ELOWE, Sabine Regulation of chromosome cohesion and genome stability by a novel Histone modification Proposal
1
IMPACT ON HEALTH: 1 in 2 Canadians will be afflicted by cancer. At present, although microtubule-targeting agents are some of the most prevalent anti-cancer drugs in the clinic; they do not distinguish between cancerous and normal cells and many patients experience severe side effects. Thus, there is an urgent need for new strategies to more effectively target heterogenic tumor populations with fewer toxic side effects. The next generation of anti-tumor agents will be more successful if they exploit tumor-specific vulnerabilities including aneuploidy, genomic instability, and disrupted checkpoints. Such agents could overwhelm cancer cells with intolerable levels of genomic instability that are incompatible with cell survival, leading to mitotic catastrophe and cell death, while non-malignant, genomically stable cells would be spared, thus providing an enhanced therapeutic index. In line with this, emerging studies show that a number of mitotic kinase inhibitors have found a measure of success in both mono and combinatorial therapy1. Targeting of a number of these kinases has been shown to sensitize cancer cells to therapeutic doses of taxanes and result in massive aneuploidy and cell death in a cancer-cell dependent manner2, 3.
A promising target being hotly pursued (in Canada and elsewhere), is the mitosis specific kinase
Haspin, one of the most upstream kinases in mitotic signaling networks that regulates chromosome
cohesion, metaphase alignment, and cell-cycle progression. Of note, Haspin is an atypical eukaryotic
protein kinase diverging significantly from others, and as such its inhibition is expected to have fewer
side and off-target effects, making it a particularly attractive target. A number of Haspin kinase
inhibitors have been developed and are currently in pre-clinical trials4, 5. Haspin protects sister
chromatid centromere cohesion during early mitosis via a completely uncharacterized mechanism that
is independent of the canonical pathway involving the Shugoshin protein (Sgo1, see below). Haspinmediated
cohesion is vital to maintaining duplicated chromosomes together in early mitosis until their
ultimate separation into each of the nascent daughter cells upon mitotic exit. This insures accurate
division of chromosomes and prevents pathogenic aneuploidy; hence, Haspin is essential for genome
integrity. We have now identified a novel Haspin target that regulates centromere cohesion, Histone
H2B T119 (H2BT119p). The proposal aims to determine the molecular mechanism through which
H2BT119p mediates sister-chromatid cohesion and the role of this pathway in cancer.
This project will contribute to the progress of biomedical knowledge in at least 2 ways: by advancing
understanding of Haspin biology through identification of the scope of its activity (aims 1,2), and by
providing invaluable information on its function and activity in normal cells and various cancers and
cancer sub-types (aims 2,3), leading potentially to identification of novel therapeutic opportunities or
disease markers. In particular, we anticipate that this project will reveal a novel pathway essential for
maintaining centromere cohesion and thus genome integrity. Understanding Haspin signaling is
critical for interpreting and effectively assessing drug efficacy during current and future
development and preclinical efforts. Our H2BT119p antibody (see below), an exquisite reader of
Haspin activity, will be instrumental in this context. Importantly, our results may be relevant to
therapeutic approaches targeting the cancer epigenome. Histone H2B ubiquitination at K120
(H2BK120ub), directly after T119 functions in histone cross-talk, transcription and DNA damage
repair, and is lost in many advanced cancers6. Moreover, Haspin cooperates with the H2AT120p-Sgo1
pathway in protection of cohesion during mitosis, although the mechanism remains unclear. Crosstalk
between these marks is unknown and will be explored. As with Haspin, inhibitors to H2BK120ub and
H2AT120p modifiers (writers and erasers) have been developed and are in pre-clinical stages of
evaluation. Our results will directly impact interpretation of their efficacy, putting it in a more
complete context. Moreover, although Haspin has been reported to be mutated in a number of cancers,
critically lacking is data on how these mutations affect Haspin activity. Scoring Haspin catalytic
activity in cancer and chromosomally stable and instable cancer lines will verify its contribution to
ELOWE, Sabine Regulation of chromosome cohesion and genome stability by a novel Histone modification Proposal
2
genome stability, making informed decisions on its therapeutic potential in various cancers more
feasible, and confirming utility of H2BT119p as a potential cancer biomarker.
INTRODUCTION and RATIONALE: Proper cohesion of duplicated chromosomes during mitosis
protects against aneuploidy and genome instability, a hallmark of cancer. Sister chromatid cohesion is
deposited after DNA duplication in S-phase until mitosis, when in vertebrates it is lost in 2 stages. In
prophase and prometaphase, cohesion is released from chromosome arms but is protected at
centromeres by Shugoshin (Sgo1) until anaphase. The current model states that Haspin activity is
required for enrichment of the chromosomal passenger complex (CPC) at centromeres which maintains
Sgo1 there during early mitosis7; Haspin phosphorylates Histone 3 at Threonine 3 (H3T3p) to recruit
the CPC which cooperates with Histone H2AT120p generated by the BUB1 kinase to establish CPC
and Sgo1 localization, and thus centromere cohesion (Fig. 1)8. This model however, does not account
for all the functions of Haspin; direct interaction between Haspin and cohesion complex subunits has
also recently been implicated in cohesion protection9, 10, but the role of substrates other than H3T3 has
not been explored. In particular evidence suggests that Haspin regulates sister chromatid cohesion,
independent of Sgo111, 12 (Fig. 2). After Haspin depletion, cohesion is lost and separation of sister
chromatids occurs, although Sgo1 remains at the centromere. Because Sgo1 RNAi also causes
separation of sister chromatids, this indicates that both Haspin and Sgo1 are independently required to
maintain centromere cohesion. Depletion of Aurora B (the catalytic subunit of the CPC), on the other
hand, causes delocalization of Sgo1 from the centromere to chromosome arms. This results in ectopic
protection of cohesion on the arms, accompanied by loosening of centromeric cohesion and loss of the
primary constriction. Co-depletion of Aurora B with Sgo1 but not its co-depletion with Haspin rescues
this phenotype indicating that the increased arm cohesion seen after Aurora B RNAi is dependent on
Sgo1 and independent of Haspin (Fig. 2) 11, 12. Collectively, these observations strongly suggest that
Haspin can regulate centromere cohesion independently of Sgo1.
To identify other potential Haspin substrates that may regulate centromere cohesion, we
reasoned that other histone targets, in addition to H3T3 may exist. Indeed, post-translational
modifications (PTMs) of core histones influence chromatin landscapes to regulate numerous cellular
processes, and are often deregulated in cancer. While exploring potential novel substrates of Haspin,
we noted, remarkably, that despite poor sequence and structural conservation, ubiquitination at similar
positions in the C-terminus of H2A (H2AK119ub) and H2B (H2BK120ub) regulates gene expression
and DNA repair. Moreover, in S.cerevisiae, DNA damage triggers phosphorylation at equivalent
positions to generate γH2AX and γH2B. To test the idea that these parallels in H2A and H2B Cterminal
PTMs extend to mitosis, we explored phosphorylation at H2BT119 (H2BT119p), the
contextual equivalent of centromeric H2AT120p, generated by the spindle checkpoint kinase BUB1,
studied in my lab. H2BT119 was particularly attractive because it matched almost perfectly the
recently described consensus motif for Haspin, and moreover, was found by phosphoproteomic
analysis to be phosphorylated in mitosis 13, 14. We thus HYPOTHESIZED that Haspin contributes to
sister chromatid cohesion and genome stability via H2BT119 phosphorylation at mitotic centromeres.
We generated a novel H2BT119p phosphospecific antibody which verified that this new histone mark
is mitotic, centromere, and Haspin-specific (Fig. 3), all in line with a function in maintaining
centromere cohesion, an idea strongly supported by additional preliminary data detailed below. Here,
we propose to analyze H2BT119p function in mitosis and study its role in cancer and genome stability.
Three major aims are envisioned to explore this idea:
Aim1: High resolution mapping of the H2BT119 phosphorylation throughout mitosis.
Aim2: Identification of H2BT119p readers.
Aim3: Exploring H2BT119p function during mitosis and in cancer.
ELOWE, Sabine Regulation of chromosome cohesion and genome stability by a novel Histone modification Proposal
3
The outcome of this project will be a more comprehensive picture of centromere identity and function,
centromere cohesion during mitosis, and Haspin activity in cancer cells. This will ultimately support
design and validation of anti-mitotic cancer drugs, and validation of a potential cancer biomarker.
Aim1: High resolution mapping of the H2BT119 phosphorylation throughout mitosis
1.1 Mapping of H2BT119p in vivo and in vitro
Depletion/inhibition of Haspin (or its activator Polo-like-kinase 1), results in H2BT119p loss, and loss
of H3T3p, the other known Haspin substrate. This strongly suggests that H2BT119 is a Haspin
substrate (Fig. 3a). Although H2BT119 adheres to the Haspin consensus, previous reports suggested
that Haspin only significantly phosphorylates H3T315, 16. We found that Haspin directly
phosphorylated recombinant H2B, as detected in an in vitro kinase assay (Fig.3b) and by Western
blotting (WB) with our H2BT119p antibody (Fig. 3c). Moreover, mutation of T119 to Alanine
completely abolished phosphorylation on H2B indicating that this site is the major Haspin target on
H2B, and confirming specificity of our antibody (Fig. 3c). Finally, we confirmed loss of H2BT119p
after Haspin depletion by IF (Fig. 3d). To explore this modification in the context of nucleosomes, we
will generate recombinant nucleosome core particles (NCPs) using combinations of H2B WT and
T119A as well as H3 WT and T3A (in collaboration with Amélie Fradet-Turcotte). These will serve as
substrates in IVKs using recombinant Haspin kinase domain (WT and KD, or WT +/- the Haspin
inhibitor 5-iodotubercidin, 5-IT). We will determine kinetic parameters (Km, Kcat, Vmax etc.) of these
reactions and explore the capacity of Haspin to phosphorylate both H3T3 and H2BT119 in NCPs.
Non-radioactive IVKs with LI-COR quantitative WB using the H2BT119p and commercial H3T3p
antibodies will be a second approach and will further verify anti-H2BT119p specificity. We will also
verify Haspin phosphorylation of H2B with native nucleosomes. Because in vivo, histone modifications
are subject to cross-talk, Haspin targeting of H2B will be also be assessed in purified histones and
mononucleosomes generated from mitotic cells (HeLa) using established protocols or commercially
purchased (e.g. from EpiCypher). Nucleosomes will be phosphorylated as above by Haspin before
being resolved for autoradiography or WB with anti-H2BT119p. Furthermore, ectopically expressing
LacI-tagged Haspin WT (wild-type) and KD (kinase dead) in cells with a 256 copy LacO-array on the
short arm of chromosome 1 will specifically target Haspin and, we anticipate, H2BT119p signals to
this locus in mitosis (Fig. 4)17, 18 which will be confirmed by quantitative immunofluorescence as
before17.
1.2 H2BT123 phosphorylation in S.cerevisiae
Preliminary data suggest that HTBT123 (the H2BT119 equivalent in S.cerevisiae HTB1 and HTB2) is
also phosphorylated (not shown) suggesting conservation of this substrate across evolution. Budding
yeast HTB1T123A, HTB2S123A single and double mutants have been obtained. In collaboration with
J.Côté, we will generate single and compound mutants of HTB1/2 and of Alk1/2 (Haspin orthologs in
yeast) and determine their role in H2B phosphorylation in by WB from mitotic yeast extracts. This will
shed light on the evolutionary conservation of this signalling pathway.
1.3 High resolution mapping of H2BT119p localization
High-resolution information on the localization of the H2BT119p is essential to understanding Haspin
function. We will use spinning disc confocal microscopy and immunofluorescence (IF) of transformed
(HeLa and U2OS) and non-transformed (RPE1) cells to map H2BT119p localization relative to other
centromere marks (candidates are listed below). IF will also be performed on chromosome spreads
which affords higher resolution and further spatial resolution may be achieved if needed by stretching
centromere DNA fibres19 (Fig. 5). Colocalization of H2BT119p with H3T3p and Haspin will be
determined. As there are currently no reliable Haspin antibodies and its subcellular localization
remains unclear20, its localization will be verified by CRISPR/Cas9-mediated in vivo tagging (with
ELOWE, Sabine Regulation of chromosome cohesion and genome stability by a novel Histone modification Proposal
4
Y.Doyon21). A functional gRNA has been identified and cell line construction is in progress.
Colocalization with CenpA, CenpAS7p, H2AT120p, Sgo and CPC components will be verified as they
are a) positionally well-defined at centromeres, and b) regulated by cross-talk with Haspin signaling
(see below). Quantitation and analysis will be done as before in ImageJ17. Alternatively, we will
perform STED microscopy (established at the CHUL) if higher resolution is required.
1.4 Investigate the cross-talk and cooperativity between mitotic histone marks
PTM crosstalk and cooperativity is a major determinant of how the histone code is read22. To explore if
this is relevant to H2BT119p, we will determine whether known proximal modifications (Ac and Ub,
Fig. 6a) regulate H2BT119p phosphorylation. Biotinylated, synthetic H2BT119 peptides, that are either
native or PTM-modified (Fig 6b, custom synthesis) will be used as substrates for Haspin in IVK
assays. After capture on streptavidin resin and washing, 32P incorporation will be measured by liquid
scintillation. Similar non-radioactive IVKs on the peptides and dot blotting with the H2BT119p
antibody will be used as a second complementary approach, which will determine anti-H2BT119p
sensitivity to adjacent PTMs, and is an essential step for proper interpretation of experiments
performed with this antibody. Of particular interest is H2BK120ub, which regulates gene expression
and DNA damage signalling6. In mitosis there is a dramatic decrease in H2BK120ub23 and we
anticipate that this facilitates H2BT119 phosphorylation. Indeed an intriguing idea is the H2BT119p
may be incompatible with H2BK120ub, and may serve to ensure its absence at centromeres during
mitosis. To address cross talk between H2A and H2B, we will overexpress, deplete by siRNA, or
inhibit Haspin, BUB1 and H2BK120ub writers/erasers24 (see list in Fig. 7, target sequences are
published for all) together with control siRNAs and determine the effect on H2BK120ub, H2BT119p,
and H2ApT120 in each condition, by IF and WB. Preliminary data shows that loss of the H2BK119ub
E2 ligase Rad6A/B, results in significant increase in H2ApT120 signals (Fig. 8).
Expected outcomes: Collectively, the results will provide a more complete picture of Haspin activity
and its crosstalk with other centromere histone PTMs. Alternative approaches are detailed throughout.
Further future experiments include exploration of crosstalk with other marks in the nucleosome (e.g.
H3 & H4 methylation) in mitosis, and functional analysis of H2BT123p in yeast.
Aim2: Identification of H2BT119p readers
We anticipate that H2BT119p associates with centromere-specific reader(s). Haspin has two known
mitotic functions: 1) H3T3 phosphorylation to recruit the CPC-Sgo1 and 2) Maintenance of centromere
cohesion via an unknown mechanism that does not appear to require the CPC-Sgo1 but may require the
kinase activity of Haspin 9, 10. We hypothesize and our preliminary data (fig. 9) shows that H2BT119p
is most likely involved in Sgo1-independent centromere cohesion, but cannot formally rule out a
cooperative role with H3T3 in CPC recruitment. Therefore, in identifying H2BT119p readers, we will
initially focus on these 2 pathways.
2.1 Candidate-based approach: The most common method for identification of histone PTM readers
is the biotinylated peptide pull-down (PD) approach25. We will thus perform PDs with H2BT119p
biotinylated peptides and controls (streptavidin beads alone and biotynylated-H2BT119) to identify
candidate binding partners (Fig. 10) from mitotic extracts by WB. Sgo1 was included in the list, but we
expect this to function as our negative control as it is not mislocalized downstream of Haspin inhibition
(Fig 2). Importantly, we have optimized conditions for the peptide PDs using a positive control,
H3T3p, which we confirmed associates with its reader survivin (Fig. 11). We are currently exploring
H2BT119p association with candidates. Next we will determine if H2BT119p regulates localization of
these candidates in vivo. We will microinject (10-20 cells/candidate) the anti-H2BT119p antibody (or
IgG control) and Alexa488-dextran and determine effect on localization of candidates by quantitative
IF. We will also generate stable inducible HCT116 (H2BT119p positive, not shown) isogenic lines
ELOWE, Sabine Regulation of chromosome cohesion and genome stability by a novel Histone modification Proposal
5
expressing siRNA resistant (target sequence validated, Fig.12) H2B-T119, -T119A, and -T119D/E,
with a C-terminal CenpB tag to ensure centromere targeting and to avoid incorporation into general
chromatin, together with a 3xFLAG tag. We have used such tags for subcellular targeting before17. Cterminal
tags are necessary as N-terminally tagging H2B interferes with its nucleosome incorporation.
C-terminal tagged H2B is functional, routinely used for chromosome imaging, and does interfere with
the cell cycle26, 27. Lines will be generated by Flp-IN recombination as before. Using these lines, and
after sensitizing the cell to mutant H2B by depleting endogenous H2B, changes in centromere
components will be assessed by quantitative IF as we have done before. This approach will
demonstrate whether H2BT119p regulates cohesion and/or CPC components. Because potential
cooperation with H2AT120p and H3T3p, H2BT119p may only partially contribute to reader
recruitment which can be tested in FRAP measurements of turnover at centromeres in each of the cell
lines, as before 17. We anticipate attenuation of the reader signal in the H2BT119A expressing cells but
a rescue in phosphomimetic H2BT119D or E expressing cells. An alternative, albeit “less clean”
method to test H2BT119 function is to simply overexpress non-targeted H2B mutants as we have done
in Fig.9 and test the localization of our candidate proteins. Although we already see mitotic defects
using this approach (Fig.9), we feel that localizing the H2BT119 mutants to the same chromosomal
location where the phosphorylation is normally observed will be a more physiologically relevant
approach. Moreover, we have tried tagging of endogenous H2B by CRISPR/Cas9, but because of
multiple H2B genes, mutating and tagging a single H2B (even the most highly expressed H2Bk),
yielded only mild expression compared to endogenous H2B signals (with Y. Doyon, data not shown),
indicating that the siRNA and rescue approach will likely be the most feasible. Indeed this approach
has been quite successful with histones in the past 28-30.
2.2 Exploratory, quantitative mass-spectrometry (MS)-based approach: H2BT119p reader(s) may
not be anticipated in our candidate list. In this case, we will take and exploratory approach. Large-scale
biotinylated peptide PDs (as in 2.1) will be done from mitotic HeLa and budding yeast extracts for
unbiased identification of H2BT119p readers. In HeLa cells, we will use stable isotope labelling in cell
culture (SILAC, Fig. 13) coupled with MS (as we have done before)17, 31, 32 at the CHUL proteomics
platform, to compare proteins associated with the phosphorylated and non-phosphorylated peptides
quantitatively after preclearing. In yeast, an essentially subtractive approach will be used as done
before33. Our MS analysis for the yeast experiments will involve a second level of quantitation using
SAINTexpress34, to score protein-protein interaction data, thus providing independent validation.
Peptide PDs may reveal indirect interactions; positive (top 5) hits from aims 2.1 and 2.2 will be verified
by peptide PDs with in vitro (bacterial or in vitro translated) produced readers to determine whether
interactions are direct or indirect. Further assays will be reader-dependent but will include verification
of reader mitotic functions after siRNA or CRISPR/Cas9 depletion/inhibition or overexpression as
above.
Alternative approaches: Cohesion and CPC recruitment may be independent of H2BT119p, or
regulators may be missed in the directed approach (2.1) but should be identified in the unbiased
approach of Aim 2.2. Complementary human and budding yeast screens maximize the chances of
reader identification using the biotinylated-peptide PD method. If the peptide PDs fail, alternative
approaches can be used. This includes PDs with NCPs harbouring H2BT119p, (with J. Côté/Amélie
Fradet-Turcotte.), or more preferably purification of 3xFlAG-2xSTREP-tagged H2B from mitotic cells
followed by mass spectrometry (with Y. Doyon, an approach he has recently optimized35).
Identification of CENPA (a centromere-specific Histone 3 variant) binding partners has been successful
using a similar approach36. T119D/E may not effectively mimic pThr, but as loss rather than gain of
readers is anticipated, the H2BT119A mutant will likely be more informative.
ELOWE, Sabine Regulation of chromosome cohesion and genome stability by a novel Histone modification Proposal
6
Aim3: Exploring H2BT119p function during mitosis
The effect of H2BT119p on global mitotic progression, CPC and most importantly cohesion function,
and its expression in cancer will be assessed.
3.1 Identification of mitotic defects caused by H2BT119p mutants
Haspin inhibition causes defects in chromosome congression and mitotic progression8, 12, 15, 20. Thus,
we will determine the mitotic phenotype after 1) anti-H2BT119p antibody microinjection, and 2)
expression of T119 mutated H2B (after siRNA depletion of endogenous H2B in the siRNA-resistant
lines generated in AIM2.1). The following assays will be used:
A) Chromosome movements (visualized with SiR-DNA dye) will be monitored by live imaging in
microinjected cells and cell lines generated in AIM 2.1. Quantifying mitotic timing (nuclear envelope
breakdown-metaphase, metaphase-anaphase), misalignments at metaphase, and anaphase lagging
chromosomes will determine H2BT119p contribution to congression. Data will be independently
verified under the same conditions by indirect IF. Preliminary data demonstrates a clear increase in
chromosome missegregation and anaphase bridges in cells expressing H2BT119A, which is
characteristic of cohesion defects. This phenotype is rescued partially in cells expressing H2BT119D
(Fig. 9). We also clearly observe premature centromere separation in chromosome spreads of cells
overexpressing H2BT119A (Fig.9a). These results validate our experimental approach and clearly
demonstrate a role for H2BT119 phosphorylation in proper mitotic progression and genome
inheritance.
B) The ultimate readout of CPC function is Aurora B target phosphorylation. We will measure by
quantitative IF the Aurora B substrates KNL1-S2437, Hec1-S4438, CenpAS7p, and MCAK
localization39. Collectively, these sites cover the spatial range of Aurora B activity from the inner
centromere to the outer kinetochore. Although we suspect a cohesion defect, rather than CPC defect
per se, these experiments will confirm CPC integrity and activity, and may further solidify a role for
H2BT119p in cohesion that is independent of the CPC.
C) Chromosome morphology and sister chromatid cohesion will be determined by mitotic chromosome
spreads to measure chromosome morphology and distances between sister chromatids. In addition we
will determine by IF the levels of cohesion-related proteins (SA2, WAPL, scc1 etc.) directly at
centromeres and chromosome arms in the cell lines from AIM2.1. Preliminary data show premature
sister chromatid separation indicative of a cohesion defect (Fig.9), justifying this line of questioning.
Importantly, we will also quantify the presence the PLK1-interacting centromere Helicase (PICH)
during anaphases as it is known to specifically associate with unresolved centromere DNA40. Because
HCT116 are chromosomally stable (CIN-), chromosome counts (measured by spreads or by centromere
marking) from individual cells after induction of expression after 5, and 10 doublings will determine if
Chromosomal Instability (CIN) has been induced.
3.2 H2BT119p in CIN+ vs CIN– cancer lines
CIN can be caused by loss of cohesion and CPC function at centromeres; restoring these functions can
correct the CIN phenotype, although the role of Haspin is unclear41. To determine if H2BT119p
correlates with CIN we will measure enrichment (relative to constitutive centromere markers e.g.
CREST) of H2BT119p in a panel of CIN+ (HeLa, A431, MCF7, U2OS, CaCo2) and CIN- (HCT116,
RPE1, DLD1, RKO) cell lines. We will distinguish between prometaphase (either aligned or nonaligned
kinetochores, KTs), and metaphase (where full KT alignment is achieved by treatment with the
proteasome inhibitor MG132 which inhibits anaphase onset). CIN+ cells display reduction in cohesion
and CPC proteins on aligned vs unaligned KTs41. Thus, by distinguishing the attachment status, a
correlation between CIN, H2BT119p, cohesion and CPC changes may emerge. In metaphase cells, we
will measure dispersal of H2BT119p signals between sister KTs which may be altered between CIN+
and CIN- cells as has been shown for cohesion proteins41.
ELOWE, Sabine Regulation of chromosome cohesion and genome stability by a novel Histone modification Proposal
7
3.3 H2BT119p expression in Cancer
GSG2, the gene coding for Haspin is mutated in a large number of cancers, and many of the mutations
are in and around the kinase domain and the N-terminal region required for Haspin localization
(Fig.14), but the effect of these mutations on Haspin activity and on carcinogenesis has not been
explored systematically. The large number of these mutations in the various cancers precludes their
characterization individually. To circumvent this issue and explore the deregulation of Haspin activity
in cancer in a more high-throughput manner, we will probe the Tissue Focus Cancer Survey Tissue
Microarray (TMA) available from origine with anti-H2BT119p and anti-H3T3p antibodies. For
TMAs, samples are selected by board-certified pathologists and include H&E images and detailed
pathology reports with histological subtypes. These include 110 tumors and 55 normal samples,
covering 11 cancer types including Breast, Colon, Lung, Kidney, Ovarian, Endometrial, Stomach,
Prostate, Melanoma, Liver, and Lymphoma. Images will be scanned on the zeiss Axio scan.Z1 slide
scanner recently acquired by the CHUL and analyzed with ImageJ (array profiler Plugins and
customized macros). The use of the phosphospecific antibodies together with high-density cancer
TMAs provide an unprecedented opportunity to rapidly and efficiently determine Haspin activity (and
not just expression) in tumours. Together with our collaborator Clémence Belleanné, we have
validated our anti-H2BpT119 for IHC, Fig. 15). H3T3 are commercial and already validated for IHC.
Anticipated results: We anticipate that antibody microinjection of H2BT119A will partially mimic
Haspin inhibition and reveal cellular functions of H2B phosphorylation. Indeed we have already shown
that overexpression of H2BT119A (but not WT H2B) affects cohesion (Fig.9). Anti-H2BT119p and
anti-H3T3 antibodies have already been validated in both WB, IHC, and IF and this dual antibody
approach will ensure success in AIMs 3.2 and 3.3. Our results will directly link deregulated Haspin
activity and H2BT119p to CIN, and will be the first to provide mechanistic insight on Haspin activity
in various types and classes of cancer where it has been found mutated. This will guide future studies
of its therapeutic potential and the utility of H2BT119p as a prognostic marker.
TIMELINE & DELIVERABLES: The identification of H2BT119p as a novel mitotic centromere
mark sets the pace for this proposal. The aims are a logical progression of this finding, and although
independent and informative on their own, they are progressive and offer potential to build on the data
generated. We anticipate 5 years for completion of the project, considering the experiments proposed
and the number and status of trainees requested, according to the timeline and milestones in Fig 16.
Considering our combined expertise and the breadth of preliminary data, this project has very high
chances of success. We anticipate three publications to emerge from this work: One on the high
resolution imaging of H2BT119p localization and its functions in mitosis, one on the role of its reader
in cohesion maintenance downstream of Haspin, and a third on the significance of H2BT119p to cancer
and CIN.
EXPERTISE, EXPERIENCE AND RESOURCES: The combination of my expertise in mitosis and
phosphosignalling, together with world-class facilities at the CHU de Québec Research Centre, provide
an ideal environment, intellectually and in terms of resources for effective execution of this project.
Proof of principle experiments that demonstrate a sound rationale and clear chance of success have
already been realised. Collectively, these all ensure that this project is poised for success.
Infrastructure and resources including equipment and dedicated, trained personnel for the proposed
experiments are all available on-site at the CHU de Québec, including the proteomics platform (with an
Orbitrap fusion tribid for phosphoproteomics) and the imaging platform (microinjectors, scanners,
FRAP etc). Importantly, we have our own, dedicated spinning disc confocal microscope (CFI-funded)
which insures timely progress. Additional imaging support is possible through the platform. We have
ELOWE, Sabine Regulation of chromosome cohesion and genome stability by a novel Histone modification Proposal
8
access to a cytospin through the Axe of Immunology, although we have budgeted for the purchase of
our own as we anticipate frequent use for this project for chromosome morphology and CIN studies.
I have a strong background in kinase signalling and functional dissection of phosphorylation events,
with a recent emphasis on mitosis, as reflected by the caliber of publications during my training42-45 and
my recent progress as a new investigator; my publications more than doubled after I started my lab,
despite significant interruptions. Our foray and experience in histone PTMs were led by our recent
report in Nature Comm. in which we showed that autophosphorylation of the BUB1 SAC kinase
regulates its proper kinetochore turnover and spatial control of H2AT120p. My productivity is not
limited to this project, and I have appended a complete manuscript that will be shortly submitted.
My role is to lead the project and direct the design and execution of the experiments in aims1-3 and
interpretation of the data generated in this proposal. I bring 17 years of experience in cell biological,
biochemical, and phosphoproteomics approaches. All mitotic assays (live cell imaging, quantitative IF,
chromosome spreads etc.) and many of the necessary reagents (antibodies, siRNAs, Flp-IN, and LacO
lines etc.) are well established in the lab, and we have sought collaborations with Jacques Côté,
Yannick Doyon, Amélie Fradet-Turcotte and Clémence Belleannée for assistance with new approaches
(see attached letters). Dr. Côté is a world leader in epigenetics with extensive experience in both yeast
and mammalian systems and will provide technical assistance with the yeast experiments. Dr. Doyon is
a talented young investigator with outstanding experience in genome editing21, 46-48 including
CRISPR/Cas9 which he has successfully introduced in his own lab. The TMA approach is rapid and
cost effective for biomarker discovery, and with Dr. Clémence Belleannée we have already optimized
our antibodies for tissue IHC. Our collaborators are fortunately all local. My lab and office is literally
between the Doyon and Belleannée labs. This will thus ensure timely assistance and problem-solving
as required. All the collaborators will spend 0.5-1hr/week on this project.
I have published multidisciplinary papers through collaborations I initiated with bioinformaticians,
structural biologists49, 50 and biophysicists49. Thus, my experience in mitotic signalling together with
my ability to successfully lead an interdisciplinary team will ensure success of this project under my
leadership. As I have minimal teaching and administrative responsibilities, I plan to commit 20
hrs/week to this project. I have one PhD student on this project (Ibrahim Alharbi (100% time
commitment), who generated most of the data shown here), and a second will be recruited (100% time
commitment) and will be trained by myself together with Ibrahim and my research associate Chantal
Garand (100% time commitment). Ibrahim will be mainly responsible for the work in Aims 1 and 3,
together with Chantal whereas Aim 2 will be performed by the new PhD student with assistance from
Ibrahim.
CONCLUSION: Centromere cohesion is crucial for maintaining genome integrity and defects in
cohesion and cohesion protection machinery have been directly shown to contribute to aneuploidy and
genome instability51. The identification of a novel pathway that regulates centromere cohesion is a
MAJOR discovery, and has the potential to transform our understanding of the mechanisms linking
cohesion, chromosomal instability, and cancer.
ELOWE, Sabine Regulation of chromosome cohesion and genome stability by a novel Histone modification Proposal
9
ELOWE, Sabine Regulation of chromosome cohesion and genome stability by a novel Histone modification Proposal
10
–
ELOWE, S Regulation of chromosome cohesion and genome stability by a novel Histone modification rev. Response
1
This grant is a resubmission of a previously unsuccessful grant that ranked 20/97 after phase 2, with 15
grants funded from its cluster. I want to thanks previous reviewers, chair and SO for their time,
insightful suggestions, and their very positive evaluations. All the comments were taken into
consideration as outlined below, and I sincerely believe this has significantly improved the proposal.
Below, I highlight the strengths noted by previous reviewers, and I address all their concerns. I thank
the current reviewers for taking these comments and our response to them into consideration.
STRENGTHS: There was considerable excitement from the panel regarding the impact of the
discovery of H2B mitotic phosphorylation, and the rationale linking it to cohesion. The implication
for genome integrity and human health was considered a strength of the proposal. For the cell cycle
field, it provides a potential explanation for contradictory literature on the role of Haspin and BUB1-
CPC-Shugoshin pathway in centromere cohesion. In addition, because of the proximity of the Haspin
H2BT119 to the K120 whose ubiquitination regulates gene expression, chromatin structure and
potentially DNA damage, the identification of phosphorylation at H2BT119 may have implications
beyond mitosis, something we plan to address in future funding opportunities. The panel also
considered the exploration of this epitope as a potential biomarker for cancer as an additional strength,
and we have added preliminary data to support feasibility of our approach (Fig. 15). Another major
strength was considered the team and the collaborations in place. We have further strengthened this
with Dr. Amélie Fradet-Turcotte (see letter and Fradet-Turcotte et al. Nature 2013, Orthwein et al,
Science 2014, Wilson et al. Nature 2016, Jacquet et al. Mol Cell 2016, Kitevsky, Fradet-Turcotte et al.
eLife 2017) who is a young investigator and expert in in vitro and in vivo nucleosome PTMs and we
have already started exploring this novel site with recombinant histones and nucleosomes with her
collaboration (Fig. 3b, c). All in all, it was considered an exciting, well-written ‘highly mechanistic
proposal’ by a ‘PI who is a master at defining mechanisms’.
WEAKNESSES:
1. It was suggested that overall, the proposal was built on a solid foundation, but it would benefit from
additional preliminary data, and this was highlighted in the SO notes. In the revised proposal, each
aim of the current application is now supported by additional evidence.
AIM1: In the revised submission there is additional data included showing purification of recombinant
histones and the direct phosphorylation of H2B by Haspin (Fig.3c). We are currently making
recombinant nuclear core particles with Amélie Fradet-Turcotte’s lab to test phosphorylation in vitro in
the context of the nucleosome. We felt this was an important assay to establish in our lab, as this will
give us the tools and expertise to assess histone cross-talk in the future. For localization of H2BT119p
signals at the centromere, the reviewers considered that it might be a challenge to identify overlap and
localization at a high enough resolution in such a small space. To address this, we are optimizing a
centromere fibre stretching approach (Fig. 5) which literally stretches out the centromere and allows
better spatial resolution of the centromere marks (ref. 19).
Aim2: It was suggested that proof-of-principle experiments demonstrating the feasibility of the
biotinylated peptide pull-down would strongly increase confidence in the use of this approach. We
have verified that the in vitro pull-down assay is functional in our hands using a positive control
peptide (H3T3p, another Haspin target) which was able to pull-down its known mitotic reader, survivin
(Fig. 11). Because the sequences surrounding H3T3 and H2BT119 are similar (indeed both are Haspin
targets and match the Haspin consensus), we anticipate that these pull-downs will be functional and
informative. Moreover, survivin did not associate with the H2BT119p peptide; thus, we also feel that
ELOWE, S Regulation of chromosome cohesion and genome stability by a novel Histone modification rev. Response
2
specificity will be achieved. Nevertheless, we have also included an alternative approach. We will use
the tandem 3xFLAG-2xSTREP tag approach reported by our collaborator Y. Doyon (ref. 21 in
proposal) for complex purification of endogenously tagged H2Bk (the most highly expressed form in
HCT116 cells). This methodology has been successfully used by our collaborators for identification of
protein-protein interactions, and is routinely used in their lab. A similar, in vivo tag-based approach
has been successful in identifying binding partners of the centromere-specific histone CENPA (ref. 36).
I am thus very confident that using the proposed strategies will allow for identification of genuine
mitotic readers of H2BT119p.
Aim3: The panel considered the link to sister chromatid cohesion as exciting but felt that evidence to
support this was lacking. I have now included chromosome spreading experiments showing that
overexpression of non-phosphorylatable H2BT119A appears to indeed cause a cohesion defect and
premature sister chromatid separation, and that this is at least partially rescued by the phosphomimetic
Asp substitution (Fig. 9). These observations strongly support the hypothesis and line of inquiry.
Furthermore, in support of the siRNA-rescue approach we proposed, an siRNA that targets a common
sequence in H2B (Fig. 12) has been verified and all the rescue plasmids generated. As this approach
has been used by us before and by others in the context of histones (Refs. 28-30), I anticipate that
feasibility will not be an issue. Finally, and in light of the significant support and excitement of the
panel for exploring the H2BT119p as a potential biomarker in cancer, we have optimized IHC
protocols for our antibody (Fig. 15). All in all, I feel that in the current rendition of the proposal, the
additional data covering all aims clearly demonstrates an excellent likelihood of success.
Minor Reviewer Comments
1. It was suggested that the budding yeast model could be further exploited to explore the function of
H2B phosphorylation. While I agree that exploring this PTM would be exciting in the budding
yeast, our focus to date has been the mammalian system and we consider experiments in yeast
somewhat premature. A proper exploration of H2B phosphorylation is beyond the scope of this
proposal and will likely form the basis of a separate proposal. Indeed, both the composition of the
CPC and the resolution of sister chromatids in budding yeast are somewhat different and slightly
less complex than in humans. While this may be considered an advantage, it may also not reflect
the complexity that we seek to explore in human cells. Nevertheless, we do intend to demonstrate
the conservation of this H2B phosphorylation in budding yeast and identify binding partners of
phosphorylated HTB1 and HTB2, all of which will form the basis of a future project.
2. It was suggested that challenges that inherently come with histone work such as incorporation of
overexpressed histone in the same way as endogenous or WT histone, and positioning of tags was
also mentioned. I am aware of and have acknowledged many of these challenges, and I have
included comments/discussion in the main body of the proposal on how we address them. I address
and justify the choice and position of our tags, and I discuss nucleosome incorporation of
exogenous histones and studying mutant histones. For example, we noted that CRISPR/Cas9 in
vivo editing of a single H2B gene will not be useful since, in our experience, expression of only a
single tagged H2B gene was significantly lower than that of total H2B.
3. Finally, some figure legends were considered to be too short. I have now added more detail to most
figure legends, directly explaining the relevance of the figure to the current proposal.
ELOWE, Sabine A novel, Haspin-dependent epigenetic mark during early mitosis Research Proposal
11
REFERENCES:
1. Dominguez-Brauer, C. et al. Targeting Mitosis in Cancer: Emerging Strategies. Mol Cell 60, 524-536
(2015).
2. Janssen, A., Kops, G. & Medema, R. Elevating the frequency of chromosome mis-segregation as a
strategy to kill tumor cells. Proceedings of the National Academy of Sciences of the United States of
America 106, 19108-19113 (2009).
3. Janssen, A., Kops, G.J. & Medema, R.H. Targeting the mitotic checkpoint to kill tumor cells. Horm
Cancer 2, 113-116 (2011).
4. De Antoni, A., Maffini, S., Knapp, S., Musacchio, A. & Santaguida, S. A small-molecule inhibitor of
Haspin alters the kinetochore functions of Aurora B. The Journal of cell biology 199, 269-284 (2012).
5. Huertas D1, S.M., Moreto J, Villanueva A, Martinez A, Vidal A, Charlton M, Moffat D, Patel S,
McDermott J, Owen J, Brotherton D, Krige D, Cuthill S, Esteller M. Antitumor activity of a smallmolecule
inhibitor of the histone kinase Haspin. Oncogene 31 (2012).
6. Cole, A.J., Clifton-Bligh, R. & Marsh, D.J. Histone H2B monoubiquitination: roles to play in human
malignancy. Endocr Relat Cancer 22, T19-33 (2015).
7. Higgins, J.M. Haspin: a newly discovered regulator of mitotic chromosome behavior. Chromosoma 119,
137-147 (2010).
8. Yamagishi, Y., Honda, T., Tanno, Y. & Watanabe, Y. Two histone marks establish the inner centromere
and chromosome bi-orientation. Science 330, 239-243 (2010).
9. Zhou, L. et al. The N-Terminal Non-Kinase-Domain-Mediated Binding of Haspin to Pds5B Protects
Centromeric Cohesion in Mitosis. Current Biology 27, 992-1004 (2017).
10. Goto, Y. et al. Pds5 Regulates Sister-Chromatid Cohesion and Chromosome Bi-orientation through a
Conserved Protein Interaction Module. Current Biology 27, 1005-1012 (2017).
11. Dai, J., Kateneva, A.V. & Higgins, J.M. Studies of haspin-depleted cells reveal that spindle-pole integrity
in mitosis requires chromosome cohesion. J Cell Sci 122, 4168-4176 (2009).
12. Dai, J., Sullivan, B.A. & Higgins, J.M. Regulation of mitotic chromosome cohesion by Haspin and Aurora
B. Dev Cell 11, 741-750 (2006).
13. Maiolica, A. et al. Modulation of the chromatin phosphoproteome by the Haspin protein kinase. Mol
Cell Proteomics 13, 1724-1740 (2014).
14. Olsen, J.V. et al. Quantitative phosphoproteomics reveals widespread full phosphorylation site
occupancy during mitosis. Sci Signal 3, ra3 (2010).
15. Wang, F. et al. Histone H3 Thr-3 phosphorylation by Haspin positions Aurora B at centromeres in
mitosis. Science 330, 231-235 (2010).
16. Kelly, A.E. et al. Survivin reads phosphorylated histone H3 threonine 3 to activate the mitotic kinase
Aurora B. Science 330, 235-239 (2010).
17. Asghar, A. et al. Bub1 autophosphorylation feeds back to regulate kinetochore docking and promote
localized substrate phosphorylation. Nat Commun 6, 8364 (2015).
18. Janicki, S.M. et al. From silencing to gene expression: real-time analysis in single cells. Cell 116, 683-698
(2004).
19. Wang, L.H., Schwarzbraun, T., Speicher, M.R. & Nigg, E.A. Persistence of DNA threads in human
anaphase cells suggests late completion of sister chromatid decatenation. Chromosoma 117, 123-135
(2008).
20. Dai, J., Sultan, S., Taylor, S.S. & Higgins, J.M. The kinase haspin is required for mitotic histone H3 Thr 3
phosphorylation and normal metaphase chromosome alignment. Genes & development 19, 472-488
(2005).
21. Dalvai, M. et al. A Scalable Genome-Editing-Based Approach for Mapping Multiprotein Complexes in
Human Cells. Cell Rep 13, 621-633 (2015).
22. Lee, J.S., Smith, E. & Shilatifard, A. The language of histone crosstalk. Cell 142, 682-685 (2010).
23. Guppy, B.J. & McManus, K.J. Mitotic accumulation of dimethylated lysine 79 of histone H3 is important
for maintaining genome integrity during mitosis in human cells. Genetics 199, 423-433 (2015).
ELOWE, Sabine A novel, Haspin-dependent epigenetic mark during early mitosis Research Proposal
12
24. Weake, V.M. & Workman, J.L. Histone ubiquitination: triggering gene activity. Molecular cell 29, 653-
663 (2008).
25. Wysocka, J. Identifying novel proteins recognizing histone modifications using peptide pull-down assay.
Methods 40, 339-343 (2006).
26. Iwasaki, W. et al. Contribution of histone N‐terminal tails to the structure and stability of nucleosomes.
FEBS Open Bio 3, 363-369 (2013).
27. Kanda, T., Sullivan, K.F. & Wahl, G.M. Histone–GFP fusion protein enables sensitive analysis of
chromosome dynamics in living mammalian cells. Current Biology 8, 377-385 (1998).
28. Sarcinella, E., Zuzarte, P.C., Lau, P.N.I., Draker, R. & Cheung, P. Monoubiquitylation of H2A.Z
Distinguishes Its Association with Euchromatin or Facultative Heterochromatin. Molecular and Cellular
Biology 27, 6457-6468 (2007).
29. Law, C. & Cheung, P. Expression of Non-acetylatable H2A.Z in Myoblast Cells Blocks Myoblast
Differentiation through Disruption of MyoD Expression. Journal of Biological Chemistry 290, 13234-
13249 (2015).
30. Wong, C.-H. et al. Apoptotic histone modification inhibits nuclear transport by regulating RCC1. Nature
Cell Biology 11, 36-45 (2008).
31. Elowe, S. et al. Uncoupling of the spindle-checkpoint and chromosome-congression functions of BubR1.
J Cell Sci 123, 84-94 (2010).
32. Elowe, S., Hummer, S., Uldschmid, A., Li, X. & Nigg, E.A. Tension-sensitive Plk1 phosphorylation on
BubR1 regulates the stability of kinetochore microtubule interactions. Genes Dev 21, 2205-2219 (2007).
33. Altaf, M. et al. NuA4-dependent acetylation of nucleosomal histones H4 and H2A directly stimulates
incorporation of H2A.Z by the SWR1 complex. The Journal of biological chemistry 285, 15966-15977
(2010).
34. Teo, G. et al. SAINTexpress: improvements and additional features in Significance Analysis of
INTeractome software. J Proteomics 100, 37-43 (2014).
35. Dalvai, M. et al. A Scalable Genome-Editing-Based Approach for Mapping Multiprotein Complexes in
Human Cells. Cell Reports 13, 621-633 (2015).
36. Foltz, D.R. et al. The human CENP-A centromeric nucleosome-associated complex. Nature Cell Biology
8, 458-469 (2006).
37. Welburn, J.P. et al. Aurora B phosphorylates spatially distinct targets to differentially regulate the
kinetochore-microtubule interface. Molecular cell 38, 383-392 (2010).
38. DeLuca, K.F., Lens, S.M. & DeLuca, J.G. Temporal changes in Hec1 phosphorylation control kinetochoremicrotubule
attachment stability during mitosis. Journal of cell science 124, 622-634 (2011).
39. Ems-McClung, S.C. et al. Aurora B inhibits MCAK activity through a phosphoconformational switch that
reduces microtubule association. Curr Biol 23, 2491-2499 (2013).
40. Baumann, C., Korner, R., Hofmann, K. & Nigg, E.A. PICH, a centromere-associated SNF2 family ATPase,
is regulated by Plk1 and required for the spindle checkpoint. Cell 128, 101-114 (2007).
41. Tanno, Y. et al. The inner centromere-shugoshin network prevents chromosomal instability. Science
349, 1237-1240 (2015).
42. Tong, J., Elowe, S., Nash, P. & Pawson, T. Manipulation of EphB2 regulatory motifs and SH2 binding
sites switches MAPK signaling and biological activity. J Biol Chem 278, 6111-6119 (2003).
43. Huusko, P. et al. Nonsense-mediated decay microarray analysis identifies mutations of EPHB2 in human
prostate cancer. Nat Genet 36, 979-983 (2004).
44. Henderson, J.T. et al. The receptor tyrosine kinase EphB2 regulates NMDA-dependent synaptic
function. Neuron 32, 1041-1056 (2001).
45. Elowe, S., Holland, S.J., Kulkarni, S. & Pawson, T. Downregulation of the Ras-mitogen-activated protein
kinase pathway by the EphB2 receptor tyrosine kinase is required for ephrin-induced neurite
retraction. Mol Cell Biol 21, 7429-7441 (2001).
46. Doyon, Y. et al. Enhancing zinc-finger-nuclease activity with improved obligate heterodimeric
architectures. Nat Methods 8, 74-79 (2011).
ELOWE, Sabine A novel, Haspin-dependent epigenetic mark during early mitosis Research Proposal
13
47. Doyon, Y. et al. Transient cold shock enhances zinc-finger nuclease-mediated gene disruption. Nat
Methods 7, 459-460 (2010).
48. Li, H. et al. In vivo genome editing restores haemostasis in a mouse model of haemophilia. Nature 475,
217-221 (2011).
49. Lee, S. et al. Characterization of spindle checkpoint kinase Mps1 reveals domain with functional and
structural similarities to tetratricopeptide repeat motifs of Bub1 and BubR1 checkpoint kinases. J Biol
Chem 287, 5988-6001 (2012).
50. Thebault, P. et al. Structural and functional insights into the role of the N-terminal Mps1 TPR domain in
the SAC (spindle assembly checkpoint). Biochem J 448, 321-328 (2012).
51. Barber, T.D. et al. Chromatid cohesion defects may underlie chromosome instability in human
colorectal cancers. Proc Natl Acad Sci U S A 105, 3443-3448 (2008).
What Students Are Saying About Us
.......... Customer ID: 12*** | Rating: ⭐⭐⭐⭐⭐"Honestly, I was afraid to send my paper to you, but you proved you are a trustworthy service. My essay was done in less than a day, and I received a brilliant piece. I didn’t even believe it was my essay at first 🙂 Great job, thank you!"
.......... Customer ID: 11***| Rating: ⭐⭐⭐⭐⭐
"This company is the best there is. They saved me so many times, I cannot even keep count. Now I recommend it to all my friends, and none of them have complained about it. The writers here are excellent."
"Order a custom Paper on Similar Assignment at essayfount.com! No Plagiarism! Enjoy 20% Discount!"
