Journal: Nature Communications

Article Title: EGR1 recruits TET1 to shape the brain methylome during development and upon neuronal activity

doi: 10.1038/s41467-019-11905-3

Figure Lengend Snippet: Methylation dynamics of EGR1 binding sites during mouse brain development. a EGR1 binding motif ( E value = 1.1e −252 ) identified from ChIP-seq data generated with mouse adult frontal cortices. b Genomic distribution of EGR1 peaks. c Distribution of histone marks H3K27ac, H3K4me1, and H3K4me3 surrounding EGR1 peaks. d Methylation dynamics of EGR1 binding sites during brain development from embryonic day 11.5 to 22 months, in neurons (NeuN+), and non-neuronal cells (NeuN−). e Correlation of methylation changes at EGR1 binding sites during mouse brain development and between cell specification. Only binding sites with at least ten methylation calls in all four methylomes were included. P -values were determined with Wilcoxon Rank Sum Test

Article Snippet: Rabbit anti-EGR1 antibody (sc-189), mouse anti-EGR1 antibody (sc-101033), rabbit normal IgG (sc-2027) and mouse normal IgG (sc-2025) were purchased from Santa Cruz Biotechnology.

Techniques: Methylation, Binding Assay, ChIP-sequencing, Generated

Journal: Nature Communications

Article Title: EGR1 recruits TET1 to shape the brain methylome during development and upon neuronal activity

doi: 10.1038/s41467-019-11905-3

Figure Lengend Snippet: Identification of the protein-protein interaction between EGR1 and TET enzymes by co-immunoprecipitation. a , b Endogenous association of EGR1 and TET proteins. EGR1 was immuno-precipitated from mouse frontal cortex, followed by western blot to detect TET1 ( a ). TET1 was immuno-precipitated from mouse frontal cortex, followed by western blot to detect EGR1 ( b ). Normal rabbit IgG served as a negative control for immunoprecipitation. IP, immunoprecipitation. c Interactions between full-length EGR1 (EGR1-FL) and TET1 deletion mutants. Flag-tagged EGR1-FL and HA-tagged TET1 deletion mutants as shown in the schematic illustration were co-expressed in HEK293T cells. d Interactions between TET1s-CD and EGR1 deletion mutants. HA-tagged TET1-CD and Flag-tagged Egr1 deletion mutants as shown in the schematic illustration were co-expressed in HEK293T cells. Protein-protein interactions were examined by IP-western blot using the antibodies indicated. Source data are provided as a Source Data file

Article Snippet: Rabbit anti-EGR1 antibody (sc-189), mouse anti-EGR1 antibody (sc-101033), rabbit normal IgG (sc-2027) and mouse normal IgG (sc-2025) were purchased from Santa Cruz Biotechnology.

Techniques: Immunoprecipitation, Western Blot, Negative Control, Protein-Protein interactions

Journal: Nature Communications

Article Title: EGR1 recruits TET1 to shape the brain methylome during development and upon neuronal activity

doi: 10.1038/s41467-019-11905-3

Figure Lengend Snippet: EGR1 recruits TET1 to its target sites. a TET1 ChIP-qPCR assay in wild-type mouse frontal cortices. b Sequential ChIP-qPCR assay in wild-type mouse frontal cortices. The first antibody used was anti-TET1; the secondary antibody used was anti-EGR1 and normal rabbit IgG. Gcg locus serves as negative control for EGR1 binding. c ChIP-qPCR assay in frontal cortices of Egr1KO and wild-type mice. TET1 enrichment is normalized to the enrichment in WT. P -values were calculated with t -test, * P < 0.05, ** P < 0.01, *** P < 0.001. n.s., not significant. Error bars ± standard deviation (s.d.) from three technical replicates. d Venn diagrams show the overlapped TET1 peaks generated with two distinct antibodies (91171 and 5D6, Active Motif) or from WT and Egr1KO frontal cortices. e The distribution of TET1 peaks relatively to their nearest EGR1 peaks. The distance of TET1 peak to its nearest EGR1 peak refers to the number of nucleotides between the centers of two peaks

Article Snippet: Rabbit anti-EGR1 antibody (sc-189), mouse anti-EGR1 antibody (sc-101033), rabbit normal IgG (sc-2027) and mouse normal IgG (sc-2025) were purchased from Santa Cruz Biotechnology.

Techniques: ChIP-qPCR, Negative Control, Binding Assay, Standard Deviation, Generated

Journal: Nature Communications

Article Title: EGR1 recruits TET1 to shape the brain methylome during development and upon neuronal activity

doi: 10.1038/s41467-019-11905-3

Figure Lengend Snippet: Cooperativity of EGR1 and TET1 modulate the enhancer activity of EGR1 binding sites. a Luciferase reporter assays for the control vector pCpGL-P and constructs with Galnt9 , Npas4 locus. Fold change was normalized to the control vector pCpGL-P. Luciferase reporter assays for unmethylated or methylated Galnt9 ( b ) and Npas4 ( c ) constructs under either Egr1 / Tet1 singularly or co-expression in primary cortical neurons. In figure b – c , fold changes were normalized to the methylated vectors without Egr1/Tet1 overexpression. Luciferase activity was measured at 48 h after transfection and normalized against the activity of a co-transfected firefly construct. mCpG represents methylated constructs. P -values were determined by t -test, * P < 0.05, ** P < 0.01. Values represent mean ± s.d. from three biological replicates

Article Snippet: Rabbit anti-EGR1 antibody (sc-189), mouse anti-EGR1 antibody (sc-101033), rabbit normal IgG (sc-2027) and mouse normal IgG (sc-2025) were purchased from Santa Cruz Biotechnology.

Techniques: Activity Assay, Binding Assay, Luciferase, Control, Plasmid Preparation, Construct, Methylation, Expressing, Over Expression, Transfection

Journal: Nature Communications

Article Title: EGR1 recruits TET1 to shape the brain methylome during development and upon neuronal activity

doi: 10.1038/s41467-019-11905-3

Figure Lengend Snippet: Correlations of DNA methylation and gene expression profiles between Egr1KO and Tet1KO frontal cortices. Methylation correlations ( a ) and gene expression correlations ( b ) between Egr1KO and Tet1KO mice. c Aberrant DNA methylation on Galnt9 and Npas4 loci. Each CpG is represented by a circle; yellow in circles indicates the percentage of methylation in each CpG site. The statistical significance of methylation differences between Egr1/Tet1KO and WT mice was evaluated with the Wilcoxon rank-sum test. d The correlations between DNA methylation levels of Galnt9, Npas4 loci and corresponding gene expression during brain development from embryonic day 11.5 (E11.5) (denoted in blue color) to 22 months (22 mo) (denoted in red color)

Article Snippet: Rabbit anti-EGR1 antibody (sc-189), mouse anti-EGR1 antibody (sc-101033), rabbit normal IgG (sc-2027) and mouse normal IgG (sc-2025) were purchased from Santa Cruz Biotechnology.

Techniques: DNA Methylation Assay, Gene Expression, Methylation

Journal: Nature Communications

Article Title: EGR1 recruits TET1 to shape the brain methylome during development and upon neuronal activity

doi: 10.1038/s41467-019-11905-3

Figure Lengend Snippet: A simplified model for EGR1 and TET1 interaction linking environmental stimuli to brain methylome programming. At birth, Egr1 -mediated and neuronal activity-induced genes are silenced with methylated EGR1 binding sites. During postnatal development and upon neuronal activity, the increase in expression of Tet1 and Egr1 leads to the demethylation of EGR1 binding sites to facilitate the binding of co-factors and shifts the genes to either “Poised” or “ON” states. DNA methylation cannot block EGR1 binding but may prevent the bindings of other transcription factors, which bind to the regions adjacent to EGR1 binding sites. Thus, the demethylation of EGR1 binding sites may facilitate the formation of stronger transcription enhanceosomes

Article Snippet: Rabbit anti-EGR1 antibody (sc-189), mouse anti-EGR1 antibody (sc-101033), rabbit normal IgG (sc-2027) and mouse normal IgG (sc-2025) were purchased from Santa Cruz Biotechnology.

Techniques: Activity Assay, Methylation, Binding Assay, Expressing, DNA Methylation Assay, Blocking Assay