BioProject

Heterochromatic regions in mammalian cells suppress recombination, silence transcription, and are crucial for maintaining cell differentiation. More...

Heterochromatic regions in mammalian cells suppress recombination, silence transcription, and are crucial for maintaining cell differentiation. Genomic and biochemical characterization of heterochromatin has relied on the associated histone modifications H3K9me3 and H3K27me3, yet these marks are also found in euchromatic regions that permit transcription. We employed a biophysical method to isolate sonication resistant heterochromatin from human somatic cells, mapped its genomic organization compared to histone modifications, and used proteomics to reveal an extensive number of heterochromatin-bound proteins. We discriminate subtypes of H3K9me3- and H3K27me3-marked domains, in sonication-resistant heterochromatin versus euchromatin, and we present a resource of hundreds of proteins that preferentially bind heterochromatin, a set enriched for RNA-binding proteins and proteins that oppose iPS reprogramming. The sonication-resistant heterochromatin landscape includes repressed genes for alternative lineages that are resistant to activation by introduced transcription factors. Depletion of identified heterochromatin-associated proteins reduces this barrier, rendering alternative-lineage genes more competent for transcriptional activation. Overall design: This study was designed to map the organization of sonication resistant heterochromatin in the human genome. Crosslinked, sonicated chromatin from human BJ fibroblasts was fractionated using sucrose gradients, to yield a Euchromatin fraction (fraction #2, containing highly sonicated fragments) and a Sonication-Resistant Heterochromatin fraction (fractions #10-17, containing larger, sonication-resistant fragments). In parallel, we performed H3K9me3-directed chromatin immunoprecipitation (ChIP), as well as H3K9me3-directed IPs performed from the Euchromatin fraction and from the Sonication-Resistant Heterochromatin fraction. Corresponding samples for both gradient analysis and ChIP were also sequenced. Purified DNAs containing larger DNA fragments were sonicated (after protein removal) to produce shorter fragments for library preparation. In total there are 7 sample types (5 purified chromatin samples and 2 types of input), with 2 biological replicates for each. Less...