Practical Guidelines for High-Resolution Epigenomic Profiling of Nucleosomal Histones in Postmortem Human Brain Tissue

Abstract

Background: The nervous system may include more than 100 residue-specific posttranslational modifications of histones forming the nucleosome core that are often regulated in cell-type-specific manner. On a genome-wide scale, some of the histone posttranslational modification landscapes show significant overlap with the genetic risk architecture for several psychiatric disorders, fueling PsychENCODE and other large-scale efforts to comprehensively map neuronal and nonneuronal epigenomes in hundreds of specimens. However, practical guidelines for efficient generation of histone chromatin immunoprecipitation followed by deep sequencing (ChIP-seq) datasets from postmortem brains are needed.

Methods: Protocols and quality controls are given for the following: 1) extraction, purification, and NeuN neuronal marker immunotagging of nuclei from adult human cerebral cortex; 2) fluorescence-activated nuclei sorting; 3) preparation of chromatin by micrococcal nuclease digest; 4) ChIP for open chromatin-associated histone methylation and acetylation; and 5) generation and sequencing of ChIP-seq libraries.

Results: We present a ChIP-seq pipeline for epigenome mapping in the neuronal and nonneuronal nuclei from the postmortem brain. This includes a stepwise system of quality controls and user-friendly data presentation platforms.

Conclusions: Our practical guidelines will be useful for projects aimed at histone posttranslational modification mapping in chromatin extracted from hundreds of postmortem brain samples in cell-type-specific manner.

Keywords: Cell type specific; ChIP-seq; Epigenomics; Postmortem human brain; PsychENCODE; Schizophrenia.

Figure 1

Chromatin immunoprecipitation followed by deep sequencing pipeline. Overview of the pipeline flow chart and corresponding quality controls (QC). FACS, fluorescence-activated cell sorting; IGV, Integrative Genomics Viewer; MNase, micrococcal nuclease; N-ChIP, native chromatin immunoprecipitation; NeuN+, neuronal; NeuN−, nonneuronal; qPCR, quantitative polymerase chain reaction.

Figure 2

Chromatin immunoprecipitation followed by deep sequencing (ChIP-seq) quality controls (QC). Quality controls include: (A) visual inspection and quantification of nuclei separated into neuronal (NeuN+) and nonneuronal (NeuN−) fraction by fluorescence-activated (cell) sorting of nuclei, including linear correlation of nuclei number with approximate prefrontal cortex gray matter tissue weight, as indicated. (B) DNA agarose gel from native chromatin digested with different amounts of micrococcal nuclease (MNase). The predominant ~150 base pair (bp) band confirms that the bulk of chromatin has been digested into mononucleosomes. (C) Peptide array containing 46 peptides representing 46 different H3 posttranslational modifications, to test specificity of the H3K4me3 antibody by dot blot (see also Supplementary Figure S1). Note no cross-reactivity other than weak activity against the dimethylated form, H3K4me2. (D) Agilent Bioanalyzer QC after ChIP confirms that predominant portion of pulldown was comprised by mononucleosomes as evidenced by sharp peak at ~148 bp. (E) ChIP-quantitative polymerase chain reaction (qPCR) confirms H3K4me3 enrichment in neuronal NeuN+ nuclei fraction (blue curve) and nonneuronal NeuN− nuclei fraction (red curve) for neuronal gene GRIN2B (upper panel) but not for negative control HBB globin sequences (lower panel). Note that the input DNA qPCR signals (dark and light green curves) are similar for these two genes. (F) Agilent Bioanalyzer QC after library preparation, confirming that large majority of DNA molecules locate to 275 bp, representing correct library ligation product (see text). (G) Early bioinformatical analyses include FASTQC, BWA, and other established programs. Note consistent GRIN2B H3K4me3 enrichment observed in the NeuN+ and NeuN− ChIP-seq tracks visualized in Integrative Genomics Viewer (IGV) browser when compared with the corresponding ChIP-qPCR signals above (E) (see text for more details). (H) FASTQC analysis of raw ChIP-seq data, represented here as the sequence quality score (y axis) versus base pair position (x axis), is an important initial step in ChIP-seq data quality control. The graph background colors separate the y axis into very good quality calls (green, score > 28), calls of reasonable quality (orange, score = 20–28), and calls of poor quality (red, score < 20). Note that our representative ChIP-seq data show remarkably high quality throughout the entire sequence, including the sequence toward the end of the read (up to 100 bp).

Figure 3

Data processing pipeline. (A) Mapped reads—the number of reads mapped to the human genome, after filtering but including duplicate reads. (B) Number of peaks called for H3K4me3 and H3K27ac in two cell types (neuronal and nonneuronal). Distribution of H3K4me3 and H3K27ac around (C) annotated promoters and (D) intergenic genome portions. Encyclopedia of DNA Elements recommended quality metrics: (E) nonredundant fraction (NRF), (F) polymerase chain reaction bottleneck coefficient (PBC), and (G) relative strand correlation (RSC). (H) Bioinformatics pipeline. kb, kilobase.

Figure 4

Cell-type-specific epigenomics. Browser tracks for eight chromatin immunoprecipitation followed by deep sequencing libraries. H3K4me3 and H3K27ac chromatin immunoprecipitation followed by deep sequencing tracks from prefrontal cortex (PFC) neuronal and nonneuronal nuclei of brains A and B, as indicated in purple and green, respectively. Tracks show 146 kilobase (kb) centered on GAD1 gamma-aminobutyric acid synthesis gene. Orange track, H3K27ac chromatin immunoprecipitation followed by deep sequencing from Roadmap Epigenomics (PFC tissue homogenate). Note cell-type-specific profiles, particularly for H3K27ac, while there is comparatively little variability between subjects.

Figure 5

The Sage Bionetworks’ Synapse System. Data generated through this effort will be disseminated to the public through the Synapse system. Synapse enables easy data access, data provenance tracking to enable reproducibility of data processing and analytical output, and tools to communicate study information and research findings.