Linker histone epitopes are hidden by in situ higher-order chromatin structure

Abstract

Background: Histone H1 is the most mobile histone in the cell nucleus. Defining the positions of H1 on chromatin in situ, therefore, represents a challenge. Immunoprecipitation of formaldehyde-fixed and sonicated chromatin, followed by DNA sequencing (xChIP-seq), is traditionally the method for mapping histones onto DNA elements. But since sonication fragmentation precedes ChIP, there is a consequent loss of information about chromatin higherorder structure. Here, we present a new method, xxChIP-seq, employing antibody binding to fixed intact in situ chromatin, followed by extensive washing, a second fixation, sonication and immunoprecipitation. The second fixation is intended to prevent the loss of specifically bound antibody during washing and subsequent sonication and to prevent antibody shifting to epitopes revealed by the sonication process. In many respects, xxChIP-seq is comparable to immunostaining microscopy, which also involves interaction of the primary antibody with fixed and permeabilized intact cells. The only epitopes displayed after immunostaining are the "exposed" epitopes, not "hidden" by the fixation of chromatin higher-order structure. Comparison of immunoprecipitated fragments between xChIP-seq versus xxChIP-seq should indicate which epitopes become inaccessible with fixation and identify their associated DNA elements. Results: We determined the genomic distribution of histone variants H1.2 and H1.5 in human myeloid leukemia cells HL-60/S4 and compared their epitope exposure by both xChIP-seq and xxChIP-seq, as well as high-resolution microscopy, illustrating the influences of preserved chromatin higher-order structure in situ. We found that xChIP and xxChIP H1 signals are in general negatively correlated, with differences being more pronounced near active regulatory regions. Among the intriguing observations, we find that transcription-related regions and histone PTMs (i.e., enhancers, promoters, CpG islands, H3K4me1, H3K4me3, H3K9ac, H3K27ac and H3K36me3) exhibit significant deficiencies (depletions) in H1.2 and H1.5 xxChIP-seq reads, compared to xChIP-seq. These observations suggest the existence of in situ transcription-related chromatin higher-order structures stabilized by formaldehyde. Conclusion: Comparison of H1 xxChIP-seq to H1 xChIP-seq allows the development of hypotheses on the chromosomal localization of (stabilized) higher-order structure, indicated by the generation of "hidden" H1 epitopes following formaldehyde crosslinking. Changes in H1 epitope exposure surrounding averaged chromosomal binding sites or epigenetic modifications can also indicate whether these sites have chromatin higher-order structure. For example, comparison between averaged active or inactive promoter regions suggests that both regions can acquire stabilized

Nucleosome reorganisation in breast cancer tissues

“…Common nucleosomes were characterised by DNA methylation enrichment, while shifted nucleosomes were depleted of DNA methylation (Figure 4B). This is consistent with previous reports that DNA methylation stabilises nucleosomes while de-methylation or hydroxymethylation makes nucleosomes more MNase-sensitive (39,51,52).…”

“…Common nucleosomes were characterised by DNA methylation enrichment, while shifted nucleosomes were depleted of DNA methylation (Figure 3B). This is 13 consistent with previous reports that DNA methylation stabilises nucleosomes while demethylation or hydroxymethylation makes nucleosomes more MNase-sensitive (Teif et al 2014;Wiehle et al 2019;Teif et al 2020). The overall distribution of DNA fragments protected from MNase digestion was more enriched with shorter sizes in cancer in comparison with normal samples (Supplemental Figure S3).…”

H3K27ac nucleosomes facilitate HMGN localization at regulatory sites to modulate chromatin binding of transcription factors

“…H3K27me3 levels correlate directly with the chromatin occupancy of histone H1 8 , 35 , an abundant protein family that binds dynamically to nucleosomes 36 and stabilizes chromatin compaction 8 . Previous ChIP analyses indicated that H1 is evenly distributed throughout the nucleus but shows low chromatin occupancy at TSS 37 , 38 . Our ChIP analyses do not show statistically significant differences between WT and DKO in H1 occupancy at a particular site; however, the aggregate plots indicate a marginally higher occupancy of H1 at enhancers and promoters in DKO, as compared to WT (Fig.…”

H3K27 Acetylated Nucleosomes Facilitate HMGN Protein Localization at Regulatory Sites to Modulate The Interaction of Transcription Factors with Chromatin

“…H3K27me3 levels correlate directly with the chromatin occupancy of histone H1 (Kim et al 2015b; Fyodorov et al 2018), an abundant protein family that binds dynamically to nucleosomes (Catez et al 2006) and stabilizes chromatin compaction (Fyodorov et al 2018). Previous ChIP analyses indicated that H1 is evenly distributed throughout the nucleus but shows low chromatin occupancy at TSS (Cao et al 2013; Teif et al 2020). Our ChIP analyses do not show statistically significant differences between WT and DKO in H1 occupancy at a particular site; however, the aggregate plots indicate a marginally higher occupancy of H1 at enhancers and promoters in DKO, as compared to WT regulatory regions (Fig.…”