Within the nucleus, DNA is packaged into chromatin states that vary in accessibility. Importantly, accessibility allows DNA binding proteins such as transcription factors, and repair and replication proteins to contact the DNA fiber. Chromatin regulators set up, maintain, and reverse chromatin states thereby controlling gene expression, chromatin structure, and genome fidelity. Understanding the mechanisms by which chromatin states are modulated is medically significant because approximately 50% of tumors have alterations in chromatin regulators. The family of chromatin regulators that is the focus of this proposal, the mammalian SWI/SNF family (also known as BAF complexes), is mutated in 20% of all tumors. BAF complexes are combinatorially assembled complexes containing 12-15 subunits encoded by 29 genes. In mammals there are 3 distinct major forms: canonical BAF (cBAF), Polybromo-associated BAF (pBAF), and GLTSCR-associated BAF (gBAF). All three share common subunits but also contain unique subunits. Of the three major BAF subtypes, the cBAF complex has the best understood role in chromatin regulation. However, much less is known about the mechanisms and function of the pBAF complex, including how common cancer mutations or subunit deletions affect the complex. A multitude of cancer genomics studies shows that alterations in cBAF and pBAF are frequent, but largely occur in cancers of different tissue origins suggesting divergent roles. On a genome-wide level cBAF most often acts at promoters and enhancers to open up chromatin leading to a transcriptionally competent locus. While pBAF is found at enhancers and promoters, it is also found along gene bodies. pBAF localization correlates with both transcriptional activation and to a larger extent than cBAF to transcriptional repression. In contrast to cBAF, pBAF may work cooperatively with polycomb during DNA repair, however the generality of this in other cell processes remains unknown.
Polycomb proteins act to oppose transcription, through histone modifications, recruitment of other proteins, and local chromatin organization changes. While the cBAF complex has many different roles in the cell, recent studies of cBAF mechanism have shown that one of the primary roles is to oppose polycomb proteins, thereby creating a more accessible locus. In most cases accessibility alone is not sufficient for transcriptional activation. However, I found that cBAF recruitment using a Cas9 based chemical induced proximity approach (FIRE-Cas9) is sufficient for transcriptional activation of the mouse Nkx2-9 locus. I can now use Nkx2-9 as a model locus of chromatin and transcription changes just minutes after inducible SWI/SNF recruitment. While it is known that cBAF and/or gBAF complexes opposes polycomb when recruited to Nkx2-9, what is not known is how recruitment of pBAF affects polycomb, the chromatin state in general or the transcriptional state of Nkx2-9.
My long-term goal is to define the mechanistic differences between the mSWI/SNF pBAF and cBAF/gBAF complexes in regard to polycomb localization, histone modifications, chromatin accessibility, transcription, and genome stability. My overall objective is to understand how the similar but distinct BAF complexes act through unique mechanisms mediated by specific subunits. Knockout of different individual subunits in mSWI/SNF complexes leads to disparate transcriptional and polycomb changes across the genome. This observation led to my central hypothesis that oncogenic alterations in subunits of similar complexes contribute to disease through distinct mechanisms. The rationale for the proposed research is that once we know mechanistic differences between similar complexes, we will have a better understanding of oncogenic mechanisms and be able to better predict points of pharmacological attack in order to restore a more normal balance of chromatin states.
I plan to test my central hypothesis by pursuing the following two specific aims:
1) Characterize genomic alterations after acute loss of the pBAF complex.
2) Compare the kinetics and mechanisms of cBAF vs. pBAF mediated chromatin changes.
With respect to outcomes, I expect the differences in mechanism between these similar, but divergent complexes will lead to a better understanding of alterations in chromatin structure that lead to an oncogenic state. This will have a positive translational impact because a better understanding of the relationship between chromatin regulators, cancer subtypes, and chemotherapeutic regimes will be important for better patient responses and outcomes. This Award will provide me the resources and training necessary to compete independently for NIH individual research grants and other external peer-reviewed support.
Jake Kirkland, Ph.D.
Assistant Member Cell Cycle and Cancer Biology