deficiency in mammalian histone h2b ubiquitin ligase bre1 ......2012/02/21 · 1 deficiency in...
TRANSCRIPT
1
Deficiency in mammalian histone H2B ubiquitin ligase Bre1
(Rnf20/Rnf40) leads to replication stress and chromosomal
instability.
Sophia B. Chernikova1, Olga V. Razorenova1, John P. Higgins2, Brock J. Sishc3, Monica
Nicolau4, Jennifer A. Dorth1, Diana A. Chernikova5, Shirley Kwok2, James D. Brooks6,
Susan M. Bailey3, John C. Game1, J. Martin Brown1, 7.
1Department of Radiation Oncology, Stanford University, Stanford, CA 94305,
2Department of Pathology, Stanford University, Stanford, CA 94305,
3Department of Environmental and Radiological Health Sciences, Colorado State University, Fort Collins, CO
80523,
4Department of Mathematics, Stanford University, Stanford, CA 94305,
5National Center for Biotechnology Information, National Library of Medicine, NIH, Bethesda, MD 20894
6Department of Urology, Stanford University, Stanford, CA 94305
7Corresponding author. Email: [email protected], phone: (650) 723-5881, FAX: (650) 723-7382
Category: Tumor and Stem Cell Biology
Running title: Mammalian Bre1 maintains genomic stability
Keywords: H3K79, carcinogenesis, recombination, R-loops, seminoma
Figures: 6
Tables: 0
Word count: 5,094
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ABSTRACT
Mammalian Bre1 complexes (BRE1A/B (RNF20/40) in humans and Bre1a/b (Rnf20/40) in
mice) function similarly to their yeast homolog Bre1 as ubiquitin ligases in monoubiquitination
of histone H2B. This ubiquitination facilitates methylation of histone H3 at K4 and K79, and
accounts for the roles of Bre1 and its homologs in transcriptional regulation. Recent studies by
others suggested that Bre1 acts as a tumor suppressor, augmenting expression of select tumor
suppressor genes and suppressing select oncogenes. In this study we present an additional
mechanism of tumor suppression by Bre1 through maintenance of genomic stability. We track
the evolution of genomic instability in Bre1-deficient cells from replication-associated double-
strand breaks (DSBs) to specific genomic rearrangements that explain a rapid increase in DNA
content and trigger breakage-fusion-bridge cycles. We show that aberrant RNA-DNA structures
(R-loops) constitute a significant source of DSBs in Bre1-deficient cells. Combined with a
previously reported defect in homologous recombination, generation of R-loops is a likely
initiator of replication stress and genomic instability in Bre1-deficient cells. We propose that
genomic instability triggered by Bre1 deficiency may be an important early step that precedes
acquisition of an invasive phenotype, as we find decreased levels of BRE1A/B and dimethylated
H3K79 in testicular seminoma and in the premalignant lesion in situ carcinoma.
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INTRODUCTION
Histone modifications are central in regulating basic processes including transcription,
gene silencing, differentiation, cell cycle progression and DNA repair (1-4). Amongst
modifications of specific histone residues, much attention has focused in the last decade on a
distinctive pathway, mediated by the E3 ligase Bre1, which monoubiquitinates histone H2B at
lysine 123 (H2BK123) in budding yeast and at lysine 120 (H2BK120) in humans. H2B
monoubiquitination plays an important role in regulation of transcription, being a prerequisite for
normal levels of methylation of histone H3 residues K4 and K79 (5-8). In addition, studies in
Saccharomyces showed that mutants deficient in H2B monoubiquitination are radiation sensitive
and defective in recombinational repair, cell cycle checkpoints, gene silencing and meiosis (see
(9) for review). Several recent publications on the mammalian homolog of Bre1
(RNF20/RNF40) suggest that mammalian Bre1 complex plays similar roles. We have reported
that loss of Bre1 in mouse cells compromised homologous recombination (HR) repair, resulting
in reduced recruitment of Rad51 to sites of radiation-induced double-strand breaks (DSBs) and
increased sensitivity to ionizing radiation and DNA-crosslinking agents (10). A subsequent study
by others concluded that human Bre1 (RNF20, or BRE1A) was involved in chromatin
reorganization, facilitating HR protein access to damaged DNA (11). This role of Bre1 appears
to be important for response to DSBs in general, as a recent study has shown that BRE1
(RNF20/40)-mediated H2B ubiquitination is induced in an ATM-dependent manner and is
essential for timely repair of DSBs (12). These findings place Bre1 at the forefront of the DNA
damage response, which serves as a barrier against cancer formation (13), and reduction in Bre1
levels would be expected to compromise stability of the genome.
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Given that Bre1 affects multiple functions involved in genome maintenance it is plausible
that Bre1 homologs have a major tumor suppressing role in higher organisms. Consistent with
this, the RNF20 promoter has been found to be hypermethylated in breast cancers (14). Shema et
al (14) showed that BRE1A restrained transcription of several proto-oncogenes, including c-myc
and c-Fos, and augmented expression of tumor suppressor genes, notably p53, and further
concluded that selective transcriptional regulation of proto-oncogenes and tumor suppressors
constituted the basis for the tumor suppressing activity of BRE1 (RNF20). According to this
hypothesis, one consequence of the loss of Bre1 would be an increased proliferation resulting
from upregulation of growth-promoting oncogenes. However, this conclusion seems to be at
odds with our current observation of impaired growth upon depletion of Bre1 (RNF20/40). Also,
the transcriptional regulation of select oncogenes and tumor suppressors does not seem to be
universal across species, as in our study we detect no reduction of p53 mRNA levels after
knockdown of Bre1 (Rnf20 and/or Rnf40) in mouse cells. Thus, additional mechanisms seem to
be at play in Bre1-depleted cells driving their transition to a malignant phenotype.
Here, we show that Bre1 acts as an important suppressor of genomic instability. We used
RNAi to deplete Bre1 from mouse cells and then followed the evolution of genomic instability in
Bre1-deficient cells from early replication stress to specific genomic rearrangements, which in
turn triggered breakage-fusion-bridge cycles, known to accelerate genomic instability. We
demonstrate that R-loops, the RNA-DNA hybrids that result from the extended pairing of
nascent mRNA with the DNA template behind the elongating RNA polymerase II, are a
significant contributor to the genomic instability phenotype in the Bre1-depleted cells. While
Bre1 homologs are highly expressed in mouse and human testis, we find reduced BRE1A and
BRE1B levels in testicular cancer and the non-malignant lesion in situ carcinoma. Collectively,
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our data demonstrate that Bre1 deficiency promotes genomic instability, which may be an early
step in carcinogenesis preceding the acquisition of an invasive phenotype.
MATERIALS AND METHODS
Cell lines. Mouse fibrosarcoma RIF-1 cells were obtained from Dr. M. J. Dorie (J. M. Brown
laboratory, Stanford University) (15) and mouse embryonic stem (MES) cells were obtained
from Dr. J. P. Murnane (University of California San Francisco) (16) in 2005. As the cell lines
were obtained directly from the source of origin the authenticity of cell lines was not tested.
RNA interference. For downregulation of Bre1a and Bre1b by RNAi we used a lentiviral RNAi
system based on the BLOCK-iT system (Invitrogen) modified by Dr. E. Campeau (17).
Quantitative RT-PCR. Quantitative RT-PCR analysis was performed as described in (18).
Human or mouse TBP was used for normalization of cDNA input. The amplification was
specific as judged by melting temperature analysis. The experiments were performed in triplicate
and repeated at least twice. Primer sequences are available upon request.
Visualization of micronuclei and anaphase bridges. The assay was performed as described in
(19). In some cases Fig.2B, S3B and S3C), cytochalasin B was not added to allow better
visualization of anaphase bridges.
Rad51 foci detection. The assay was performed as described in (10).
RNase H1 transfection. GFP-RNase H1 plasmid was obtained from Dr. Olivier Sordet and
transfection performed using procedures similar to described in (20) and in Supplemental
Information.
More detailed Materials and Methods can be found in Supplemental Information.
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RESULTS
Bre1a/Bre1b-depletion impairs cell growth. To investigate the effects of loss of function of the
Bre1a/Bre1b complex in mouse cells we created lentivirus-based shRNA constructs targeting
either of the two subunits of the complex. As a control, we used an shRNA construct targeting
GFP. Expression of Bre1a and Bre1b shRNAs in mouse fibrosarcoma RIF-1 cells resulted in a
significant reduction of corresponding mRNA transcript levels after seven days of antibiotic
selection, as shown by quantitative real-time polymerase chain reaction (qRT-PCR) (Fig. 1A).
Protein levels of Bre1a and Bre1b were also reduced (Fig. 1B). Importantly, the levels of Bre1b
protein were decreased in the knockdown of Bre1a (Fig. 1B, top panel), while the levels of
Bre1b mRNA were not affected by the knockdown of Bre1a (Fig. 1A), suggesting that stability
of Bre1b depends on the formation of Bre1a/Bre1b heterodimer.
In agreement with previous studies (21), we show that downregulation of either Bre1a or
Bre1b leads to reduction in ubiquitination of H2B (Fig. 1C) and subsequent reduction of uH2B-
dependent methylation of H3K79 and H3K4 (Fig. 1B). The reduction in H3K4me3 was evident
in all knockdowns, but was substantially less pronounced than the reduction in H3K79me2 (Fig.
1B), which closely followed the reduction in Bre1a or Bre1b levels in all experiments.
Knockdown of Bre1a or Bre1b significantly impaired cell growth in different mouse cell
types including RIF-1, C3H 10T1/2 and MES cells (Fig. 1D and Fig. S1A), while cells grew well
after knockdown of either GFP, or tumor suppressor Rb (Fig. S1A). In order to monitor the
effects of disruption of the Bre1a/Bre1b complex over time, we created RIF-1-derived cell lines,
in which the knockdown of Bre1a or Bre1b could be initiated by addition of doxycycline. A
doxycycline-inducible GFP shRNA system was used as a control. A detailed description of the
Bre1 shRNA-inducible system is presented in the Supplemental Materials and Methods and in
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Fig. S1B and S1C. Note that the Bre1b shRNA inducible cell line has a slightly reduced level of
Bre1b even in the absence of doxycycline compared to the control cell line with GFP shRNA
(Fig. S1C, lanes 1 and 4, respectively), indicating that the doxycycline-inducible system is leaky
and a partial knockdown of Bre1b occurs even when doxycycline is not present. Similarly to
cells with the constitutive Bre1b knockdown (Fig. 1B), the inducible Bre1b shRNA cell line
demonstrated significant reduction in growth rate upon addition of doxycycline (Fig. 1E, panel
a). Consistent with the leaky phenotype, the growth rate of the inducible Bre1b shRNA cells was
slightly reduced even in the absence of doxycycline when compared to the control inducible GFP
shRNA cell line (Fig. 1E). We were able to obtain a more tightly regulated cell line by
modifying stringencies of antibiotics’ selection (shBre1b2 in Fig. 2A), which corrected the
problem of reduced cell growth in the absence of doxycycline. The availability of the leaky cell
line shBre1b1 with partial knockdown of Bre1b gave us an advantage to investigate weaker
and/or long-term effects of Bre1b depletion (shown further).
Apoptosis contributes to growth impairment in Bre1-depleted cells. To explain the reduction
in growth rate observed in the Bre1-deficient cells we investigated the possibility of increased
apoptosis. Apoptosis frequencies after Bre1b knockdown were increased by up to five-fold
compared to uninduced cells, and to both induced control and uninduced control cells (Fig. 1E
(panel b) and Fig. S2A). Consistent with the leaky phenotype, low level of apoptosis was present
in the shBre1b1 cells even in the absence of doxycycline (Fig. 1E, panel b). Apoptosis also
increased after depletion of Bre1 in MES cells (Fig. S2B). We conclude that the function of
Bre1 is critical for homeostasis and that apoptosis contributes to the reduced growth phenotype
in cell cultures with low levels of Bre1a and Bre1b.
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Bre1 loss compromises genomic integrity. Reduction in growth rate and increased apoptosis
also closely correlated with increased frequencies of micronuclei (MN) (Fig. 1E, F) supporting
the conclusion that the function of the Bre1a/Bre1b complex is essential for maintenance of
DNA integrity. Importantly, we observed a tight correlation between H2B ubiquitination and
MN levels (Fig. S3A and S3B).
Progressive genomic instability in Bre1-deficient cells. While significant loss of Bre1 was
catastrophic to the cells and lead to cessation of proliferation and cell death (Fig. 1E), more
subtle changes in Bre1b expression due to the partial knockdown in the uninduced shBre1b1 cell
line allowed cells to proliferate and manifest dramatic genomic instability associated with Bre1
deficiency after longer culture periods. When cultured for 3 weeks and longer under conditions
of partial knockdown (Fig. 2A), the shBre1b1 cells displayed MN frequencies comparable to that
observed on day 5 after complete Bre1b knockdown in the shBre1b2 cells (Fig. 2B).
Additionally, partial knockdown shBre1b1 cells at higher passages were characterized by the
appearance of anaphase bridges (Fig. 2C and Fig.S3C), many of which contained telomere
signal(s) (Fig. 2C), and by an increased DNA content (Fig. S4). To differentiate between fusions
resulting from loss of telomere end protection (22, 23) and fusions resulting from DSBs (24, 25),
we performed telomere FISH analysis on metaphase chromosomes from the Bre1-deficient and
control cell lines. Since no telomere signal was observed interstitially at fusion points,
chromosome analysis provided no evidence for the loss of telomere end capping function,
suggesting that anaphase bridges resulted from DSBs.
We also found that loss of Bre1 led to the formation of three main types of chromosome
aberrations, providing insight into mechanisms underlying the formation of telomere-positive
anaphase bridges and to the increased DNA content in the Bre1-knockdown cells. The
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chromosome aberrations predominantly found in the Bre1-knockdown cells were Robertsonian-
like translocations with amplified centromeric heterochromatin (RTCH) (Fig. 2D), aberrations
involving large regions of amplified centromeric heterochromatin at pericentric and interstitial
locations (ACH), and chromatid-type aberrations (CA) (Fig. 2E, F), aberrations associated with
instability. The amplified regions of centromeric heterochromatin observed in the metaphase
chromosomes of the Bre1-knockdown cell line were sometimes involved in dicentric
chromosomes, which lead to the generation of anaphase bridges. These bridges contained
telomere signals not associated with any telomere end protection defect and subsequent
chromosomal end fusions, but rather telomeres associated with chromosomes pulled into the
nucleoplasmic tube of an anaphase bridge, providing explanation for the presence of telomere
signals within the anaphase bridges of the Bre1-knockdown cell line.
The chromosomal instability phenotype initiated by partial Bre1b knockdown (shBre1b1
cells grown without doxycycline) for three weeks and longer was more dramatic than the one
initiated by complete short-term knockdown of Bre1b at early passage (Fig. 2E and F).
Additionally, cells at higher passages accumulated chromatid-type aberrations, which were not
present in the lower passage cells, as well as very complex aberrations, which appeared to result
from disruption/amplification/decompaction of centromeric heterochromatin (Fig. 2E and F).
Bre1 deficiency leads to replication stress. To elucidate how loss of Bre1 contributes to
increased DSBs and genomic rearrangements, we measured γH2AX levels after Bre1
knockdown. Consistent with previous reports (26, 27), we found two distinct subpopulations of
cells with elevated γH2AX. In the first, an increase in γH2AX signal was detected as a shift of
the main cell population in the FACS profile (Fig. 3A) that could be accounted for by both an
increase in the intensity of γH2AX foci and/or in the number of cells with γH2AX foci at sites of
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DSBs (Fig. 3B, 3C and 3D (panels a and b)). The other population of cells had dramatically
elevated pan-nuclear staining of γH2AX (Fig. 3D (panel c)), which identified apoptotic or pre-
apoptotic cells (26, 27). FACS analysis showed that while γH2AX signal (DSBs) was elevated
throughout the cell cycle (Fig. 3A), cells with pan-nuclear staining of γH2AX (apoptosis) were
restricted to S-phase cells after knockdown of Bre1 (Fig. 3E). To investigate how Bre1-deficient
cells dealt with replication stress we treated cells with 2mM hydroxyurea (HU), which stalls
replication forks by inhibiting ribonucleotide reductase essential for the synthesis of DNA
precursors. A prolonged (24 hr) treatment with 2 mM HU leads to replication fork collapse,
which requires HR for repair of resulting DSBs (28). We found that prolonged treatment of
control cells with 2mM HU led to a distribution of γH2AX signal reported previously (29), with
γH2AX-positive cells in three distinct clusters corresponding to G1, S, and G2/M cells (Fig. 3F).
Consistent with increased apoptosis during replication, we also found that loss of Bre1 resulted
in depletion of cells primarily from the cluster corresponding to the γH2AX-positive S-phase
cells (Fig. 3F). Replication stress and occurrence of chromatid aberrations are consistent with
defective homologous recombination in Bre1-depleted cells we reported previously, and we
show that spontaneous Rad51 foci formation was significantly affected by loss of Bre1 (Fig.
S4C).
Co-transcriptional formation of R-loops contributes to replication stress in Bre1-depleted
cells. To explore additional factors contributing to DSB formation in Bre1-deficient cells we
performed gene expression analysis on Bre1a and Bre1b knockdown cells. Since growth-
affecting changes were observed after longer times of Bre1 knockdown (Fig. 1D), we performed
microarray analysis after 7 days of RNAi. Consistent with both subunits of Bre1 complex being
required for the H2B ubiquitination, we observed a high correlation between expression profiles
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in the Bre1a and Bre1b knockdowns (Fig. S5). Despite the general role of Bre1 in transcriptional
regulation, loss of Bre1 downregulated a distinct set of genes and upregulated another without
affecting majority of genes (Tables S1 and S2). Further analysis revealed that the group of genes
upregulated after Bre1 knockdown was dramatically enriched for genes involved in RNA
processing/splicing, showing a response typical of the one to co-transcriptional formation of
recombinogenic RNA-DNA hybrids (R-loops) (Fig. 4A). R-loops often arise during perturbed
mRNA processing as a result of the extended pairing of nascent messenger RNA (mRNA) with
the transcribed DNA strand behind the elongating Pol II, creating DSBs and leading to increased
recombination and genomic instability (30, 31). In a genome-wide siRNA screen for genes
whose deregulation leads to elevated levels of γH2AX (32), the mRNA processing module
represented the most significantly enriched category of genes, suggesting that abnormal mRNA
processing was the most common and direct source of genomic instability. To determine if R-
loop formation contributed to increased formation of DSBs in Bre1-deficient cells, we tested
whether overexpression of RNase H1, which degrades RNA in R-loops, would reduce levels of
γ-H2AX. Fig. 4B shows that overexpression of RNase H1 decreased the number of cells with γ-
H2AX foci in the Bre1-depleted cells, while it did not have an effect on cells in which Bre1
RNAi was not induced. In addition, RNase H1 expression reduced the average number of γ-
H2AX foci per cell (Fig. 4C, D). These data suggest a mechanism for the generation of DSBs
through the R-loop formation in the Bre1-depleted cells.
We also found that the gene subset upregulated after depletion of Bre1 was strongly
enriched for histone genes from the compact chromatin cluster on 13qA3.1 (p<0.0001) (Table
S3). Normally, replication-dependent histone mRNAs do not have poly(A) tails, but since we
relied on poly(A)-dependent amplification of mRNA for hybridization to our microarray chip,
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overrepresentation of histone genes from the 13qA3.1 cluster is a compelling evidence for the
presence of mRNA that has been polyadenylated. These data confirm the previous finding by
Pirngruber et al (33) showing that BRE1(RNF20/40)-dependent H2Bub1 acts as a marker for
correct recognition of the histone mRNA 3’-end cleavage site. Due to the massive synthesis of
histones during replication, defective processing of replication-associated histone mRNA in
BRE1(RNF20/40)-deficient cells may be the main contributor to the formation of DSBs and
hence replication stress.
BRE1A/B and H3K79me2 levels are lower in human seminoma than in normal testis. We
hypothesized that tissues with high expression of Bre1 homologs would depend on the function
of the complex for maintenance of genomic integrity. Western blot analysis showed that among
various mouse organs including brain, heart, kidney, liver, lung, muscle, skin, testis, spleen, and
bladder, expression of Bre1b was highest in testis and spleen (Fig. 5A), suggesting that the
Bre1a/Bre1b complex may play essential roles in these organs. We observed strong Bre1b
staining in spermatogonia (Fig. 5B, left panel). We also found a strong signal for H3K79me2 and
H3K4me3, the two modifications affected by ubiquitination of H2B. H3K79me2 was highest in
middle meiosis (Fig. 5B), while H3K4me3 was strongest at the beginning and in the middle of
meiosis (Fig. 5B), consistent with the roles these modifications play during yeast meiosis (34-
36). Importantly, the staining for total histone H3 was uniform throughout the sections (Fig. 5B).
Having demonstrated the importance of Bre1 homologs to maintenance of genomic
stability, we hypothesized that suboptimal expression of Bre1 homologs in testis might be
associated with testicular cancer. To test this supposition, we analyzed BRE1A (RNF20) and
BRE1B (RNF40) expression data available from public databases (37, 38) and found
significantly lower levels of BRE1A mRNA in human seminoma compared to normal testicular
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tissue (Fig. S6). Seminoma is a type of testicular germ cell cancer that is the most common solid
tumor in otherwise healthy men aged 15-35 years. It is widely accepted that seminoma arises
from the precursor lesion carcinoma in situ (CIS, also known as intratubular germ cell neoplasia)
and can grow rapidly and metastasize (39). Seminomas are characterized by high levels of
genome instability and gains of chromosome 12p, which are not present in CIS. Although
formerly often lethal, seminoma is highly sensitive to ionizing radiation therapy and
chemotherapy and most are now cured.
To assess BRE1A/B protein levels we stained tissue microarrays containing tissue
sections from normal testis and seminoma. Levels of both BRE1B protein and BRE1A/B-
dependent dimethylation of H3K79 and trimethylation of H3K4 were significantly lower in
seminoma compared to normal tissue (Fig. 5C). Further analysis of publicly available gene
expression arrays (37, 38) demonstrated that among different testicular cancers seminoma
displayed the lowest levels of BRE1A (Fig. S6), and by staining the testis tissue microarrays we
found that amongst testicular cancers, H3K79me2 was also lowest in seminoma (Fig. 5D, S7 and
Table S4). Importantly, staining of BRE1A and H3K79me2 in CIS was also lower than in
normal tissue from the same patients (Fig. 5E), suggesting that deficiency in BRE1A/B and in
methylation of H3K4 and H3K79 occurs before acquisition of an invasive phenotype.
DISCUSSION
We demonstrate that Bre1 (human BRE1A/B (RNF20/40) and mouse Bre1a/b
(Rnf20/40)) acts as an important suppressor of chromosomal instability (CIN). This finding
complements the previously suggested mechanism for Bre1 tumor suppression through
transcriptional regulation of select oncogenes and tumor suppressor genes (14). The types of
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chromosomal aberrations we observed after knockdown of Bre1 indicated that a defect in
homologous recombination (HR) contributes to CIN in the Bre1-deficient cells (40, 41). This
conclusion is consistent with our previous observation that reduced monoubiquitination of H2B
in Saccharomyces bre1null mutants and in mouse cells leads to defective recombinational repair
of double-strand breaks (DSBs) (10, 42). We show that R-loops, the RNA-DNA hybrid
structures usually formed behind elongating RNA polymerase II when mRNA processing is
disturbed (30-32), constitute a significant source of DSBs in Bre1-deficient cells. Overall, our
data support a model in which reduction in Bre1-dependent ubiquitination of histone H2B
increases genomic instability through increased generation of DSBs resulting from a defect in
correct processing of canonical histone mRNA and through inhibition of HR needed for DSB
resolution (Fig. 6).
It should be noted that while we interpret the observed Bre1 knockdown phenotype as
arising from an impact on the well-known role of Bre1 in H2B ubiquitination, it is formally
possible that additional targets for the Bre1 ubiquitin ligase exist, which could contribute to the
knockdown phenotype. Testing this with an H2B K120 substitution mutant is not
straightforward in mammals, because their genomes contain at least 17 H2B genes (43).
However studies using overexpressed ectopic H2BK120R mutant gene in cells with wild-type
chromosomal copies of H2B have shown that effects of overexpression of H2BK120R mimic
those of loss of BRE1, including the effect on the formation of γH2AX foci, which we used in
our study to detect DSBs (11, 12). Also, in Saccharomyces, where chromosomal copies of the
H2B gene number only two and can be deleted, H2BK123R mutants do mimic the bre1 deletion
phenotype (reviewed in (9)), implying that the phenotypes are likely to be conferred through
effects on the H2B target. Questions remain, however, as to whether the phenotypes we
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observed after loss of Bre1 are mediated via the well-known impact of H2B ubiquitination on
methylation of histone H3, and specifically whether H3K4me3 and or H3K79me2 are involved.
Detailed investigation of the role that H3K4 methylation plays in different Bre1 phenotypes is
hampered by the fact that there are many SET1 homologues in mammals, some of which may
not be uH2B-dependent (reviewed in (9)). Also, knocking down the H3K79 methyltransferase
Dot1 would completely eliminate methylation of H3K79, while loss of Bre1-mediated H2B
ubiquitination only eliminates the higher states of methylation of H3K79, so additional
phenotypes may be conferred by the Dot1 knockdown beyond those mediated by the impact of
uH2B on H3K79. Although the effects of Dot1 knockdown in mice (44, 45) paralleled the
impaired cell growth, increased ploidy and centromeric abnormalities we observed after Bre1
knockdown, specific clarification of these issues requires further study.
A crucial unresolved question in cancer biology is whether CIN represents an early event
and is therefore a driving force of carcinogenesis. Our results support models in which CIN
drives tumorigenesis rather than being its consequence. We provide a comprehensive
demonstration of a stepwise accumulation of chromosomal changes that start with
downregulation of Bre1. Consistent with the CIN model, we also show that BRE1A/B deficiency
accompanies early steps of testicular cancer development.
Correct processing of replication-associated histone mRNA is particularly relevant in
connection to testicular carcinogenesis. In the testis a massive synthesis of histone variants
accompanies dramatic reorganization of the genome, during which the majority of the histones
are replaced by transition proteins and protamines. Lack of Bre1 leading to abnormal presence of
polyadenylated histone mRNAs, which are not rapidly degraded at the end of S-phase, could
interfere with proper incorporation of the variant histones into chromatin, and lead to testicular
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16
dysgenesis. We demonstrated that low levels of BRE1A and BRE1B, and low H3K79me2 are
found in intratubular cell neoplasia (CIS), as well as in seminomas. Seminomas show
chromosomal changes similar to those found in CIS and therefore are considered a default
pathway from the CIS precursor lesion to invasive testicular germ cell tumors (TGCT) (39). Like
all TGCTs, seminomas are characterized by high levels of CIN and aneuploidy, and a gain of
chromosome 12p (46). Gain of 12p is not present in CIS, and so it is believed that
overexpression of gene(s) on 12p is pertinent to invasive growth. Thus, we can speculate that
genomic instability initiated by the abnormal downregulation of BRE1A/BRE1B function may
facilitate gain of 12p and thus constitute one possible route to seminoma. Seminomas
recapitulate the undifferentiated and pluripotent primordial germ cell (PGC) phenotype, and are
thought to arise when a block in maturation of PGCs prevents them from forming
spermatogonia. Downregulation of BRE1A/BRE1B may be associated with such a maturation
block (39), and thus may lead to infertility. In fact, men from families with fertility problems are
known to have an elevated risk of testicular cancers, especially seminoma (47-49). Hence,
deficiency in BRE1A/B may be among etiological factors in common for both infertility and
testicular cancer. In addition, mutation of BRE1 (RNF20) has been found among other CIN
genes mutated in colorectal cancers (50) suggestive of a more general role of Bre1 in CIN.
In conclusion, we propose that the mammalian homologs of the yeast BRE1 gene serve as
tumor suppressors by preventing replication stress and chromosomal instability that arise from
DSBs associated with incorrect processing of replication-associated histone mRNAs and
inefficient HR. In addition to clarifying basic cellular mechanisms, the identification of Bre1 as a
CIN gene may have specific relevance for estimation of risk and diagnosis of testicular cancer.
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17
ACKNOWLEDGEMENTS
We are grateful to Xin Huang (University of Pittsburgh School of Medicine), Elena Seraia
(Stanford Functional Genomics Facility), and Janos Demeter (Stanford Microarray Database) for
help with image processing and microarray analysis. The work was supported by grant CA67166
awarded to JMB by the National Cancer Institute.
REFERENCES:
1. Osley MA, Fleming AB, & Kao CF (2006) Histone ubiquitylation and the regulation of transcription. Results and problems in cell differentiation 41:47-75 .
2. Shilatifard A (2006) Chromatin modifications by methylation and ubiquitination: implications in the regulation of gene expression. Annual review of biochemistry 75:243-269.
3. Shilatifard A (2008) Molecular implementation and physiological roles for histone H3 lysine 4 (H3K4) methylation. Current opinion in cell biology 20(3):341-348 .
4. Escargueil AE, Soares DG, Salvador M, Larsen AK, & Henriques JA (2008) What histone code for DNA repair? Mutation research 658(3):259-270 .
5. Sun ZW & Allis CD (2002) Ubiquitination of histone H2B regulates H3 methylation and gene silencing in yeast. Nature 418(6893):104-108.
6. Ng HH, Xu RM, Zhang Y, & Struhl K (2002) Ubiquitination of histone H2B by Rad6 is required for efficient Dot1-mediated methylation of histone H3 lysine 79. J Biol Chem 277(38):34655-34657.
7. Briggs SD, et al. (2002) Gene silencing: trans-histone regulatory pathway in chromatin. Nature 418(6897):498.
8. Dover J, et al. (2002) Methylation of histone H3 by COMPASS requires ubiquitination of histone H2B by Rad6. J Biol Chem 277(32):28368-28371.
9. Game JC & Chernikova SB (2009) The role of RAD6 in recombinational repair, checkpoints and meiosis via histone modification. DNA repair 8(4):470-482 .
10. Chernikova SB, Dorth JA, Razorenova OV, Game JC, & Brown JM (Deficiency in Bre1 Impairs Homologous Recombination Repair and Cell Cycle Checkpoint Response to Radiation Damage in Mammalian Cells. Radiation research 174(5) .
11. Nakamura K, et al. (Regulation of homologous recombination by RNF20-dependent H2B ubiquitination. Molecular cell 41(5):515-528 .
12. Moyal L, et al. (Requirement of ATM-dependent monoubiquitylation of histone H2B for timely repair of DNA double-strand breaks. Molecular cell 41(5):529-542 .
13. Halazonetis TD, Gorgoulis VG, & Bartek J (2008) An oncogene-induced DNA damage model for cancer development. Science (New York, N.Y 319(5868):1352-1355 .
14. Shema E, et al. (2008) The histone H2B-specific ubiquitin ligase RNF20/hBRE1 acts as a putative tumor suppressor through selective regulation of gene expression. Genes & development 22(19):2664-2676 .
Research. on August 20, 2021. © 2012 American Association for Cancercancerres.aacrjournals.org Downloaded from
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Author Manuscript Published OnlineFirst on February 21, 2012; DOI: 10.1158/0008-5472.CAN-11-2209
18
15. Twentyman PR, et al. (1980) A new mouse tumor model system (RIF-1) for comparison of end-point studies. J. Natl. Cancer Inst. 64(3):595-604.
16. Pedram M, et al. (2006) Telomere position effect and silencing of transgenes near telomeres in the mouse. Molecular and cellular biology 26(5):1865-1878 .
17. Campeau E, et al. (2009) A versatile viral system for expression and depletion of proteins in mammalian cells. PloS one 4(8):e6529 .
18. Razorenova OV, et al. (VHL loss in renal cell carcinoma leads to up-regulation of CUB domain-containing protein 1 to stimulate PKC{delta}-driven migration. Proceedings of the National Academy of Sciences of the United States of America .
19. Chernikova SB, Wells RL, & Elkind MM (1999) Wortmannin sensitizes mammalian cells to radiation by inhibiting the DNA-dependent protein kinase-mediated rejoining of double-strand breaks. Radiation research 151(2):159-166 .
20. Sordet O, et al. (2009) Ataxia telangiectasia mutated activation by transcription- and topoisomerase I-induced DNA double-strand breaks. EMBO reports 10(8):887-893 .
21. Kim J, et al. (2009) RAD6-Mediated transcription-coupled H2B ubiquitylation directly stimulates H3K4 methylation in human cells. Cell 137(3):459-471 .
22. Crabbe L, Jauch A, Naeger CM, Holtgreve-Grez H, & Karlseder J (2007) Telomere dysfunction as a cause of genomic instability in Werner syndrome. Proceedings of the National Academy of Sciences of the United States of America 104(7):2205-2210 .
23. van Steensel B, Smogorzewska A, & de Lange T (1998) TRF2 protects human telomeres from end-to-end fusions. Cell 92(3):401-413 .
24. Bailey SM, et al. (1999) DNA double-strand break repair proteins are required to cap the ends of mammalian chromosomes. Proceedings of the National Academy of Sciences of the United States of America 96(26):14899-14904 .
25. Bailey SM, Cornforth MN, Kurimasa A, Chen DJ, & Goodwin EH (2001) Strand-specific postreplicative processing of mammalian telomeres. Science (New York, N.Y 293(5539):2462-2465 .
26. de Feraudy S, Revet I, Bezrookove V, Feeney L, & Cleaver JE (A minority of foci or pan-nuclear apoptotic staining of gammaH2AX in the S phase after UV damage contain DNA double-strand breaks. Proceedings of the National Academy of Sciences of the United States of America 107(15):6870-6875 .
27. Kurose A, et al. (2006) Effects of hydroxyurea and aphidicolin on phosphorylation of ataxia telangiectasia mutated on Ser 1981 and histone H2AX on Ser 139 in relation to cell cycle phase and induction of apoptosis. Cytometry A 69(4):212-221 .
28. Petermann E, Orta ML, Issaeva N, Schultz N, & Helleday T (Hydroxyurea-stalled replication forks become progressively inactivated and require two different RAD51-mediated pathways for restart and repair. Molecular cell 37(4):492-502 .
29. de Feraudy S, et al. (2007) Pol eta is required for DNA replication during nucleotide deprivation by hydroxyurea. Oncogene 26(39):5713-5721 .
30. Li X & Manley JL (2006) Cotranscriptional processes and their influence on genome stability. Genes & development 20(14):1838-1847 .
31. Aguilera A & Gomez-Gonzalez B (2008) Genome instability: a mechanistic view of its causes and consequences. Nat Rev Genet 9(3):204-217 .
32. Paulsen RD, et al. (2009) A genome-wide siRNA screen reveals diverse cellular processes and pathways that mediate genome stability. Molecular cell 35(2):228-239 .
Research. on August 20, 2021. © 2012 American Association for Cancercancerres.aacrjournals.org Downloaded from
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Author Manuscript Published OnlineFirst on February 21, 2012; DOI: 10.1158/0008-5472.CAN-11-2209
19
33. Pirngruber J, et al. (2009) CDK9 directs H2B monoubiquitination and controls replication-dependent histone mRNA 3'-end processing. EMBO reports 10(8):894-900 .
34. San-Segundo PA & Roeder GS (2000) Role for the silencing protein Dot1 in meiotic checkpoint control. Molecular biology of the cell 11(10):3601-3615.
35. Sollier J, et al. (2004) Set1 is required for meiotic S-phase onset, double-strand break formation and middle gene expression. The EMBO journal 23(9):1957-1967.
36. Yamashita K, Shinohara M, & Shinohara A (2004) Rad6-Bre1-mediated histone H2B ubiquitylation modulates the formation of double-strand breaks during meiosis. Proceedings of the National Academy of Sciences of the United States of America 101(31):11380-11385.
37. Korkola JE, et al. (2006) Down-regulation of stem cell genes, including those in a 200-kb gene cluster at 12p13.31, is associated with in vivo differentiation of human male germ cell tumors. Cancer research 66(2):820-827 .
38. Sperger JM, et al. (2003) Gene expression patterns in human embryonic stem cells and human pluripotent germ cell tumors. Proceedings of the National Academy of Sciences of the United States of America 100(23):13350-13355 .
39. Looijenga LH, Gillis AJ, Stoop HJ, Hersmus R, & Oosterhuis JW (2007) Chromosomes and expression in human testicular germ-cell tumors: insight into their cell of origin and pathogenesis. Annals of the New York Academy of Sciences 1120:187-214 .
40. Tinline-Purvis H, et al. (2009) Failed gene conversion leads to extensive end processing and chromosomal rearrangements in fission yeast. The EMBO journal 28(21):3400-3412.
41. Nakamura K, et al. (2008) Rad51 suppresses gross chromosomal rearrangement at centromere in Schizosaccharomyces pombe. The EMBO journal 27(22):3036-3046 .
42. Game JC, Williamson MS, Spicakova T, & Brown JM (2006) The RAD6/BRE1 histone modification pathway in Saccharomyces confers radiation resistance through a RAD51-dependent process that is independent of RAD18. Genetics 173(4):1951-1968.
43. Marzluff WF, Gongidi P, Woods KR, Jin J, & Maltais LJ (2002) The human and mouse replication-dependent histone genes. Genomics 80(5):487-498 .
44. Jones B, et al. (2008) The histone H3K79 methyltransferase Dot1L is essential for mammalian development and heterochromatin structure. PLoS Genet 4(9):e1000190.
45. Barry ER, et al. (2009) ES cell cycle progression and differentiation require the action of the histone methyltransferase Dot1L. Stem Cells 27(7):1538-1547 .
46. Looijenga LH & Oosterhuis JW (2002) Pathobiology of testicular germ cell tumors: views and news. Analytical and quantitative cytology and histology / the International Academy of Cytology [and] American Society of Cytology 24(5):263-279 .
47. Walsh TJ, Croughan MS, Schembri M, Chan JM, & Turek PJ (2009) Increased risk of testicular germ cell cancer among infertile men. Archives of internal medicine 169(4):351-356 .
48. Jacobsen R, et al. (2000) Risk of testicular cancer in men with abnormal semen characteristics: cohort study. BMJ (Clinical research ed 321(7264):789-792 .
49. Raman JD, Nobert CF, & Goldstein M (2005) Increased incidence of testicular cancer in men presenting with infertility and abnormal semen analysis. The Journal of urology 174(5):1819-1822; discussion 1822 .
Research. on August 20, 2021. © 2012 American Association for Cancercancerres.aacrjournals.org Downloaded from
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Author Manuscript Published OnlineFirst on February 21, 2012; DOI: 10.1158/0008-5472.CAN-11-2209
20
50. Barber TD, et al. (2008) Chromatid cohesion defects may underlie chromosome instability in human colorectal cancers. Proceedings of the National Academy of Sciences of the United States of America 105(9):3443-3448 .
FIGURE LEGENDS
Fig 1. Depletion of Bre1a or Bre1b impairs cell growth. (A) qRT-PCR showing reduced
mRNA levels of Bre1a or Bre1b in the knockdowns of Bre1a or Bre1b, respectively. (B)
Knockdown of Bre1a or Bre1b results in decrease in H3K4me3 and H3K79me2. (C) Depletion
of Bre1a or Bre1b decreases monoubiquitination of histone H2B. (D) Depletion of Bre1a and
Bre1b results in slow growth of RIF-1 cells. (E) Conditional expression of Bre1b shRNA in
mouse RIF-1 cells results in (a) slow growth, (b) increased apoptosis (as measured by annexin-
FITC staining), and (c) increased induction of micronuclei. Error bars are standard errors of the
mean. Experiments were repeated more than three times. (+) and (-) show presence or absence of
doxycycline in the medium. (F) Representative microphotograph showing micronuclei in RIF-1
cells after knockdown of Bre1a (panel a) or Bre1b (panel b).
Fig. 2. Evidence for ongoing genomic instability in Bre1-depleted cells. (A, B) Bre1b
depletion leads to increased frequency of micronucleus (MN) formation. Note different levels of
Bre1b, uH2B and H3K79me2 in two doxycycline-inducible cell lines, shBre1b1 and shBre12.
shBre1b1, a Bre1b shRNA doxycycline-inducible cell line shows a partial Bre1b knockdown
even in the absence of doxycycline. shBre1b2 is derived from early passage shBre1b1 cells line
by increasing the stringency of blasticidin selection (from 5 ug/ml to 10 ug/ml). The (+) and (-)
show presence or absence of doxycycline in the medium. (C) Anaphase bridges in Bre1b-
depleted cells contain telomeric sequences (arrows). (D) Bre1b-depleted cells are characterized
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21
by appearance of aberrations with amplified centromeric heterochromatin (stained by DAPI).
Shown is a Robertsonian-like Translocation with Amplified Centromeric Heterochromatin
(RTCH). (E) Long-term partial knockdown of Bre1b in the shBre1b1 cell line leads to multiple
chromosomal abnormalities, which increase in frequency and complexity under. Abbreviations:
CA – chromatid-type aberrations; RT – classic Robertsonian Translocations; RTCH –
Robertsonian-like Translocations involving Centromeric Heterochromatin; ACH - complex
aberrations involving multiple amplification of heterochromatin. Note that RTCH frequencies
are much higher in shBre1b1 cells at later passages. Significance analysis is provided in Table
S5. (F) Representative examples of observed chromosome aberrations: panels a and b –
chromatid-type (CA) aberrations, panels c, d and e – complex aberrations involving
heterochromatin (ACH). Arrowhead points to RTCH in (F) panel e.
Fig. 3. Bre1 knockdown leads to increase in double-strand breaks and to replication stress.
(A) FACS analysis shows that loss of Bre1b elevates γH2AX. A representative experiment is
shown. Increase in γH2AX signal results from an increase in intensity of γH2AX foci (B) and
from increased number of cells counted as γH2AX-positive (C). (D). Panel (a) shows cells with
low γH2AX signal and panels (b) and (c) show cells with more intense γH2AX foci after Bre1
knockdown. Panel (c) shows a pre-apoptotic cell with very bright γH2AX signal. (E) Bre1-
deficient cells show more cells with high γH2AX signal specifically in S-phase. For quantitation
purposes FACS settings were adjusted so that the cells were in the same location on the plots.
Note that in the corresponding Western blot % of γH2AX –positive cells is inversely
proportional the levels of uH2B in the Bre1a or Bre1b knockdowns. (F) 24 hour treatment with
hydroxyurea leads to different distribution of γH2AX signal in the Bre1 knockdown cells. (+)
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22
and (-) show presence or absence of doxycycline in medium. Experiments were repeated more
than three times.
Fig. 4. Defect in mRNA processing contributes to double-strand break formation in Bre1-
depleted cells. (A) Classification enrichment in gene sets down-regulated and upregulated after
Bre1 knockdown. Pathway/Process enrichment analysis was performed using DAVID
bioinformatics database (Supplemental Methods). The statistical threshold was applied for –
log(p=0.05) shown in dashed line. (B) Overexpression of RNase H1 in the shBre1b cells results
in reduction of cells with γH2AX foci. (C) Overexpression of RNase H1 in Bre1b-depleted cells
reduces number of γH2AX foci per cell. (D) A representative photograph of an experiment from
(C). The cells were transfected with GFP-RNase H1 or GFP-Nuc (control) on day 3 after the
induction of knockdown and were analysed on day 6 after the induction of knockdown. In (B)
the GFP-expressing cells were FACS sorted on day 5 after the induction of knockdown and
plated for analysis to be performed next day. The FACS sorting experiments were performed
twice. In (C) cells transfected with GFP-RNase H1 were analysed without prior sorting.
Asterisks denote a significant difference from cells with Bre1 depletion, but not expressing
RNase H1 (*** correspond to p<0.001, t-test).
Fig. 5. Loss of BRE1A/B is associated with development of seminoma. (A) Bre1b is highly
expressed in mouse testis. (B) Bre1b levels are the highest in meiotic prophase cells (depicted by
arrows). Staining of Bre1b, H3K79me2, H3K4me3 and of total histone H3 is shown. (C) Protein
levels of BRE1B, H3K79me and H3K4me3 are lower in seminoma compared to normal testis.
(D) H3K79me2 is significantly lower in seminoma compared to normal testis and non-
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23
seminomatous cancers of testis. (E) Representative pictures show that loss of Bre1A/B and
H3K79me2 occur early in seminoma development. Normal testis, CIS – carcinoma in situ, and
seminoma sections are taken from same patients. Arrows point to CIS. Asterisks denote a
significant difference from normal tissue section (*** correspond to p<0.001, and ** correspond
to p<0.01, t-test).
Fig. 6. A model depicting sources of genomic instability in Bre1-deficient cells. Loss of Bre1
leads to increase in DSBs due to defects in homologous recombination and in the processing of
canonical histone mRNAs. Incorrect processing of replication-dependent histone mRNA 3’-ends
facilitates co-transcriptional formation of R-loops, which block replication forks and result in
DSB.
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Chernikova_Fig_1
0.5
1
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Chernikova_Fig_2
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Chernikova_Fig_3
B)A)
ount
s shGFP(-) shGFP(+)
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C)
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shGFP shBre1b shBre1a- + - + - +
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Chernikova_Fig_4
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Chernikova_Fig_5
A)Bre1b
Gapdh
B)Bre1b H3K79me2 H3K4me3 panH3
ning
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Chernikova_Fig_6
Loss of BRE1 (RNF20/40)
Reduced H2B monoubiquitination
Homologous Defect in histone Homologous recombination
defect
mRNA processing
R-loops
Replication-associated DSBs
Cell death
Genomic instability
CancerCell death Cancer
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Published OnlineFirst February 21, 2012.Cancer Res Sophia B. Chernikova, Olga V. Razorenova, John P. Higgins, et al. instability(Rnf20/Rnf40) leads to replication stress and chromosomal Deficiency in mammalian histone H2B ubiquitin ligase Bre1
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