Figure 3. REST regulates hypoxia repressed genes.

(A) Overview of the RNA-seq approach used to reveal the contribution of REST to gene repression in hypoxia. (B) REST mRNA expression from the 3 independent RNA-seq experiments after treatment with hypoxia or REST-RNAi treatment. (C) REST-RNAi treatment on cells exposed to normoxia (21% oxygen) for 24 hours led to a significant increase in the expression of well-characterised REST target genes (shown as Cuffdiff mean fold changes (log base 2) of 3 independent experiments). (D) RNA-Seq analysis revealed 201 REST-Dependent genes (Red) among the repressed genes (Blue). Data shown as a Volcano plot showing changes in gene expression due to hypoxia (1% O₂) plotted against significance. Only genes with statistically significant fold changes are shown. Each dot represents the mean fold change for a single gene with induced-genes as green dots (“Hypoxia induced”), repressed-genes as blue (“Hypoxia repressed”), and the genes that were not repressed by hypoxia in the presence of siRNA against REST shown as red dots (“REST-Dependent”). (E) Heat-map showing genes repressed by hypoxia (left column, Ctrl-RNAi), that where not repressed upon REST-RNAi treatment (right column, REST-RNAi). Data is presented as fold change to hypoxia of the top 20 genes most repressed by hypoxia among the REST-dependent genes. (F) Summary of the gene clusters found to be uniquely repressed by REST-Independent mechanisms (Blue), REST-Dependent mechanisms (Red) and the common clusters between the two mechanisms (Grey). Clusters were obtained using DAVID Functional Annotation Clustering together with manual curation of the results. For a detailed description of the annotation terms and gene groups associated with each cluster see Table S2.