Abstract
H1 linker histones facilitate higher-order chromatin folding and are essential for mammalian development. To achieve high-resolution mapping of H1 variants H1d and H1c in embryonic stem cells (ESCs), we have established a knock-in system and shown that the N-terminally tagged H1 proteins are functionally interchangeable to their endogenous counterparts in vivo. H1d and H1c are depleted from GC- and gene-rich regions and active promoters, inversely correlated with H3K4me3, but positively correlated with H3K9me3 and associated with characteristic sequence features. Surprisingly, both H1d and H1c are significantly enriched at major satellites, which display increased nucleosome spacing compared with bulk chromatin. While also depleted at active promoters and enriched at major satellites, overexpressed H10 displays differential binding patterns in specific repetitive sequences compared with H1d and H1c. Depletion of H1c, H1d, and H1e causes pericentric chromocenter clustering and de-repression of major satellites. These results integrate the localization of an understudied type of chromatin proteins, namely the H1 variants, into the epigenome map of mouse ESCs, and we identify significant changes at pericentric heterochromatin upon depletion of this epigenetic mark.
Highlights
In all eukaryotes, nuclear DNA is packaged into chromatin by its association with histones [1]
Analysis of histone extracts of chromatin prepared from cis-targeted H1dFLAG cells by HPLC and immunoblotting indicated that FLAG-H1d was associated with chromatin and eluted in the same fraction as the endogenous H1d, suggesting that FLAG-H1d has the same hydrophobicity as the endogenous H1d (Figure 1C and 1D)
The nucleosome repeat length (NRL) at major satellites had a value of 200 bp, which was,13 bp and,8 bp longer than the NRLs of respective bulk chromatin and minor satellites in embryonic stem cells (ESCs) (Figure 5B). These results suggest that the enrichment of H1d and H1c at major satellite repeats may contribute to the increase of NRL in the pericentromeric region compared with bulk ESC chromatin
Summary
Nuclear DNA is packaged into chromatin by its association with histones [1]. The nucleosome, the building block of chromatin, consists of an octamer of four core histones (H2A, H2B, H3 and H4) wrapped by 147 bp of DNA [2]. Linker histone H1 binds to DNA entering and exiting nucleosome core particles as well as the linker DNA between nucleosomes, facilitating folding of chromatin into higher order structure [1,3,4,5]. 11 different H1 variants have been characterized in mammals, including somatic H1 variants (H1a to H1e), the replacement H1 (H10), germ cell specific H1s (H1t, H1T2, HILS1 and H1oo), as well as the recently characterized variant H1x [6]. Deletion of three major somatic H1 variants (H1c, H1d and H1e) together leads to a 50%
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