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.
Method development and modeling
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.
To interpret the data we collect, we are also developing in silico simulations to model the interaction of radiation-generated hydroxyl radicals with chromatin and infer chromatin conformation ensembles from RICC-seq data.
X Chromosome Inactivation
One of the two X chromosomes in female mammalian cells is stably inactivated by chromatin-based mechanisms, silencing transcription of most, but not all, of its genes. Reactivation of inactive X chromosomes is a feature of some cancers, but partial reactivation is also a promising avenue toward therapies for some genetic diseases.
Using epigenomic methods including RICC-seq, ATAC-seq and microscopy, we are working to understand how local interactions between nucleosomes change when a whole chromosome is silenced, dissect the contribution of protein complexes involved in X inactivation to such architectural differences, and understand how short-range chromatin folding contributes to transcriptional silencing.
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.