Functional annotation clustering allows the clas sification of regulated genes according to their functional relevance. Each of the six sub clusters obtained in the hierarchical clustering was independently annotated. moreover An overview of the various functional categories for the six sub clusters is shown in Table 2, Gene Ontology annota tions of individual clusters can be found in Additional file 1 Tables S11 S21. Strong differences in the func tional categories arose upon comparison of up and down regulated gene clusters. Within the gene cluster including genes upregulated after TSA treatment, functional cat egories like antigen processing, metabolism, cell mem brane and cell adhesion were enriched, the cluster of downregulated genes in cluded functional categories related to chromosome organization, transcriptional processes, metabolism, and posttranslational processes.

In the case of BMP2 treatment, the gene cluster of upre gulated genes was enriched for functional categories associated with cell communication, cell membrane, extracellular matrix, differentiation and development. Genes downregulated after BMP2 treatment were enriched in the functional categories related to cell communication and signal trans duction. The functional annotation of the sub cluster containing genes upregu lated after both treatments showed an enrichment of categories related to extracellular matrix and cell adhe sion, whereas the sub cluster of downregulated genes comprised categories related to differentiation and devel opment.

As well from the list of individual genes as from the functional cluster analysis it was apparent that BMP2 and TSA treatment resulted in independent gene profiles. While TSA treat ment mainly led to a regulation of transcriptional pro cesses, BMP2 treatment rather resulted in a regulation of signal transduction processes. Even though both treat ments primarily led to a different expression of genes, the downregulation of certain genes seems to reflect the similar phenotype which we had observed in both TSA and BMP2 treated neurosphere cultures. While only a few primary target genes of TSA and BMP2 were clus tered within the sub clusters containing genes regulated after both treatments, it is obvious that a variety of genes involved in neural development were present, such as the oligodendrocyteproteins Mag, Mal, Mog, Omg, Mbp, and Mobp, which were downregulated in one or both treatments.

Since the functional annotation clustering did not dis close an enrichment of direct target genes of TSA or BMP2, and because we detected the strongest overlap of regulated genes between TSA and BMP2 treatment after 24 h, we decided to perform an additional DAVID Entinostat analysis including genes regulated significantly after different times of treatment. Figure 4 summarizes the clustered functional categories obtained from TSA 6 h, TSA 24 h, BMP2 6 h or BMP2 24 h experiment.