XB-IMG-158342
Xenbase Image ID: 158342
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Figure S1, related to Figures 1-2: Analysis of M18BP1 nucleosome binding
A) G1 localization of M18BP1 in human cells is unaffected by CENP-C depletion.
Representative images of mRuby-Flag-M18BP1 localization in human DLD1 cells in the
presence (top row) and absence (bottom row) of CENP-C. Both alleles of CENP-C were tagged
with an auxin-inducible degron (AID)-EYFP tag in cells stably expressing the exogenous F-box
protein TIR1. Following treatment with 1 mM indole-3-acetic acid (IAA), CENP-C protein is
efficiently degraded and colocalization of Flag with anti-centromere antibodies (ACA)
visualized. Early G1 cells were identified by midbody (tubulin) staining. Scale bar, 10 μm.
B) Quantification of mRuby-Flag-M18BP1 signal at G1 centromeres from experiment in (A).
After 24 h of CENP-C depletion, M18BP1 levels at centromeres remain largely unchanged. Plot
shows mean ± SEM from three independent experiments. At least 12 pairs of early G1 cells were
quantified per experiment.
C) Representative Western blot showing efficient and sustained degradation of M18BP1
following addition of 1 mM IAA to tissue culture medium. Whole cell extracts were collected at
the indicated times after addition of IAA, sonicated, and resolved to visualize total CENP-C and
YFP. Tubulin is a loading control.
D) M18BP1-1 binds CENP-A nucleosomes via a region homologous to M18BP1-2747-944
.
Binding of M18BP1-1 truncations to CENP-A chromatin was assayed as in Figure 1. M18BP1-
1
750-1094 is homologous to M18BP1-2747-1125
.
2
E) Mutation of R774A in the CENP-C motif of M18BP1-1 causes a 35 ± 5% reduction at
centromeres. Xenopus sperm nuclei were assembled in interphase extract depleted of endogenous
M18BP1 and complemented with wild-type or R774A mutant M18BP1-1. Average centromere
intensity is quantified relative to wild-type localization. All graphs show the mean ± SEM of at
least two experiments.
F) Representative anti-Flag Western blot of reactions shown in B showing equivalent translation
of wild-type and R774A mutant M18BP1-1.
G) Human M18BP1 does not bind CENP-A nucleosomes directly. Binding of in vitro translated
Xenopus or human M18BP1 protein to CENP-A or H3 chromatin was assessed by
immunofluorescence. Unlike Xenopus M18BP1 (also shown in Figure 1B,C), human M18BP1
did not show enhanced binding to CENP-A chromatin.
H) Schematic of the histone chimeras used and the amino acid residues of human CENP-A
(pink) and histone H3 (blue), respectively.
I) Nucleosome binding by M18BP1-2 requires both the CATD and the CAC. Representative
images of M18BP1-2 binding to chromatin comprising the CENP-A/H3 chimeras indicated to
the left. Immunolocalized proteins indicated above. Scale bar, 5 μm. Quantification shown in
Figure 2D.
J) Nucleosome binding is required for M18BP1-2 recruitment to interphase centromeres.
Representative images of Xenopus sperm nuclei incubated in interphase egg extracts depleted of
endogenous M18BP1 and complemented with M18BP1-2 proteins indicated on the left.
Immunolocalized protein is indicated above. Scale bar, 10 μm. Insets magnified 3X.
Quantification shown in Figure 2E. Image published in: French BT et al. (2017) Copyright © 2017. Image reproduced with permission of the Publisher, Elsevier B. V. Larger Image Printer Friendly View |