Article
Extended Data Fig. 1 | Analysis of phenotypes and gene expression after
H2AFZ knockdown, and analysis of the interaction between H2A.Z and
ORC1. a, Statistical outcome of FACS analysis of apoptosis of cells treated with
siNC or siH2A.Z oligonucleotides. Annexin is used as a marker of apoptotic
cells; DAPI is 4′,6-diamidino-2-phenylindole, a nuclear marker. b, Representative
images of cells undergoing senescence (within boxed regions), shown by
β-galactosidase staining. c, Cell-cycle analysis of siNC or siH2A.Z HeLa cells.
Cells were pulse labelled with BrdU and stained with propidium iodide (PI), and
then analysed by FACS. d, Western blots showing the H2A.Z level when cells are
released from arrest at G2/M phase after treatment with IA A. AID, auxin-
inducible degron. e, Volcano plot showing genome-wide expression dynamics
after H2AFZ knockdown; n = 33,835. Genes with a log2(fold change) of 1 or more
and P-values of less than 0.01 were selected as differentially expressed genes.
f, Real-time PCR analysis of gene expression in siNC or siH2A.Z cells. The
expression level was normalized to that of the glyceraldehyde-3-phosphate
dehydrogenase gene (GAPDH). g, Left, diagram showing the LacO/LacI
targeting system in A03_1 cells. An interaction between a LacI–Cherry-tagged
histone and an enhanced green f luorescent protein (EGFP)-tagged ORC protein
would result in an overlap of green f luorescence with red f luorescence. Right,
there is no interaction in this experiment between ORC1 and H2A or H2A.Z.
Scale bar, 5 μm. h, Western blot analysis of the enrichment of H4K20me2,
H3K36me3 (negative control), SUV420H1, ORC1, ORC2 and MCM2 on H2A or
H2A.Z nucleosomes from G1-phase-synchronized cells. Data in panel a are
means; n = 2 biological replicates, with dot plots overlaid. Data in panels c, f are
means ± s.e.m.; n = 3 biological replicates; two-tailed unpaired t-test. The
β-galactosidase staining in panel b, the FACS results in c, the western blots in d,
h, and the f luorescence image in g were independently repeated three times
with similar results. H4 was used as a loading control and sample processing
control in d and h. For gel source data, see Supplementary Fig. 1. For imaging
source data, see Supplementary Fig. 2. For the FACS gating strategy, see
Supplementary Fig. 3.