Bridge/Pilot/Limited Term Research Project Funding ...campbell//resources/REC_example_proposa… · HP1 chromodomain is a nearly ideal binding module for the proposed applications

September 15, 2005

Bridge/Pilot/Limited Term Research Project Funding Application

REVIEW CRITERIA

Statement of Objective(s)

- Is the objective(s) of the proposal stated clearly

Background Information and Literature Review

- Is the information well organized and presented clearly and concisely

- Has important and relevant material been overlooked

Design

- Is the experimental procedure specified (i.e. the collection and/or analysis of data, scientific research, demonstrationwith evaluation, preliminary development)

- Is the type of data specified (physical, biological, sociological - economic)

- Are sample sizes adequate in terms of numbers, types of cases, disease entities, behavioral habits, etc.

- Are the sampling procedures adequately described

- Is a hypothesis being tested

- Is the hypothesis clearly stated and will the proposed design adequately test it

Instrumentation

- Are the measuring instruments proposed for the project well known or clearly described

Analysis

- Is it evident that the appropriate statistical methods of analysis have been included

Research Personnel

- Is there a clear description of their role in the project

Budgets

- Is the proposed budget well justified and are the listed expenses relevant to the goals of the project

Resubmission’s

- If this is a resubmission, has a rebuttal been provided to the reviewers’ comments?

ACB- Bridge/Pilot/Limited Term Projects Page 10

September 15, 2005

Development of versatile and highly specific molecular recognition modules that bind to

trimethylated lysine 9 and lysine 27 of histone H3 in living cells.

Overview. An emerging paradigm in carcinogenesis is that alterations in the regulation of cell growth

most commonly occur through epigenetic mechanisms (1), where the expression of tumor suppressor

proteins and proteins required for the maintenance of genomic stability are altered through chromatin-

dependent changes in gene regulation (2-6). Indeed, many oncogenic translocations involved in the

genesis of leukemias and lymphomas involve the generation of fusion proteins with histone

methyltransferases (HMTs) and histone acetyltransferases (HATs) (4,7-11). The critical importance of

epigenetic mechanisms in carcinogenesis is exemplified by the fact that the Rb pathway, which has a

major role in gene-specific targeting of histone modifications, is defective in ~90% of solid tumors (12).

Furthermore, mice that lack the ability to methylate lysine 9 of histone H3 (H3K9) display genomic

instability and a high probability of tumor formation (10). Epigenetic changes, unlike mutations in the

DNA sequence, can be reversed through inhibition of the enzymes that are responsible for the

establishment and maintenance of the “epigenome”. It is therefore not surprising that pharmaceutical

companies are investing considerable effort in the development of anti-cancer drugs that selectively

target epigenetic machinery. For example, inhibitors of histone deacetylases and DNA

methyltransferases have shown very promising results in stage I and stage II clinical trials (1,13,14).

Aberrant methylation of the N-terminal tails of the core histones (Figure 1A), due to the up or down

regulation or improper targeting of HMTs, is one particular epigenetic change that has recently been

implicated in a surprisingly diverse array of human cancers (4,9-11,8,15). However, for the majority of

these cases, it is still an open question as to whether aberrant histone methylation is a cause or an effect

of malignancy. Despite this uncertainty, one clear conclusion has emerged from these studies: the

pattern of histone methylation and acetylation provides an ‘epigenetic signature’ that can both identify

cancer cells (8) and predict the aggressiveness and probability of recurrence (16). A recent and striking

example of the use of histone modifications as a prognostic marker for prostate cancer recurrence relied

on the quantitative detection of acetylation at H3K9, H3K18, and H4K12, and dimethylation at H4R3

(where R = arginine) and H3K4 (16). Based on the intensity of the immunohistochemical staining for

each histone modification in tissue samples, the researchers were able to distinguish 2 groups of patients

with distinct probabilities of tumor recurrence. Given that the H3K9-specific HMT RIZ1 is silenced in

many types of tumors (e.g. breast, liver, colon, melanoma, and lung), and that the H3K27-specific HMT

EZH2 is overexpressed in a wide variety of cancers (e.g. prostate, lymphomas, and breast), quantitation

of methylated H3K9 and H3K27 (Figure 1B) in malignant tissue would be an excellent diagnostic or

prognostic marker for many types of cancer (17). The fact that detection of these modifications has not

yet been used as a cancer biomarker is almost certainly due to the lack of reliable and specific

polyclonal antibody reagents.

While the lack of reliable antibodies is slowing the development of clinical epigenetic-based

diagnostics and prognostics, it is also handicapping academic researchers working to decipher the

biological mechanisms and consequences of histone posttranslational modifications. For example, the

standard method for detecting trimethylated H3K9 in cell culture is to use polyclonal anti-H3Me3K9

antibodies and traditional immunofluorescence techniques with fixed cells. Despite the fact that

monoclonal antibody technology was introduced about 30 years ago (18), most of the commercially

available antibodies for research purposes, including all anti-H3Me3K9 antibodies, are polyclonal.

Unfortunately, these polyclonal antibodies are notorious for their considerable batch-to-batch variability

and polyclonals that can faithfully discriminate tri- from di-methylated lysines are exceedingly rare (see

collaboration letter from Dr. Michael Hendzel). In reading the epigenetic literature, one is often struck

by the degree of suspicion that accompanies any experiment in which antibody-based methods with a

polyclonal antibody are used to identify a specific histone modification (see Ref. (19) for examples).

For almost all conceivable applications, small protein binding domains that combine the exquisite

specificity of monoclonal antibodies with the ability to be genetically manipulated and recombinantly


September 15, 2005

expressed in living cells, are preferable to antibodies. We propose to undertake a proof-of-principal pilot

project to develop small, versatile, and highly specific recombinant molecular recognition modules that

bind to trimethylated H3K9 (Figure 1B, n = 3) and trimethylated H3K27, respectively. To achieve these

aims, we will use phage display and directed evolution to engineer versions of the chromodomain of

HP1 with the desired specificity. The next stage of the project (beyond the scope of this grant) will be

the functionalization of these domains to develop reagents for diagnostic immunohistochemistry;

chromatin immunoprecipitation (Dr. J. Davie, U. of Manitoba); localization and function blocking in

interphase (Dr. M. Hendzel); and localization and function blocking in mitosis (Dr. G. Chan).

HP1 chromodomain as a candidate molecular recognition module. In recent years it has become

clear that posttranslational modification of specific amino acid residues in the N-terminal portions of the

core histones (Figure 1A) creates unique protein docking sites for regulatory protein complexes (20-22).

Perhaps the best-characterized posttranslational modification that functions through this mechanism is

the trimethylation of H3K9 (Figure 1B). This modification provides a binding site for heterochromatin

protein 1 (HP1), a structural protein with an essential role in maintenance of the heterochromatin state

and gene repression (23-25). HP1 is a modular protein composed of two homologous domains (known

as chromodomains), connected by a flexible linker region (26,27). The first chromodomain of HP1 has

been extensively characterized and the atomic structure of this domain in complex with a trimethylated

H3K9 peptide (Figure 2AB) has been reported (28,29). The peptide sequence recognized by this

chromodomain extends only to the 3 residues preceding trimethylated lysine 9 (threonine 6, alanine 7,

and arginine 8) and the one residue following trimethylated lysine 9 (serine 10). Interestingly, the

Polycomb chromodomain is highly homologous to the HP1 chromodomain (54% identity) but binds

preferentially to a similar, but distinct, sequence at trimethylated H3K27 (see Table 1) (30). The altered

binding specificities of the highly homologous HP1 and Polycomb chromodomains supports the notion

that we could introduce mutations into the HP1 chromodomain that could result in ‘unnaturally’

improved specificity for a particular target sequence.

Hypothesis: We will be able to identify a mutated variant of the HP1 chromodomain that has

greater specificity for binding to trimethylated H3K9 than the wild-type domain.

Due to its small size and its ability to recognize the trimethylated H3K9 with moderate specificity, the

HP1 chromodomain is a nearly ideal binding module for the proposed applications. However, this

binding module does not have sufficient specificity for trimethylated H3K9 as evidenced by its

promiscuous binding to dimethylated H3K9 and trimethylated H3K27 (see Table 1). As a rough

benchmark, we hope to achieve at least 10! greater specificity for H3Me3K9 (and H3Me3K27) over all

closely related sequences. Based on the reported peptide binding specificity of HP1 chromodomain (see

Table 1) we expect that this level is achievable. We note that by this criterion, HP1 already has sufficient

specificity for H3Me3K9 (4 µM) versus H3Me3K27 (64 µM) but not versus H3Me2K9 (7 µM). The

highly homologous Polycomb is better able to discriminate between H3Me3K27 (5 µM) and H3Me2K27

(28 µM), suggesting that the poor selectivity of HP1 is not the result of some chemical limitation, but

rather a result of evolutionary optimization for its particular biological function. To engineer a variant of

this domain with improved selectivity towards trimethylated H3K9, we propose to subject this

chromodomain to ‘unnatural’ selection pressures through the use of phage display. Our goal of

engineering a chromodomain with improved specificity towards H3K9 can be broken down into two

specific aims as elaborated below. All work to achieve Specific Aims 1 and 2 will be done by in the

Campbell lab by an experienced research technician and graduate student Carine Lafaille.

Specific Aim 1: Improve the specificity for trimethylated vs. dimethylated H3K9. During the

summer of 2005, an undergraduate researcher in the Campbell lab, Wallis Rudnick, cloned the gene

encoding the HP1 chromodomain into phagemid pCANTAB 5E (GE Healthcare) as an in frame fusion

with gene 3. She went on to express and purify the phage particles displaying the protein as a fusion to

phage coat protein 3 (p3). The next step of this project will be to create an HP1 chromodomain gene


September 15, 2005

library (CDlib1) by overlap extension PCR with semi-degenerate primers followed by subcloning into

the phagemid pCANTAB 5E. The CDlib1 gene library will contain amino acid diversity in the expected

vicinity of the N" of lysine 9 (refer to Figure 2A). The CDlib1 libraries of chromodomain variants will

be expressed as p3 fusions on the surface of filamentous phage particles and specific binders of

H3Me3K9 will be selected following the protocol shown in Figure 3. This process will be repeated with

increasing selection stringency until single clones exhibiting the desired specificity are identified by

ELISA. To increase the stringency of selection, the concentration of the target H3Me3K9-biotin peptide

will be kept constant at 10 pmol while the concentration of the non-biotinylated 'dummy' H3Me2K9

peptide is increased to >100 pmol. This 10:1 ratio of competitor to target will ensure that that the

selected binding modules have at least a 10! greater specificity for H3Me3K9 over all closely related

sequences. This level of specificity is the expected minimum required for our intended in vivo

applications. To prevent selection of phage that interact with the streptavidin coated beads themselves, a

negative selection step of adding, incubating, and removing streptavidin coated beads will be done prior

to addition of the biotinylated target. To identify specific clones from the enriched library, we will

employ an ELISA assay against the immobilized biotinylated peptides. An HRP conjugated anti-phage

antibody (GE Healthcare) and an appropriate chromogenic substrate will be used for detection. Clones

that exhibit the greatest difference in signal between target peptide and the ‘dummy’ peptides will be

sequenced and the protein expressed in soluble form for purification and further characterization of the

binding domain. This selection protocol will be performed in parallel with a target H3Me3K27-biotin

peptide and a non-biotinylated ‘dummy’ H3Me2K27 peptide. Our expectation is that these experiments

will be redundant and will result in identification of similar sequences that discriminate between tri- and

di-methylated lysines independent of whether the H3K9 or H3K27 flanking sequence is present.

Specific Aim 2: Improve specificity for H3Me3K9 vs. H3Me3K27 and vice versa. The X-ray

structures of the HP1 and Polycomb chromodomains bound to trimethylated peptides clearly shows that

there is no interaction between the binding domain and residues in positions greater than n + 1 (where n

is the methylated lysine) (28-30). Selectivity depends most strongly on interactions with residues in the

n - 4 and n - 5 positions (sequence of KQ for H3K9 versus TK for H3K27, refer to Figure 1B) (30).

Starting with the chromodomain variant that exhibits the highest specificity for trimethylated lysines, we

will construct a second HP1 chromodomain gene library (CDlib2) with degenerate codons encoding

amino acid diversity at residues that are known to contact positions n - 4 and n - 5 of the peptide (refer to

Figure 2B). The selection strategy will focus on selecting variants that bind preferentially to H3Me3K9

over H3Me3K27 and vice versa. The strategy will be similar to that described for Specific Aim 1 except

that the target and ‘dummy’ peptides will be H3Me3K9-biotin and H3Me3K27 or H3Me3K27-biotin and

H3Me3K9 in the two separate panning experiments to be undertaken in parallel.

Long Term Objectives. The collaborations section of this application describes a number of mid- to

long-term objectives to be pursued primarily by our collaborators. Due to the fact that our in vitro

selections will be done on isolated peptides while the in vivo target of the binding domains are histone

tails in the context of chromatin, it is possible that the in vitro specificity will not accurately predict the

in vivo specificity. For this reason we feel that one particularly important mid-term objective will be in

vivo validation experiments using mice embryonic fibroblasts that will lack the H3Me3K9 modification

(see Hendzel letter). Beyond that, the Campbell lab intends to continue building a complete library of

modification specific binding modules that can be employed in a wide range of clinical and

experimental applications. Other target sequences will include dimethylated H3K9 and H3K27 and

trimethylated H4K20. We are also interested in making acetylated lysine-specific reagents based on the

bromodomain scaffold. Furthermore, we will explore the use of our engineered domains as therapeutic

anti-cancer agents where they would behave as dominant-negative proteins that inhibit the functional

consequences of the histone modification when delivered to tumors as either a genetic fusion to a

translocating peptide such as TAT (31), or by an adenovirus-mediated gene therapy (32).


September 15, 2005

Chromodomain Me1 H3K9 Me2 H3K9 Me3 H3K9 Me1 H3K27 Me2 H3K27 Me3 H3K27

HP1 46 µM 7 µM 4 µM n.d. n.d. 64 µM

Polycomb >1000 µM >1000 µM 125 µM 20 µM 28 µM 5 µM

Table 1. Binding affinities of two highly conserved chromodomains for methylated peptidescorresponding to H3K9 and H3K27. Data adapted from Fischle et al. 2003 (30).

Name Sequence

H3Me2K9 GTKQTAR[KMe2]STGGGYC

H3Me3K9 GTKQTAR[KMe3]STGGGYC

H3Me2K27 GATKAAR[KMe2]SAPAGYC

H3Me3K27 GATKAAR[KMe3]SAPAGYC

H3Me2K9-biotin GTKQTAR[KMe2]STGGGYC-biotin

H3Me3K9-biotin GTKQTAR[KMe3]STGGGYC-biotin

H3Me2K27-biotin GATKAAR[KMe2]SAPAGYC-biotin

H3Me3K27-biotin GATKAAR[KMe3]SAPAGYC-biotin

Table 2. Synthetic peptides that will be used in this project.

H2A

H3

H4 H4

H3

H2B

H2A

H2B

N

N

N

N

N

N

N

N

HN

Nterm-ARTKQTAR

O

STGGKAPRKQLATKAARKSAPATGGVK

N

CH3n

lysine 9 lysine 27

H3

A

B

Figure 1. Representation of H3K9 and H3K27 in the context of the nucleosome. A. Cartoonrepresentation of a single nucleosome composed of 8 histones around which 146 base pairs of DNA iswrapped. B. The N-terminal sequence of histone H3 with emphasis on the location of lysine 9 which canbe posttranslationally methylated (n = 0,1,2 or 3). Methylation of lysine 9 is associated with changes inchromatin structure. Note the identical amino acid sequences (ARKS) immediately surrounding bothlysine 9 and lysine 27.


September 15, 2005

Tyr 24

Gly 47

Tyr 48

Pro 49

Glu 52Thr 54

Gln 64

Asp 62Asp 65

Cys 63B

A

Figure 2. The structure of the chromodomain of HP1 complexed with the residue 5 to 10 peptidefragment corresponding to H3K9 and neighboring amino acids (PDB ID 1KNE). The backbone ofthe peptide fragment is shown as a dark grey coil. Side chains of the bound peptide other than thetrimethylated lysine are not shown. A. Labeled residues (side chains represented with white bonds withwhite atoms) are in the immediate vicinity of N" of lysine 9 (side chain represented with grey bondswith black atoms) and will be targeted for diversification in the phage-displayed library CDlib1.Specifically, we will construct a library with the following diversity: Tyr 24 and Tyr 48 to Tyr or Phe,Glu 52 to Glu or Asp, Thr 54 to Thr or Ser, and Gly 47 and Pro 49 to all 20 amino acids. B. Labeledresidues are in the immediate of the n-4 and n-5 residues of the bound peptide and have been proposedto be important for binding specificity. These residues will be targeted for diversification in phage-displayed library CDlib2. Specifically, we will construct a library in which Asp 62, Cys 63, Gln 64, andAsp 65 are mutated to all 20 possible amino acids.


September 15, 2005

Figure 3. Overview of the phage display and selection protocol. The phage library (constructed usingmanufacturer’s protocols) will be purified from infected E. Coli TG1 bacterial culture and then mixedwith a solution containing biotinylated and trimethylated target peptide, H3Me3K9-biotin (Table 2), andan equal concentration of the non-biotinylated and dimethylated peptide, H3Me2K9 (Table 2). Theparticular phage expressing chromodomain variants that bind preferentially to trimethylated H3K9 willbecome non-covalently associated with the biotin label. Upon mixing with streptavidin coated magneticbeads (Dynabeads M-280 Streptavidin), those phage will bind to the beads, while non-binding phage, orphage that preferentially associate with the non-biotinylated 'dummy' H3Me2K9 peptide, will remainfree in solution. A powerful magnet (Dynal MPC®-S, Dynal Biotech) will then be used to hold thebeads firmly to the side of the tube while the supernatant is decanted and the beads thoroughly washed.Phage will be eluted with standard conditions (100 mM triethylamine) and used to reinfect E. coli TG1.In the first round of panning, each 1 ml reaction will contain ~10 pmol (~20 ng) each of peptide-biotinconjugate and non-biotinylated ‘dummy’ peptide, ~17 pmol phage (~1013 individual phage), and 1 mg ofmagnetic beads with a total binding capacity of ~700 pmol free biotin or ~10 pmol phage-peptide-biotincomplex due to steric crowding effects (33).


September 15, 2005

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2. Balch, C., Huang, T. H., Brown, R., Nephew, K. P., (2004) “The epigenetics of ovarian cancer drugresistance and resensitization”, Am J Obstet Gynecol 191, 1552-72.

3. Jones, P. A., (2003) “Epigenetics in carcinogenesis and cancer prevention”, Ann N Y Acad Sci 983,213-9.

4. Kim, K. C., Huang, S., (2003) “Histone methyltransferases in tumor suppression”, Cancer Biol Ther2, 491-9.

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10. Peters, A. H., O'Carroll, D., Scherthan, H., Mechtler, K., Sauer, S., Schofer, C., Weipoltshammer,K., Pagani, M., Lachner, M., Kohlmaier, A., Opravil, S., Doyle, M., Sibilia, M., Jenuwein, T.,(2001) “Loss of the Suv39h histone methyltransferases impairs mammalian heterochromatin andgenome stability”, Cell 107, 323-37.

11. Schneider, R., Bannister, A. J., Kouzarides, T., (2002) “Unsafe SETs: histone lysinemethyltransferases and cancer”, Trends Biochem Sci 27, 396-402.

12. Hake, S. B., Xiao, A., Allis, C. D., (2004) “Linking the epigenetic 'language' of covalent histonemodifications to cancer”, Br J Cancer 90, 761-9.

13. Marks, P. A., Richon, V. M., Kelly, W. K., Chiao, J. H., Miller, T., (2004) “Histone deacetylaseinhibitors: development as cancer therapy”, Novartis Found Symp 259, 269-81; discussion 281-8.

14. Shabbeer, S., Carducci, M. A., (2005) “Focus on deacetylation for therapeutic benefit”, IDrugs 8,144-54.

15. Nguyen, C. T., Weisenberger, D. J., Velicescu, M., Gonzales, F. A., Lin, J. C., Liang, G., Jones, P.A., (2002) “Histone H3-lysine 9 methylation is associated with aberrant gene silencing in cancercells and is rapidly reversed by 5-aza-2'-deoxycytidine”, Cancer Res 62, 6456-61.

16. Seligson, D. B., Horvath, S., Shi, T., Yu, H., Tze, S., Grunstein, M., Kurdistani, S. K., (2005)“Global histone modification patterns predict risk of prostate cancer recurrence”, Nature 435,1262-6.

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18. Winter, G., Milstein, C., (1991) “Man-made antibodies”, Nature 349, 293-9.19. Cao, R., Zhang, Y., (2004) “The functions of E(Z)/EZH2-mediated methylation of lysine 27 in

histone H3”, Curr Opin Genet Dev 14, 155-64.20. Felsenfeld, G., Groudine, M., (2003) “Controlling the double helix”, Nature 421, 448-53.21. Jenuwein, T., Allis, C. D., (2001) “Translating the histone code”, Science 293, 1074-80.22. Iizuka, M., Smith, M. M., (2003) “Functional consequences of histone modifications”, Curr Opin

Genet Dev 13, 154-60.23. Lachner, M., O'Carroll, D., Rea, S., Mechtler, K., Jenuwein, T., (2001) “Methylation of histone H3

lysine 9 creates a binding site for HP1 proteins”, Nature 410, 116-20.24. Cheutin, T., McNairn, A. J., Jenuwein, T., Gilbert, D. M., Singh, P. B., Misteli, T., (2003)

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25. Bannister, A. J., Zegerman, P., Partridge, J. F., Miska, E. A., Thomas, J. O., Allshire, R. C.,Kouzarides, T., (2001) “Selective recognition of methylated lysine 9 on histone H3 by the HP1chromo domain”, Nature 410, 120-4.

26. Brehm, A., Tufteland, K. R., Aasland, R., Becker, P. B., (2004) “The many colours ofchromodomains”, Bioessays 26, 133-40.

27. Jones, D. O., Cowell, I. G., Singh, P. B., (2000) “Mammalian chromodomain proteins: their role ingenome organisation and expression”, Bioessays 22, 124-37.

28. Jacobs, S. A., Khorasanizadeh, S., (2002) “Structure of HP1 chromodomain bound to a lysine 9-methylated histone H3 tail”, Science 295, 2080-3.

29. Nielsen, P. R., Nietlispach, D., Mott, H. R., Callaghan, J., Bannister, A., Kouzarides, T., Murzin, A.G., Murzina, N. V., Laue, E. D., (2002) “Structure of the HP1 chromodomain bound to histoneH3 methylated at lysine 9”, Nature 416, 103-7.

30. Fischle, W., Wang, Y., Jacobs, S. A., Kim, Y., Allis, C. D., Khorasanizadeh, S., (2003) “Molecularbasis for the discrimination of repressive methyl-lysine marks in histone H3 by Polycomb andHP1 chromodomains”, Genes Dev 17, 1870-81.

31. Wadia, J. S., Dowdy, S. F., (2005) “Transmembrane delivery of protein and peptide drugs by TAT-mediated transduction in the treatment of cancer”, Adv Drug Deliv Rev 57, 579-96.

32. Hitt, M. M., Graham, F. L., (2000) “Adenovirus vectors for human gene therapy”, Adv Virus Res 55,479-505.

33. Pini, A., Viti, F., Santucci, A., Carnemolla, B., Zardi, L., Neri, P., Neri, D., (1998) “Design and useof a phage display library. Human antibodies with subnanomolar affinity against a marker ofangiogenesis eluted from a two-dimensional gel”, J Biol Chem 273, 21769-76.

34. Wysocka, J., Swigut, T., Milne, T. A., Dou, Y., Zhang, X., Burlingame, A. L., Roeder, R. G.,Brivanlou, A. H., Allis, C. D., (2005) “WDR5 associates with histone H3 methylated at K4 andis essential for H3 K4 methylation and vertebrate development”, Cell 121, 859-72.

35. Fischle, W., Wang, Y., Allis, C. D., (2003) “Binary switches and modification cassettes in histonebiology and beyond”, Nature 425, 475-9.

36. Daujat, Sylvain, Zeissler, Ulrike, Waldmann, Tania, Happel, Nicole, Schneider, Robert, (2005)“HP1 binds specifically to K26 methylated histone H1.4, whereas simultaneous S27phosphorylation blocks HP1 binding”, J. Biol. Chem., C500229200.


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14. If this is a RESUBMISSION, please outline below your responses to the reviewers’

comments provided to you from your previous submission. Your response should notexceed the space provided on this page. Nothing smaller than 12-point font should beused with margins no less than .75 inch. Page reduction is not allowed. Anythingexceeding this limit will be withdrawn from the competition. Please format this pageappropriately.

This grant is a resubmission of Project #21957 which was submitted to the May 01, 2005 PilotProjects Application Competition #4624. The original application received favorable and encouraginganonymous reviews and a final score of 3.7. Building upon the insightful suggestions of the anonymousreviewers has significantly strengthened this current grant application. In the paragraphs below, theaccompanying changes are explained in greater detail:

1. Regarding the committee’s concern about the timeframe for achieving the stated aims. Ihave attempted to more carefully demarcate the research to be undertaken in the Campbell researchgroup from research to be undertaken in the Hendzel research group (see comment 3 below). We feelthat the in vitro selection of H3Me3K9- and H3Me3K27-specific binding domains (Aims 1 and 2,respectively) can be undertaken simultaneously and completed within 1 year.

2. Regarding reviewer #1’s concern that there is no backup plan if HP1 chromodomains withthe requisite binding specificity are not found through phage display. The backup plan is to engineerrecombinant antibody fragments (single chain variable fragments or ScFv’s) with the requisitespecificity. The Campbell lab has the necessary expertise and reagents for generating antibodyfragments from cDNA libraries derived from the spleens of immunized animals. Yet another (distant)possibility would be to use the 7-bladed WD40 #-propeller protein, WDR5, as a scaffold from which to

create libraries of variants and select for high-specificity binders. WDR5 has recently been shown tospecifically bind to di- and tri-methylated H3K4 (34) but the peptide-binding site has not beenidentified. Until the binding site is characterized, attempting to change the binding specificity of WDR5would present a significant challenge.

3. Regarding reviewer #1’s concern that Aim 3 was not well described. Aim 3 describes thevalidation of the H3Me3K9-specific binding domains in live cells. This section has been removed fromthe body of the grant and is now described in greater detail in the ‘Collaboration’ section as well as inthe letter of support from Dr. Hendzel. The scope of the grant is now limited to the in vitro generationand characterization of the binding modules in the Campbell research group.

4. Regarding reviewer #1’s concern that the amount of peptide required was not specified. Inthe legend for Figure 3 I have included the requested experimental details.

5. Regarding reviewer #2’s suggestion that the importance of trimethylated H3K9 to cancershould receive more emphasis and that specific examples should be provided. I have strengthenedthe discussion of the relationship between abnormal patterns of histone modification and cancer. Inparticular, I emphasize the use of histone modifications in cancer diagnostics and prognostics.Furthermore, I have extended the scope of the proposal to cover the engineering of binding modules fortrimethylated H3K27, a modification that is implicated in cancers from a range of tissues.

6. Regarding reviewer #2’s request for further discussion about the specificity required, thepossibility that HP1 has undergone evolutionary selection for specificity, and the relationshipbetween in vitro and in vivo specificity. I have provided additional discussion in the proposal sectionstitled ‘HP1 chromodomain as a candidate molecular recognition module’ and ‘Long term objectives’.

7. Regarding reviewer #2’s request for further discussion about the possible role of post-translational modification of adjacent residues (specifically phosphorylation of serine 10) that mayinfluence the binding of HP1. The reagent(s) that we are proposing to develop will recognizeH3Me3K9 only if serine 10 is not phosphoylated. It has been proposed (and evidence is accumulating tosupport the proposal) that K9/S10 is a binary switch that can exist in two mutually exclusivetranscription states: a methylated ‘off’ state or a phosphorylated ‘on’ state (35,36). At this point we areinterested in detecting the methylated and silenced ‘off’ regions of heterochromatin. However, apossible future direction for this project would be to develop reagents for the detection oftranscriptionally active ‘on’ regions by recognition of phosphorylated H3S10.


September 15, 2005

15. Summarize briefly what your FUTURE PLANS are regarding the continuation of the

proposed research project and how this will be achieved (limit to half a page). If this is abridging grant application, indicate what the funds requested will be bridging from and to (e.g.NCIC to RIP).

The goal of this pilot project is to generate proof-of-principle results that will enable my collaborators

and I to secure longer-term funding from NSERC, CIHR, or NCIC. With support of this ACB Pilot

project grant, we will generate highly specific binding modules for trimethylated H3K9 and H3K27 and

validate the use of these domains in live cells. With these validated binding domains in hand, we will

have ‘opened-the-door’ to a wide variety of applications and experimental approaches that extend far

beyond the financial scope of this pilot grant. Financial support for the subsequent 3 to 5 years for these

ongoing experiments will likely come from one of the following team project grants: Collaborative

Health Research Projects (CIHR and NSERC), Team Grant Program (CIHR), Strategic Project Grants

(NSERC), or Program project grants (NCIC).

16. If applicable, comment on the NATURE OF THE COLLABORATION and the

specific role of each principal investigator with respect to the project (limit of one, single-spacedpage). Append letters of collaboration from all others who will be involved in the project (i.e.consultants)

All work on developing the binding modules (Specific Aims 1 and 2) will be done in the

Campbell research group. The subsequent stage of this project, (which is beyond the scope of this

grant) will be the functionalization of these domains in order to develop and optimize specialized

reagents that will be validated and applied in a wide range of experimental applications. This work will

be distributed between three collaborating laboratories that will develop and/or test reagents designed

for immunofluorescence applied to localization and function blocking in interphase (Dr. Michael

Hendzel), for chromatin immunoprecipitation (Dr. James Davie), and for in vivo fluorescence imaging

for the study of mitosis (Dr. Gordon Chan).

(i) Immunofluorescence and Immunohistochemistry in the Hendzel Research group. The

Hendzel Research group will be largely responsible for the in vivo validation of the specificity of the

engineered chromodomain variants. The critical test of specificity in live cells will be an in vivo

function blocking activity assay. Briefly, we will use immortalized mouse embryonic fibroblasts

obtained from mice lacking the suv39h1/h2 histone methyltransferases. These proteins are normally

responsible for trimethylating H3K9. A binding module that is specific for the trimethylation H3K9

will therefore not associate with pericentromeric heterochromatin sequences in this cell line. In

contrast, the matched wild type control cell line should show strong binding of the binding module. The

Hendzel laboratory will define these associations by quantifying the association with these domains by

localization and the affinity of binding by fluorescence recovery after photobleaching. It is anticipated

that the entire process of binding module creation and validation will be iterative, with the Campbell

laboratory continuing to refine the binding modules as the Hendzel laboratory tests them in vivo. In

subsequent work, they will employ these binding modules for quantification and intragenomic

localization analysis in fluorescence microscopy and histochemical applications in tumors isolated form

patients. The direct coupling of fluorescent tags for quantitative immunofluorescence and horseradish

peroxidase for application in histochemical staining of tissue sections will generate reagents with

distinct advantages relative to conventional antibody-based approaches.


September 15, 2005

(ii) Chromatin immunoprecipitation Experiments in the Davie Research group. For these

experiments, the binding module will be constructed as a bacterial expression vector using a cold-shock

promoter and an N-terminal or a C-terminal 6xhistidine tag. The purified reagent will then be mixed

with formaldehyde-crosslinked chromatin that has been sheered to an average of 500 bps in length of

DNA sequence and bound sequences will be isolated by metal affinity chromatography. The

immunoprecipitated DNA will be analyzed for enrichment in major satellite and telomere sequences.

(iii) In vivo chromatin binding specificity and affinity in the Chan Research group. The

Chan research group will use the mammalian Lumio Gateway expression for in situ protein labeling of

the recombinant binding module with a cell-permeable dye. The binding module will be transiently

transfected into cells, and subsequently fluorescently labeled with FlAsH or ReAsH, in order to

determine the localization, effects on mitotic progression, and its effect on the integrity of the mitotic

checkpoint. Through the use of various live cell fluorescence microscopy techniques, the effects of the

binding module on mitotic entry, mitotic chromosome condensation, centromere and kinetochore

protein dynamics, centromere tension, and mitotic checkpoint integrity will be examined. The effect of

overexpressing the binding module in live cells (and presumably blocking HP1 binding to trimethylated

H3K9) will be analyzed by immunofluorescence of various centromere/kinetochore assembly markers.

This work will build upon recent results from the Chan lab that demonstrate that centromere structure is

altered in suv39h1/h2 knockout cells (Chan, Rattner, and Hendzel, in preparation).


September 15, 2005

20. (a) NON-TECHNICAL SUMMARYThe ACB often needs to supply information to the media or other lay organizations. Please providea 150-200 word summary, in simple, non-technical language, which explains the rationale of the

investigation and the relevance to cancer.

The human genome is defined as the sequence of the 3 billion bases of DNA that is present within our

chromosomes. The technology to ‘read’ and ‘decode’ this sequence is very well established and due to

the efforts of the Human Genome Project, the complete genome of a few individuals has now been read

in its practical entirety. Although it is not common knowledge, it has become evident in recent years

that the proteins (the histones) that are associated with DNA carry an orthogonal code that is

established by enzymes that add small chemical groups at specific sites in these proteins. This "histone

code", or “epigenetic code’, is involved in determining which genes are expressed, where they are

expressed, and when they are expressed. It is now believed that alterations in this code account for at

least 50% of the changes that occur to generate human cancers. In this study, we propose to develop

tools that will enable the epigenetic content of the genome to be determined. These tools may have

immediate application in the clinical diagnosis and prognostic assessment of individual cancers.

(b) CASE FOR SUPPORTFunding for this program is made possible from donations to the Alberta Cancer Foundation.Expanding on the above summary, in 150-200 words, describe the reasons why a prospective donorshould consider supporting this project. Include tangible benefits the proposed initiatives have forstudents, faculty, staff, patients, Alberta, Canada and beyond. Describe the benefits of this initiativeto the donor.

Epigenetic changes are thought to be at least as important as genetic changes in the development of

human cancers. In contrast to the genetic code, the epigenetic code cannot be easily ‘read’ or ‘decoded’

with current technology. In this study, we propose to develop tools that will enable the epigenetic

content of the genome to be determined. These tools may have immediate application in the clinical

diagnosis and prognostic assessment of individual cancers. For example, it is not unreasonable to

expect that epigenetic differences between patients will be predictive of the responsiveness of patients

to epigenetic therapies once these drugs are in widespread use. They will also provide a tool that will

have a substantial impact upon our ability to study, and thereby expand our understanding of,

epigenetic processes. Genome biologists working to understand and catalogue the combinatorial

complexity of histone posttranslational modifications are faced with a monumental task but also a great

opportunity. Once it is fully understood, we may be able to exploit the epigenome to, for example,

reverse transform cancers.

(c ) RELEVANCE TO CANCERIn 150-200 words, describe the objective of the proposed research and its relevance to cancer.

Chemotherapeutic approaches that target epigenetic changes are in various stages of clinical trials and

are likely to emerge as a new wave of chemotherapies that may offer new hope for individuals

diagnosed with terminal cancers. The binding modules that we will generate will be candidates for

altering the epigenetic code through the combinatorial use of binding modules and functional domains

that can be targeted using the binding modules constructed in our studies. In addition, once gene

therapy methods have evolved sufficiently that they are generally applicable and successful in the

treatment of patients, these modules and the functionalization of these modules will offer a powerful

tool for the site-selective delivery of molecules that can directly impact on the function of

trimethylation of lysine 9 for therapeutic intervention. For example, we may be able to de-repress

epigenetically silences tumor suppressor proteins using this approach.

Documents

Bridge/Pilot/Limited Term Research Project Funding ...campbell//resources/REC_example_proposa… · HP1 chromodomain is a nearly ideal binding module for the proposed applications