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Effect of Bmi1 over-expression on gene expression in adult and embryonic murine neural stem cells

Ganapathi, Mythily, Boles, Nathan C., Charniga, Carol, Lotz, Steven, Campbell, Melissa, Temple, Sally, Morse, Randall H.·2018·Scientific Reports
DOI 10.1038/s41598-018-25921-8PMC PMC5945652
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Abstract

The ability of isolated neural stem cells (NSCs) to proliferate as neurospheres is indicative of their competence as stem cells, and depends critically on the polycomb group (PcG) member Bmi1: knockdown of Bmi1 results in defective proliferation and self-renewal of isolated NSCs, whereas overexpression of Bmi1 enhances these properties. Here we report genome-wide changes in gene expression in embryonic and adult NSCs (eNSCs and aNSCs) caused by overexpression of Bmi1. We find that genes whose expression is altered by perturbations in Bmi1 levels in NSCs are mostly distinct from those affected in other multipotent stem/progenitor cells, such as those from liver and lung, aside from a small core of common targets that is enriched for genes associated with cell migration and mobility. We also show that genes differing in expression between prospectively isolated quiescent and activated NSCs are not affected by Bmi1 overexpression. In contrast, a comparison of genes showing altered expression upon Bmi1 overexpression in eNSCs and in aNSCs reveals considerable overlap, in spite of their different provenances in the brain and their differing developmental programs.

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Figure 1 Gene ontology enrichment among genes affected by Bmi1 overexpression in aNSCs. Categories enriched for genes down-regulated (A) or up-regulated (B) at least two-fold upon Bmi1 overexpression in aNSCs are indicated along with −log of the corrected p-value for each category. All categories having corrected p-value <10−5 are shown except for unannotated genes, which were enriched among down-regulated genes.
Figure 1 Gene ontology enrichment among genes affected by Bmi1 overexpression in aNSCs. Categories enriched for genes down-regulated (A) or up-regulated (B) at least two-fold upon Bmi1 overexpression in aNSCs are indicated along with −log of the corrected p-value for each category. All categories having corrected p-value <10−5 are shown except for unannotated genes, which were enriched among down-regulated genes.CC BY · PubMed Central
Figure 2 K-means clustering of gene expression in active and quiescent NSCs and aNSCs. K-means clustering (K = 10) of gene expression for active and quiescent aNSCs32, and for control and Bmi1-overexpressing aNSCs, was performed using genes that exhibited at least four-fold change in expression between active and quiescent adult NSCs. Expression values from microarray analyses were adjusted by subtraction of the mean value to allow values to center around zero. Genes belonging to the 10 clusters are listed in Supplementary Table S5.
Figure 2 K-means clustering of gene expression in active and quiescent NSCs and aNSCs. K-means clustering (K = 10) of gene expression for active and quiescent aNSCs32, and for control and Bmi1-overexpressing aNSCs, was performed using genes that exhibited at least four-fold change in expression between active and quiescent adult NSCs. Expression values from microarray analyses were adjusted by subtraction of the mean value to allow values to center around zero. Genes belonging to the 10 clusters are listed in Supplementary Table S5.CC BY · PubMed Central
Figure 3 Effect of elevated Bmi1 expression in aNSCs, hepatic precursor cells, and lung stem cells. (A) Hierarchical clustering of 4202 genes showing altered expression of at least 1.5 fold in at least one of the three data sets indicated. (B) Overlap among genes up- or down-regulated by at least 1.5 fold in the indicated data sets. (C) Hierarchical clustering was performed on gene expression changes caused by elevated Bmi1 levels in aNSCs, hepatic precursor cells, and lung stem cells using 64 genes down-regulated at least 2 fold in aNSCs overexpressing Bmi1 and belonging to the GO category of nervous system development. The expanded region shows a cluster of genes most of which are also down-regulated in lung stem cells. Highlighted genes encode known and probable transcription factors.
Figure 3 Effect of elevated Bmi1 expression in aNSCs, hepatic precursor cells, and lung stem cells. (A) Hierarchical clustering of 4202 genes showing altered expression of at least 1.5 fold in at least one of the three data sets indicated. (B) Overlap among genes up- or down-regulated by at least 1.5 fold in the indicated data sets. (C) Hierarchical clustering was performed on gene expression changes caused by elevated Bmi1 levels in aNSCs, hepatic precursor cells, and lung stem cells using 64 genes down-regulated at least 2 fold in aNSCs overexpressing Bmi1 and belonging to the GO category of nervous system development. The expanded region shows a cluster of genes most of which are also down-regulated in lung stem cells. Highlighted genes encode known and probable transcription factors.CC BY · PubMed Central
Figure 4 Effect on gene expression of Bmi1 overexpression in eNSCs compared to aNSCs. (A) Comparison of transcript levels measured by RNA-seq (reads per kilobase per million reads mapped, RPKM) in embryonic and adult NSCs (empty vector control, average of two biological replicates). (B) Gene ontology categories enriched for genes down-regulated (top) or up-regulated (bottom) upon Bmi1 overexpression in eNSCs (red bars) or aNSCs (blue bars). Categories having P < 10−6 (hypergeometric test, corrected for multiple category testing) are shown for down-regulated genes and having P < 10−4 for up-regulated genes; GO categories having more than 1624 member genes (the number in nervous system development) are not shown. (C) Hierarchical clustering was performed for genes down-regulated at least two-fold by Bmi1 overexpression with p < 0.01 in either eNSCs or aNSCs and belonging to the GO category Neurogenesis (left) or by at least two-fold in eNSCs with p < 0.01 and belonging to Cell Adhesion (right). The right-hand two lanes in each panel depict as heat maps log2 of the change in expression between cells overexpressing Bmi1 and control cells, while the left-hand lanes indicate relative expression levels in control cells (transduced with empty vector). Reads (RPKM) were divided by 6 prior to clustering to allow them to be visualized on the same scale as the log2 changes in expression. Red dots mark genes expressed at a level of at least 3 RPKM (before correction) and differing at least two-fold in expression level between eNSCs and aNSCs. Note that the color scales are different for the two clustering plots.
Figure 4 Effect on gene expression of Bmi1 overexpression in eNSCs compared to aNSCs. (A) Comparison of transcript levels measured by RNA-seq (reads per kilobase per million reads mapped, RPKM) in embryonic and adult NSCs (empty vector control, average of two biological replicates). (B) Gene ontology categories enriched for genes down-regulated (top) or up-regulated (bottom) upon Bmi1 overexpression in eNSCs (red bars) or aNSCs (blue bars). Categories having P < 10−6 (hypergeometric test, corrected for multiple category testing) are shown for down-regulated genes and having P < 10−4 for up-regulated genes; GO categories having more than 1624 member genes (the number in nervous system development) are not shown. (C) Hierarchical clustering was performed for genes down-regulated at least two-fold by Bmi1 overexpression with p < 0.01 in either eNSCs or aNSCs and belonging to the GO category Neurogenesis (left) or by at least two-fold in eNSCs with p < 0.01 and belonging to Cell Adhesion (right). The right-hand two lanes in each panel depict as heat maps log2 of the change in expression between cells overexpressing Bmi1 and control cells, while the left-hand lanes indicate relative expression levels in control cells (transduced with empty vector). Reads (RPKM) were divided by 6 prior to clustering to allow them to be visualized on the same scale as the log2 changes in expression. Red dots mark genes expressed at a level of at least 3 RPKM (before correction) and differing at least two-fold in expression level between eNSCs and aNSCs. Note that the color scales are different for the two clustering plots.CC BY · PubMed Central
Figure 5 Comparison of gene expression changes in embryonic and adult NSCs caused by Bmi1 overexpression. (A) Reads per kilobase per million reads (RPKM) from RNA-seq data from aNSCs and eSNCs is plotted for 4296 genes having expression altered at least two-fold by Bmi1 overexpression in either eNSCs or aNSCs. (B) Genes plotted in (A) were broken down according to their relative expression in eNSCs and aNSCs as indicated.
Figure 5 Comparison of gene expression changes in embryonic and adult NSCs caused by Bmi1 overexpression. (A) Reads per kilobase per million reads (RPKM) from RNA-seq data from aNSCs and eSNCs is plotted for 4296 genes having expression altered at least two-fold by Bmi1 overexpression in either eNSCs or aNSCs. (B) Genes plotted in (A) were broken down according to their relative expression in eNSCs and aNSCs as indicated.CC BY · PubMed Central

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Metadata

Authors
Ganapathi, M., Boles, N. C., Charniga, C., Lotz, S., Campbell, M., Temple, S., & Morse, R. H.
ORCID
Morse, Randall H.
Venue
Scientific Reports
Publisher
Springer Science and Business Media LLC
Published
2018-05-10
Volume
8
Issue
1
ISSN
2045-2322
DOI
10.1038/s41598-018-25921-8
PMC
PMC5945652
Cited by
24
References
2
Language
en
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