Metzakopian, E.; (2010) Genome wide identification of transcriptional targets of Foxa2 in midbrain dopaminergic cells by ChIP-Seq. Doctoral thesis , UCL (University College London).

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Abstract

Midbrain dopaminergic (mDA) neurons are involved in the regulation of movement and behavior, and their loss causes severe neurological disorders, such as Parkinson's disease. Foxa1 and Foxa2 (Foxa1/2), members of the Foxa family of forkhead/winged helix transcription factors, are expressed in mDA neurons throughout their development and display overlapping functions. Previously, it has been shown that Foxa1/2 regulate specification and differentiation of mDA neuron development. During specification, Foxa1/2 are required for the expression of Lmx1a, an intrinsic determinant of mDA identity. Recent data strongly suggests that Foxa2 cooperate with Lmx1a and Nurr1 (Nr4a2) in subsequent feed forward loops to regulate differentiation of mDA neurons. However, Foxa2 regulated direct targets and the mechanisms underlying its roles in mDA development are largely unknown. In this study, we performed chromatin immunoprecipitation (ChIP) and massively parallel Illumina 2G sequencing (ChIP-seq) using in vitro and in vivo DA systems. We produced a genome wide profile of Foxa2 binding sites at two stages of mDA neuron development: specification (in vitro), and differentiation (E12.5 and E14.5 in vivo tissue). Foxa2 binding was observed on known regulated elements, the Shh brain enhancer and the Foxa2 floor plate enhancer in both in vivo and in vitro data sets. Validation of candidate targets was carried out by independent in vivo ChIP-qPCR analysis and reverse transcriptase-qPCR expression assays using ventral midbrain tissue from both wild type and transgenic Foxa1;Foxa2 null mice. Furthermore, genomic regions in the Lmx1a and Lmx1b loci identified in our ChIP-seq analysis were validated for enhancer activity by transgenic LacZ reporter mice. These results strongly suggest that Foxa2 directly regulates the Lmx1a and Lmx1b enhancers emphasizing its key role in mDA specification. In addition, luciferase reporter assays in P19 cells demonstrate the combinatorial role of Foxa2 with Lmx1a and/or Nurr1 in regulating candidate enhancer regions of genes expressed in mDA neurons. These results confirm the quality of our data sets in predicting Foxa2 regulated target genes.

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