Research

nuc_dynamics-01Nucleosomes are constantly disrupted and reformed throughout our genome, shaping the accessible DNA landscape. Nucleosomes form due to multiple strong contacts between the histone octamer and 147 bp of DNA. I want to understand how these contacts break and reform during critical processes like transcription and replication.

I also want to understand how DNA-binding proteins and chromatin remodelers modify nucleosome structure to modulate DNA accessibility.

I am interested in these questions in the context of the cell’s nucleus and currently, I am using biochemistry and genomics to probe these questions.

Another aspect of the nucleosome landscape that I am interested in is memory. Chromatin states persist through dilution during replication and disruption during transcription. How is this cellular memory maintained? There are several elegant models for cellular memory of chromatin states, which I want to test by developing experimental methods that track chromatin states temporally.

Some of my published work on chromatin dynamics:

Mapping In vivo Nascent Chromatin using EdU and sequencing (MINCE-seq)

mince_schematic-01

Passage of the replication fork disrupts every nucleosome in the genome. It had been believed that nucleosomes reassemble in the same position following replication fork passage. However, the chromatin landscape after fork passage was never mapped. To map the newly replicated chromatin landscape, we developed Mapping In vivo Nascent Chromatin with EdU and sequencing (MINCE-seq). MINCE-seq can generate a high-resolution map of newly replicated and maturing chromatin without the need for cell sorting or synchronization. Using MINCE-seq, we discovered that nucleosomes replace transcription factors at active promoters and enhancers genome-wide after the passage of the replication fork in Drosophila cells. The characteristic nucleosome landscape emerges from a uniformly packaged genome post-replication by the action of TFs, RNA-Polymerase II, and chromatin remodelers after replication fork passage. This study showed that the distinctive chromatin landscapes that reflect cellular identity are erased genome-wide during replication and need to be reestablished post-replication every cell cycle.

  1. Ramachandran, S., Henikoff, S. “Transcriptional regulators compete with nucleosomes post-replication”, Cell, 165(3):580-92 (2016) pubmed     journal
  2. Ramachandran, S., and Henikoff, S. “Replicating nucleosomes”, Science Advances 1, e1500587 (2015)  pubmed    journal

 Nucleosome dynamics during chromatin remodeling in vivo

rsc_model_gradient-01For transcription factor binding and for transcription to take place, nucleosomes would have to be disrupted and reformed at active regions of the genome. However, little is known about how the nucleosome structure is altered during these processes. We constructed a molecular model for H4-S47C-anchored chemical cleavage mapping, which was originally developed to determine nucleosome positions genome-wide at base-pair resolution. Applying my molecular model to genome-wide H4-S47C-anchored chemical cleavage data, we identified nucleosomes with asymmetric histone-DNA contacts that were highly enriched at positions proximal to promoters. Using a combination of H4S47C-anchored cleavage mapping, MNase cleavage mapping, and ChIP-seq, we determined that the asymmetric loss of histone-DNA contacts was due to the formation of a nucleosome-remodeler complex that featured partial unwrapping and protection of nucleosomal DNA during chromatin remodeling. This study demonstrated the ability to map alternate nucleosome structures formed during active processes in vivo.

  1. Ramachandran, S., Zentner G. E., Henikoff, S. “Asymmetric nucleosomes flank promoters in the budding yeast genome”, Genome Research, 25(3):381-90 (2015) pubmed     journal
  2. Ramachandran S., and Henikoff, S. “Nucleosome dynamics during chromatin remodeling in vivo”, Nucleus 7(1):20-6 (2016)  pubmed    journal