Journal: bioRxiv

Article Title: MeCP2 Interacts with the Super Elongation Complex to Regulate Transcription

doi: 10.1101/2024.06.30.601446

Figure Lengend Snippet: (A) Endogenous MeCP2 interacts with endogenous SEC components (AFF4, AF9, ENL, and ELL2) and RNA pol II in HEK293T cells. Normal mouse IgG was used as a negative control. (B) Endogenous MeCP2 interacts with SEC components (AFF4 and ELL2) and RNA pol II in the cortex of WT mouse at 7-weeks of age. (C) Reverse IP of endogenous AFF4 from cortical lysates of Camk2a-Cre/+ control (Cre) and Camk2a-Cre> Aff4 fl/fl ( Aff4 cKO) mice at 7-weeks of age. Mice were injected with either saline (baseline) or kainic acid (KA).

Article Snippet: 2μg of spike-in antibody (Active Motif; 61686) was added to each sample with one of the following antibodies for immunoprecipitations: 5μg of AFF4 antibody (Bosterbio; M03824) and 5μl of RNA pol II antibody (Cell Signaling Technology; 14958S).

Techniques: Negative Control, Control, Injection, Saline

Journal: bioRxiv

Article Title: MeCP2 Interacts with the Super Elongation Complex to Regulate Transcription

doi: 10.1101/2024.06.30.601446

Figure Lengend Snippet: (A) shRNA-mediated knockdown of AFF4 in HEK293T cells abolishes the interaction between MeCP2 and the remaining components of the SEC (ENL and ELL2). In contrast, knockdown of ENL or ELL2 does not affect the interaction between MeCP2 and the remaining components of the SEC. NT sh is a non-targeting short-hairpin negative control. (B) In vitro binding assay between recombinant FLAG-tagged MeCP2 and HA-tagged SEC components. (C) In vitro binding assay between FLAG-tagged MeCP2 fragments and HA-tagged AFF4. The domains critical for interaction are highlighted in bold. Diagram on the right shows a map of the MeCP2 fragments that were tested. NTD = N-terminal domain; MBD = methyl-CpG-binding domain; ID = intervening domain; TRD = transcriptional repression domain; CTD = C-terminal domain. (D) In vitro binding assay between AFF4 and MeCP2 with deletions in the NTD, ID, TRD, ID and TRD, and CTD. The domains critical for interaction are in bold.

Article Snippet: 2μg of spike-in antibody (Active Motif; 61686) was added to each sample with one of the following antibodies for immunoprecipitations: 5μg of AFF4 antibody (Bosterbio; M03824) and 5μl of RNA pol II antibody (Cell Signaling Technology; 14958S).

Techniques: shRNA, Knockdown, Negative Control, In Vitro, Binding Assay, Recombinant

Journal: bioRxiv

Article Title: MeCP2 Interacts with the Super Elongation Complex to Regulate Transcription

doi: 10.1101/2024.06.30.601446

Figure Lengend Snippet: (A) Heatmap of log occupancy of AFF4 in the cortex of WT and Mecp2 null mice. (B) Heatmap of log fold change of AFF4 occupancy in Mecp2 null mouse compared to WT mouse. (C) Heatmap of log occupancy of RNA pol II in the cortex of WT and Mecp2 null mice. (D) Heatmap of log fold change of RNA pol II occupancy in Mecp2 null mouse compared to WT mouse. 8696 RNA pol II-bound genes are represented in all heatmaps.

Article Snippet: 2μg of spike-in antibody (Active Motif; 61686) was added to each sample with one of the following antibodies for immunoprecipitations: 5μg of AFF4 antibody (Bosterbio; M03824) and 5μl of RNA pol II antibody (Cell Signaling Technology; 14958S).

Techniques:

Journal: bioRxiv

Article Title: MeCP2 Interacts with the Super Elongation Complex to Regulate Transcription

doi: 10.1101/2024.06.30.601446

Figure Lengend Snippet: (A) Heatmap showing the log fold change of AFF4 and RNA pol II binding in the Mecp2 null cortex based on hierarchical clustering. (B) Two-dimensional plot showing the change in AFF4 vs. RNA pol II binding in Mecp2 null animals across each cluster. Color scale indicates the gene count. Data shown is the average of three biological replicates. Spearman’s correlation values: cluster I π=0.14, p=1.5e-10; cluster II π=0.09, p=8.2e-10; cluster III π=0.09, p=0.1. (C) Violin plot showing the average expression value of all genes in each cluster relative to WT cluster I. Data shown is the average of six biological replicates. Statistical significance was assessed using the Wilcoxon Rank Sum Test. (D) Gene ontology analysis of cluster III genes. All terms are FDR < 0.05. (E) Track examples of AFF4 and RNA pol II binding and RNA expression levels in WT (grey) and Mecp2 null (orange) mouse.

Article Snippet: 2μg of spike-in antibody (Active Motif; 61686) was added to each sample with one of the following antibodies for immunoprecipitations: 5μg of AFF4 antibody (Bosterbio; M03824) and 5μl of RNA pol II antibody (Cell Signaling Technology; 14958S).

Techniques: Binding Assay, Expressing, RNA Expression