576 | Nature | Vol 577 | 23 January 2020
Article
H2A.Z facilitates licensing and activation of
early replication origins
Haizhen Long1, 2 ,1 0, Liwei Zhang1,1 0, Mengjie Lv3,1 0, Zengqi Wen1, 2 ,1 0, Wenhao Zhang^4 ,
Xiulan Chen2,5, Peitao Zhang^6 , Tongqing Li^7 , Luyuan Chang1,2, Caiwei Jin2,3, Guozhao Wu1,2,
Xi Wang^8 , Fuquan Yang2,5, Jianfeng Pei^7 , Ping Chen^1 , Raphael Margueron^9 , Haiteng Deng^4 ,
Mingzhao Zhu2,3* & Guohong Li1,2*DNA replication is a tightly regulated process that ensures the precise duplication of
the genome during the cell cycle^1. In eukaryotes, the licensing and activation of
replication origins are regulated by both DNA sequence and chromatin features^2.
However, the chromatin-based regulatory mechanisms remain largely
uncharacterized. Here we show that, in HeLa cells, nucleosomes containing the
histone variant H2A.Z are enriched with histone H4 that is dimethylated on its lysine
20 residue (H4K20me2) and with bound origin-recognition complex (ORC). In vitro
studies show that H2A.Z-containing nucleosomes bind directly to the histone lysine
methyltransferase enzyme SUV420H1, promoting H4K20me2 deposition, which is in
turn required for ORC1 binding. Genome-wide studies show that signals from
H4K20me2, ORC1 and nascent DNA strands co-localize with H2A.Z, and that depletion
of H2A.Z results in decreased H4K20me2, ORC1 and nascent-strand signals
throughout the genome. H2A.Z-regulated replication origins have a higher firing
efficiency and early replication timing compared with other origins. Our results
suggest that the histone variant H2A.Z epigenetically regulates the licensing and
activation of early replication origins and maintains replication timing through the
SUV420H1–H4K20me2–ORC1 axis.In eukaryotes, DNA-replication origins are first licensed in G1 phase
by the pre-replication complex^3 ; the licensed origins are then selec-
tively activated during S phase^1. In budding yeast, the ORC recognizes
autonomously replicating sequences (ARSs) to achieve origin licens-
ing^4. In metazoans, which lack ARSs, replication origins are determined
by both DNA sequence and chromatin-associated factors^2. Of these
chromatin features, it has been reported^5 that the histone modifica-
tion H4K20me2 is recognized by ORC1. Given the broad distribution
of H4K20me2 across the genome^6 , however, other factors must be
involved to precisely define the function of H4K20me2 in DNA replica-
tion. Genome-wide studies have shown that the histone variant H2A.Z is
also enriched at replication origins^7 ,^8. However, whether the enrichment
of H2A.Z has a functional role during DNA replication remained unclear.
H4K20me2 and ORC1 recruitment by H2A.Z
We first found that knocking down the H2AFZ genes in HeLa cells results
in cell growth defects (Fig. 1a), but not in apoptosis or senescence
(Extended Data Fig. 1a, b). Further analysis of H2AFZ-knockdown cells
revealed a defect in incorporation of the replication marker bromo-
deoxyuridine (BrdU), along with a decreased proportion of S-phase
and an increased proportion of G1-phase cells (Extended Data Fig. 1c),
indicating that the cells are arrested at the G1/S boundary. Next, we
synchronized cells at G2/M phase and induced H2A.Z degradation
using a knock-in auxin-inducible degron (AID) tag^9. Six hours after
release from G2/M arrest, H2A.Z-depleted cells (with depletion having
been triggered with the auxin indole-3-acetate, or IAA; Extended Data
Fig. 1d) showed a lower proportion of S-phase cells (dashed green line)
and a higher proportion of G1-phase cells (dashed red line) (Fig. 1b).
This finding supports the idea that cells become arrested at the G1/S
boundary after H2A.Z depletion.
Although we identified some genes that are differentially expressed
after H2AFZ knockdown (Extended Data Fig. 1e), we did not find any
enriched terms relating to cell-cycle regulation. Reverse transcrip-
tion–polymerase chain reaction (RT–PCR) analysis of genes involved
in S-phase cell-cycle control (including CCNE1 and CDK2) and DNA
replication (including ORC1, ORC2, the DNA helicase MCM2, CDC6,
PCNA and RPA1) revealed that H2AFZ knockdown did not change the
expression of these genes by very much (Extended Data Fig. 1f ). Next,
using mass spectrometry, we found that the subunits of the pre-rep-
lication complex, including ORC1, ORC2 and MCM2–7, were enriched
on H2A.Z nucleosomes compared with H2A nucleosomes (Fig. 1c andhttps://doi.org/10.1038/s41586-019-1877-9
Received: 5 December 2018
Accepted: 31 October 2019
Published online: 25 December 2019
(^1) National Laboratory of Biomacromolecules, CAS Center for Excellence in Biomacromolecules, Institute of Biophysics, Chinese Academy of Sciences, Beijing, China. (^2) University of Chinese
Academy of Sciences, Beijing, China.^3 Key Laboratory of Infection and Immunity, Institute of Biophysics, Chinese Academy of Sciences, Beijing, China.^4 Ministry of Education (MOE) Key
Laboratory of Bioinformatics, School of Life Sciences, Tsinghua University, Beijing, China.^5 Laboratory of Proteomics, Institute of Biophysics, Chinese Academy of Sciences, Beijing, China.
(^6) Department of Cell Biology, Tianjin Medical University, Tianjin, China. (^7) Center for Quantitative Biology, Academy for Advanced Interdisciplinary Studies, Peking University, Beijing, China.
(^8) Department of Immunology, School of Basic Medical Sciences, Capital Medical University, Beijing, China. (^9) Institut Curie, PSL Research University, Paris, France. (^10) These authors contributed
equally: Haizhen Long, Liwei Zhang, Mengjie Lv, Zengqi Wen. *e-mail: [email protected]; [email protected]
There are amendments to this paper