Impact of Nucleosomal DNA Sequences on SWR Remodeler Activity and Histone H2A.Z Targeting

The variant histone H2A.Z is deposited into nucleosomes immediately downstream of promoters, where it plays a critical role in transcription. The site-specific deposition of H2A.Z is catalyzed by the SWR complex, a conserved chromatin remodeler with affinity for promoter-proximal nucleosome-depleted regions (NDRs) and histone acetylation. By comparing the genomic distribution of H2A.Z in wild-type and SWR-deficient cells, we found that SWR is also responsible for depositing H2A.Z at thousands of non-canonical sites not directly linked to NDRs or histone acetylation. To understand the targeting mechanism of H2A.Z, we presented SWR to a library of canonical nucleosomes isolated from yeast and analyzed the preferred substrates. Our results revealed that SWR preferentially deposited H2A.Z into a subset of endogenous H2A.Z sites, which are overrepresented by polyadenine tracks on the top strands of the DNA duplex at the nucleosomal entry-exit sites. Insertion of polyadenine sequences into recombinant nucleosomes near the outgoing H2A-H2B dimer enhanced SWR’s affinity for the nucleosomal substrate and increased its H2A.Z insertion activity. These findings suggest that the genome encodes sequence-based information that facilitates remodeler-mediated targeting of H2A.Z. To determine the genomic locations of SWR-dependent H2A.Z nucleosomes, we compared the ChIP-seq profiles of H2A.Z in WT and swc2∆ cells. Experimentally, bulk chromatin was isolated from spheroplasted cells followed by fragmentation into mainly mono-nucleosomes by limited micrococcal nuclease (MNase) digestion. The nucleosomes were subjected to immunoprecipitation with anti-FLAG agarose targeting the 2xFLAG epitope tag on the C-terminus of H2A.Z. The immunoprecipitated DNA fragments were sequenced using the Illumina platform and mapped to the yeast genome (version R64-1-1). This experimental design applies to samples: GSM8008562, GSM8008561, GSM8008560, and GSM8008559. LP001.bw, LP004.bw, LP007.bw, and LP010.bw are the processed input DNA associated with the ChIP-seq samples GSM8008562, GSM8008561, GSM8008560, and GSM8008559, respectively. LP002.bw, LP005.bw, LP008.bw, and LP011.bw are the processed flow-through DNA associated with the ChIP-seq samples GSM8008562, GSM8008561, GSM8008560, and GSM8008559, respectively. LP157.bw, LP159.bw, LP161.bw, and LP163.bw are MNase-seq data for the indicated cell lines. The experimental design for LP157.bw, LP159.bw, LP161.bw, and LP163.bw are the same as indicated above, except that the nucleosomes were not subjected to immunoprecipitation.