Journal: bioRxiv

Article Title: The histone deacetylase complex MiDAC regulates a neurodevelopmental gene expression program

doi: 10.1101/2020.04.07.029751

Figure Lengend Snippet: MiDAC binds to and modulates the expression of genes that regulate neural differentiation and neurite outgrowth. A Heatmaps displaying the genome-wide distribution of all DNTTIP1 binding sites on promoters (TSS) and promoter distal elements in WT and Dnttip1 KO1 mESCs sorted by enrichment in descending order in WT mESCs and compared to H3K4me1, H3K4me3, H3K27ac and H3K27me3 occupancy in WT and Dnttip1 KO1 mESCs. The color scale depicts the normalized ChIP-seq signal intensity (log 2 CPM per 20 bp bin). B, C Occupancy plots displaying the average DNTTIP1 enrichment over gene bodies stratified by B transcriptionally down-(FC < −1.5, p < 0.01) or C upregulated (FC > 1.5, p < 0.01) genes in WT versus Dnttip1 KO1 mESCs. D De novo motif analysis of DNTTIP1-bound genes that are down- or upregulated in Dnttip1 KO1 versus WT mESCs with motif-enrichment plot for ELK1 and RBFOX2. E, F ChIP-seq profiles of the E Ntn1 and F Id1 loci for DNTTIP1 in WT, Dnttip1 KO1 and Elmsan1 KO1 mESCs and for HDAC1 in WT mESCs. Promoter and putative enhancer regions used for manual ChIP experiments in and Figure S6 are highlighted by orange boxes.

Article Snippet: Experiments to rescue the neurite outgrowth defects of Dnttip1 KO neurons with axon guidance ligands were carried out as described above except that recombinant SLIT3 (R&D Systems, 9296-SL-050), NTN1 (R&D Systems, 6419-N1-025) or a combination of SLIT3/NTN1 were added daily to differentiation medium 3 (at a concentration of 0.5 and 0.25 ug/ml for SLIT3 and NTN1 respectively) from day 6 of differentiation onward.

Techniques: Expressing, Genome Wide, Binding Assay, ChIP-sequencing

Journal: bioRxiv

Article Title: The histone deacetylase complex MiDAC regulates a neurodevelopmental gene expression program

doi: 10.1101/2020.04.07.029751

Figure Lengend Snippet: MiDAC directly targets positive and negative regulators of neurite outgrowth during neural differentiation . A-J qPCR from manual ChIP experiments against A, F DNTTIP1, B, G ELMSAN1, C, H HDAC1, D, I H3K27ac and E, J H4K20ac from WT, Dnttip1 KO1 and Elmsan1 KO1 NE targeting select promoter, putative enhancer and intragenic control regions of A-D the Ntn1 or E-H the Id1 loci as highlighted in Figure S5E and F. IgG was used as a control antibody. Unpaired t-test was performed throughout where **, p ≤ 0.01; and ns, p > 0.05 is not significant.

Article Snippet: Experiments to rescue the neurite outgrowth defects of Dnttip1 KO neurons with axon guidance ligands were carried out as described above except that recombinant SLIT3 (R&D Systems, 9296-SL-050), NTN1 (R&D Systems, 6419-N1-025) or a combination of SLIT3/NTN1 were added daily to differentiation medium 3 (at a concentration of 0.5 and 0.25 ug/ml for SLIT3 and NTN1 respectively) from day 6 of differentiation onward.

Techniques: Control

Journal: bioRxiv

Article Title: The histone deacetylase complex MiDAC regulates a neurodevelopmental gene expression program

doi: 10.1101/2020.04.07.029751

Figure Lengend Snippet: MiDAC does not transcriptionally regulate the receptors Robo3 and Unc5b of the SLIT3 and NTN1 signaling pathways. A, B ChIP-seq profiles of the A Robo3 and B Unc5b loci for DNTTIP1 in WT, Dnttip1 KO1 and Elmsan1 KO1 mESCs and for HDAC1 in WT mESCs. Promoter regions used for manual ChIP experiments in Figure S7C and E are highlighted by orange boxes. C qPCR from manual ChIP experiments against DNTTIP1, ELMSAN1 and HDAC1 from WT, Dnttip1 KO1 and Elmsan1 KO1 NE targeting the promoter of the Robo3 locus as highlighted in Figure S7A. IgG was used as a control antibody. D qRT-PCR for Robo3 mRNA in WT, Dnttip1 KO1 and Elmsan1 KO1 mESCs. Expression was normalized to Gapdh. E qPCR from manual ChIP experiments against DNTTIP1, ELMSAN1 and HDAC1 from WT, Dnttip1 KO1 and Elmsan1 KO1 NE targeting the promoter of the Unc5b locus as highlighted in Figure S7B. IgG was used as a control antibody. F qRT-PCR for Unc5b mRNA in WT, Dnttip1 KO1 and Elmsan1 KO1 mESCs. Expression was normalized to Gapdh .

Article Snippet: Experiments to rescue the neurite outgrowth defects of Dnttip1 KO neurons with axon guidance ligands were carried out as described above except that recombinant SLIT3 (R&D Systems, 9296-SL-050), NTN1 (R&D Systems, 6419-N1-025) or a combination of SLIT3/NTN1 were added daily to differentiation medium 3 (at a concentration of 0.5 and 0.25 ug/ml for SLIT3 and NTN1 respectively) from day 6 of differentiation onward.

Techniques: Protein-Protein interactions, ChIP-sequencing, Control, Quantitative RT-PCR, Expressing

Journal: bioRxiv

Article Title: The histone deacetylase complex MiDAC regulates a neurodevelopmental gene expression program

doi: 10.1101/2020.04.07.029751

Figure Lengend Snippet: MiDAC regulates neurite outgrowth via the SLIT3/ROBO3 and NTN1/UNC5B signaling pathways. A Experimental outline of assay to rescue neurite outgrowth defects in Dnttip1 KO1 or Elmsan1 KO1 NE as described in -C and 6B-D. B Experimental outline of the chamber assay to rescue the neuronal network formation defects of granule neuron progenitor (GNP)-derived neurons co-cultured with Dnttip1 KO1 or Elmsan1 KO1 NE as described in Figure S8C, D or CM of Dnttip1 KO1 or Elmsan1 KO1 NE as described in Figure S8E, F. C Chamber assay utilizing GNPs that were co-cultured in the lower chamber in parallel with differentiating WT, Dnttip1 KO1 or Elmsan1 KO1 NE in the upper chamber comprising a time window from day 7-12 of differentiation to induce neuronal network formation from GNPs. Defined differentiation-inducing GNP medium was used as a positive and CM from mouse embryonic fibroblasts (MEFs) as a negative control. TUBB3 IF staining of GNP-derived neuronal networks was performed after 6 days of differentiation. Nuclei were stained with DAPI. The white scale bar represents 50 µm. D Quantification of neuronal network formation from the TUBB3 IF staining in Figure S8C using ImageJ. The percentage of formed neuronal networks within the total population of TUBB3-positive neurons is displayed. A neuronal network was scored when a closed local circuit was detected around an individual neuron. Neuronal network formation was assessed for 100 neurons per sample in triplicate. E Chamber assay to rescue the network formation defects of GNP-derived neurons that were supplemented with CM of Dnttip1 KO1 NE. CM of Dnttip1 KO1 NE was supplemented with the recombinant signaling ligands SLIT3 and/or NTN1 from day 7-12 without or with pre-blocking of GNP-derived neurons with control IgG or signaling receptor antibodies against ROBO3 and/or UNC5B. TUBB3 IF staining was performed after 12 days of differentiation. Nuclei were stained with DAPI. The white scale bar represents 50 µm. F Quantification of neuronal network formation from the TUBB3 IF staining in Figure S8E using ImageJ. The percentage of formed neuronal networks within the total population of TUBB3-positive neurons is displayed. A neuronal network was scored when a closed local circuit was detected around an individual neuron. Neuronal network formation was assessed for 100 neurons per sample in triplicate. Unpaired t-test was performed throughout where ***, p ≤ 0.01.

Article Snippet: Experiments to rescue the neurite outgrowth defects of Dnttip1 KO neurons with axon guidance ligands were carried out as described above except that recombinant SLIT3 (R&D Systems, 9296-SL-050), NTN1 (R&D Systems, 6419-N1-025) or a combination of SLIT3/NTN1 were added daily to differentiation medium 3 (at a concentration of 0.5 and 0.25 ug/ml for SLIT3 and NTN1 respectively) from day 6 of differentiation onward.

Techniques: Protein-Protein interactions, Boyden Chamber Assay, Derivative Assay, Cell Culture, Negative Control, Staining, Recombinant, Blocking Assay, Control

Journal: bioRxiv

Article Title: The histone deacetylase complex MiDAC regulates a neurodevelopmental gene expression program

doi: 10.1101/2020.04.07.029751

Figure Lengend Snippet: MiDAC regulates neurite outgrowth via the SLIT3/ROBO3 and NTN1/UNC5B signaling pathways. A WB for signaling components of the SLIT3/ROBO3 and NTN1/DCC/UNC5B signaling axes from CM and total cell lysates of WT and Dnttip1 KO1 NE after 12 days of differentiation. To enrich SLIT3 and NTN1 from CM, IPs were performed with SLIT3 and NTN1 antibodies from CM of WT and Dnttip1 KO1 NE. Actin is the loading control for the total cell lysates. B Assay to rescue the neurite outgrowth defects in Dnttip1 KO1 NE. CM of Dnttip1 KO1 NE was supplemented with the recombinant signaling ligands SLIT3 and/or NTN1 from day 7-12 without or with preblocking of Dnttip1 KO1 NE with IgG or signaling receptor antibodies against ROBO3 and/or UNC5B. MAP2 IF staining was performed after 12 days of differentiation. Nuclei were stained with DAPI. To facilitate analysis the neuronal cell body (blue) and its neurites were manually traced with ImageJ software and for each sample one traced neuron is displayed in the inlet. The white scale bar represents 50 µm. C, D Quantification of C neurite length and D the total number of neurites per neuron from the MAP2 IF staining in using ImageJ. C Neurite length was divided into two categories of short neurites < 50 µm (green box plots) and longer neurites ≥ 50 µm (white box plots). C, D The neurites of 200 neurons were assessed per sample. One-way ANOVA was performed throughout where ***, p ≤ 0.001; and ns, p > 0.05 is not significant.

Article Snippet: Experiments to rescue the neurite outgrowth defects of Dnttip1 KO neurons with axon guidance ligands were carried out as described above except that recombinant SLIT3 (R&D Systems, 9296-SL-050), NTN1 (R&D Systems, 6419-N1-025) or a combination of SLIT3/NTN1 were added daily to differentiation medium 3 (at a concentration of 0.5 and 0.25 ug/ml for SLIT3 and NTN1 respectively) from day 6 of differentiation onward.

Techniques: Protein-Protein interactions, Control, Recombinant, Staining, Software

Journal: bioRxiv

Article Title: The histone deacetylase complex MiDAC regulates a neurodevelopmental gene expression program

doi: 10.1101/2020.04.07.029751

Figure Lengend Snippet: Model of MiDAC function in neurite outgrowth and morphogenesis. MiDAC directly binds to regulatory elements of pro-neural genes such as those of the axon guidance ligands SLIT3 and NTN1 resulting in their activation while simultaneously inhibiting expression of genes of negative regulators of neurogenesis such as SPRY4 and ID1 by removing H3K27ac from their promoters and enhancers.

Article Snippet: Experiments to rescue the neurite outgrowth defects of Dnttip1 KO neurons with axon guidance ligands were carried out as described above except that recombinant SLIT3 (R&D Systems, 9296-SL-050), NTN1 (R&D Systems, 6419-N1-025) or a combination of SLIT3/NTN1 were added daily to differentiation medium 3 (at a concentration of 0.5 and 0.25 ug/ml for SLIT3 and NTN1 respectively) from day 6 of differentiation onward.

Techniques: Activation Assay, Expressing