Linker histone H1 is present in centromeric chromatin of living human cells next to inner kinetochore proteins

Abstract

The vertebrate kinetochore complex assembles at the centromere on α-satellite DNA. In humans, α-satellite DNA has a repeat length of 171 bp slightly longer than the DNA in the chromatosome containing the linker histone H1. The centromere-binding protein CENP-B binds specifically to α-satellite DNA with properties of a centromeric-linker histone. Here, we analysed if linker histone H1 is present at or excluded from centromeric chromatin by CENP-B. By immunostaining we detected the presence, but no enrichment or depletion of five different H1 subtypes at centromeric chromatin. The binding dynamics of H1 at centromeric sites were similar to that at other locations in the genome. These dynamics did not change in CENP-B depleted cells, suggesting that CENP-B and H1 co-exist in centromeric chromatin with no or little functional overlap. By bimolecular fluorescence complementation (BiFC) and Förster resonance energy transfer (FRET), we revealed that the linker histone H1 subtypes H1° and H1.2 bind to centromeric chromatin in interphase nuclei in direct neighbourhood to inner kinetochore proteins.

Down the rabbit hole of centromere assembly and dynamics

“…Two new methods, lifetime-FRET and biFC, offer the distinct advantage of coupling fluorescently tagged proteins with live imaging to track kinetochore dynamics in situ. Such experiments independently confirm interactions between CENP-A and CENP-B/C/N, support the view that CENP-W/T bind H3 not CENP-A [58, 70–71], and unveil a new role for H1 at the centromere [72]. Thus, these kinds of studies will allow visualization of kinetochore protein binding to centromeric domains in situ.…”

Dynamics of CENP-N kinetochore binding during the cell cycle

“…However, whether CENP-A and CENP-N are closely associated in vivo is not known. To analyse the binding of cellular CENP-N to the CENP-Acontaining nucleosome in centromeric chromatin, we performed a FRET-based approach, which we have previously utilised to study protein proximities and interactions in cells (Hellwig et al, 2009;Orthaus et al, 2008;Orthaus et al, 2009). We expressed a full-length human CENP-N fusion with EGFP and confirmed that the EGFP-CENP-N colocalised to kinetochores with CENP-A in human HEp-2 cells (Fig.…”

“…This vector was digested with AgeI-BsrGI. Into the resulting purified 4.433 bp fragment, we ligated a 713 bp AgeI-BsrGI fragment resulting from the vector pmCherry-C1 (Orthaus et al, 2009). For expressing human CENP-N fused to the C-terminus of EGFP, we used vector pH-G-CENP-N (Hellwig et al, 2009).…”

“…Here, the FRET pair EGFP-mCherry was used. FRET experiments were conducted as described (Orthaus et al 2008;Orthaus et al, 2009). If the orientation of the fluorophore dipole moment of the acceptor relative to that of the donor were known, or if at least one of them rotated freely faster than nanoseconds, a more detailed distance between donor and acceptor could be deduced from the measured FRET efficiency (E FRET ) values.…”

“…Experiments were carried out as described (Orthaus et al, 2009). In short, the donor fluorescence lifetime was determined by time-correlated single photon counting (TCSPC) in living human HEp-2 cells.…”