Screening for Novel Regulators of Embryonic Stem Cell
Identity
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Citation
Subramanian, Vidya, Carla A. Klattenhoff, and Laurie A. Boyer.
“Screening for Novel Regulators of Embryonic Stem Cell
Identity.” Cell Stem Cell 4, no. 5 (May 2009): 377-378. © 2009
Elsevier Inc.
As Published
http://dx.doi.org/10.1016/j.stem.2009.04.006
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Elsevier B.V.
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Final published version
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Wed May 25 19:02:23 EDT 2016
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http://hdl.handle.net/1721.1/82941
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Detailed Terms
Cell Stem Cell
Previews
Screening for Novel Regulators
of Embryonic Stem Cell Identity
Vidya Subramanian,1,2 Carla A. Klattenhoff,1,2 and Laurie A. Boyer1,*
1Department
of Biology, Massachusetts Institute of Technology, 77 Massachusetts Avenue, Cambridge, MA 02139, USA
authors contributed equally to this work
*Correspondence: lboyer@mit.edu
DOI 10.1016/j.stem.2009.04.006
2These
Two recent studies, including one in this issue of Cell Stem Cell, have identified novel regulators of embryonic
stem cell (ESC) self-renewal using genome-wide RNAi screens in mouse ESCs (Ding et al., 2009; Hu et al.,
2009) and have further expanded the repertoire of factors that regulate ESC identity.
Embryonic stem cells (ESCs) are characterized by their ability to self-renew and
to differentiate into any cell type in the
organism. This potential could be used
to develop treatments for a variety of
human diseases. Elucidating the molecular mechanisms responsible for maintaining ESC identity will lead to a better
understanding of both development and
disease, and will contribute to advances
that enable therapeutic use of these cells.
In an effort to systematically identify new
stem cell regulators, two groups performed genome-wide RNAi screens in
mouse ESCs (Ding et al., 2009; Hu et al.,
2009). These studies reveal additional
transcription modules that may function
to maintain the balance between selfrenewal and differentiation.
RNAi screens have proven to be a
powerful genetic method for the identification of pathway components in a variety
of systems. The success of these screens
relies not only on an appropriate reporter
system but also on stringent validation
criteria. In the current studies, both
groups used the same reporter cell line
to monitor Oct4 expression levels and
hence the differentiation status of ESCs
(Figure 1A). Importantly, Oct4 is an essential pluripotency regulator that functions
in a transcriptional circuit with Sox2 and
Nanog as well as within a larger network
that includes a growing list of transcription factors (Loh et al., 2008). While these
studies each confirmed the roles of
several previously known genes in selfrenewal, Ding et al. (2009) identified
various components of the Paf1C
complex from 296 candidates, whereas
Hu et al. (2009) focused their efforts on
the characterization of Cnot3 and Trim28
from their initial list of 104 high-confi-
dence target genes. The targets in each
study were similarly validated using independent siRNAs and by analysis of cell
morphology and of the expression of ESC
markers including Oct4 and Nanog upon
depletion of these factors.
The screen by Ding and colleagues
yielded members of the highly conserved
Paf1C complex (Ctr9 and Rtf1), and the
authors then tested that additional
components (Paf1, Leo1, Cdc73) of this
complex were also required to maintain
ESC identity (Ding et al., 2009). While the
role of Paf1C in mammalian development
is not known, prior studies in yeast
suggest that it plays a role in transcription
initiation and elongation by recruitment of
the H3K4 methyltransferase to RNAPol2
promoters as well as in mRNA processing (reviewed in Rosonina and Manley,
2005). Consistent with a role in early
lineage decisions, the levels of Paf1C
subunits decreased during ESC differentiation, and overexpression of Ctr9
blocked exit from self-renewal, maintaining the population in an undifferentiated
state. The authors also found that Paf1C
occupied the promoters of pluripotency
genes and is required to maintain
H3K4me3 patterns and expression of
its target genes. Paf1C depletion led to
gene expression changes similar to losses of Oct4 or Nanog and, surprisingly,
to lineage-restricted differentiation, as
only endoderm differentiation was impaired in these cells. These data suggest
that Paf1C may coordinate signals from
the core regulatory network and provide
further clues as to how this network
may be robustly maintained in an active
state.
The work by Hu et al. also identified
several
transcriptional
regulators,
including Cnot3 and Trim28 (Hu et al.,
2009). Cnot3 (Ccr4-Not transcription
complex, subunit 3) is a component of
the Ccr4-Not complex, which has been
shown to regulate gene expression at
both transcriptional and posttranscriptional levels (Collart and Timmers, 2004).
Because other members of the complex
were not identified in this screen, the
authors propose that Cnot3 might have
functions independent of the Ccr4-Not
complex in ESCs. It is interesting to note
that the screen by Ding and colleagues
detected Cnot1, suggesting that perhaps
other components of the complex are
also involved in ESC regulation. Thus,
a systematic analysis of Ccr4-Not complex will be required to determine the
role of each of these factors in ESC selfrenewal. Trim28/Kap-1 (tripartite motifcontaining 28) was previously identified
as important for self-renewal in an independent screen for regulators of ESC
identity (Fazzio et al., 2008).
Consistent with their proposed role in
self-renewal, both Cnot3 and Trim28 are
highly expressed in ESCs and downregulated during differentiation. Chromatin
immunoprecipitation revealed that these
factors co-occupied a similar set of target
genes and shared many common targets
with two other components of the ESC
regulatory circuitry, namely c-Myc and
Zfx (Wang et al., 2006), but not with
Oct4, Sox2, and Nanog. Interestingly,
the common target genes include
regulators of cell cycle and cell survival,
suggesting that these factors function
cooperatively to maintain self-renewal.
The authors propose that Cnot3, Trim28,
c-Myc, and Zfx form a unique module
distinct from the Oct4-Sox2-Nanog
network to maintain self-renewal through
Cell Stem Cell 4, May 8, 2009 ª2009 Elsevier Inc. 377
Cell Stem Cell
Previews
the regulation of genes
lineage commitment. This adinvolved in cell cycle and cell
vance may lead to improved
survival. It has recently been
protocols for directing the
suggested that the Ccr4-Not
differentiation of ESCs tocomplex functions to faciliward particular lineages and
tate histone methylation by
for the generation of ES-like
recruitment of the PAF
cells from somatic cells. It
complex (Mulder et al.,
will be essential to integrate
2007), indicating that the tranthe new components into the
scription modules identified
broader network and to underin the two independent
stand how signaling pathways
screens may converge to
communicate with these facorchestrate
ESC
selftors to control cell fate. Imporrenewal. Collectively, these
tantly, regulatory networks
findings uncover a new set
controlling self-renewal in
of factors that may influence
stem cells could also be active
transcriptional and chromatin
in certain cancers (Ben-Porath
states, cell-cycle progreset al., 2008; Wong et al., 2008),
sion, and the execution of
and so their further charactercell-fate decisions in ESCs.
ization may uncover novel
While the two genometherapeutic targets or diagwide screens were similarly
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Figure 1. Screening for Novel Regulators of ESC Identity
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