Supplementary Material from Epigenetic regulation of transcriptional plasticity associated with developmental song learning

Ethologists discovered over 100 years ago that some lifelong behavioural patterns were acquired exclusively during restricted developmental phases called critical periods (CPs). Developmental song learning in zebra finches is one of the most striking examples of a CP for complex learned behaviour. After Posthatch day 65, whether or not a juvenile male can memorize a ‘tutor's’ song depends on his experiences in the month prior. If he experienced a tutor, he can no longer learn, but if he has been isolated from hearing a tutor the learning period is extended. We aimed to identify how tutor experience alters the brain and controls the ability to learn. Epigenetic landscapes are modulated by experience and are able to regulate the transcription of sets of genes, thereby affecting cellular function. Thus, we hypothesized that tutor experiences determine the epigenetic landscape in the auditory forebrain, a region required for tutor song memorization. Using ChIPseq, RNAseq and molecular biology, we provide evidence that naturalistic experiences associated with the ability to learn can induce epigenetic changes, and propose transcriptional plasticity as a mediator of CP learning potential

Supplementary Material from Epigenetic regulation of transcriptional plasticity associated with developmental song learning

Ethologists discovered over 100 years ago that some lifelong behavioural patterns were acquired exclusively during restricted developmental phases called critical periods (CPs). Developmental song learning in zebra finches is one of the most striking examples of a CP for complex learned behaviour. After Post-hatch day 65, whether or not a juvenile male can memorize the song of a ‘tutor' depends on his experiences in the month prior. If he experienced a tutor, he can no longer learn, but if he has been isolated from hearing a tutor the learning period is extended. We aimed to identify how tutor experience alters the brain and controls the ability to learn. Epigenetic landscapes are modulated by experience and are able to regulate the transcription of sets of genes, thereby affecting cellular function. Thus, we hypothesized that tutor experiences determine the epigenetic landscape in the auditory forebrain, a region required for tutor song memorization. Using ChIPseq, RNAseq and molecular biology, we provide evidence that naturalistic experiences associated with the ability to learn can induce epigenetic changes, and propose transcriptional plasticity as a mediator of CP learning potential

An Alternative Approach to ChIP-Seq Normalization Enables Detection of Genome-Wide Changes in Histone H3 Lysine 27 Trimethylation upon EZH2 Inhibition

<div><p>Chromatin immunoprecipitation and DNA sequencing (ChIP-seq) has been instrumental in inferring the roles of histone post-translational modifications in the regulation of transcription, chromatin compaction and other cellular processes that require modulation of chromatin structure. However, analysis of ChIP-seq data is challenging when the manipulation of a chromatin-modifying enzyme significantly affects global levels of histone post-translational modifications. For example, small molecule inhibition of the methyltransferase EZH2 reduces global levels of histone H3 lysine 27 trimethylation (H3K27me3). However, standard ChIP-seq normalization and analysis methods fail to detect a decrease upon EZH2 inhibitor treatment. We overcome this challenge by employing an alternative normalization approach that is based on the addition of <i>Drosophila melanogaster</i> chromatin and a <i>D</i>. <i>melanogaster-</i>specific antibody into standard ChIP reactions. Specifically, the use of an antibody that exclusively recognizes the <i>D</i>. <i>melanogaster</i> histone variant H2Av enables precipitation of <i>D</i>. <i>melanogaster</i> chromatin as a minor fraction of the total ChIP DNA. The <i>D</i>. <i>melanogaster</i> ChIP-seq tags are used to normalize the human ChIP-seq data from DMSO and EZH2 inhibitor-treated samples. Employing this strategy, a substantial reduction in H3K27me3 signal is now observed in ChIP-seq data from EZH2 inhibitor treated samples.</p></div

Reduced H3K27me3 binding is detected by ChIP-qPCR.

<p><b>(A)</b> ChIP was performed using chromatin from KARPAS-422 cells treated with the EZH2 inhibitor CPI-360. qPCR using the positive control primer <i>MYT1</i> showed reduced H3K27me3 occupancy in the presence of the inhibitor. <b>(B)</b> ChIP was performed using chromatin from PC9 cells treated with the EZH2 inhibitor GSK126. qPCR using the positive control primer <i>MYT1</i> showed reduced H3K27me3 occupancy in cells treated with the inhibitor. (<b>C</b>) Libraries were generated from KARPAS-422 cells using 15 cycles of PCR amplification. Library DNA was diluted and qPCR was performed using positive control primers for <i>MYT1</i> and <i>CCND2</i>. (<b>D</b>) Libraries were generated from PC9 cells as described in (C) and library DNA was used for qPCR using positive control primers for <i>MYT1</i> and <i>CCND2</i>. All experiments are represented as the mean of two independent experiments with qPCRs performed in triplicate ±SD. The <i>ACTB</i> promoter served as a negative control for all experiments.</p

<i>D</i>. <i>melanogaster</i> tag counts from H3K27me3 ChIP-seq reactions are elevated in EZH2 inhibitor treated samples.

<p>H2Av bound regions of the <i>D</i>. <i>melanogaster</i> genome were determined using the H2Av antibody in ChIP-seq reactions containing <i>D</i>. <i>melanogaster</i> S2 or OSS chromatin. <i>D</i>. <i>melanogaster</i> tags from ChIP-seq spike-in reactions were mapped only to these pre-defined H2Av regions. <b>(A)</b> H3K27me3 ChIP-seq reactions with <i>D</i>. <i>melanogaster</i> spike-in in KARPAS-422 cells have a substantial increase in <i>D</i>. <i>melanogaster</i> tags in spike-in libraries prepared from CPI-360 treated cells both at 4 days and 8 days after treatment. <b>(B)</b> The increase was not observed in the control H3K9me3 reactions. <b>(C)</b> H3K27me3 ChIP-seq reactions with <i>D</i>. <i>melanogaster</i> spike-in in PC9 cells have a substantial increase in <i>D</i>. <i>melanogaster</i> tags in spike-in libraries prepared from GSK126 treated cells. <b>(D)</b> The substantial increase in tags was not observed in the control H3K4me3 ChIP-seq spike-in reactions.</p

Schematic representation of the ChIP-seq spike-in protocol.

<p>ChIP-seq spike-in reactions are set up by adding the test chromatin of interest (human or other), the target antibody of interest, a small portion of <i>D</i>. <i>melanogaster</i> chromatin and the <i>D</i>. <i>melanogaster-</i>H2Av-specific antibody. The <i>D</i>. <i>melanogaster</i> spike-in chromatin is added in equal amounts and the H2Av antibody functions to pull down a small portion of the <i>D</i>. <i>melanogaster</i> chromatin in each reaction. After sequencing, tags are mapped to the genome corresponding to the test chromatin as well as to the <i>D</i>. <i>melanogaster</i> genome. The total number of tags uniquely mapping to the <i>D</i>. <i>melanogaster</i> genome are counted for each sample and used to generate correction factors (DMSO tags/inhibitor tags). The test chromatin tag counts are then normalized using the correction factors.</p

EZH2 inhibition reduces global H3K27me3 levels, however standard ChIP-seq methods do not reveal the reduction.

<p><b>(A)</b> Western blot showing reduced global H3K27me3 levels in KARPAS-422 cells treated with 1.5 μM CPI-360 for 4 and 8 days. Whole cell extracts were resolved by SDS page and immuno-blotted with anti-H3K27me3. Anti-H3 immuno-blots show equal levels of total H3. <b>(B)</b> Western blot showing reduced global H3K27me3 levels in PC9 cells treated with 1 μM of GSK126 for 5 days. Whole cell extracts were resolved by SDS page and immuno-blotted with anti-H3K27me3. Anti-H3 immuno-blots show equal levels of total H3. <b>(C, D)</b> Representation of H3K27me3 ChIP-seq data using IGV. No obvious differences are detected in CPI-360 (C) and GSK126 (D) treated KARPAS-422 and PC9 cells when compared to vehicle-treated controls. <b>(E, F)</b> Genome-wide data from H3K27me3 ChIP-seq experiments under different treatment conditions are represented as scatter plots.</p