How To Write a Proposal

How To Write a Proposal

Your proposal should be organized as follows:

Title: Should be Concise and relate to your hypothesis.

“Investigating the Role of BRCA2 in Telomere Replication and Homology-Directed Repair”

Table of Contents: List all sections and correct page number

Abstract: The abstract should summarize your proposal. Include one sentence to introduce the problem you are investigating, why this problem is significant, the hypothesis to be tested, a brief summary of experiments that you wish to conduct and a single concluding sentence. An abstract should not have any cited material, that is it should be all in your own words. There are no figures or tables that should be included in your abstract.

“The correct function of telomeres is necessary for genomic integrity and is dependent on their proper replication and their protection by the shelterin complex. Defects in shelterin cause telomeres to be recognized as double-stranded breaks (DSBs) leading to the activation of DSB repair pathways including the homologous recombination (HR) and non-homologous end-joining (NHEJ) pathways. This can cause the formation of tumorigenic chromosome fusions. The breast cancer susceptibility protein BRCA2 is a necessary component of the HR pathway of DSB repair, but its role in HR at dysfunctional telomeres has remained largely unstudied. BCRA2 has additional roles in the stabilization and processing of stalled replication forks, although the mechanistic details of this are unclear. Telomeres present a major challenge to replication machinery due to their propensity to form complex secondary structures, and several HR proteins have been implicated in their proper replication. Here, we propose an investigation into the role of BRCA2 in HR at dysfunctional telomeres and in the proper replication of telomeric regions in response to replication stress. We hypothesize that BRCA2 depletion will inhibit the formation of HR-mediated chromosomal aberrations in the context of telomere dysfunction. We also predict that BRCA2 will be important in the restart of stalled replication forks at telomeres.”

Introduction: The introduction discusses the background and significance of the problem you are investigating. Lead the reader from the general to the specific. For example, if you want to write about the role that Brca1 mutations play in breast cancer pathogenesis, talk first about the significance of breast cancer as a disease in the US/world population, then about familial breast cancer as a small subset of breast cancers in general, then about discovery of Brca1 mutations in familial breast cancer, then Brca1’s normal functions in DNA repair, then about how Brca1 mutations result in damaged DNA and onset of familial breast cancer, etc. Definitely include figures with properly labeled text boxes (designated as Figure 1, Figure 2, etc) here to better illustrate your points and help your reader wade through unfamiliar science.Each aim (a total of three aims) should include background material so the reader understands what you are basing your hypothesis and aims on.

Hypothesis: Formulate a hypothesis that will be tested in your grant proposal. Remember, you are doing hypothesis-driven research so there should be a hypothesis to be tested! The hypothesis should be focused, concise and flow logically from the introduction.

“We propose an investigation to clarify the role of BRCA2 in HR at dysfunctional telomeres and elucidate its mechanistic role in telomere replication. We hypothesize that BRCA2 depletion will inhibit the recruitment of RAD51 to dysfunctional telomeres and decrease the frequency of HR-mediated chromosomal aberrations. We also expect BRCA2-depleted cells to exhibit sensitivity to replication stress-inducing agents, reduced fork regression, and delayed fork restart.

Our findings could potentially reveal two context-specific roles for BRCA2 in oncogenesis: as a promoter of genome instability that mediates chromosome fusion when shelterin is defective, and as a guardian of chromosome stability that prevents telomere replication defects.”

Based on your hypothesis, your Specific Aims section should be geared to support it. The hypothesis is stated in one sentence in the proposal.

Aims, Rationale, Methods, Predicted Outcome, Potential Pitfalls and Alternative Strategies:In the grading rubric this comes under Method/Specific Aims and Expected results with discussion for each aim.

Specific Aims (listed as Specific Aim 1, Specific Aim 2etc…): This is where you will want to work with your mentor to craft the experimental portion of your proposal. Propose three original specific aims to test your hypothesis. In the example presented, Specific Aim 1 might be “To determine the oncogenic potential of Brca1 null cell lines expressing wild type Brca1 cDNA”. Specific aim 2 might be “To determine the metastatic potential of Brca1 null cells that express WT Brca1”. You do not have to go into extensive technical details, just enough for the reader to understand what you propose to do. The best aims yield mechanistic insights-that is, experiments proposed address some mechanisms of biology. A less desirable aim proposes correlative experiments that does not address mechanistically how BRCA1 mutations generate cancer. It is also very important that the two aims are related but NOT interdependent. What this means is that if Aim 1 doesn’t work, Aim 2 is not automatically dead. For example, say you propose in Aim 1 to generate a BRCA1 knockout mouse model, and in Aim 2 you will take tissues from this mouse to do experiments. If knocking out BRCA1 results in early embryonic death, you will never get a mouse that yields tissues for Aim 2. You can include some of your data here as “Preliminary data”. Remember to carefully cite all your sources.

Do not forget to include data interpretation of your predicted results.

Potential pitfalls and alternative strategies for each aim: This is a very important part of any proposal. This is where you want to discuss the experiments you propose in your aims. Remember, no experiment is perfect. Are there any reasons why experiments you proposed might not work? Why? What will you do to resolve this? What are other possible strategies you might use if your experiments don’t work?

“Aim 1: Investigating the Role of BRCA2 in Homologous Recombination at Dysfunctional Telomeres

Rationale: BRCA2 is known to be required for the recruitment of Rad51 and the stabilization of the Rad51 filament at genomic DSBs. This is a critical step for HR-mediated repair of DSBs. We expect that BRCA2 will play a similar role in promoting aberrant HR at dysfunctional telomeres with shelterin defects. 

We will deplete BRCA2 expression in an HCT116 human colorectal carcinoma cell line by stably expressing a short-hairpin RNA (shRNA) against BRCA2 using retroviral transduction. We will then induce telomere dysfunction by overexpressing TPP1ΔRD, which is a dominant-negative defective shelterin component that cannot recruit POT1, leading to ssDNA damage signaling at telomeres that causes robust HR activity (Guo et al. 2007). 

We will then use Immunofluorescence-Fluorescence in-situ hybridization (IF-FISH) to evaluate the telomeric localization of BARD1, BRCA2, and RAD51. BARD1 is the stabilizing binding partner of BRCA1, which is involved in the 5’ resection stage of HR upstream of BRCA2 and RAD51. It therefore serves as an internal control that should not be affected by BRCA2 depletion. We expect the shBRCA2-expressing groups to exhibit lower localization of RAD51 due to the decreased BRCA2 expression levels. Telomeres will be visualized using a telomeric peptide nucleic acid (PNA) probe. 

In addition, we will use chromosome-orientation FISH (CO-FISH) on metaphase spreads to look at the effects of BRCA2 depletion on the frequency and nature of chromosomal aberrations in the TPP1ΔRD-expressing cells. We expect the BRCA2-depleted cells to exhibit a lower frequency of chromosome end-to-end fusions due to decreased HR activity. In addition, we predict that BRCA2 depletion will increase the frequency of the Multiple Telomeric Signals (MTS) phenotype. MTS are indicative of telomere fragility due to spontaneous replication stress (Sfeir et al. 2010). Due to its putative role in replication fork stability, we expect BRCA2 to suppress this phenotype. Finally, we predict the BRCA2-depleted cells to exhibit a lower frequency of telomere sister-chromatid exchanges (T-SCEs), which are indicative of crossover HR events at telomeres.

Potential Pitfalls and Alternative Strategies: There is a possibility that the BRCA2 depletion may be lethal to the cells and cause rapid apoptosis before Tpp1ΔRD can be expressed. We do not expect this to be the case since BRCA2-knockout cell lines are routinely used in the literature (FradetTurcotte et al. 2016). If this occurs, however, we could attempt to use a cell line other than HCT116, or to use a conditional knockout BRCA2 cell line and induce BRCA2 knockdown after Tpp1ΔRD expression. In addition, we may experience double-labelling issues in the CO-FISH experiment that prevent chromosome orientation from being distinguished. If this is the case, a different cell line could be used (such as U2OS). Alternatively, a PNA-FISH could be used that allows chromosomes and telomeres to be visualized but would not provide data on T-SCEs”

Timetable: Prediction for the completion of your project.

References: Cite all references, including published data in the body of your proposal. If it is not your original idea, then you must cite!!

Appendix: This is where your data figures and tables will go.