Nucleosomes, composed of DNA and histone proteins, represent the fundamental repeating unit of the eukaryotic genome; posttranslational modifications of these histone proteins influence the activity of the associated genomic regions to regulate cell identity. Traditionally, trimethylation of histone 3-lysine 4 (H3K4me3) is associated with transcriptional initiation, whereas trimethylation of H3K27 (H3K27me3) is considered transcriptionally repressive. The apparent juxtaposition of these opposing marks, termed “bivalent domains”, was proposed to specifically demarcate a small set of transcriptionally-poised lineage-commitment genes that resolve to one constituent modification through differentiation, thereby determining transcriptional status. Since then, many thousands of studies have canonized the bivalency model as a chromatin hallmark of development in many cell types. However, these conclusions are largely based on chromatin immunoprecipitations (ChIP) with significant methodological problems hampering their interpretation. Absent direct quantitative measurements, it has been difficult to evaluate the strength of the bivalency model. Here, we present reICeChIP, a calibrated sequential ChIP method to quantitatively measure H3K4me3/H3K27me3 bivalency genome-wide, addressing the limitations of prior measurements. With reICeChIP, we profile bivalency through the differentiation paradigm that first established this model: from naïve mouse embryonic stem cells (mESCs) into neuronal progenitor cells (NPCs). Our results cast doubt on every aspect of the bivalency model; in this context, we find that bivalency is widespread, does not resolve with differentiation, and is neither sensitive nor specific for identifying poised developmental genes or gene expression status more broadly. Our findings caution against interpreting bivalent domains as specific markers of developmentally poised genes.

Next-generation sequencing allows researchers to efficiently determine the sequences of hundreds of millions of short DNA fragments from an experiment. To find the origins of those fragments, the corresponding sequences are aligned to the genome; these alignments can then be used in downstream analyses. However, this alignment process is complicated by the fact that the genome has many highly similar and repetitive sequences, making it difficult or impossible to unambiguously assign some sequences to a single genomic location. To address this problem, we have developed SmartMap, which serves to process and appropriately weight the alignments of reads that map to more than one genomic location. This enables us to examine many genomic regions that were previously “invisible” to analysis and helps us draw new insights into the regulation and function of repetitive elements of the genome.

Chromatin immunoprecipitation coupled to next-generation sequencing (ChIP-seq) has served as the central method for the study of histone modifications for the past decade. Here we provide a detailed protocol for internally calibrated ChIP (ICeChIP), a method we recently developed to resolve these problems by spike-in of defined nucleosomal standards within a ChIP procedure.

Histone post-translational modifications (PTMs) are important genomic regulators often studied by chromatin immunoprecipitation (ChIP), whereby their locations and relative abundance are inferred by antibody capture of nucleosomes and associated DNA. Here, we use histone peptide arrays and internally calibrated ChIP (ICeChIP) to characterize 52 commercial antibodies purported to distinguish the H3K4 methylforms (me1, me2, and me3, with each ascribed distinct biological functions).