Timing Mechanism Dependent on
Cell Division Is Invoked by Polycomb
Eviction in Plant Stem Cells
Bo Sun, Liang-Sheng Looi, Siyi Guo, Zemiao He, Eng-Seng Gan, Jiangbo Huang, Yifeng Xu,
Wan-Yi Wee, Toshiro Ito*
Introduction: In plants, leaves and flowers originate from the shoot apical meristem. In an indeterminate shoot apical meristem, stem cells persist for the life of the plant. In a determinate meristem,
a certain number of organs are produced before the meristem is terminated; this characterizes the
floral meristem derived from the shoot apical meristem. In Arabidopsis, stem cell identity is sustained by expression of the gene WUSCHEL. Expression of WUSCHEL can be terminated by the zinc
finger protein KNUCKLES (KNU), with the result that stem cell identity is inactivated. KNU expression is induced by the floral homeotic protein AGAMOUS (AG), but that induction process requires
~2 days and invokes modification of histones resident at the KNU locus. Here, we show that the
2-day time lag is a consequence of a regulated molecular mechanism and that this mechanism can
be embedded in a synthetic regulatory system to invoke a similar time lag.
Methods: For transgenic Arabidopsis plants, we accelerated or inhibited cell cycles with pharmacological agents and studied the resulting KNU expression in response to AG induction. We used
chromatin immunoprecipitation to study the presence of Polycomb proteins on the KNU locus at
specific times during flower development. We used insertional mutagenesis to alter the function
of the Polycomb response element (PRE) and analyzed the response from a heterologous promoter
in Arabidopsis cell cultures. We constructed a synthetic mimic in Arabidopsis floral buds of the AG
function by using the DNA binding domain of the lactose operon repressor (lacI) with its cognate
binding sites. To test the logic that the delay in downstream gene induction was caused by the need
to evict Polycomb group (PcG) proteins from their residence, we simulated the competition between
PcG proteins and DNA binding proteins by using lacI, designed transcription activator–like effector
DNA binding proteins, and synthesized promoters in Arabidopsis cell lines.
Results: AG induces KNU with a time delay regulated by epigenetic modification. In wild-type
plants, KNU expression begins in the center of the floral meristem and follows cell cycle progression.
The binding sites for AG in the KNU upstream region are located within the PRE sequences required
for the repressive histone modification. Binding of AG displaces PcG proteins, leading to the failure
to maintain the repressive histone methylation. The combination of lacI operator sequences with
a chimeric protein that contained the lacI DNA binding domain but lacked the activation domain
was able to mimic the AG activity in Arabidopsis floral buds. We also reconstituted the cell division–
dependent delayed-induction circuit in cell lines.
Discussion: Our results indicate that flower development in Arabidopsis employs cell division
to provide stem cells with a window of opportunity to change fate. The competition we observed
between repressive PcG proteins and an activating transcription factor may reflect a general mechanism. The logic of the molecular circuit we have uncovered here may impose timing control on
diverse growth and differentiation pathways in plants and animals.
FIGURES IN THE FULL ARTICLE
Fig. 1. KNU induction timing is
Fig. 2. Polycomb group protein binding
on KNU and cis activities.
Fig. 3. Polycomb response element
and simulation of AG.
Fig. 4. Synthetic epigenetic timer
and eviction model.
Figs. S1 to S21
Tables S1 to S4
Full Reference List
RELATED ITEMS IN SCIENCE
X. Zhang, Delayed gratification—Waiting to
terminate stem cell identity. Science 343,
498–499 (2014). DOI: 10.1126/science.1249343
Induction of KNU in Arabidopsis floral meristems. Synchronized inflorescences imaged by confocal microscopy and reconstructed into three-dimensional projections (red stains by a fluorescence dye
show the shapes of developing flowers). In an
Arabidopsis line that has been engineered so that its floral
development is both inducible and synchronized, KNU
expression (green) begins 1 to 3 days after the activation of flower development. The delay is mediated by
repressive histone methylation at the KNU locus.
Upon activation, the transcription factor AG displaces
Polycomb proteins, and the repressive histone marks
are lost with cell cycle progression. Scale bar, 100 µm.
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Cite this article as B. Sun et al.,
Science 343, 1248559 (2014).
The list of author affiliations is available in the full article online.
*Corresponding author. E-mail: firstname.lastname@example.org
RESEARCH ARTICLE SUMMARY
www.sciencemag.org SCIENCE VOL 343 31 JANUARY 2014 505