Native and tagged CENP-A histones are functionally inequivalent

Abstract

Background: Over the past several decades, the use of biochemical and fluorescent tags has elucidated mechanistic and cytological processes that would otherwise be impossible. The challenging nature of certain nuclear proteins includes low abundancy, poor antibody recognition, and transient dynamics. One approach to get around those issues is the addition of a peptide or larger protein tag to the target protein to improve enrichment, purification, and visualization. However, many of these studies were done under the assumption that tagged proteins can fully recapitulate native protein function.

Results: We report that when C-terminally TAP-tagged CENP-A histone variant is introduced, it undergoes altered kinetochore protein binding, differs in post-translational modifications (PTMs), utilizes histone chaperones that differ from that of native CENP-A, and can partially displace native CENP-A in human cells. Additionally, these tagged CENP-A-containing nucleosomes have reduced centromeric incorporation at early G1 phase and poorly associates with linker histone H1.5 compared to native CENP-A nucleosomes.

Conclusions: These data suggest expressing tagged versions of histone variant CENP-A may result in unexpected utilization of non-native pathways, thereby altering the biological function of the histone variant.

Keywords: Atomic force microscopy; CENP-A; Centromere; Chromatin; Chromatin immuno-precipitation; Deep sequencing; Epitope tags; Immuno-fluorescence; Post-translational modifications.

Fig. 1

CpA-TAP has poor affinity for CENP-C and altered post-translational modifications (PTM) signature. a) Native CpA protein and fusion CpA-TAP protein consisting of CpA + S protein + TEV cleavage site + Protein A. b) CoIF of HeLa cells transiently expressing CpA-TAP with native CENP-C. Scale bar = 5 μm. c) Immuno-precipitation of native CpA versus CpA-TAP (S-tag IP) (see Fig. S1a) followed by Western detection for CENP-C, and quantification of the ratio of CENP-C enrichment normalized against CpA ChIP. Error bar represent SEM. HC = heavy chain. d) Long TAU (L-TAU) Western comparing control HeLa cells and cells with transiently transfected CpA-TAP (merged panel below). e) AFM heights for IP’ed native CpA versus CpA-TAP nucleosomes. AFM measurements were done in air mode. Bulk = extracted input chromatin and α-S ChIP = immuno-precipitated CpA-TAP chromatin. Scale bar = 50 nm

Fig. 2

ChIPseq analysis of native CpA an CpA-TAP in control HeLa cells. a) Venn diagram depicting native CpA versus CpA-TAP total, centromeric, and non-centromeric hotspots. b) Categorical dissection of incorporated sites for native CpA versus CpA-TAP. c) Heat map of promoter occupancy for native CpA unique, CpA-TAP unique, and common sites

Fig. 3

ChIPseq analysis of native CpA-TAP in DAXX KO HeLa cells. a) Western confirmation that DAXX is knock-out and that CpA-TAP is esxpressed (rDAXX: recombinant DAXX protein, AbCam cat #ab131785). b) CoIF of CpA-TAP and native CENP-C during interphase and mitosis (left panel), and native CpA versus S-tag ChIP followed by CENP-C Western (right panel). Scale bar = 5 μm. c) Venn diagram detailing total, centromeric, and non-centromeric hotspots for native CpA and CpA-TAP. d) Categorical dissection of native CpA versus CpA-TAP incorporated sites

Fig. 4

Karyoplot analysis of native CpA versus CpA-TAP deposition in both control (untransfected) HeLa and DAXX KO cells

Fig. 5

Karyoplot analysis of native CpA in control HeLa cells, versus in the presence of CpA-TAP, and versus in the presence of CpA-TAP + DAXX KO.

Fig. 6

Native CpA have differing deposition profiles compared to N-terminally tagged GFP-CpA and C-terminally tagged TAP. a) Triple Venn diagram highlighting overlapping and non-overlapping sites among native CpA, CpA-TAP, and GFP-CpA. b) Percentage of centromeric versus non-centromeric sites of native CpA, CpA-TAP, and GFP-CpA under various treatments. c) Peak snapshots of several genic regions for native CpA, CpA-TAP, and GFP-CpA

Fig. 7

Tagged and untagged histone variants differ in genome wide distribution and nucleosomal interactions. a) Previously reported H1.5 and H1.5-HA ChIPseq sites were compared to native CpA ChIPseq sites from this study. b) ChIP performed against native CpA, GFP-CpA, and HA-CpA mono-nucleosomes and probed for histone H1.5 (Invitrogen Cat #711,912). HC = heavy chain. c) Ratios of H1.5/native or tagged CpA, normalized against H1.5/native CpA, from 2–5 independent experiments. Error bars = SEM