OLIG2 regulates lncRNAs and its own expression during oligodendrocyte lineage formation

Abstract

Background: Oligodendrocytes, responsible for axon ensheathment, are critical for central nervous system (CNS) development, function, and diseases. OLIG2 is an important transcription factor (TF) that acts during oligodendrocyte development and performs distinct functions at different stages. Previous studies have shown that lncRNAs (long non-coding RNAs; > 200 bp) have important functions during oligodendrocyte development, but their roles have not been systematically characterized and their regulation is not yet clear.

Results: We performed an integrated study of genome-wide OLIG2 binding and the epigenetic modification status of both coding and non-coding genes during three stages of oligodendrocyte differentiation in vivo: neural stem cells (NSCs), oligodendrocyte progenitor cells (OPCs), and newly formed oligodendrocytes (NFOs). We found that 613 lncRNAs have OLIG2 binding sites and are expressed in at least one cell type, which can potentially be activated or repressed by OLIG2. Forty-eight of them have increased expression in oligodendrocyte lineage cells. Predicting lncRNA functions by using a "guilt-by-association" approach revealed that the functions of these 48 lncRNAs were enriched in "oligodendrocyte development and differentiation." Additionally, bivalent genes are known to play essential roles during embryonic stem cell differentiation. We identified bivalent genes in NSCs, OPCs, and NFOs and found that some bivalent genes bound by OLIG2 are dynamically regulated during oligodendrocyte development. Importantly, we unveiled a previously unknown mechanism that, in addition to transcriptional regulation via DNA binding, OLIG2 could self-regulate through the 3' UTR of its own mRNA.

Conclusions: Our studies have revealed the missing links in the mechanisms regulating oligodendrocyte development at the transcriptional level and after transcription. The results of our research have improved the understanding of fundamental cell fate decisions during oligodendrocyte lineage formation, which can enable insights into demyelination diseases and regenerative medicine.

Keywords: Histone modification; LncRNAs; OLIG2; Oligodendrocyte development; Regulation after transcription; Transcriptional regulation.

Fig. 1

Experimental scheme and the genome-wide binding of OLIG2 in NSCs, OPCs, and NFOs. a Experimental scheme and data integration. b Genomic distribution of OLIG2 called peaks in NSCs, OPCs, and NFOs. c Overlapping OLIG2 binding genes in NSCs, OPCs, and NFOs. d Functional enrichment of specific genes in NSCs, OPCs, and NFOs

Fig. 2

OLIG2 activates or represses gene expression during transitions from NSCs to OPCs and OPCs to NFOs. a, b Heatmaps of DEGs in OPCs compared with NSCs (a) and NFOs compared with OPCs (b). c, d Overlap of DEGs bound by OLIG2 in OPCs compared with NSCs (c) and NFOs compared with OPCs (d). e, f Gene set enrichment of downregulated (e) or upregulated (f) genes that are also bound by OLIG2 in NFOs compared with OPCs. g Some examples of top binding motifs of TFs that are enriched in the OLIG2 called peak regions. h Gene expression of TFs

Fig. 3

LncRNAs bound by OLIG2 in NSCs, OPCs, and NFOs. a K-means clustering of lncRNAs identified in NSCs, OPCs, and NFOs. b The potential function of 48 lncRNAs in cluster 7. Group1 genes (39 lncRNAs) have OLIG2 called peaks in NSCs but not in OPCs or NFOs; group 2 genes (9 lncRNAs) have OLIG2 called peaks in OPCs and NFOs but not in NSCs. Color depth represents NES (normalized enrichment score) calculated by GSEA and indicates association strength. c ChIP-Seq and gene expression of AC140285.1 in NSCs, OPCs, and NFOs

Fig. 4

Distribution of H3K4me3 (K4) and H3K27me3 (K27) in NSCs, OPCs, and NFOs. a Genomic distribution of H3K4me3 and H3K27me3 called peaks in NSCs, OPCs, and NFOs. b Heatmap of H3K4me3 (K4) and H3K27me3 (K27) ChIP-Seq data in NSCs, OPCs, and NFOs in the interval from − 3 kb to + 3 kb from the TSS. c The percentage of genes in NSCs, OPCs, and NFOs associated with only H3K4me3, bivalent marks, only H3K27me3, or other states. d–f. Box plot of expression values of genes from different epigenomic categories in NSCs (d), OPCs (e), and NFOs (f). g Profiles of H3K4me3 and H3K27me3 marks and RNA-Seq visualized using IGV (Nkx2-2)

Fig. 5

Bivalent genes in NSCs, OPCs, and NFOs. a Overlaps of bivalent genes in NSCs, OPCs, and NFOs. b Functional enrichment of overlapping bivalent genes in NSCs, OPCs, and NFOs, based on MSigDB. c Dynamic regulation of NSCs bivalent genes in OPCs and NFOs. d, e H3K4me3 and H3K27me3 binding peaks and gene expression of Sox10 (d) and Zfp488 (e)

Fig. 6

OLIG2 is involved in regulation of its own expression at the transcriptional level and after transcription. a Experimental overview. b ChIP-Seq result shows that OLIG2 binding to its promoter region, potentially self-regulates its own transcription. c Binding of OLIG2 to Olig2 mRNA in NSCs shown by RIP-Seq. IgG was used as control. d In vitro transcribed biotinylated Olig2 RNA retrieves OLIG2 protein. The profiles were established by RNA protein pull-down using NSCs protein extract. Retrieved OLIG2 protein (32 kDa) was detected by Western blot assay. e Addition of the Olig2 3′ UTR to the ZsGreen1 reporter C1 end substantially reduces expression of ZsGreen1 in Olig2 overexpressing cells. Flow cytometric analyses on MFI of ZsGreen1 fluorescence signal in 293FT cells 48 h after transfection. The MFI of ZsGreen1 fluorescence signal was normalized by dividing the MFI of mRuby fluorescence signal in each replicate. The error bars show standard errors of the means (error bars) (n = 6 in each assay) f. The normalized protein level of OLIG2 in NSC cells 48 h after transfected ZsGreen1 reporter constructs with or without Olig2 3′ UTR. The protein level of OLIG2 was examined by Western blot and normalized to corresponding GAPDH levels. All quantified data were presented as mean ± SEM (standard error of the mean). N = 3; t test analysis *, p < 0.05; **, p < 0.01

Fig. 7

Diagram summarizing the main findings. a The first layer indicates the up- and downregulated of lncRNAs and TFs with OLIG2 called peaks. The second layer shows some examples of the TFs potentially cooperating with OLIG2 in the regulation of gene expression. The third layer indicates the dynamically regulated genes with histone marks in NSCs, OPCs, and NFOs and some examples mentioned in the manuscript. b The proposed involvement of OLIG2 in regulating its own gene expression at the transcriptional level as a transcriptional factor, and after transcription through Olig2 3′ UTR directly or indirectly to influence OLIG2 expression