10.1101/gad.16962311Access the most recent version at doi:
2011 25: 2158-2172Genes Dev.
Dalibor Blazek, Jiri Kohoutek, Koen Bartholomeeusen, et al.
regulation of expression of DNA damage response genes
The Cyclin K/Cdk12 complex maintains genomic stability via
Material
Supplemental http://genesdev.cshlp.org/content/suppl/2011/10/19/25.20.2158.DC1.html
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The Cyclin K/Cdk12 complex maintains
genomic stability via regulation of expression
of DNA damage response genes
Dalibor Blazek,1,2,9,10
Jiri Kohoutek,3,9
Koen Bartholomeeusen,1
Eric Johansen,4
Petra Hulinkova,3
Zeping Luo,1
Peter Cimermancic,5,6,7
Jernej Ule,8
and B. Matija Peterlin1,9
1
Department of Medicine, Microbiology, and Immunology, Rosalind Russell Medical Research Center, University of California
at San Francisco (UCSF), San Francisco, California 94143, USA; 2
Central European Institute of Technology, Masaryk University,
62500 Brno, Czech Republic; 3
Department of Toxicology, Pharmacology, and Immunotherapy, Veterinary Research Institute,
62100 Brno, Czech Republic; 4
UCSF Sandler-Moore Mass Spectrometry Core Facility, UCSF Helen Diller Family
Comprehensive Cancer Center, University of California at San Francisco (UCSF), San Francisco, California 94143, USA;
5
Department of Bioengineering and Therapeutic Sciences, University of California at San Francisco, San Francisco, California
94158, USA; 6
Department of Pharmaceutical Chemistry, University of California at San Francisco, San Francisco, California
94158, USA; 7
California Institute for Quantitative Biosciences, University of California at San Francisco, San Francisco,
California 94158, USA; 8
MRC, Laboratory of Molecular Biology, Cambridge CB20QH, United Kingdom
Various cyclin-dependent kinase (Cdk) complexes have been implicated in the regulation of transcription. In this
study, we identified a 70-kDa Cyclin K (CycK) that binds Cdk12 and Cdk13 to form two different complexes
(CycK/Cdk12 or CycK/Cdk13) in human cells. The CycK/Cdk12 complex regulates phosphorylation of Ser2 in the
C-terminal domain of RNA polymerase II and expression of a small subset of human genes, as revealed in
expression microarrays. Depletion of CycK/Cdk12 results in decreased expression of predominantly long genes
with high numbers of exons. The most prominent group of down-regulated genes are the DNA damage response
genes, including the critical regulators of genomic stability: BRCA1 (breast and ovarian cancer type 1
susceptibility protein 1), ATR (ataxia telangiectasia and Rad3-related), FANCI, and FANCD2. We show that CycK/
Cdk12, rather than CycK/Cdk13, is necessary for their expression. Nuclear run-on assays and chromatin
immunoprecipitations with RNA polymerase II on the BRCA1 and FANCI genes suggest a transcriptional defect
in the absence of CycK/Cdk12. Consistent with these findings, cells without CycK/Cdk12 induce spontaneous
DNA damage and are sensitive to a variety of DNA damage agents. We conclude that through regulation of
expression of DNA damage response genes, CycK/Cdk12 protects cells from genomic instability. The essential
role of CycK for organisms in vivo is further supported by the result that genetic inactivation of CycK in mice
causes early embryonic lethality.
[Keywords: P-TEFb; CTD kinase; Cdk9; camptothecin; mitomycin C; g-H2AX]
Supplemental material is available for this article.
Received May 5, 2011; revised version accepted September 12, 2011.
Eukaryotic transcription can be divided into several stages,
including preinitiation complex formation and productive
elongation (Sims et al. 2004; Fuda et al. 2009). For the
regulation of transcription, the phosphorylation status of
the C-terminal domain (CTD) of RNA polymerase II
(RNAPII) is essential (Buratowski 2009; Fuda et al. 2009;
Munoz et al. 2010). In humans, the CTD consists of 52
repeats of a heptapeptide, YSPTSPS, in which individual
serine residues can be differentially phosphorylated
(Phatnani and Greenleaf 2006; Egloff and Murphy 2008).
Phosphorylation of serine at position 2 (Ser2) is thought to
be responsible for productive transcriptional elongation
and synthesis of full-length mature mRNA (Peterlin and
Price 2006; Bres et al. 2008). Cyclin-dependent kinase 9
(Cdk9), a subunit of positive transcription elongation
factor b (P-TEFb) is considered to be the main Ser2 kinase
(Chao et al. 2000); however, recent studies suggest that
other Ser2 kinases exist in cells (Gomes et al. 2006;
Bartkowiak et al. 2010).
Human Cyclin K (CycK), a 40-kDa and 357-amino-acid
protein, was considered to be the cyclin subunit of Cdk9,
together with CyclinT1 (CycT1) and CycT2 (Fu et al.
1999; Peterlin and Price 2006). It was found to be
associated with RNAPII and potent CTD kinase activity
(Edwards et al. 1998), and, in functional studies, it acti-
9
These authors contributed equally to this work.
10
Corresponding author.
E-mail dblazek@med.muni.cz.
Article is online at http://www.genesdev.org/cgi/doi/10.1101/gad.16962311.
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vated transcriptional elongation (Lin et al. 2002). Cdk12
and Cdk13 are 1490- and 1512-amino-acid-long proteins,
respectively, with a conserved CTD kinase domain (Ko
et al. 2001; Even et al. 2006). A recent study suggested
that Cdk12 in Drosophila, with Cdk12 and Cdk13 in
mammals, is a homolog of Ctk1 in yeast (Bartkowiak
et al. 2010). It also showed that Drosophila Cdk12 and
human Cdk12 have a Ser2 kinase activity, and found
Drosophila Cdk12 associated with CycK (Bartkowiak
et al. 2010). However, many basic facts about these Cdks
and their cyclin partners remain unknown or poorly
understood.
DNA damage response (DDR) is an evolutionarily
conserved mechanism that detects and signals the presence
of DNA lesions and mediates their repair (Harper
and Elledge 2007; Jackson and Bartek 2009; Ciccia and
Elledge 2010). Impaired DDR leads to the accumulation
of DNA lesions, which results in genomic instability and
can be followed by the malignant transformation of a cell
(Motoyama and Naka 2004). DNA double-stranded
breaks (DSBs) and DNA interstrand cross-links (ICLs)
are among the most severe DNA lesions (Harper and
Elledge 2007; Bonner et al. 2008; Jackson and Bartek 2009;
Moldovan and D’Andrea 2009). DDR operates through
a large network of proteins; however, some proteins lie at
the core of DDR mechanisms and regulate responses to
several types of lesions (Matsuoka et al. 2007; Jackson and
Bartek 2009; Ciccia and Elledge 2010). For example,
ataxia telangiectasia and Rad3-related (ATR) kinase phosphorylates
hundreds of targets and signals for DNA
replication and repair (Matsuoka et al. 2007). Similarly,
breast and ovarian cancer type 1 susceptibility protein 1
(BRCA1) acts on several types of DNA lesions, and its
pivotal task is the maintenance of genomic stability
(Huen et al. 2010). The FANCD2 and recently discovered
FANCI proteins are the central players in the Fanconi
anemia (FA) pathway, which repairs ICLs and protects
against genomic instability (Smogorzewska et al. 2007;
Moldovan and D’Andrea 2009).
In this study, we report the identification of a 70-kDa
CycK that associates with Cdk12 and Cdk13 in two
separate, functionally distinct complexes. We show that
the CycK/Cdk12 complex protects cells from genomic
instability via the regulation of expression of DDR genes.
Results
Human CycK is a 70-kDa protein with a C-terminal
proline-rich region and does not associate with Cdk9
We were initially interested in potential differences
among three Cdk9 complexes formed by different cyclin
subunits: CycT1, CycT2, or CycK. Our attention was
brought to a single band of 70-kDa protein identified by
Western blotting, with an antibody directed against the
N-terminal cyclin box of CycK (Fig. 1A). Although the
molecular mass of the originally identified CycK protein
was ;40 kDa (Edwards et al. 1998), we did not find any
protein of such size in cell lysates from various cell lines
tested with different CycK antibodies (Fig. 1A; data not
shown). Interestingly, next to the published 40-kDa and
357-amino-acid human CycK protein (CycK-357) (Edwards
et al. 1998), the Ensembl database (http://www.ensembl.
org) provides information on a 580-amino-acid human
CycK protein with a predicted size of ;70 kDa. This form
of CycK differs from the original one in the C-terminal
proline-rich region (Fig. 1B).
Next, we generated stable cell lines expressing the Flag
epitope-tagged CycK (CycK-Flag), CycT1 (Flag-CycT1),
357-amino-acid form of CycK (Flag-CycK-357), and
empty plasmid vector (Flag-ev) in 293 cells. Western
blotting of the cell lysates probed with an antibody
against CycK revealed two proteins next to each other—
one belonging to the endogenous CycK, and another
belonging to CycK-Flag—confirming that this form of
CycK is expressed in cells (Fig. 2A, bottom panel, lane 2;
data not shown). To determine whether CycK interacts
with Cdk9, we immunoprecipitated CycK-Flag from
cells, but there was no detectable association of CycK
with Cdk9 (Fig. 1C, lane 8). Also, we could not observe
any association of the Flag-CycK-357 with Cdk9 or
Hexim1 (another P-TEFb-associated protein) (Fig. 1C,
lane 7), while the interaction of Flag-CycT1 with Cdk9
and Hexim1 was observed (see Fig. 1C, lane 6).
To investigate which kinases are associated with CycK,
we immunoprecipitated CycK-Flag stably expressed in
293 cells and identified associated proteins by highperformance
liquid chromatography (HPLC) electrospray
ionization mass spectrometry (ESI-MS) and tandem MS
(MS/MS) techniques. This experiment provided several
Figure 1. Identification of CycK as 70-kDa protein in cells. (A)
Detection of the 70-kDa form of CycK in cell lysates from 293
cells with an antibody directed against the N-terminal cyclin
box of CycK (Sigma, HPA000645). (B) Schematic representation
of 40-kDa CycK-357 (Edwards et al. 1998) and the 70-kDa forms
of CycK. (C) CycK and CycK-357 do not bind Cdk9. Flag-CycT1,
CycK-Flag, Flag-CycK-357, and Flag-ev were immunoprecipitated
from 293 cells and probed with antibodies to Cdk9 (right
middle panel) and Hexim1 (right bottom panel) by Western
blotting. (Left panels) Expression of the Cdk9, Hexim1, and Flag
epitope-tagged proteins was measured with appropriate antibodies
and represents 5% input of cell lysates.
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peptides corresponding to Cdk12 and Cdk13 (Supplemental
Table S1A). To confirm that Cdk12 and Cdk13 interact
with CycK, immunoprecipitations of endogenous Cdk12
and HA epitope-tagged Cdk13 (Cdk13-HA) proteins from
293 cells were carried out. The Coomassie blue-stained
gel provided a band of 70-kDa protein in both immunoprecipitations
(data not shown). The HPLC ESI-MS and
MS/MS procedure identified that peptides isolated from
the 70-kDa bands represented the sequences of CycK
(Supplemental Fig. S1A). For the detailed list of peptides
Figure 2. CycK binds Cdk12 and Cdk13 in two separate complexes. (A) CycK interacts with Cdk12 and Cdk13, but not with Cdk9.
(Lanes 4–6) Flag epitope-tagged proteins or empty plasmid vector (ev) were immunoprecipitated from lysates of 293 cells,
immunoprecipitations were resolved by Western blotting, and bound proteins were identified with antibodies indicated on the right.
Lanes 1–3 represent 5% input of cell lysates. (B) Cdk12 and Cdk13 interact with CycK, rather than with CycL. (Lanes 5–8) HA epitopetagged
proteins or empty plasmid vector (ev) were immunoprecipitated from lysates of 293 cells, immunoprecipitations were resolved
by SDS-PAGE, and bound proteins were identified with antibodies indicated on the right by Western blotting. Lanes 1–4 represent 5%
input of the cell lysates to the immunoprecipitation. Stars indicate the positions of HA epitope-tagged proteins. (C) CycK, Cdk12, and
Cdk13 comigrate in glycerol gradient. Lysates of 293 cells from control (left panel) or actinomycin D-treated (right panel) cells were
divided into 13 fractions by a glycerol gradient centrifugation. Amounts of endogenous proteins were assessed with antibodies indicated
on the inner sides of both panels by Western blotting. Numbers above and below the panels refer to the glycerol gradient fraction.
(LMM) Low molecular mass; (HMM) high molecular mass; (ActD) actinomycin D; (C) control. Five percent input of lysates in glycerol
gradients is presented in the panel on the left. (D) CycK colocalizes with Cdk12 and Cdk13. Expression of GFP- and Cherry-tagged
proteins in HeLa cells was visualized by confocal microscopy either alone or merged as indicated above the pictures. DAPI depicts for
the staining of the nucleus. (E) The CycK/Cdk13 complex is free of Cdk12. (Lanes 3,4) Cdk13-HA or HA-ev were immunoprecipitated
from the lysate of 293 cells and immunoprecipitations were resolved by SDS-PAGE followed by Western blotting, where bound proteins
were identified with antibodies indicated on the right. Lanes 1 and 2 represent 5% input of cell lysate. (F) The CycK/Cdk12 complex is
free of Cdk13. Endogenous Cdk12 or control (C) immunoprecipitation without antibody was carried out as in E. (Lanes 3,4) Proteins
identified by Western blotting are indicated on the right. Lanes 1 and 2 represent 5% input of cell lysate. (G) CycK stabilizes CycK/
Cdk12 and CycK/Cdk13 complexes. Proteins were knocked down in HCT116 cells by the indicated siRNAs and lysates were separated
by SDS-PAGE followed by Western blotting with the antibodies indicated on the side of the panels.
Blazek et al.
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of CycK found in Cdk12 or Cdk13 immunoprecipitations,
see Supplemental Table S1B. Notably, CycK is evolutionarily
conserved, mainly in its N-terminal cyclin box and
also in the newly identified C-terminal proline-rich region,
suggesting an important function of this protein in
the organisms (see Supplemental Fig. S1B). These experiments
established the existence of the 70-kDa CycK,
which is the CycK form referred to throughout the
present study. In addition, we did not find any evidence
for the existence of CycK-357 at the protein level even
though our antibody should, in principle, recognize both
forms of the protein.
CycK associates with Cdk12 and Cdk13 to form two
separate complexes: CycK/Cdk12 and CycK/Cdk13
To confirm that Cdk12 and Cdk13 indeed bind CycK, we
used 293 cells stably expressing CycK-Flag, Flag-CycT1,
and Flag-ev. We immunoprecipitated these Flag epitopetagged
proteins and followed the binding of Cdks by
Western blotting (Fig. 2A). As expected, CycK-Flag bound
the endogenous Cdk12 and Cdk13 proteins but not Cdk9
and Cdk11 (Fig. 2A, lane 5), and Flag-CycT1 immunoprecipitated
only endogenous Cdk9 protein, rather than
Cdk11, Cdk12, or Cdk13 (Fig. 2A, lane 6). Also, the Flag
epitope-tagged proteins were properly immunoprecipitated
(Fig. 2A, two bottom panels, lanes 5,6).
To further examine the association of kinases with
cyclins, we used 293 cells stably expressing Cdk13-HA,
Cdk12-HA, and Cdk9-HA proteins and empty plasmid
vector. HA epitope-tagged kinases were immunoprecipitated,
and Western blots were reprobed with antibodies
against CycK and CycT1 (Fig. 2B). Cdk9 associated only
with CycT1 rather than with CycK (Fig. 2B, lane 8); in
contrast, Cdk12 and Cdk13 associated only with CycK
(Fig. 2B, lanes 6,7). Also, all HA epitope-tagged proteins
were efficiently immunoprecipitated (Fig. 2B, top panel,
lanes 6–8). As overexpressed CycL was also shown to be
an interacting partner of Cdk12 and Cdk13 (Chen et al.
2006, 2007), we reprobed our Western blot with anti-CycL
antibody. However, none of the tested kinases interacted
with endogenous CycL (Fig. 2B, lanes 5–8). Coincidently,
with a recently published study (Bartkowiak et al. 2010),
we also did not recover any CycL peptides in Cdk12 and
Cdk13 immunoprecipitations subjected to LC-MS/MS.
To elucidate further the biochemical and functional
characteristics of these new CycK kinases, cell lysates
were fractionated on a 10%–30% glycerol gradient (Fig.
2C). As predicted, antibodies to CycK, Cdk12, and Cdk13
revealed significant overlap among these proteins in
fractions 5–9, while Cdk9 and Hexim1 proteins were
found in fractions 2–13 (Fig. 2C, left panel). Fractionation
of lysates from cells treated with actinomycin D, which
disrupts the large inactive complex of P-TEFb (Li et al.
2005; Yang et al. 2005), revealed almost no shift of
the CycK/Cdk12 and CycK/Cdk13 complexes; in contrast,
Hexim1 and Cdk9 shifted to a lower molecular
mass fraction, as expected (Fig. 2C, right panel). These
experiments further confirm that CycK/Cdk12 and
CycK/Cdk13 form different complexes from P-TEFb in
cells. Moreover, fluorescence microscopy experiments
analyzing GFP-fused CycK and Cherry-fused Cdk12 and
Cdk13 proteins also support our data that CycK is a bona
fide partner of these kinases, as CycK-GFP colocalized
with Cdk12-Cherry and Cdk13-Cherry (Fig. 2D).
Furthermore, we were interested in determining whether
CycK exists in mutually exclusive complexes with Cdk12
or Cdk13. We immunoprecipitated either Cdk13-HA or
endogenous Cdk12 from 293 cells and followed the Cdk12
or Cdk13 binding, respectively, by Western blotting (Fig.
2E,F). While Cdk13 did not coimmunoprecipitate with
Cdk12, CycK was efficiently recovered (Fig. 2E, lane 4).
Similarly, while the endogenous Cdk12 did not immunoprecipitate
Cdk13, its interaction with CycK was detected
(Fig. 2F, lane 4). This experiment indicates that CycK
forms two separate complexes with Cdk12 and Cdk13.
Interestingly, fluorescence microscopy analysis of Cdk12GFP
and Cdk13-Cherry demonstrated colocalization between
most of these two proteins; however, in certain
regions, Cdk12 and Cdk13 seem to exist independently of
each other (Supplemental Fig. S2A).
Taking into account that cyclin and Cdk subunits
usually stabilize each other in the heterodimer complex,
we explored this possibility with CycK/Cdk12 and CycK/
Cdk13 (Fig. 2G). We knocked down CycT1 in cells and
observed no significant change in Cdk12 and Cdk13
levels, while Cdk9 and Hexim1 levels decreased (although
not completely, due to the stabilization effect of
CycT2 on Cdk9) (Fig. 2G, lane 2). Knockdown of CycK led
to a significant decrease of levels of Cdk12 and a slight
decrease of levels of Cdk13 (Fig. 2G, lane 3). The loss of
Cdk12 resulted in a significant decrease in the amount of
CycK (Fig. 2G, lane 4). A similar situation occurred after
Cdk13 was depleted from cells (Fig. 2G, lane 5). The
knockdown of Cdk12 and Cdk13 together led to the
complete loss of CycK (Fig. 2G, lane 6). Complementing
these results, individual overexpression of Cdk12 or
Cdk13 led to their inefficient expression; however, when
coexpressed with CycK, their protein levels increased
significantly, as these Cyc/Cdk complexes were stabilized
(Supplemental Fig. S2B, cf. lanes 1,4 and 2,3,5,6,
respectively). These experiments defined the existence of
two separate complexes: CycK/Cdk12 and CycK/Cdk13.
CycK/Cdk12 can activate transcription
and phosphorylate Ser2 in the CTD of RNAPII
Since Cdk12 has a kinase domain similar to Cdk9
(Malumbres et al. 2009), and work on CycK-357 indicated
its role in transcription (Edwards et al. 1998; Fu et al.
1999; Lin et al. 2002), we were interested in whether the
CycK/Cdk12 can also regulate transcription (Fig. 3). To
begin to answer this question, we used an RNA-tethering
assay (Fig. 3A) that measures transcriptional activity (Fig.
3B). Indeed, when we coexpressed the Rev-Cdk9 chimera
with the SLIIB-CAT reporter plasmid, the levels of chloramphenicol
acetyltransferase (CAT) activity increased
>20-fold compared with when the reporter was coexpressed
with the empty plasmid vector (Fig. 3B, cf. lanes 2
and 1). This increase was not detected when the kinaseCycK/Cdk12
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inactive mutant of Cdk9, Rev-Cdk9D167N, was coexpressed
(Fig. 3B, cf. lanes 1 and 3), confirming that an
increase in CAT activity, and thus transcription, is the
result of the kinase activity of Cdk9. Rev-Cdk9 and RevCdk9D167N
expressed equally (Fig. 3B, cf. lanes 2 and 3).
Coexpression of Rev-Cdk12 with SLIIB-CAT resulted in
>10-fold increase in transcriptional activity (Fig. 3B, cf.
lanes 1 and 4), indicating that Cdk12 could also direct
transcription. Of note, expression levels of Rev-Cdk12
were lower than Rev-Cdk9 (Fig. 3B, cf. lanes 2 and 4).
Thus, CycK/Cdk12 can affect transcription.
As phosphorylation of Ser2 in the CTD of RNAPII is
thought to be critical to overcome an early elongation
block and for subsequent processivity of transcribing
polymerases (Peterlin and Price 2006; Egloff and Murphy
2008; Buratowski 2009), we examined whether the absence
of CycK complexes affects its global levels (Fig. 3C).
We used 3E10 and H5 antibodies, both of which recognize
phosphorylated Ser2. While 3E10 predominantly recognizes
heptapeptides phosphorylated solely on the Ser2,
H5 is more specific for the phosphorylated Ser2 in the
context of phosphorylated neighboring Ser5 residues
(Chapman et al. 2007). Indeed, CycK knockdown resulted
in a significant decrease of phosphorylation of Ser2 when
evaluated by both antibodies (Fig. 3C, cf. lanes 1 and 2),
and depletion of Cdk12 provided the same result (Fig. 3C,
cf. lanes 1 and 3). Knockdown of Cdk13 did not change the
levels of phosphorylation of Ser2 significantly (Bartkowiak
et al. 2010; data not shown).
To determine whether Cdk12 is able to directly phosphorylate
Ser2 in the CTD of RNAPII, we performed an in
vitro kinase assay (Fig. 3D). For this purpose, we purified
HA-ev, Cdk12-HA, and Cdk9-HA proteins and incubated
them with the recombinant GST-CTD fusion protein. As
predicted, addition of increasing amounts of purified
Cdk12 and Cdk9 led to dose-dependent phosphorylation
of GST-CTD (Fig. 3D, lanes 5,6 and 8,9 respectively). We
conclude that the CycK/Cdk12 complex can phosphorylate
Ser2 in the CTD domain of RNAPII and control
transcription in human cells.
Depletion of CycK/Cdk12 changes the expression
of a small subset of genes
Since the depletion of CycK and Cdk12 had an effect on
levels of phosphorylation of Ser2 (Fig. 3C), we explored
the role of the CycK/Cdk12 complex in the regulation of
eukaryotic gene expression (Fig. 4). For this purpose, we
Figure 3. CycK/Cdk12 regulates transcription and phosphorylates Ser2 in the CTD of RNAPII. (A) Schematic depiction of
heterologous RNA tethering assay. Plasmid reporter used to test transcription contains modified HIV-LTR promoter (blue square)
followed by TAR (transactivation response RNA) (red square) with inserted stem–loop IIB (SLIIB) (yellow line in RNA) from the HIV Rev
response element (RRE). Nascent RNA (black dashed line) synthetized by RNAPII (violet oval) forms a dsRNA loop with the SLIIB
element. Rev-cdk fusion proteins tethered via SLIIB to paused RNAPII can release RNAPII when phosphorylate Ser2 (P) (red circle) in its
CTD (violet line), which results in the transcription of CAT reporter gene (yellow square). (B) Rev-Cdk12 activates transcription from
the SLIIB-CAT plasmid. The activity of the CAT reporter SLIIB-CAT coexpressed with empty plasmid vector (C) was set as 1, and bars
represent relative CAT activity obtained by the cotransfection of the reporter with indicated plasmids in HeLa cells. Western blotting
shows expression of Rev fusion proteins and actin. (C) Knockdown of CycK/Cdk12 decreases the global phosphorylation of Ser2 in the
CTD of RNAPII. HCT116 cell were transfected with the indicated siRNAs, and the levels of proteins were followed with the indicated
antibodies by Western blotting. (D) CycK/Cdk12 phosphorylates Ser2 in the GST-CTD in vitro. Ten nanograms of GST-CTD was
incubated with increasing amounts (indicated by the triangle) of purified HA-ev, Cdk12-HA, and Cdk9-HA. The amounts of
phosphorylated Ser2 (P-Ser2) were monitored by Western blotting with the indicated anti-Ser2 phospho-specific antibody.
Blazek et al.
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employed expression microarrays in cells depleted of
CycK. The data revealed that 3.9% of the genes were
down-regulated, 2.6% were up-regulated, and 93.5% were
unaffected (Fig. 4A). The RT-qPCR with selected genes
confirmed the validity of the microarray data, as ;85% of
the RT-qPCR data mirrored the microarray results (for
examples of validated genes, see Supplemental Fig. S3A).
Furthermore, expression microarrays in Cdk12-depleted
cells showed that 2.14% of the genes were down-regulated,
0.53% were up-regulated, and 97.33% were not affected
(Supplemental Fig. S3B). A high Pearson correlation coefficient
(r = 0.64) among the genes affected in both microarrays
further supports the hypothesis that CycK/Cdk12
acts as a complex (Supplemental Fig. S3C). For the numbers
of commonly affected genes in both microarrays, see
Supplemental Table S2. We were interested in the regulation
of expression by the CycK/Cdk12 complex and focused
our attention on the group of down-regulated genes.
Figure 4. CycK/Cdk12 knockdown changes expression of a small subset of human genes. (A) Distribution of genes either differentially
expressed or with no change of expression after CycK knockdown in HeLa cells. (B) The graph presents the distributions of the length of
all human genes (red) and genes down-regulated in expression microarray in Cdk12-depleted cells (green). Human gene length data were
obtained from the University of California at Santa Cruz (UCSC) Genome Browser. (C) The graph presents the distributions of the
number of exons in all human genes (red) and genes down-regulated in expression microarray in Cdk12-depleted cells (blue). Data about
the number of exons were obtained from the UCSC browser. (D) The classification of genes with reduced expression after CycK
knockdown is based on their enrichment relative to total numbers in their specific category. Significance is expressed as Àlog (P-value)
with a threshold value of 1.3 = Àlog (P = 0.05), and was calculated by the Ingenuity program using right-tailed Fischer’s exact test. (E)
Depiction of the BRCA1 network. The network was generated by the Ingenuity program from genes down-regulated after CycK
knockdown. (Gray shapes) Genes down-regulated in the microarray; (white shapes) genes not found down-regulated in the microarray;
(oval) transcriptional regulator; (diamond) enzyme; (triangle) kinase; (trapezoid) transporter. A solid line represents direct interaction,
a dashed line indicates indirect interaction, a line without arrows indicates binding, and an arrow from protein A to protein B means
that protein A acts on protein B.
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We compared gene length-based distributions of all
human genes with the down-regulated genes in Cdk12depleted
cells (Fig. 4B). The group of down-regulated
genes showed higher frequencies of long genes in comparison
with their representation among all human genes
(Fig. 4B, cf. both distributions). Analysis of the downregulated
genes from the CycK microarray provided
a similar result (Supplemental Fig. S3D). For the comparison
of the representation of genes longer than 10 kb and
50 kb among all human genes and the down-regulated
genes in the CycK and Cdk12 microarrays, see Supplemental
Table S3. The genes with reduced expression after
Cdk12 or CycK knockdowns have higher numbers of
exons in comparison with all human genes (Fig. 4C;
Supplemental Fig. S3E, respectively). These analyses
suggest that CycK/Cdk12 directs expression of predominantly
long and complex genes. The identified downregulated
genes in the CycK microarray were further
classified into gene ontology groups (Fig. 4D; for information
about the pathways enriched in the up-regulated
genes, see Supplemental Fig. S3F). In the classification
according to molecular and cellular function, DNA
replication, recombination, and repair scored the highest.
Network modeling using the Ingenuity Program identified
as the top network the BRCA1 module. This centers
the BRCA1 protein in functional connection with the
key regulators of the FA pathway—proteins FANCI
and FANCD2, DNA damage sensor kinase ATR, and a subunit
of the SWI/SNF remodeling complex, SMARCC2)—
participating in the regulation of the DDR (Fig. 4E). These
proteins are the key components of several DDR pathways
(Motoyama and Naka 2004; Harper and Elledge
2007; Jackson and Bartek 2009; Huen et al. 2010). The
complete list of DDR genes down-regulated in the microarray
after depletion of CycK is in Supplemental Table S4.
Most of these DDR genes were also down-regulated in the
microarray from Cdk12-depleted cells (Supplemental
Table S4, the Cdk12 column). These data demonstrated
that depletion of CycK/Cdk12 from cells resulted in disrupted
expression of a small subset of genes and in the
down-regulation of predominantly long, complex genes
and a group of DDR genes.
CycK/Cdk12 directs transcription of key DDR genes
Given the importance of genes from the BRCA1 module
for the DDR, we focused our attention on elucidating the
mechanism by which CycK/Cdk12 contributes to their
expression (Fig. 5). To confirm the microarray data for
these genes, we knocked down CycK, Cdk12, and Cdk13
in cells and measured mRNA levels of BRCA1, ATR,
FANCI, FANCD2, and SMARCC2 genes by RT-qPCR
(Fig. 5A). Cells with depleted CycK had significantly
lower levels of mRNA for all five genes in comparison
with cells with control siRNA (Fig. 5A, cf. bars for control
and CycK siRNAs). Depletion of Cdk12 provided the
same result except for the ATR gene (Fig. 5A, cf. bars for
control and Cdk12 siRNAs), while knockdown of Cdk13
resulted in no significant change in mRNA levels for all
five genes (Fig. 5A, cf. bars for control and Cdk13 siRNA).
To determine whether knockdown of CycK, Cdk12, and
Cdk13 alters the protein levels of BRCA1, ATR, FANCI,
and FANCD2, we performed Western blotting (Fig. 5B).
Indeed, knockdown of CycK and also of Cdk12 resulted in
significantly lower levels of these proteins (Fig. 5B, lanes
2,3), in contrast to when Cdk13 was depleted (Fig. 5B, lane
4). These results suggest that CycK/Cdk12 rather than
CycK/Cdk13 regulates expression of these genes and
show that the CycK/Cdk12 and CycK/Cdk13 complexes
have different functions in cells.
P-TEFb is considered to be the major Ser2 kinase and
was implicated in the regulation of transcriptional elongation
of most, if not all, protein-coding genes (Chao et al.
2000; Nechaev and Adelman 2008; Rahl et al. 2010). To
determine how depletion of P-TEFb and the CycK/Cdk12
complex affects BRCA1 protein levels and global phosphorylation
of Ser2, we knocked down both cyclin subunits
of P-TEFb and the CycK/Cdk12 complex (Fig. 5C).
As expected, loss of CycK/Cdk12 but not of P-TEFb
resulted in lowered BRCA1 levels (Fig. 5C, cf. lanes 1,2
and 3,4). Although depletion of CycK/Cdk12 and P-TEFb
had a similar effect on the levels of phosphorylation of
Ser2 when measured by the 3E10 antibody (Fig. 5C, cf.
lanes 1–3 and 4), the phosphorylation of Ser2 detected by
the H5 antibody was reproducibly less affected by the loss
of P-TEFb than the CycK/Cdk12 complex (Fig. 5C, cf.
lanes 1,2 and 3). It can be concluded that BRCA1 expression
is not sensitive to P-TEFb depletion.
To address the discrepancy between the significant
drop of global phosphorylation of Ser2 (Figs. 3C, 5C) and
few detected changes of gene expression (Fig. 4A; Supplemental
Fig. S3B), we performed global nascent RNA analysis
experiments (Lin et al. 2008) to determine whether
CycK/Cdk12 affects global transcription (Fig. 5D,E). To
measure nascent transcripts, cells were labeled with
3
H-uridine and total RNA (processed and unprocessed)
was precipitated with trichloracetic acid. The rate of
global transcription was correlated in the amount of
3
H-uridine labeling of newly synthetized RNA. Knockdown
of CycK/Cdk12 did not reduce the global rate of
transcription in comparison with control siRNA, while
treatment with the transcriptional inhibitor actinomycin
D resulted in its significant decrease (Fig. 5D). Similarly,
the amounts of 3
H-uridine-labeled polyadenylated nascent
mRNA purified with oligodTsepharose from CycK/Cdk12depleted
cells did not exhibit any decrease within the total
polyadenylated RNA pool in comparison with control
siRNA (Fig. 5E), suggesting that RNAPII transcription
was not globally impaired in cells without CycK/Cdk12.
To examine whether the defect in the expression of
DDR genes (Fig. 5A,B) after CycK/Cdk12 depletion is transcriptional,
we performed nuclear run-on assays and
measured the levels of nascent RNA of BRCA1 and FANCI
genes by RT-qPCR (Fig. 5F). As a control, we measured the
levels of nascent RNA of the TNSK1BP1 gene that was not
differentially expressed in microarrays after CycK/Cdk12
knockdown. Levels of nascent RNA of BRCA1 and FANCI
genes were significantly reduced when CycK or Cdk12
were silenced, but not when Cdk13 or control siRNA were
transfected. Importantly, treatment with actinomycin D
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Figure 5. CycK/Cdk12 regulates transcription of key DDR genes. (A) Knockdown of CycK/Cdk12 results in decreased mRNA levels of
key DDR genes in HCT116 cells. Graphs present levels of mRNA of described genes in cells transfected with control (C), CycK, Cdk12,
or Cdk13 siRNA. RT-qPCR results are normalized to the mRNA of GAPDH. (B) Knockdown of CycK/Cdk12, rather than of CycK/
Cdk13, depletes protein levels of the DDR genes. HCT116 cell were transfected with indicated siRNAs, and protein levels were
measured with indicated antibodies by Western blotting. (C) Knockdown of P-TEFb does not decrease expression of the BRCA1 gene.
HCT116 cells were transfected with the depicted siRNAs, and protein levels were measured by Western blotting with the indicated
antibodies. (D,E) Depletion of CycK/Cdk12 in cells does not affect the global transcription rate in nascent RNA analysis. HeLa cells were
transfected with indicated siRNA for 48 h or treated with actinomycin D for 1 h. The graphs present the ratio of RNA-incorporated 3
Huridine
in precipitated nascent RNA (D) or in polyadenylated nascent mRNA (E) to free 3
H-uridine as a measure of the amount of cells.
Representative experiments are shown. (F) Reduction of nascent transcript of BRCA1 and FANCI in nuclear run-on from CycK/Cdk12depleted
cells. HCT116 cells were transfected with indicated siRNA or treated with actinomycin D. Graphs show relative abundance of
nascent RNA transcripts for indicated genes in nuclear run-on measured by RT-qPCR. RT-qPCR results are normalized to the nascent
mRNA of GAPDH, and error bars indicate the standard deviation from three independent transfections. (G) Depletion of the CycK leads
to lower levels of the RNAPII on the promoters of BRCA1 and FANCI genes. ChIP analysis for the occupation of RNAPII on the regions
of indicated genes after transfection with mock or CycK siRNAs. Schemes depicting BRCA1 and FANCI genes show the position of ChIP
primers. Their location is marked by the arrows, and the number indicates their approximate distance in kilobases from the transcription
start site. (PR) Promotor; [CR(5)] 59 prime of the coding region; [CR(M)] coding region; (ST) stop codon; (3Down) region down of the stop
codon; (PolyA) polyadenylation signal; [IR(3)] intergenic region. IgG corresponds to the empty beads control. Experiments are the results
of four (BRCA1 and FANCI) or three (TNSK1BP1 and GAPDH) independent transfections of HCT116 cells, and qPCR was performed in
triplicate for each transfection. Statistical significance of each pairwise comparison is depicted with a star; (*) P < 0.05.
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decreased the abundance of nascent transcripts for both
genes (Fig. 5F, cf. bars for BRCA1 and FANCI genes). In
contrast, the amount of nascent RNA transcript for the
TNKS1BP1 gene was not affected in CycK/Cdk12-depleted
cells (Fig. 5F, cf. bars for TNKS1BP1 gene).
To further address the transcriptional defect on these
genes, we performed chromatin immunoprecipitation
(ChIP) with RNAPII on BRCA1 and FANCI genes (Fig.
5G). ChIPs were performed in cells transfected with
mock or CycK siRNA using an antibody that recognizes
RNAPII and primers covering several regions of both
genes. In cells where CycK was depleted, we observed
significantly less RNAPII on the promoter of FANCI and
BRCA1 compared with mock-transfected cells, while its
levels on the other regions did not change significantly
(Fig. 5G, cf. bars). Likewise, RNAPII occupancy on the
ATR promoter was also reduced significantly in the absence
of CycK (data not shown). In contrast, amounts of
RNAPII on the promoters of CycK/Cdk12-independent
genes TNKS1BP1 and glyceraldehyde 3-phosphate dehydrogenase
(GAPDH) were not affected significantly
(Fig. 5G). These experiments established that the depletion
of CycK/Cdk12 does not affect global transcriptional
rates, but CycK/Cdk12 is the limiting factor that
affects the transcription of a small subset of genes,
including some key DDR genes.
CycK/Cdk12 is required for the maintenance
of genomic stability
Since the CycK/Cdk12 complex regulates the expression
of several important DDR genes, we hypothesized that it
should play a broad role in the cellular response to DNA
damage and in the maintenance of genomic stability (Fig.
6). We knocked down CycK in HeLa cells and measured
their sensitivity to various DNA-damaging agents in
survival assays (Smogorzewska et al. 2007; O’Connell
et al. 2010). As a positive and negative control, we used
BRCA1 and ATR siRNAs and scrambled siRNA, respectively
(Fig. 6A). Loss of CycK resulted in a strong and
dose-dependent sensitivity of cells to DSB inducers
etoposide and camptothecin, an effect comparable with
cells with depleted BRCA1 and ATR proteins (Fig. 6A).
Knockdown of CycK also led to moderate sensitivity to
mitomycin C, an agent causing DNA ICLs, indicating
that CycK may play a more general role in cellular response
to DNA damage (Fig. 6A). Consistent with this
finding, CycK-depleted cells show increased levels of
mitomycin C-induced chromosome instability (data not
shown). As expected, knockdown of Cdk12 rather than of
Cdk13 caused sensitivity of cells to etoposide, camptothecin,
and mitomycin C (Supplemental Fig. S4), confirming
the essential function of the CycK/Cdk12 but not the
Figure 6. CycK/Cdk12 is required for the maintenance of genomic stability. (A) CycK-depleted cells are sensitive to a variety of DNA
damage agents. The graphs represent the results of survival assays of HeLa cells transfected with siRNA directed to the shown targets
and treated with either mitomycin C (MMC), etoposide (ETO), or camptothecin (CMT), or left untreated. Mean and standard deviation
values represent the result of at least three independent transfections. Cell viability was normalized to relative growth of cells
transfected with specific siRNA. (B) Depletion of CycK/Cdk12 induces DNA damage signaling. The graph shows percentage of positive
cells 3 median fluorescence intensity of HeLa cells transfected with indicated siRNAs or treated with etoposide (ETO), labeled with
g-H2AX antibody, and analyzed by fluorescence-activated cell counting. The results represent the values of four independent
experiments. (C,D) Knockdown of the CycK/Cdk12 complex causes spontaneous DNA damage. (C) U2OS cells transfected with the
indicated siRNAs or treated with etoposide (ETO) were labeled with 53BP1 antibody or DAPI, and images showing 53BP1 foci were
visualized by indirect immunofluorescence. (D) Graph represents the quantification of cells positive for 53BP1 foci from three
independent experiments. Cells with at least five foci were considered positive, and a minimum of 300 cells were analyzed for each
condition in each experiment.
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CycK/Cdk13 complex in the cellular response to exogenous
DNA damage.
To address the role of the CycK/Cdk12 complex in the
maintenance of genomic stability in the absence of
exogenous DNA damage, we measured the levels of
g-H2AX (Fig. 6B) and the number of 53BP1 foci (Fig.
6C,D) in cells depleted with either CycK or Cdk12.
Knockdown of CycK or Cdk12 in HeLa cells caused an
increase in the amount of g-H2AX protein. This increase
was similar to the situation in which BRCA1 was depleted
from cells and indicated that the loss of the CycK/
Cdk12 complex in the absence of the exogenous DNA
damage induces DNA damage signaling (Fig. 6B). Since
53BP1 accumulates at the g-H2AX foci, it can be used as
another marker of DSBs in cells (Bonner et al. 2008); we
observed the formation and counted the number of 53BP1
foci in U2OS cells without CycK and Cdk12 using
immunofluorescence microscopy (Fig. 6C,D). Silencing
of CycK or Cdk12 resulted in the formation of strong
53BP1 foci (five or more foci per cell) in ;20% of cells, in
comparison with cells treated with control siRNA, where
only ;5% of cells developed the foci. BRCA1-depleted
and etoposide-treated cells scored the strong foci in 20%
and 50% of cells, respectively (Fig. 6C,D). Cell cycle
analysis of CycK and Cdk12-depleted cells showed increased
numbers of accumulated cells in G2–M phase
(Supplemental Fig. S5), indicating the activation of this
DNA damage cell cycle checkpoint (Lobrich and Jeggo
2007) and supporting the role of CycK/Cdk12 in the
maintenance of genomic stability. These data show that
CycK/Cdk12 regulates resistance of cells to the exogenous
DNA-damaging agents and is essential for the
maintenance of genomic stability.
CycK is required for early embryonic development
To define the function of CycK in an organism, we
generated a CycK knockout mouse (Fig. 7). Mouse embryonic
stem cells with b-geo gene trap insertion (plasmid)
within intron 1 of the CycK gene were obtained from
the German Gene Trap Consortium and were used to
generate chimeric mice (Fig. 7A). Indeed, the trap could be
detected using primers to intron 1 (WTf and WTr) and
b-Geo (TRf and TRr) sequences (Fig. 7B). Since translation
of CycK mRNA is initiated from the second exon, the
insertion of the trap vector within the first intron resulted
in the loss-of-function allele of the CycK gene. Although
our wild-type and heterozygous CycK+/À
mice were
healthy, appeared normal, and reproduced well, their
mating did not generate any homozygous CycKÀ/À
offFigure
7. Genetic inactivation of CycK leads to early embryonic lethality. (A) Schematic representation of wild-type (WT) and trapped
(TR) (CycK.b-geo) alleles. Wild-type and trapped loci contain one to 11 and one to four exons, respectively. The integration of the gene
trap vector into intron 1 of CycK resulted in the inactivation of the CycK gene. The insertion also led to an in-frame fusion of CycK
with the b-geo (encoding b-galactosidase and neomycin phosphotransferase) reporter gene. Arrows below the scheme represent primer
sets for wild-type (WT) (WTf and WTr) and trapped (TR) (TRf and TRr) alleles used in B. (B) Representative result of PCR genotyping.
PCR genotyping of genomic DNA from the adult mouse was performed with two sets of primers—WTf+WTr and TRf+TRr (shown in A,
below the mutant allele of the CycK gene)—to detect wild-type (lower band) and trapped (upper band) alleles of the CycK gene.
Genomic DNA was isolated from 3-wk-old mice. For the primers used, see the Supplemental Material. (C) The table presents the
results of genotyping of specified CycK embryos at indicated embryonic days. (D) Expression of CycK during mouse mid-gestation and
early embryogenesis. LacZ staining of heterozygous embryos was used to visualize spatiotemporal expression of CycK.b-geo, which
reflects transcription from the endogenous CycK promoter. Wild-type (+/+) and heterozygous (+/À) embryos were isolated at the given
embryonic day and stained with X-Gal. CycK was expressed in forming tissues and organs in E8.5 and E11.5 embryos. Importantly, the
expression of CycK was localized to the embryonic region and ectoderm in E6.5 and E7.5 embryos.
CycK/Cdk12 maintains genomic stability
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spring. To examine further the function of CycK during
mouse development, embryos at embryonic day 4.5 (E4.5)
(blastocyst), E6.5, E7.5, E8.5, and E14.5 were genotyped (Fig.
7C). Importantly, no CycKÀ/À
embryos were detected at
any given time period, suggesting that CycK might play an
important role during early mouse development (Fig. 7C).
To further explore the spatiotemporal expression of
CycK in developing mouse embryos, LacZ staining was
monitored in E6.5, E7.5, E8.5, and E11.5 embryos. Since
its transcription is driven by the endogenous CycK promoter,
the expression of the hybrid CycKÁb-Geo protein
reflects the expression of CycK (Fig. 7D). The b-Galactosidase
activity, monitored by blue staining, was detected
in the embryonic region (E6.5), embryonic ectoderm
(E7.5), and throughout the forming embryonic tissues
and organs (E8.5 and E11.5) (Fig. 7D).
Discussion
In this study, we report the identification of a 70-kDa
form of the human CycK and found Cdk12 and Cdk13 as
its bona fide partners. We show that depletion of the
CycK/Cdk12 complex affects expression of a small subset
of genes, predominantly relatively longer and complex
ones. The most prominent group of genes down-regulated
in the absence of CycK/Cdk12 belongs to the group of
DDR genes such as BRCA1, ATR, FANCI, and FANCD2.
Consistent with this finding, CycK/Cdk12-depleted cells
are sensitive to a variety of DNA damage agents and
develop spontaneous DNA damage signaling. We propose
that the CycK/Cdk12 complex maintains genome stability
via regulation of expression of DDR genes.
We were unable to identify the previously described 40kDa
CycK-357 at the level of mRNA (data not shown) or
protein (Fig. 1A), and could not confirm any association of
CycK (both 40-kDa or 70-kDa forms) with Cdk9 (Fig. 1C;
Fu et al. 1999; Lin et al. 2002). However, it is still possible
that CycK-357 exists in other cells or during certain
developmental stages of an organism to play alternative
functions to CycK; for example, as a splicing variant.
Silencing of CycK or Cdk12 resulted in an at least 50%
decrease of phosphorylation of bulk Ser2 (Figs. 3C, 5C). Of
note, the yeast homolog of Cdk12, Ctk1, is considered to
be the major Ser2 kinase, and Bur1 (homolog of P-TEFb)
partially contributes to bulk Ser2 phosphorylation (Cho
et al. 2001; Qiu et al. 2009). Since elongating RNAPII is
primarily phosphorylated at Ser2, it is surprising that
CycK/Cdk12 silenced cells did not show global transcription
defect (Figs. 4A, 5D). We can speculate that absence
of CycK/Cdk12 can transform the cell into a different
pattern of CTD phosphorylation compatible with the
productive transcription on most of the genes. Alternatively,
modifications of neighboring residues of phosphorylated
Ser2 in the CTD can render phosphorylated Ser2
undetectable by the phospho-Ser2-specific antibodies. Of
note, yeast deficient of Ctk1 or phosphorylated Ser2 do
not exhibit transcription defects (Cho et al. 2001; Ahn
et al. 2004; Kim et al. 2010).
Data from the microarray demonstrate that a subset of
genes have reduced expression in the absence of CycK.
Interestingly, these genes were the ones associated with
the primary functions of the cell, such as DNA replication/repair
or lipid/nucleic acid metabolism (Fig. 4D). We
present evidence that CycK/Cdk12 is crucial for the
expression of many DDR genes, and for at least BRCA1,
FANCI, and ATR, the defect is transcriptional. The knockdown
of CycK resulted in lower amounts of RNAPII
on the promoters of BRCA1, FANCI, and ATR genes
(Fig. 5G; data not shown), and amounts of their nascent
mRNA was reduced (Fig. 5F; data not shown). Since a
common feature of these and other CycK/Cdk12-regulated
DDR genes is their length (tens of kilobases) and
high complexity (presence of many exons), it is attractive
to speculate that phosphorylation of Ser2 by Cdk12
throughout the whole transcription unit might be the
major ‘‘bottleneck’’ for not only their efficient transcription,
but also proper splicing. However ChIP experiments
with phospho-Ser2-specific antibodies did not give any
signal on the body of the BRCA1 or FANCI genes (data
not shown). We did not observe accumulation of RNAPII
on the body of these genes in the absence of CycK (Fig.
5G) that would be indicative of aberrant transcriptional
processing or polyadenylation (Ahn et al. 2004; Kim et al.
2010). Of note, splicing-sensitive microarrays in the absence
of CycK or Cdk12 did not show any splicing defect
for BRCA1, FANCI, ATR, FANCD2, and SMARCC2 (data
not shown). As long genes are expressed at low levels
(Castillo-Davis et al. 2002), it might be that it is technically
unfeasible to detect existing changes in the density/
modification of RNAPII on the body of these genes, as
their levels there are very low (Fig. 5G). Cdk12 was
localized on several genes in Drosophila and its amounts
culminated in the middle and end of transcription units,
indicating that Cdk12 might indeed be responsible for the
phosphorylation of Ser2 in the middle and at the 39 end of
some genes (Bartkowiak et al. 2010). What might cause the
drop of RNAPII levels at the promoters of these predominantly
long and complex genes? Several features of the
TATA-box core promoter elements were correlated to the
gene length, the number and size of exons, and the strength
of gene expression (Moshonov et al. 2008). The mechanistic
link of these correlations is unknown, but could explain
the less efficient loading of the RNAPII on the promoters
of these long and complex CycK/Cdk12-dependent genes.
Transcriptional regulation of BRCA1 and FA proteins is
significant in the development and progression of cancer;
however, the mechanism is largely unknown. Lower
expression of BRCA1 was identified in sporadic breast
cancer patients and was correlated with poor prognosis
(Thompson et al. 1995; Wilson et al. 1999). Reduced expression
of FANCD2 protein was also reported in ovarian
cancer samples that showed genomic instability when
exposed to mitomycin C (Pejovic et al. 2006). CycK was
identified in a genome-wide shRNA screen as one of the
major proteins responsible for the resistance to the DNA
damage-inducing drug camptothecin (O’Connell et al.
2010). The strength of the CycK phenotype is comparable
with the effect of known regulators of camptothecin
resistance ATR and BRCA1, and also with newly identified
protein MMS22L. This study also demonstrates that
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MMS22L is required for the resistance to mitomycin C
and the maintenance of genomic stability (O’Connell et al.
2010). Notably, we found decreased expression of MMS22L
in our microarrays when CycK or Cdk12 was depleted
(Supplemental Table S4). These data support our finding
that CycK/Cdk12 directs DDR and maintenance of genomic
stability via regulation of expression of key DDR genes.
The role of CycK/Cdk9 in the pathways that maintain
genomic stability in response to replication stress has
been suggested recently (Yu et al. 2010; Yu and Cortez
2011). This work found that knockdown of CycK/Cdk9
causes impaired cell cycle recovery after challenge with
hydroxyurea and induction of g-H2AX in the absence of
exogenous DNA damage. However, expression defects of
DDR genes were excluded as a causative mechanism,
since depletion of Cdk9 did not show any changes in
expression of DDR genes in their transcriptional genomewide
microarray. Consistently, transcriptional changes of
DDR genes were also not observed for the kinase-dead
mutant of Bur1 (yeast homolog of Cdk9) (Clausing et al.
2010). Our data show the down-regulation of many key
DDR genes in expression microarrays after knockdown of
CycK or Cdk12 (Supplemental Table S4). Supported by
the biochemical and mechanistic characterization of
CycK complexes, we suggest that CycK/Cdk12 is a master
regulator of expression of DDR genes and an important
player in cellular response to DNA damage. Since a recent
study found Cdk12 among significantly mutated genes in
ovarian cancer, evidence pointing to its significant clinical
relevance is also accumulating (Bell et al. 2011).
We also analyzed mice with genetic inactivation of
CycK. We observed the lethal phenotype during early
mouse development. This implies that CycK/Cdk12 and/
or CycK/Cdk13 kinases play a major role during embryo
development, perhaps by the modulation of phosphorylation
of the CTD of RNAPII during expression of the
genes regulating the development. Similarly, genetic inactivation
of CycT2 led to embryonic lethality during the
preimplantation period (Kohoutek et al. 2009). These
results emphasize the importance of CTD kinases for
the regulation of early stages of development. Also, given
that CycK regulates expression of DDR genes and other
genes participating in the primary processes of the cell
(Fig. 4D), it is not surprising that the mouse knockout has
an early embryonic-lethal phenotype. Notably, BRCA1
and ATR knockout mice also have embryonically lethal
phenotypes (Hakem et al. 1996; de Klein et al. 2000).
In summary, this study identifies new players in
the emerging field of CTD kinases. It establishes
CycK/Cdk12-directed transcription as a critical regulatory
step in DDR and maintenance of genomic stability.
These findings should provide a foundation for future
research and potential medical therapeutic applications.
Materials and methods
Antibodies
Cdk11 and CycL antibodies were generous gifts of Dr. J. Lahti
(Loyer et al. 2008); BRCA1 antibody was a generous gift from Dr.
K. Gardner (De Siervi et al. 2010). For more information on
antibodies, see the Supplemental Material.
Immunoprecipitation
Cells were lysed in buffer with 150 mM NaCl, 2 mM EDTA, 1%
NP-40, 10 mM Tris-HCl (pH 7.4), and protease (Sigma, P8340)
and phosphatase (Sigma, P00044) inhibitor. Lysate was precleared
with G-Sepharose (GE Healthcare) for 1 h and then incubated
with either HA- or Flag-conjugated agarose (Sigma) or
a relevant antibody, followed by 1 h of incubation with the
G-Sepharose. Immunoprecipitates were washed three times with
0.5 mL of the lysis buffer, eluted from the beads with SDS sample
buffer, and boiled for 3 min. Immunoprecipitates were then
resolved on SDS-PAGE gel followed by Western blotting.
Constructs
For cloning purposes, the cDNA sequence for Cdk12 was PCRamplified
from a eukaryotic expression vector kindly provided
by Dr. J. Pines (Ko et al. 2001). The Cdk13 cDNA was PCRamplified
for subsequent cloning from a eukaryotic expression
plasmid kindly provided by Dr. A.-M. Genevie`re (Even et al.
2006). The Rev fusion assay reporter plasmid encoding CAT and
pSLIIB-CAT, and plasmids encoding Rev-Cdk9 and RevCdk9D167N
were described previously (Lin et al. 2002).
A CycK expression plasmid encoding the human 580-aminoacid
CycK with a C-terminal Flag tag was purchased from
Origene (pCMVentryCCNK, RC202024, cloned into SgflI and
MluI restriction sites), here termed pCycK-Flag. Eukaryotic
expression plasmids encoding C-terminally HA-tagged fusion
proteins were produced by ligating Cdk12 and Cdk13 PCRamplified
cDNA into NheI and AflII restriction sites in the
pcDNA3.1 HA plasmid (Invitrogen), rendering pCdk12-HA and
pCdk13-HA, respectively. Expression plasmids encoding C-terminally
Flag-tagged Cdk12 and Cdk13 fusion proteins were
produced by oligo ligation of a 3xFlag tag sequence into the
pCdk12-HA and pCdk13-HA plasmids using restriction sites
AflII–XbaI and AflII–XhoI, respectively, while removing the HA
tag, rendering pCdk12-Flag and pCdk13-Flag. An expression
plasmid encoding a C-terminal eGFP fusion of CycK was produced
by ligating PCR-amplified eGFP cDNA into the MluI and
XhoI sites of pCycK-Flag, rendering pCycK-GFP. An expression
plasmid encoding untagged CycK was produced by restriction
linearization, fill-in, and religation of the pCycK-Flag plasmid,
removing the Flag tag, rendering pCycK. Expression plasmids
encoding a C-terminal mCherry fusion of Cdk12 and Cdk13
were produced by ligation of mCherry cDNA into the pCdk12HA
and pCdk13-HA plasmids using restriction sites AflII–XbaI
and AflII–XhoI, respectively, rendering pCdk12-mCherry and
pCdk13-mCherry. An expression plasmid encoding a C-terminal
eGFP fusion of Cdk12 was produced by ligating PCR-amplified
eGFP cDNA into the AflII and XbaI sites of pCdk12-HA,
rendering pCdk12-GFP. Rev fusion expression plasmid for
Cdk12 was produced by inserting PCR-amplified cDNA for
Cdk12 into the pRev expression plasmid at restriction sites NheI
and SalI, rendering pRev-Cdk12. The integrity of the DNA
sequence of the expression plasmids was verified by DNA
sequencing, and a 123-base-pair (bp) deletion in the N-terminal
region of the Cdk13 CDS was noted in plasmids encoding HAtagged
and mCherry-tagged Cdk13. This in-frame deletion from
base pair 417 to 540 removes a 44-amino-acid region that is
outside the kinase domain and does not remove any known functional
protein region. To prepare the eukaryotic expression plasmid
expressing the N-terminally Flag-tagged short form of CycK-
357, the corresponding DNA sequence was cloned into the BamHI
CycK/Cdk12 maintains genomic stability
GENES & DEVELOPMENT 2169
Cold Spring Harbor Laboratory Presson October 19, 2011 - Published bygenesdev.cshlp.orgDownloaded from
and XhoI restriction sites, resulting in the vector, here rendering
pFlag-CycK-357. The cDNA of the human cycT1 was inserted
into the pcDNA3.1Flag vector (Invitrogen) by the PCR sitedirected
insertion, resulting in N-terminally Flag-tagged CycT1,
here rendering pFlag-CycT1.
Stable cell lines
293 cells were transfected with appropriate plasmids using
Fugene6 (Roche) and were selected with either G418 (Flag-ev,
Flag-CycT1, Flag-CycK-357, CycK-Flag, and HA-ev) or hygromycin
(Cdk9-HA, Cdk12-HA, and Cdk13-HA). The cell lines
carrying the stably integrated plasmid were expanded from the
single colony.
Rev fusion assay
HeLa cells were plated in 96-well format at 10,000 cells per well.
A total of 300 ng of plasmid was transfected using Fugene6
(Roche) according to the manufacturer’s protocol (30 ng of
pSLIIB-CAT and 270 ng of Rev fusion or empty control plasmid
as indicated in the text). After 48 h, CAT activity in the cell
lysate was measured by a scintillation counter. The protein
content of whole-cell lysate for normalization was determined
using the BCA protein determination kit (Pierce).
In vitro kinase assay
HA-tagged proteins were immunoprecipitated from the established
stable cell lines using HA agarose as described. Immunoprecipitations
were extensively washed, and bound HA-tagged
proteins were eluted with 1.5 mg of HA peptide (Sigma, I2149).
The kinase reaction was performed in the volume of 30 mL
containing 20 mM HEPES (pH 7.8), 400 mM ATP, 1.5 mg of BSA,
phosphatase inhibitor (1:1000; Sigma), and 10 ng of GST-CTD
(Proteinone, P4016-02), and with 0 mL, 1 mL, or 3 mL of eluted
immunoprecipitation. The kinase reaction was carried out for 30
min at 30°C and the reaction was stopped with 30 mL of 23 SDS
sample buffer. Reactions were resolved on SDS-PAGE gel and the
amount of phosphorylation of Ser2 was measured by Western
blotting with Ser2 phospho-specific antibody (Covance, H5).
Fluorescence-activated cell counting
Fluorescence cell counting after antibody staining was performed
as described previously (Krutzik et al. 2011). Phosphorylated
H2AX was detected using rabbit anti-phospho-H2AX
(20E3, Cell Signaling) as the primary antibody and Alexa594-labeled
donkey anti-rabbit (Molecular Probes Invitrogen) as the secondary
antibody. Cell counting was performed using a BD-FACSAria II
(Becton-Dickinson) installed with the BD-FACSDiva software, and
data were subsequently analyzed using FlowJo software.
Survival assay
HeLa cells were transfected with indicated siRNAs as described
in the Supplemental Material. The next day, the cells were
counted and equal numbers of cells were seeded, followed by
incubation with DNA-damaging agents mitomycin C, camptothecin,
or etoposide for 6 d. The ratio of cells transfected with
specific siRNA and control siRNA in untreated cells was used to
normalize for the relative cell growth. The relative survival of
control siRNA-transfected cells after treatment with a DNA damage
agent was set to 100% as described previously (Smogorzewska
et al. 2007; O’Connell et al. 2010).
RT-qPCR
HeLa cells were transfected with control or CycK siRNA as
described in the Supplemental Material. After 72, cells were
harvested and total RNA was isolated by the miRNeasy minikit.
A total of 0.5 mg of RNA was used for reverse transcription with
the M-MLV reverse transcriptase system and random primers
(Invitrogen, 28025-013 and 48190-011). Then, an equal amount
of cDNA was mixed with the LightCycler 480 probes master mix
(Roche Diagnostic, 4707494001). The amplifications of the
samples were carried out in a final volume of 20 mL in a reaction
mixture containing 5 mL of probes Master (Roche Diagnostics),
6 mL of H2O, 0.2 mL of forward primer, 0.2 mL of reverse primer,
0.2 mL of probe, and 3 mL of cDNA. The final concentration of
each primer was 1.0 mM, and the final concentration of the probe
was 1 mM. The amplifications were run on the LightCycler480II
(Roche Diagnostics) using the following conditions: initial activation
step for 5 min at 95°C, followed by 40–45 cycles of 15 sec
at 95°C, and 35 cycles of 60 sec at 60°C and 1 sec 72°C. Changes
in gene expression were calculated using the comparative
threshold cycle method with GAPDH. All primers were synthesized
at VBC-Biotech, and probes were used from the universal
probe library (Roche Diagnostics).
Glycerol gradients
Glycerol gradients were performed as described previously
(Blazek et al. 2005). The cells were treated with 1 mg/mL actinomycin
D (Sigma) for 1 h.
Expression microarray
HeLa cells were transfected with control, CycK, or Cdk12 siRNA
and left for 72 h. The total RNA from cells was purified using
miRNeasy minikit (Qiagen). A total of six samples were used:
three from the siRNA control and three from knockdown.
The high-resolution AltSplice expression microarrays were produced
by Affymetrix, and the cDNA samples were prepared
using GeneChip WT cDNA Synthesis and Amplification kit
(Affymetrix 900673), followed by GeneChip Hybridization,
Wash, and Stain kit (Affymetrix 900720) using the Affymetrix
Gene Chip Fluidics Station 450, and were scanned on an
Affymetrix GeneChip Scanner 3000 7G. The resulting .CEL files
were first analyzed with version 3 of ASPIRE software (Konig
et al. 2010). The dTrank threshold, which combines fold transcript
level change (dT) and Student’s t-test, was calculated by ASPIRE
software and was applied to rank the genes. The genes were
considered to be differentially expressed when dTrank was >1. To
validate the changes in the expression of the genes predicted by
the microarray, RT-qPCR was performed with selected downregulated
or up-regulated genes. Ingenuity program was used for
the gene ontology term and network modeling.
Correlation analysis
Correlation analysis was performed with all genes with dTrank
of >1 in CycK and Cdk12 microarrays (total of 1713 genes). The
Pearson correlation coefficient (r) was calculated among their dT
values in both data sets.
More Materials and Methods and a list of all primers are
available in the Supplemental Material.
Acknowledgments
We thank the members of Peterlin’s laboratory for their helpful
discussions. We also thank Kevin Gardner, Ann-Marie Geneviere,
Blazek et al.
2170 GENES & DEVELOPMENT
Cold Spring Harbor Laboratory Presson October 19, 2011 - Published bygenesdev.cshlp.orgDownloaded from
Jill Lahti, and Jonathon Pines for generously providing the
reagents; Melis Kayikci for analyzing the microarray data; Hana
Paculova for help with experiments; Audrey Low for helping
with the preparation of the figures; and Matjaz Barboric, Tomas
Brdicka, Jim Cleaver, Wendy Fantl, Tina Lenasi, and Yien-Ming
Kuo for their comments on the manuscript. The work was
supported by the following grants: from the NIH (IP50 GM082250,
R01 AI049104, and RO1 AI49104 ARRA supplement) to B.M.P.;
from the Czech Science Foundation (P305/11/1564), SoMoPro
(SRGA454), and the Ministry of Education, Youth, and Sports
(ME09047) to D.B.; and from the Czech Ministry of Agriculture
(MZE0002716202) and the Czech Science Foundation (301/09/
1832) to J.K..
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