Research Overview

The nearly two meters (six feet) of DNA making up the genome of every human cell is packaged by nucleosomes into chromatin. Chromatin not only physically compacts the DNA by wrapping it and helping to neutralize its negative charge, but also helps to organize it into functional compartments. Nucleosomes can be chemically modified in a way that is thought to define functional states, turning genes “on” and “off” in terms of their transcription from DNA to RNA, the first step of gene expression. Nucleosomes also interact with each other, creating more and less compact states of chromatin that are known to be associated with different transcriptional states. At longer length scales, regulatory regions of the genome like enhancers interact with their target genes to drive transcription, through a mechanism that is not yet well understood. Their associations are circumscribed by long-range organization of the genome into topological domains and compartments.

We probe this structural complexity of chromatin using DNA sequencing-based assays, in vitro reconstitution, microscopy, and modeling. By focusing on chromatin architecture as a phenotype that is proximal to the molecules that reshape chromatin to control transcription and other genome functions, we hope to understand how the many concurrent molecular processes that reshape chromatin work together.

Probing short-range chromatin folding

The high density of chromatin in the cell nucleus presents a major experimental challenge, making it difficult to probe the conformation of chromatin at short length scales and creating a molecular environment that is difficult to replicate with the dilute solutions that are amenable to in vitro manipulation. RICC-seq (Radiation-Induced Correlated Cleavage with sequencing) is a new method that can probe DNA-DNA contacts in intact or fixed cells using ionizing radiation. It is orthogonal to enzyme-based methods for probing chromatin structure and minimally perturbative to the nuclear environment.

We use RICC-seq in combination with a variety of other epigenomic and light microscopy-based technologies to probe the conformation of chromatin in cells as the cells undergo key developmental transitions or stress responses. ATAC-seq is a method for quantifying the exposure of genomic DNA to binding by exogenous proteins and is effective in identifying putative regulatory regions. ChAR-seq is a recently developed method for mapping all RNA-DNA contacts in the genome simultaneously.

Methods development

We are developing new DNA sequencing-based methods to target RICC-seq to subsets of the genome and increase coverage at single loci. We are also working on data analysis pipelines for RICC-seq data.

Computational methods

We utilize computational simulations in order to validate findings from RICC-seq experiments via integration of genomic data with simulated snapshots. These findings will be used to determine the conformational space of chromatin for various epigenetic states, which will explain how changes in these states can give rise to longer-range chromatin interactions.

We are integrating this work with nanoscale simulations of heavy-ion irradiation in collaboration with NASA JSC, funded by a HERO grant from the NASA Human Research Program.

Molecular regulators of chromatin architecture

Using epigenomic methods including RICC-seq, ATAC-seq, ChAR-seq and microscopy, we are working to understand how local interactions between nucleosomes, long-range chromatin looping, and chromatin-RNA interactions change are regulated by the essential histones linker histone H1 and macro-H2A.

Our work on macroH2A is founded by a Rita Allen Scholar Award from the Rita Allen Foundation.

Chromatin organization, cell cycle exit, and robustness of the epigenome

We are interested in understanding how the organization of chromatin in the nucleus changes when cells exit the cell cycle in response to stressors like DNA damage or cell cycle inhibitors used to treat certain cancers.

Liposarcoma is a cancer that affects approximately 1000 new people per year in the United States and primarily targets adults over the age of 50. Although some cases are successfully cured with surgery if caught early, patients traditionally had few options if the cancer came back or if surgery did not eradicate it, because standard chemotherapy and radiation therapy were not effective. A new class of drugs called CDK4/6 inhibitors has recently begun to change the prospects of these patients. These drugs stop cancer cells from dividing without killing them. In some patients, the same drugs cause the cells to enter what is called senescence: the cells never resume dividing, even when the drug is removed. Senescence normally occurs in cells whose DNA has been damaged, so this exciting new form of senescence called SAGA (senescence after growth arrest), that is triggered by a CDK4/6 inhibitor, is not as well understood. In collaboration with Andrew Koff at MSKCC, we are using our mapping tools to study how the structure of the genome in liposarcoma cells helps cells to decide whether to enter or stay in SAGA, what genes to turn on, and how we might control these genes using other drugs that can be combined with CDK4/6 inhibitors.

This work is funded by a V Scholar Grant from the V Foundation for Cancer Research.