FIGURE SUMMARY
Title

Multi-Dimensional Transcriptome Analysis Reveals Modulation of Cholesterol Metabolism as Highly Integrated Response to Brain Injury

Authors
Gourain, V., Armant, O., Lübke, L., Diotel, N., Rastegar, S., Strähle, U.
Source
Full text @ Front. Neurosci.

Injury-induced changes in level of polyadenylated RNAs. (A) Similarities between transcriptome of RNASeq samples were assessed by hierarchical clustering on the Euclidean distances computed between samples. The RNASeq samples were consistently grouped in their condition, control and injured. (B) RNAs were quantified for all annotated genes in the zebrafish reference genome GRCz11 (“Tested,” n = 32,520), genes expressed in the adult zebrafish telencephalon (“Expressed,” n = 17,301) or genes differentially expressed upon injury (“Differentially expressed”). The blue color depicts increased levels of transcript after injury (n = 1,946) and the yellow color decreased levels of transcript after injury (n = 3,043). (C) The sensitivity of the RNASeq analysis was further evaluated with significant changes in level of RNAs of relevant markers for regenerative neurogenesis. These markers were, selected based on literature, were grouped according to respective biological functions relative to the repair of damaged adult zebrafish telencephalon (color code). *adjp = 0.05, **adjp < 10– 02, ***adjp < 10– 04.

Selected enriched biological functions. To gain information about functions of genes with significant changes in their levels of transcript, the enrichment of ontologies (GO terms), signaling pathways (KEGG pathways) and metabolism pathways (Reactome pathways) was tested and up-regulated genes (red) and down-regulated genes (green) were detailed. *adjp = 0.05, **adjp < 0.01, ***adjp < 0.001 (For complete list see Supplementary Table 2).

Alteration in cholesterol metabolism in response to brain injury. (A) Increases in level of transcripts coding for cholesterol synthesizing enzymes (green) and decreased level of transcripts coding for transporter involved in ferrying cholesterol through the body and across membrane (red) were identified. Products and substrates are represented in blue boxes and enzymatic reactions by blue arrows. The double black arrow represents flow across membrane. (B,C) Changes in levels of mRNA were validated comparing the quantification by qRT-PCR of mRNAs encoding three selected enzymes synthesizing cholesterol (B) and two transporters (C) in three independent control (yellow) and injured (blue) telencephalic hemispheres (n = 3). *p-value = 0.05, **p-value < 10– 03.

Sterol Regulatory Element (SRE) motif analysis. (A) Two mammalian SRE motifs were retrieved from the literature (left and middle panels). From the mapping of these two consensus sequences one SRE motif derived in the zebrafish genome (right panel). (B) The SRE motifs were mapped in the promoter of genes involved in cholesterol metabolism. The promoter sequence was defined from 1 kb upstream of the transcription start site and the SRE motif were mapped in both forward (+) and reverse (–) strands. (C) Genes harboring a SRE motif in their 1-kb promoter (underlined) were identified in the cholesterol synthesis pathway, including genes coding for two upstream regulators (srebf2 and insig1). For further details see also legend to Figure 3A.

Injury-induced changes in levels of miRNAs. (A) The consistency of small RNASeq samples was tested by hierarchical clustering on Euclidean distances as for the RNASeq samples (see Figure 1A). The small RNASeq samples were consistently grouped into their respective condition, control or injured. (B) Changes in level of miRNAs were assessed comparing injured and uninjured telencephalic hemispheres. Significant differences in level of miRNA were tested (red; adjp < 0.05). (C) Targets of miR-182, miR155, miR-146a, and miR-31 were identified in the cholesterol synthesis pathway. For further details see also legend to Figures 3A, 4C.

Differentially expressed lncRNAs selected for their association with cholesterol synthesis or transport. LncRNAs annotated in the zebrafish genome, and with significant changes in their respective levels upon injury, were localized in the direct vicinity of genes encoding cholesterol-related proteins. Color indicates pairs of coding and non-coding RNAs. *adjp = 0.05, **adjp < 10– 02, ***adjp < 10– 04, ns: not significant. See legend of Figure 7B for the position of the genes in the cholesterol pathway.

Alternative splicing of RNAs related to cholesterol metabolism in response to injury. (A) Splicing isoforms of RNAs encoding proteins of the cholesterol synthesis and transport pathway were first reconstructed and then quantified in both uninjured and injured telencephalic hemispheres. The color blue depicts a decrease in the number of supporting reads while yellow an increase. A number of splice isoforms were not yet annotated in the genome (unannotated). *adjp = 0.05, **adjp < 10–02, ***adjp < 10–03. (B) All results about cholesterol metabolism were finally integrated, including up-regulated transporters (red) down-regulated synthesizing enzymes (green) and genes encoding mRNA affected by alternative splicing (indicated by +) or predicted targets of microRNA or lncRNA (indicated by *). Underlined names depict genes harboring the SRE motif in their 1-kbp promoter.

Acknowledgments
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