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. 2022 Apr 25;5(8):e202201385.
doi: 10.26508/lsa.202201385. Print 2022 Aug.

Nutrient sensitive protein O-GlcNAcylation modulates the transcriptome through epigenetic mechanisms during embryonic neurogenesis

Shama Parween  1 , Thilina T Alawathugoda  1 , Ashok D Prabakaran  2 , S Thameem Dheen  3 , Randall H Morse  4 , Bright Starling Emerald  2   5 , Suraiya A Ansari  6   5
Affiliations

Nutrient sensitive protein O-GlcNAcylation modulates the transcriptome through epigenetic mechanisms during embryonic neurogenesis

Shama Parween et al. Life Sci Alliance. .

Abstract

Protein O-GlcNAcylation is a dynamic, nutrient-sensitive mono-glycosylation deposited on numerous nucleo-cytoplasmic and mitochondrial proteins, including transcription factors, epigenetic regulators, and histones. However, the role of protein O-GlcNAcylation on epigenome regulation in response to nutrient perturbations during development is not well understood. Herein we recapitulated early human embryonic neurogenesis in cell culture and found that pharmacological up-regulation of O-GlcNAc levels during human embryonic stem cells' neuronal differentiation leads to up-regulation of key neurogenic transcription factor genes. This transcriptional de-repression is associated with reduced H3K27me3 and increased H3K4me3 levels on the promoters of these genes, perturbing promoter bivalency possibly through increased EZH2-Thr311 phosphorylation. Elevated O-GlcNAc levels also lead to increased Pol II-Ser5 phosphorylation and affect H2BS112O-GlcNAc and H2BK120Ub1 on promoters. Using an in vivo rat model of maternal hyperglycemia, we show similarly elevated O-GlcNAc levels and epigenetic dysregulations in the developing embryo brains because of hyperglycemia, whereas pharmacological inhibition of O-GlcNAc transferase (OGT) restored these molecular changes. Together, our results demonstrate O-GlcNAc mediated sensitivity of chromatin to nutrient status, and indicate how metabolic perturbations could affect gene expression during neurodevelopment.

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Conflict of interest statement

The authors declare that they have no conflict of interest.

Figures

Figure 1.
Figure 1.. Effect of elevated O-GlcNAcylation on transcriptome during embryonic cortical neurogenesis.
(A) Human embryonic stem cells (H9) were differentiated into cortical neurons using dual SMAD inhibition protocol in DMEM/F12 media with knockout serum replacement (KSR) containing LDN-193189 (BMP signalling inhibitor) and SB-431542 (TGF-β Receptor Kinase inhibitor) and treated with ThiametG or DMSO as control. The cells were immunostained for stage-specific markers, OCT4 (pluripotency), PAX6 (neural stem cell), and β-TUBULIN III (early-born neuron), collected on days 0, 12, 30, and 70 of differentiation and processed for RNA-Seq (two biological replicates). (B) Hierarchical clustering analysis and corresponding heat map of differentially expressed genes in control and ThiametG (TMG) treated cells from four stages of neural differentiation. Hierarchical clustering was performed using Euclidean distance to visualize the expression of genes across groups that were significant in at least one inter-stage comparison with false discovery rate < 5%. (C) Number of genes up/down-regulated in TMG-treated cells compared to control with different P-values and false discovery rate. (D) Venn diagram of the number of genes that are significantly affected (P-value ≤ 0.5) due to increased O-GlcNAc levels from four-stages of neural differentiation. (E) Ingenuity pathway analysis was performed on the list of genes significantly affected (P-value < 0.5) in TMG-treated cells compared to control on D12, D30, and D70 of neural differentiation. The red colour indicates activated pathways, and the blue colour indicates inhibited pathways. (F) GAD disease association on the list of genes affected significantly (≥2-fold with P-value ≤ 0.5) in TMG-treated cells compared with controls on D12, D30, and D70 of neural differentiation.
Figure 2.
Figure 2.. Elevated O-GlcNAc levels de-repress neural transcription factor genes known to be regulated by EZH2.
(A) Heat map of select transcription factor genes identified by Ingenuity Pathway Analysis from the list of significantly affected genes due to ThiametG treatment from all four stages of neural differentiation. (B) Chromatin immunoprecipitation assay was performed to analyze the enrichment of EZH2, H3K27me3, H3K27Ac, H3K4me3, and H3 at the promoters (two sites, around −1,000 bp of transcription start site) of TBR1, NEUROD1, EOMES, FOXG1, FOXP2, PAX3, TBX3, and HOPX using specific antibodies as indicated in ThiametG-treated and DMSO control samples (days 30 and 49 of cortical differentiation). Data represent the mean of three biological replicates ± SD. Statistical analyses (unpaired t test) were made between control and treated samples using GraphPad Prism Software. Asterisks represent differences being significant (*P < 0.05, **P < 0.01, ***P < 0.001).
Figure S1.
Figure S1.. Transcription factor (TF) enrichment analysis identifies EZH2 enriched at differentially expressed genes due to elevated O-GlcNAc levels.
(A, B) ENCODE ChIP-seq significance tool was used to identify TF enrichment on the list of all genes and (B) lineage-specific TF genes significantly affected (≥2-folds with P ≤ 0.5) due to O-GlcNAc elevation during D12, D30, and D70 of cortical neuronal differentiation of human embryonic stem cells.
Figure 3.
Figure 3.. Effect of elevated O-GlcNAc levels on EZH2 and histone modifications.
(A) Co-immunoprecipitation was performed on cell lysate from ThiametG-treated and DMSO control samples from D0, D12, D30, and D49 of cortical differentiation using anti-EZH2 antibody followed by Western blotting of IP’d samples using anti-O-GlcNAc and anti-phospho(Ser/Thr) antibodies. IP with IgG was used as control. Input is the total cell lysate. (B) Densitometric quantitation of Western blots from panel (B). Data shown are mean ± SD of three biological replicates. (C) Western blotting was performed for O-GlcNAc, EZH2, EZH2-Thr487p, EZH2-Thr311p, H3K27me3, H3K4me2, H3K4me3, MLL1, MLL2, LSD1, and GAPDH on ThiametG-treated and DMSO control samples from D0, D12, D22, D30, D49, and D70 of cortical differentiation of H9 cells. The expression of GAPDH was used as the loading control. (D) Densitometric quantitation of Western blots from panel (C). The values are expressed as a percentage relative to the values for D0-DMSO samples set as 100%. Data represent the mean of three biological replicates ± SD. Statistical analyses (unpaired t test) were made between control and treated samples using GraphPad Prism Software. Asterisks represent differences being significant (*P < 0.05, **P < 0.01, ***P < 0.001).
Figure 4.
Figure 4.. Effect of maternal hyperglycemia on the expression of markers of embryonic neurodevelopment.
(A) Protein expression analysis by Western blotting was performed on embryo brain cortices from E14.5, E16.5, and E18.5 of hyperglycemia and control rats using anti-O-GlcNAc, PCNA, β-Tubulin III, Pax6, Eomes, Tbr1, and Ezh2 antibodies. The expression of β-Actin was used as the loading control. The results are representative of three independent biological replicates. (A, B) Densitometric quantitation of Western blots from panel (A). Data shown are mean ± SD of three biological replicates. Statistical analyses (unpaired t test) were made between control and hyperglycemia samples for each developmental stage using GraphPad Prism Software. Asterisks represent differences being significant (*P < 0.05, **P < 0.01, ***P < 0.001).
Figure 5.
Figure 5.. Effect of maternal hyperglycemia on promoter histone methylation during embryonic neurodevelopment.
(A, B, C) Embryo brain cortices from E14.5, E16.5, and E18.5 of hyperglycemic (Hg) and control (Ctrl) rats were used to perform chromatin immunoprecipitation assay for H3, H3K4me3 and H3K27me3 using chromatin immunoprecipitation grade antibodies. ChIP’d DNA was analyzed by real-time qPCR for the promoter regions of Tbr1, Neurod1, Eomes, Foxg1, Foxp2, Tbx3, and Ngn2. Data represent the mean of three biological replicates ± SD. Statistical analyses (unpaired t test) were made between control (Ctrl) and hyperglycemia (Hg) samples using GraphPad Prism Software. Asterisks represent differences being significant (*P < 0.05, **P < 0.01, ***P < 0.001).
Figure 6.
Figure 6.. Effect of high O-GlcNAc levels on promoter H2B O-GlcNAcylation/mono-ubiquitination and Pol II-Serine5/2 phosphorylation.
(A, B, C, D) Chromatin immunoprecipitation assay was performed to analyze the enrichment of histone, H2B, H2BS112O-GlcNAc and H2BK120Ub1at the promoter (A, B) and unmodified Pol II, and Pol II-Ser5p (C, D) at 5′ of the coding region of the gene of TBR1, NEUROD1, EOMES, FOXG1, FOXP2, PAX3, NR2E1, HES5, and FEZF1 using chromatin immunoprecipitation grade antibodies as indicated in ThiametG-treated and DMSO control samples (day 30 and 49 of cortical differentiation). Data represent the mean of three biological replicates ± SD. Statistical analyses (unpaired t test) were made between control and treated samples using GraphPad Prism Software. Asterisks represent differences being significant (*P < 0.05, **P < 0.01, ***P < 0.001).
Figure 7.
Figure 7.. Effect of maternal hyperglycemia on histone H2B O-GlcNAcylation/mono-ubiquitination during embryonic neurodevelopment.
(A) Protein expression analysis by Western blotting was performed on embryo brain cortices from E14.5, E16.5, and E18.5 of hyperglycemia (HG) and control (C) rats using anti-H2B, H2BS112O-GlcNAc, and H2BK120ub1 antibodies. The expression of β-Actin was used as the loading control. (B) Densitometric quantitation of Western blots from panel (B). Data shown are mean ± SD of three biological replicates. Statistical analyses (unpaired t test) were made between control and hyperglycemia samples for each developmental stage using GraphPad Prism Software. Asterisks represent differences being significant (*P < 0.05, **P < 0.01, ***P < 0.001). (C, D, E) Embryo brain cortices from E14.5, E16.5, and E18.5 of hyperglycemic (Hg) and control (Ctrl) rats were used to perform chromatin immunoprecipitation assay for H2B, H2BS112O-GlcNAc and H2BK120ub1 using chromatin immunoprecipitation grade antibodies. ChIP’d DNA was analyzed by real-time qPCR for the promoter regions of Tbr1, Neurod1, Eomes, Foxg1, Foxp2, Tbx3, and Ngn2. Data shown are mean ± SD of three biological replicates.
Figure 8.
Figure 8.. Effect of maternal hyperglycemia on Pol II-Serine5/2 phosphorylation during embryonic neurodevelopment.
(A, B) Embryo brain cortices from E14.5, E16.5, and E18.5 of hyperglycemic (Hg) and control (Ctrl) rats were used to perform chromatin immunoprecipitation assay for unmodified Pol II and Pol II-Ser5p using chromatin immunoprecipitation grade antibodies. ChIP’d DNA was analyzed by real-time qPCR to analyze recruitments at 5′ of the coding region of the gene of Tbr1, Neurod1, Eomes, Foxg1, Foxp2, Tbx3, and Ngn2. Data represent the mean of three biological replicates ± SD. Statistical analyses (unpaired t test) were made between control (Ctrl) and hyperglycemia (Hg) samples using GraphPad Prism Software. Asterisks represent differences being significant (*P < 0.05, **P < 0.01, ***P < 0.001).
Figure 9.
Figure 9.. The effect of O-GlcNAc transferase inhibition on maternal hyperglycemia-mediated molecular changes in developing embryo brains.
(A) Protein expression analysis by Western blotting was performed on embryo brain cortices from E14.5 of control (C), hyperglycemic (HG), and hyperglycemic treated with O-GlcNAc transferase inhibitor, ST045849 (ST) rats using anti-O-GlcNAc, Ezh2-Thr311p, Ezh2-Thr487p, Ezh2, H3K4me2, H3K4me3, H3K27me3, H3, Mll1, Mll2, and Lsd1 antibodies. The expression of β-Actin was used as the loading control. The results are representative of three independent biological replicates. (A, B) Densitometric quantitation of Western blots from panel (A). Data shown are mean ± SD of three biological replicates. Statistical analyses (unpaired t test) were made between C versus HG samples and HG versus ST samples using GraphPad Prism Software. Asterisks represent differences being significant (*P < 0.05, **P < 0.01, ***P < 0.001).
Figure S2.
Figure S2.. O-GlcNAc transferase inhibition restores maternal hyperglycemia-mediated changes in the expression of neuronal markers in developing embryo brains.
(A) Protein expression analysis by Western blotting was performed on embryo brain cortices from E14.5 of control (C), hyperglycemia (HG), and hyperglycemia treated with O-GlcNAc transferase inhibitor ST045849 (ST) rats using anti-Eomes, Tbr1, and β-Tubulin III antibodies. The expression of β-Actin was used as the loading control. The results are representative of two biological replicates. (A, B) Densitometric quantitation of Western blots from panel (A).
Figure 10.
Figure 10.. Model connecting hyperglycemia and elevated O-GlcNAc levels to epigenetic dysregulation and gene expression changes during in vitro and in vivo embryonic neurogenesis.
Based on our findings, pharmacological up-regulation of O-GlcNAc levels during human embryonic stem cells’ neuronal differentiation in vitro and maternal hyperglycemia in rats which elevates global O-GlcNAc levels in the developing embryo brain cortex leads to increased phosphorylation of EZH2 at Thr311, resulting in decreased H3K27me3 and subsequent transcriptional up-regulation through increased Pol II Ser5-p, H3K4me3, H2BS112-O-GlcNAc, and H2BS120-Ub1 levels.
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