Lab In The News
The Sarma Laboratory
The Sarma laboratory is interested in the mechanisms of epigenetic gene regulation, or how the dynamic modifications of the architecture of chromatin, the complex of DNA and proteins within the nucleus of our cells, impacts gene expression and cellular function. The lab investigates consequences of epigenetic alterations in neuronal cancers and neurodegenerative diseases using a combination of biochemistry, cell and molecular biology with genome wide approaches to gain mechanistic insight into how chromatin architecture is modified in disease. The goal is to identify new pathways and interactions that can be targeted to correct these epigenetic perturbations.
Anna Bieluszewska, Ph.D.
Phillip Wulfridge, Ph.D.
Qingqing Yan, Ph.D
We are interested in understanding the molecular mechanisms of RNA-mediated epigenetic gene regulation. Aberrations in epigenetic gene silencing can be a causal mechanism of numerous human diseases and developmental syndromes.
The eukaryotic genome is organized into chromatin, a mixture of histone proteins, DNA and RNAs (Figure 1). In recent years, RNAs have emerged as important factors that play critical roles in epigenetic gene regulation and also dictate chromatin architecture. It has also become clear that many protein factors that regulate gene expression interact with RNAs. Our goal is to elucidate the molecular mechanisms and functional implications of RNA interactions in gene regulation and in genome organization.
Our research has focused on an RNA binding chromatin remodeler called ATRX. ATRX is an epigenetic regulator that is mutated in both neurodevelopmental disorders and cancers. Despite its importance in normal development and maintenance of genome instability, how it contributes to these processes is not well understood. ATRX belongs to a unique class of proteins with the ability to bind the three major chromatin components – DNA, histones, and RNA. While its action on DNA and histones have been studied to some extent, the functional significance of its interactions with RNA remains largely unexplored. Our previous studies have shown that ATRX interacts with the Xist long non-coding RNA and plays an important role in the process of X chromosome inactivation. ATRX also regulates the association of the Polycomb repressive complex 2 (PRC2) with the inactive X chromosome as well as many polycomb gene targets genome-wide (Figure 2).
We have recently discovered an RNA binding region (RBR) within ATRX. We hypothesize that ATRX is initially recruited to heterochromatic regions through its interactions with RNAs that reside at these loci. ATRX is stabilized at these regions through both histone and DNA contacts and functions in transcription repression at these sites to maintain genome stability (Figure 3). Some questions that we are addressing include:
- What RNAs interact with ATRX?
- Do RNA interactions dictate ATRX genomic targeting in vivo?
- How does loss of RNA interactions affect ATRX function in gene regulation and genome stability?
Another area of investigation in the lab is the impact of RNA associations on chromatin structure. Here, we focus on triplex nucleic acid structures known as R-loops, which are comprised of a DNA:RNA hybrid and displaced ssDNA. R-loops are formed during transcription when the mRNA invades dsDNA (forming the DNA:RNA hybrid) and exposes a ssDNA that can then adopt a G quadruplex (G4) structure (Figure 4). Transcription from G-rich repetitive regions results in the formation of G4 DNA that impedes the reannealing of DNA strands, promotes DNA:RNA hybridization, and stabilizes R-loops. In addition to known regulatory roles, R-loops are closely linked to increased DNA damage and genome instability. Stable aberrant R-loops have also been discovered in several neurological disorders, neurodegenerative diseases, and cancers.
Discovering the genome-wide locations of R-loops is challenging because of the requirement for large sample size and inefficient enrichment using the monoclonal antibody that recognizes the RNA:DNA hybrid within R-loops. We have developed a new antibody-independent approach, called MapR, to identify native R-loops genome-wide. Some questions that we are interested in exploring are:
- Where do R-loops form in specific disease states?
- How do unscheduled R-loops contribute to neurodegenerative diseases and cancers?
- What are the protein factors that function in R-loop resolution and stabilization?
- How can R-loops impact gene regulation and genome organization in disease states?
For our studies we use a combination of biochemical, cell biological and functional genomics approaches in embryonic stem cell, neural stem cell, and cancer cell models.
Wulfridge, P., Sarma, K. "A nuclease- and bisulfite-based strategy captures strand-specific R-loops genome-wide." Elife. 2021 Feb 23;10:e65146. doi: 10.7554/eLife.65146.
Ren, W., Medeiros, N., Warneford-Thomson, R., Wulfridge, P., Yan, Q., Bian, Y. Sidoli, S., Garcia, B.A., Skordalakes, E., Joyce, E., et al. "Disruption of ATRX-RNA interactions uncovers roles in ATRX localization and PRC2 function." Nat Commun. 2020 May 6;11(1):2219. doi: 10.1038/s41467-020-15902-9.
Yan. Q., Sarma, K. "MapR: A Method for Identifying Native R-Loops Genome Wide." Curr Protoc Mol Biol. 2020 Mar;130(1):e113. doi: 10.1002/cpmb.113.
Yan, Q., Shields, E., Bonasio, R., Sarma, K. "Mapping native R-loops genome-wide using a targeted nuclease approach." Cell Rep. 2019 Oct 29;29(5):1369-1380.e5. doi: 10.1016/j.celrep.2019.09.052.
Sarma, K., Cifuentes-Rojas, C., Ergun, A., Del Rosario, A., Jeon, Y., White, F., Sadreyev, R., Lee, J.T. "ATRX directs binding of PRC2 to Xist RNA and Polycomb targets." Cell. 2014 Nov 6;159(4):869-83. doi: 10.1016/j.cell.2014.10.019.
Joseph Salvino, Ph.D.
Professor, Molecular & Cellular Oncogenesis Program, The Wistar Institute Cancer Center
Scientific Director, Molecular Screening & Protein Expression Facility