-homeodomain transcription factor essential for development. Putative LMX1B target genes have been identified through mouse gene targeting studies; however, in the absence of in vivo molecular characterization of their regulation, their identity as direct LMX1B targets remains hypothetical. We describe here the first molecular characterization of LMX1B target gene regulation. A tetracycline-inducible expression system and microarray analysis showed that a subset of NF-kappa B target genes, including IL-6 and IL-8 are upregulated in LMX1B-expressing HeLa cells. Chromatin immunoprecipitation assays revealed that LMX1B binds to the proximal promoter region of IL-6 and IL-8 in vivo, in the vicinity of the characterized kappa B site, and that LMX1B recruitment correlates with an increased NF-kappa B DNA association. Inhibition of NF-kappa B activity by short interfering RNA-mediated knock-down of p65 impairs LMX1B-dependent induction of NF-kappa B target genes, while activation of NF-kappa B activity by TNF-alpha results in a synergistic induction of these genes by LMX1B. IL-6 promoter-driven reporter assays showed that the kappa B site and an adjacent putative LMX1B binding motif are both involved in LMX1B-mediated transcription. Expression of a number of NF-kappa B target genes is affected in the kidney of Lmx1b-/- knock-out mice, thus supporting the biological relevance of the data obtained in the human cell line. Together, these data demonstrate for the first time that LMX1B directly regulates transcription of a subset of NF-kappa B target genes in cooperation with nuclear p50/p65 NF-kappa B. Human subset (GSM303547-GSM303550): Two biological replicates of non-expressing HtTA-LMX1B cells (grown in doxycycline-containing medium) and of LMX1B-expressing HtTA-LMX1B cells (grown for 4 days in doxycycline-free medium) were processed for gene expression array analyses using an Affymetrix platform. Mouse subset (GSM304379-GSM304384): Three kidney samples from newborn wild-type mice and from newborn Lmx1b-/- knock-out mice were processed for gene expression array analyses using an Agilent platform.

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RNA-Seq analysis of mouse dopamine neurons identifies transcriptional repression of PlexinC1 by Lmx1a and Lmx1b

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