Ultraconserved elements in the Olig2 promoter

Abstract

Background: Oligodendrocytes are specialized cells of the nervous system that produce the myelin sheaths surrounding the axons of neurons. Myelinating the axons increases the speed of nerve conduction and demyelination contributes to the pathology of neurodegenerative diseases such as multiple sclerosis. Oligodendrocyte differentiation is specified early in development by the expression of the basic-helix-loop-helix transcription factor Olig2 in the ventral region of the neural tube. Understanding how Olig2 expression is controlled is therefore essential for elucidating the mechanisms governing oligodendrocyte differentiation. A method is needed to identify potential regulatory sequences in the long stretches of adjacent non-coding DNA that flank Olig2.

Methodology/principal findings: We identified ten potential regulatory regions upstream of Olig2 based on a combination of bioinformatics metrics that included evolutionary conservation across multiple vertebrate genomes, the presence of potential transcription factor binding sites and the existence of ultraconserved elements. One of our computational predictions includes a region previously identified as the Olig2 basal promoter, suggesting that our criterion represented characteristics of known regulatory regions. In this study, we tested one candidate regulatory region for its ability to modulate the Olig2 basal promoter and found that it represses expression in undifferentiated embryonic stem cells.

Conclusions/significance: The regulatory region we identified modifies the expression regulated by the Olig2 basal promoter in a manner consistent with our current understanding of Olig2 expression during oligodendrocyte differentiation. Our results support a model in which constitutive activation of Olig2 by its basal promoter is repressed in undifferentiated cells by upstream repressive elements until that repression is relieved during differentiation. We conclude that the potential regulatory elements presented in this study provide a good starting point for unraveling the cis-regulatory logic that governs Olig2 expression. Future studies of the functionality of the potential regulatory elements we present will help reveal the interactions that govern Olig2 expression during development.

Figure 1. A) Frequency distribution of potential transcription factor binding sites in 2 Kb windows across the Olig2 upstream region.

Sequences upstream of Olig2 were scanned using Patser , for any potential binding site for any of the mammalian transcription factors in TRANSFAC . Total lengths of potential TFBS were summed up for each 2 kb window. B) Frequency distribution of percent identity in the Olig2 promoter. The average percent identity was calculated for every 2 kb window spanning the multiple alignments of sixteen species in the regions upstream of Olig2.

Figure 2. Expected and observed length distribution of non-coding sequences with perfect conservation across human, mouse, and rat reference genomes of Olig2.

The expected length distribution was generated by randomizing the columns in the human, mouse, and rat multiple alignments containing only non-coding regions.

Figure 3. A) Distribution of fluorescence intensity in 28 P-clones in the undifferentiated state. B) Distribution of fluorescence intensity in 48 UP-clones in the undifferentiated state.

P- and UP-clones were created by transfecting ES cells using two different constructs: one with a basal Olig2 promoter upstream of GFP and the other with ULTRA placed in front of the Olig2 promoter and GFP. Integration events were selected by applying neomycin. Clones were expanded and analyzed for GFP expression by flow cytometry. The distributions of fluorescence intensity for P- and UP-clones were statistically different (P-value<0.05, Wilcoxon rank sum test).

Figure 4. A) Distribution of fluorescence intensity in 25 P-clones in the differentiated state. B) Distribution of fluorescence intensity in 46 UP-clones in the differentiated state.

ES cells were grown in suspension to form embryonic bodies and retinoic acid and Shh agonist Hh-Ag 1.4 were added to the medium after 2 days. Differentiated cells were detected using Olig2 antibodies. The distributions of differentiated P- and UP-clones were not statistically different (P value >0.05, Wilcoxon rank sum test).