Project 2: Molecular Modulation of Chromatin and Nuclear Structure in Cancer
Ross Levine, Medicine, Memorial Sloan Kettering Cancer Center
Job Dekker, Biochemistry and Molecular Pharmacology, University of Massachusetts Medical School
Lucy Godley, Medicine, University of Chicago
This project seeks to elucidate the role of enhancer dysfunction in modulating the spatio-temporal organization of chromatin and information transfer in cancer. Genome sequencing efforts in recent years have revealed the presence of recurrent somatic mutations in epigenetic regulators in myelodysplasia (MDS) and acute myeloid leukemia (AML), such as loss of function mutations in EZH2 and TET2. The effects of these mutations on chromatin structure and function are beginning to be elucidated. Meanwhile with the advent of the ENCODE data and genome wide chromatin precipitation-sequencing studies it has become apparent that a large fraction of gene regulatory events are governed at enhancer sites located tens to hundreds of kilobases away from the start site of transcription. Such sites are brought into apposition to gene promoters to stimulate gene expression through the formation of chromatin loops. More recently, genomic surveys showed the presence of mutations in proteins linked to enhancer function, such as loss of function mutations of MLL3 (found within the common chromosome 7q36.1 deletion region in MDS and AML). These enzymes encode histone methyltransferases that lead to the monomethylation of histone 3 lysine 4, a modification characteristic of enhancers. p300, a histone acetyltransferase that acetylates these enhancers is mutated in MDS as well. Mutation of TET2 in MDS signifies the importance of the hydroxymethylation modification, which our data indicate is present in enhancer sequences. Finally the cohesin complex allows promoters and enhancers to loop together, facilitating gene expression. Mutations in the genes of the cohesin assembly such as SMC3, STAG2, and RAD21 are found in MDS and AML. Collectively these data suggest that a subset of cases of AML and MDS suffers from enhancer dysfunction.
We hypothesize that enhancer dysfunction leads to an altered physical state of chromatin, including an abnormal state of 3-D looping, leading to aberrant formation of transcriptional, initiating and elongating complexes and destroying the normal regulation of gene expression in the cell. This leads to the ineffective hematopoiesis and clonal expansion characteristic of MDS precursors. We will use emerging CRISPR technology, advanced imaging (PWS/STORM microscopy) to visualize and monitor chromatin loop formation in response to oncogenic stimuli. We will use sophisticated chromatin confirmation capture and bioinformatics methods to create models of the physical state of chromatin in response to loss of key chromatin regulators. Genome wide chromatin surveys will elucidate the aberrant structure and function of chromatin in hematologic malignancies. Our work on the functional consequences of changes in chromatin structure at the kilobase-scale will interlock with an understanding of how ion concentrations alter chromatin structure and how folding mediated by condensins and cohesins affects chromatin structure and function at the megabase and chromosomal scale. We will use genetically edited cell models, genetically faithful animal models of AML and human specimens in xenografts to cross validate our work. Finally we will determine if aberrant chromatin configurations lead to enhanced susceptibility to epigenetically targeted drugs.
Our Specific Aims will ask:
- What are the biological effects of MLL3 or cohesin inactivation?
- Create genetically matched cell systems to examine the effects of inactivation of MLL3 and SMC3 on chromatin using CRISPR
- Measure the effects of MLL3 and SMC deficiency on chromatin stability, DNA damage response and epigenetic agents
- Does loss of function mutations in enhancer chromatin modifiers and the cohesin complex lead to altered 3D-chromatin conformations? How does this lead to aberrant gene expression?
- Determine the effects of inactivation on 3D chromatin looping on a genome-wide basis as ascertained by high throughput chromosome conformation capture
- Examine changes in histone modifications and DNA hydroxymethylation at enhancers and promoters and correlate with altered gene expression using next gen sequencing technologies
- Visualize loops in wild-type and MLL3/SMC3 mutant cells using a CRISPR- mediated locus tagging strategy
- How critical are changes in the chromatin loopscape in vivo? How does aberrant loop formation correlate with the development of disease in vivo?
- Analyze chromatin looping and modification during normal myelopoiesis using normal human CD34+ cells
- Analyze the loopscape of human tumors grown as xenografts
- Determine whether murine Mll3 and Smc3 loss/haplosufficiency collaborates with signaling mutations such as FLT3-ITD to generate tumors and how this relates to productive chromatin loops.
- Determine if MLL3 or cohesin mutations sensitize cells to EZH2 inhibitors.