Periodic Report Summary 2 - HINLOD (Roles of homeoproteins in lymphoid organ development)
1) identification of the growth-related regulatory networks and target genes controlled by homeoproteins during organogenesis;
2) validation and functional characterisation of candidate targets during organ development.
Over the past two years, we performed a comprehensive DNA microarray analysis and used Gene set enrichment analysis (GSEA) for interpreting gene expression data, a computational method that allows focusing on set of genes (instead of single gene) that share common biological functions. By employing this approach, we found statistically significant differences in the expression profiles connected with several developmental pathways. In particular, we found that loss of Hox11 is associated with up-regulation of several pathways including retinoc acid, Wnt and BMP. In addition, to determine whether Hox11 and Pbx1 directly controls these genes / pathways, we performed chromatin immunoprecipitation (ChIP) analysis coupled to deep sequencing using and identified several genomic regions bound by these transcription factors. We selected a few genes belonging to the retinoic acid pathways and confirmed, by ChIP and qPCR analyses, they are direct target genes of Hox11 and Pbx1. By using an in vitro organ culture system, we functionally validated a few targets belonging to the RA pathways and are currently extending our analysis to other target genes / pathways.