The Transcription Factor Nerve Growth Factor-Inducible Protein A Mediates Epigenetic Programming: Altering Epigenetic Marks by Immediate-Early Genes

This eLetter provides a guide to the experimental process used to generate autoradiograph images in Weaver et al. 2007. The original autoradiograph documentation and image analysis for this study are located at the following OSF webpage: https://osf.io/5yp42

The images result from Southern blot transfer of ChIP-PCR products to autographic film. The autoradiographic films were exposed to nitrocellulose membranes labeled with a radioactive labelled probe to detect specific DNA fragments that had been separated by electrophoresis on an agarose gel. These experiments were designed to examine subtle changes in band intensity across each experimental condition, lane ‘A’ designating the ‘Antibody’ lane. Importantly, it is this lane alone that is intended to reflect variation across samples. Much time is spent to run equal amounts of each sample, to ensure that agarose gels of PCR products are very consistent for characterisation of the chemical and physical system (not biological variation). Assays require the Input ‘I’ and Non-immune IgG ‘N’ lane is the same across all gels.

In summary, the ‘I’ and ‘N’ bands should be very similar, with variation only in Lane ‘A’, as this is needed for confident analysis of the true signal. Herein, when comparing the ‘I’ or ‘A’ bands with each other, they are similar but differ qualitatively and quantitively.

Please note, Fig. 3(e) and 4(b) only partially differ between the DNA sequence used to bind NGFI-A protein. Lanes 1-6 in Fig. 4(b) simply show a repeat of the same experiment shown in lanes 1-6 in Fig. 3(e), the purpose of which was to be able to compare NGFI-A association with wildtype non-methylated verse 5’ and 3’ mutated exon 1_7 GR promoter in the same experiment. Lanes 7-12 in Fig. 3(e) and 4(b) result from NGFI-A ChIP – wildtype methylated or 5’ mutated exon 1_7 GR promoter—luciferase reporter plasmid PCR +/- NGFI-A, respectively. Mutation/methylation of the DNA sequence can disrupt NGFI-A protein association with gene promoter regions. Given the same molecular outcome of DNA methylation/mutation, highly similar NGFI-A association patterns under the same experimental condition is to be expected.

The assays in Fig. 4(c) and 5(d) only differ between the type of antibody used to probe the proteins. Both assays contain wildtype verse 5’ or 3’ mutated and methylated exon 1_7 GR promoter—luciferase reporter plasmid. DNA mutation and methylation can disrupt NGFI-A and H3K9Ac protein association with gene promoter regions. Given their molecular functions and dependence on each other, highly similar NGFI-A/H3K9Ac association patterns under the same experimental condition is to be expected and indeed was the predicted outcome.

Lastly, the assays in Fig. 5(a,b) only differ between the type of antibody used to probe the proteins. Both assays contain wildtype verse 5’ or 3’ mutated exon 1_7 GR promoter—luciferase reporter plasmid. DNA mutation can disrupt CBP and H3K9Ac protein association with gene promoter regions. Given their molecular functions and dependence on each other, highly similar CBP/H3K9Ac association patterns under the same experimental condition is to be expected, and again predicted.