LTP-Related Gene Networks Figure 3. Validation of differentially expressed genes by quantitative PCR (qPCR). Differential expression of selected genes was validated by qPCR. (A) Genes previously reported to be LTP-regulated: HOMER, homer homolog 1; TIMP1, tissue inhibitor of metalloproteinase 1; APP, amyloid precursor protein. (B) Novel LTP-regulated genes: LINGO1, leucine rich repeat and Ig domain containing; SP1 specificity protein 1; BHLHE22, basic helix-loop-helix family, member e22; HDAC1, histone deacetylase 1; KCNE2, potassium voltage-gated channel, Isk-related family, member 2 and DNAJB5, DnaJ (Hsp40) homolog, subfamily B, member 5. Results are expressed as mean fold change 6 SEM. *p#0.05, Student’s t-test (n = 5–9). qPCR samples were normalised to the housekeeping gene HPRT using the 22 DDCT method. doi:10.1371/journal.pone.0040538.g003 between the 3 data sets by heatmap (Figure 4B) demonstrates that there is a close relationship between the 20 min and 5 h data sets; however, the heatmap includes data that have not reached statistical significance and only comprises a small proportion of the differentially expressed genes within each timepoint. Thus we conclude that the three LRG sets represent distinct temporally defined datasets. Remarkably, there is only one gene in common between all 3 data sets, the class II histone deacetylases (HDACs)- interacting protein, DNAJB5 [20]. DNAJB5 is postulated to act as a chaperone involved in nuclear localisation of HDACs, which regulate gene transcription by removal of acetyl groups from e-N- acetylated histones. This enhances histone-DNA association and prevents access of transcriptional machinery to DNA. Interesting- ly, also present within the intersection between the 5 h and 24 h gene sets is the class I histone deacetylase HDAC1, GMNN (Geminin), a molecule that has recently been shown to regulate the conformation of chromatin around neural genes [21] and MLL4, a histone methyltransferase which also regulates acetylation [22]. These findings highlight potential epigenetic control of LTP- related gene expression. 2. Temporal analysis of LRG sets To explore the potential contribution that our temporally defined LRG datasets make to the late phases of LTP, the relationships between the contributing genes were analysed using Ingenuity Pathway Analysis (as described [13]) (IPA; Canonical Pathways, Biological Function and Network Analysis), and DAVID software. Our 20 min-LRG data set [13] was reanalysed alongside those from 5 h and 24 h to examine LTP-associated PLoS ONE | www.plosone.org 3 July 2012 | Volume 7 | Issue 7 | e40538