Following are public and technical abstracts for the SETD2 project funded by the Department of Defense Kidney Cancer Research Program (KCRP) for 2017.
Principal Investigator: Durga Tripathi
Institution: Baylor College of Medicine
Funding Mechanism: Concept Award
Award Amount: $118,861
Public Abstract
Background: According to the American Cancer Society annual report, there are more than 60,000 new cases of kidney cancer and more than 14,000 deaths from this disease every year in the United States. Chromatin remodelers are proteins that determine how DNA and its associated proteins (chromatin) are packaged, remodeled, and interpreted for the expression of genes. Genes encoding chromatin remodelers such as SETD2, PBRM1, and SMARCC1 are among the most frequently mutated, or otherwise inactivated, in kidney cancer. Our group has recently discovered that the chromatin remodeler SETD2 has two important functions in the cell, one of which we were already aware (chromatin remodeling) and a surprising new one remodeling the cytoskeleton by serving as a “writer” that places a mark on the cytoskeleton. The cytoskeleton maintains the shape and structure of the cell and controls key aspects of cancer biology, such as the integrity of the genome and cell movement, an important component of metastasis. Therefore, in order to fully understand and target chromatin remodeler defects in kidney cancer, we will need to focus on their effects on both chromatin and the cytoskeleton. Toward this goal, we must identify who “reads” (and how) the mark placed by SETD2 on the cytoskeleton. Both PBRM1 and SMARCC1 are candidate “readers” because they contain the type of molecular domains that would be expected to interact with the SETD2 mark.
Hypothesis: We hypothesize that PBRM1 and/or SMARCC1 are the readers of the mark placed by SETD2 on the cytoskeleton. To test this concept and potentially identify other candidate “readers,” we propose the following experiments:
Use biochemical and genetic approaches to determine if PBRM1 and/or SMARCC1 recognize and bind to the SETD2 mark on the cytoskeleton.
Perform an unbiased screening to determine if other proteins that are often mutated in kidney cancer contain molecular domains that can recognize and interact with the SETD2 mark on the cytoskeleton.
Identify the exact areas within these proteins that are necessary and sufficient to specifically recognize the SETD2 mark on the cytoskeleton.
Innovation: The effect of chromatin remodelers on the cytoskeleton is poorly understood. The proposed project will address the innovative hypothesis that these genes have a dual role on both chromatin and the cytoskeleton. In addition, from a technical standpoint, we will use an innovative, unbiased approach taking advantage of protein-domain microarray technology to screen for candidate “readers” of the SETD2 mark on the cytoskeleton.
Impact: This project will generate crucial data, which would otherwise be lacking, that can guide future efforts to develop targeted therapies for kidney cancer. Even if our hypothesis is incorrect, the proposed experiments will provide valuable information on how defects in chromatin remodeler genes found in kidney cancer can affect the biology of cancer cells.
Technical Abstract
Background: Defects in chromatin remodeler genes encoding the “readers, writers, and erasers” of the epigenome have emerged as an important new class of drivers of RCC. These chromatin remodelers include genes such as PBRM1 (a “reader” in the SWI/SNF ATPase complex) and SETD2 (a “writer” of histone methyl marks), which are the second and third most commonly mutated genes, respectively, in RCC. Components of the SWI/SNF chromatin remodeling complex are mutated in 20% of all cancers and 40% of kidney cancers, with PBRM1 and SMARCC1 the most frequently mutated SWI/SNF subunits in clear cell RCC. These “readers, writers, and erasers” of histone methyl marks control key chromatin features including conformation, integrity, and transcription. Recently, a novel discovery from our group revealed that one of these, the “writer” SETD2, methylates the cytoskeleton apart from chromatin, and this methyl mark on microtubules is required for proper chromosome segregation during mitosis, with defects in methylation causing genomic instability. This finding opens an entirely new area for investigation in RCC exploring how both chromatin and cytoskeletal defects caused by alterations in chromatin remodelers drive oncogenesis.
Hypothesis and Objective: A key knowledge gap for the field is identification of “readers” for the SETD2 methyl mark on microtubules, and understanding how defects in chromatin remodelers linked to kidney cancer influence key cytoskeletal drivers for oncogenesis, such as migration, invasion, and genomic instability. Filling this knowledge gap will uncover new ways that chromatin remodeler defects drive RCC via cytoskeletal alterations. To begin to fill this knowledge, we will test the hypothesis that SWI/SNF complexes (and possibly others) are dual-function chromatin and cytoskeleton remodelers that “read” methyl marks on microtubules to remodel the cytoskeleton.
Specific Aims: (1) Test the hypothesis that PBRM1 and/or SMARCC1 recognize the SETD2 methyl mark on microtubules (alpha-TubK40me) and direct the SWI/SNF complex to these cytoskeletal elements. (2) Using unbiased methyl-domain binding arrays, determine if other proteins with methyl binding domains specifically recognize alpha-TubK40me3 of microtubules and exhibit defects in RCC. (3) Identify the specific domains in methyl “readers” that recognize/bind methyl marks and determine if the ATPase (or other catalytic) activity is required for activity at microtubules.
Study Design: To identify “readers,” we will use both a targeted approach and unbiased screening of methyl-domain binding protein microarrays to find candidate methyl “reader” proteins. For our targeted approach, we have selected two candidate proteins, PBRM1 and SMARCC1, which are both part of the SWI-SNF complex, and which contain methyl-binding BAH and chromo domains, respectively; both PBRM1 and SMARCC1 are frequently mutated in RCC. We will interrogate PBRM1, SMARCC1, and others identified in our unbiased screen, binding to methylated microtubules using biochemical and genetic approaches in a panel of normal kidney cells (HKC) and kidney cancer cell lines (786-O and RCC4). Further, we will introduce cancer-associated pathogenic mutations into methyl-binding domains of PBRM1 and SMARCC1 to identify domains required for microtubule function, using genomic stability (mitotic chromosome abnormalities and micronuclei) as readouts.
Innovation: The proposed concept that SWI/SNF chromatin remodelers “read” this new methyl mark of microtubules is conceptually novel as this complex has never been assigned a cytoskeletal function. If correct, this would be the basis for a major conceptual shift in the fields of both chromatin and cell biology. Importantly, this would be the first demonstration of convergence at the cytoskeleton for multiple chromatin remodeler defects (SETD2, PBRM1, SMARCC1, and possibly others) in RCC.
Impact: Knowledge of remodeling activity at the cytoskeleton of chromatin remodelers that drive kidney cancer is critical for understanding how defects in SWI/SNF (and others) drive RCC, and for the development of effective therapies targeting both chromatin and cytoskeleton alterations associated with high-frequency chromatin remodeler defects in RCC.