from an epidermal cell lineage (Fig. 1A) (4, 5).
Two populations of stomatal precursor stem cells,
meristemoid mother cells and meristemoids, have
limited self-renewing properties and proliferate without the benefit of a stem cell niche (4–6).
These stem cells are created through the post-embryonic activity of SPEECHLESS (SPCH) in a
subset of protodermal cells (7, 8). SPCH is a control point through which developmental, environmental, and phytohormone signals are integrated
(4, 5). However, no targets of SPCH have been reported, and thus the sphere of its regulatory influence is unknown. Here, we develop a ChIP method
optimized for rare developmental regulators and
profile the genome-wide binding of SPCH in vivo.
In combination with multiple transcriptional response data sets, our ChIP-sequencing (ChIP-seq)
data indicate that SPCH programs an entire lineage
by promoting fate transitions and asymmetric cell
divisions (ACDs). SPCH also modulates the sensitivity of stomatal lineage cells to hormone and
peptide/receptor–mediated signaling. Our results
suggest how this lineage exhibits considerable
autonomy while still coordinating with the overall organ development program.
Like many developmental regulators, SPCH
expression is transient and limited to few cells
(Fig. 1A). Standard ChIP assays on SPCH yielded
only modest target enrichment (~4-fold, Fig. 1C,
blue box), and thus we needed improved ChIP
sensitivity for the detection of endogenously weak
signals. We hypothesized that if background sig-
nals in a ChIP assay could be kept low, increasing
the experimental scale would lead to a dispropor-
tional increase in signals from targets (true signal)
over background (Fig. 1B). Therefore, performing
ChIP at a large scale may achieve high target en-
richment even for low-abundance proteins. We
tested this hypothesis with ChIPs at three differ-
ent scales on a spch mutant line bearing a com-
plementing, Myc-tagged SPCH variant driven by
its native promoter (SPCHpro:SPCH2-4A-MYC)
(fig. S1). The scales represented 4, 8, and 16 times
(or 6, 12, and 24 g) the input materials used in a
typical Arabidopsis ChIP experiment. ChIP-qPCR
(quantitative polymerase chain reaction) assays of
SPCH on the promoter of TOO MANY MOUTHS
(TMM) showed that scale increase improves tar-
get enrichment up to 600-fold at 16x (or a >30-
fold increase in enrichment with a 4-fold scale
increase) (Fig. 1C, three rightmost columns). Thus,
weak signals can be enhanced by maximizing in-
put. We termed this method Maximized Objects
for Better Enrichment (MOBE)–ChIP.
To profile genome-wide binding events of
SPCH, we performed and pooled six MOBE-ChIPs on SPCHpro:SPCH2-4A-MYC and on a
wild-type (WT) control for high-throughput sequencing (scale, 16x; total, 144 grams per genotype)
(Fig. 1C, red box, and fig. S2B). For comparison,
standard ChIP-seq was also included [pooled from
nine independent ChIPs on SPCHpro:SPCH2-
4A-YFP (yellow fluorescent protein) and nucGFP
(green fluorescent protein) at 4x] (Fig. 1C, blue
box, and fig. S2A). MOBE-ChIP-seq confirmed
the ChIP-qPCR results at the TMM promoter, revealing a single peak with an enrichment score of
178 [–log10(q value), 1.2 × 106]; the corresponding peak
from our 4x run had a score of 1.2 [–log10(q-value),
5.7] (Fig. 1D). Low background signal is also a
genome-wide trend. Using the peak-calling algorithm ChIP-seq Analysis in R (CSAR) (9), we
detected peaks with an enrichment score as low
as 1.62 at a false discovery rate (FDR) of 1 × 10−6,
in contrast to other studies whose peaks above
threshold scores of 1.85 and 79.6 were detected
at FDRs of 0.01 and 0.001, respectively (table S1)
(10, 11). The ability to identify these low-coverage
peaks is indicative of the power of signal enrichment. Thus, through MOBE-ChIP-seq, we generated a comprehensive in vivo genome-wide
binding map of SPCH.
Using two complementary peak-calling pipelines, we identified 8327 SPCH-bound regions
(tables S2 and S3). Seventy percent of the SPCH
binding peaks are associated with gene promoters,
mostly within 500 base pairs upstream of the
transcriptional start site (Fig. 2A and fig. S3).
De novo discovery of enriched motifs in the binding peaks identified CDCGTG as the top-scoring
motif; this variant of the E-box (CACGTG), to
which basic helix-loop-helix (bHLH) proteins
typically bind, is enriched at the summit of the
SPCH peaks (Fig. 2B and fig. S4).
To focus on loci most likely to respond transcriptionally to SPCH binding, we generated a
“high-confidence” subset of peaks that were non-intergenic with enrichment scores ≥10 (table S2).
Among the high-confidence targets, Gene Ontology (GO) terms for genes involved in regulation
of transcription, signaling, response to stimulus,
and regulation of hormone levels were significantly enriched (Fig. 2E, fig. S5, and table S4). This
suggests that in the initiation of the stomatal
lineage, SPCH could act as a mediator of environmental and hormone inputs that are translated into further downstream transcriptional
and signaling networks. The enrichment of the
GO term “protein targeting to membrane” is interesting given the membrane-associated polarization of stomatal lineage proteins BASL and
POLAR during asymmetric divisions (12, 13).
To correlate SPCH binding with transcriptional
responses on a genome-wide scale, we compared
the high-confidence SPCH targets to data sets representing genes expressed in response to SPCH
induction (fig. S6 and table S5) and those enriched for genes preferentially expressed in the
stomatal lineage (13) (fig. S7). Significant enrichment of the SPCH targets was found among
genes both up- and down-regulated in response
to SPCH induction (27 and 20%, respectively) (Fig.
2C) and in plants with excess or no meristemoids
(31 and 12%) (Fig. 2D). By chance, SPCH would be
predicted to bind to ~4.5% of genes in the data
sets (1517 targets out of 33,602 Arabidopsis genes).
Overall, these comparisons indicate that nearly a
1606 26 SEPTEMBER 2014 • VOL 345 ISSUE 6204
1Department of Biology, Stanford University, Stanford, CA
94305, USA. 2Howard Hughes Medical Institute, Stanford
University, Stanford, CA 94305, USA. 3Carnegie Institution
for Science, Stanford, CA 94305, USA.
*Present address: Department of Genetics, Stanford Medical
School, Stanford, CA 94305, USA. †Present address: Agricultural
Research Service, 800 Buchanan Street, Albany, CA 94710, USA.
‡Corresponding author. E-mail: firstname.lastname@example.org
100 bp TMM
4.2 3.3 F
SPCH2-4A-MYC; spch3 180
cells Meristemoid Protodermal cell Meristemoid mother cell
EnXXL >> EnL
Self- renewal Asymmetric
In low background systems:
Fig. 1. Chromatin immunoprecipitation (ChIP) optimized for cell-type–specific studies in vivo. (A) Arabidopsis stomatal development scheme. SPCH
controls the initiation and proliferation of the stem cell–like stomatal lineage precursors (pink and red cells). (B) Model for improving target enrichment in ChIPs
through increasing experimental scale. (C and D) ChIPs at larger scales improve target enrichments. ChIP-qPCR assays of a SPCH variant on the TMM promoter
performed at the indicated conditions (C). SPCH ChIP-seq profiles at TMM (D) generated from ChIPs at 4x and 16x [blue and red box in (C), respectively].
The y axis represents the enrichment values; note scales. Dashed box marks the SPCH-binding region.