Research Interest
Despite coordinating incredible morphological complexity, developmental patterning is remarkably robust. We are interested in uncovering the properties that allow complex biological processes, like development, to occur so reproducibly. One attractive system to study these ideas is the production of flat leaf architecture. The leaves of many species emerge from the stem cell niche as radially symmetric bumps, then develop into long and wide, but very shallow, structures. Leaves have solved this difficult biological problem by using the boundary between their dorsal (adaxial or top) and ventral (abaxial or bottom) sides as a guide to orient their growth. Ensuring the dorsoventral axis is rigorously specified and maintained is thus key to the robust nature of flat leaf production. We exploit the complex, gene regulatory network underlying dorsoventral patterning to assess the determinants – and their interactions – that lead to robust developmental outcomes in multicellular organisms.
A parallel but overlapping project involves the CLASS III HOMEODOMAIN LEUCINE ZIPPER (HD-ZIPIII) proteins. This ancient family of transcription factors arose at least 700 million years ago, and was repeatedly co-opted to drive several evolutionarily-important innovations, including flat leaf production, stem cell maintenance, and vascular patterning. In addition to DNA-binding and dimerization domains, HD-ZIPIII proteins contain a StAR-related transfer (START) domain, raising the intriguing possibility that HD-ZIPIII activity may be under direct control of a lipophilic ligand. Determining how HD-ZIPIII proteins are able to function in such different developmental contexts, and identifying their putative ligands, are central goals of the lab. Given their broad and deep conservation throughout the plant kingdom, we are also considering these ideas through the lens of evolution.
Attending Physician, Children’s Hospital of Philadelphia, Division of Genetics
Research Scientist, Children’s Hospital of Philadelphia, Center for Childhood Cancer Research
Director , Beckwith-Wiedemann Syndrome Clinic, Children’s Hospital of Philadelphia
Director, Program of Excellence in Beckwith-Wiedemann Syndrome, Orphan Disease Center, University of Pennsylvania
Our lab demonstrated for the first time that a transcription factor called TCF-1 has an exceptional property to unwrap DNA from nucleosomes and create open chromatin, establishing the epigenetic identity of T cells (Johnson et al., Immunity, 2018). Strikingly, we found that the ectopic expression of TCF-1 in fibroblasts can unwrap DNA from nucleosomes even at stretches with the most refractory chromatin states, leading to gene expression. Most recently, we extended the role of this transcription factor to the three-dimensional (3D) genome organization (Wang et al, Nature Immunology, 2022). Using high-resolution molecular and optical mapping of the 3D genome, we found that TCF-1 is linked to changes in the structure of topologically associating domains in T cell progenitors that lead to interactions between previously insulated regulatory elements and target genes at late stages of T cell development. To the best of our knowledge, this is the first report describing the role of a transcription factor in dismantling topologically associating domains during a developmental trajectory. Whether other lineage-determining transcription factors act in a way similar to TCF-1 to enable interactions between regulatory elements and the genes required for various developmental pathways remains to be studied. Our ongoing focus on this transcription factor aims to elucidate the precise epigenetic mechanisms through which TCF-1 interacts with chromatin remodeling enzymes and the specific domain of this protein which is required to unwrap DNA from nucleosomes and bend DNA for genome folding, thus leading to a highly orchestrated cascade of gene expression events that drive T cell development and function.
Although the main function of T cells is to protect us from infectious agents, many medically important diseases are associated with abnormal T cell responses directed against proteins produced by our own body’s tissues. This broad category of immune-mediated diseases is referred to as autoimmune disorders and includes diseases such as type 1 diabetes (T1D), inflammatory bowel disease, rheumatoid arthritis, and multiple sclerosis. In the case of T1D, interactions between T lymphocytes and insulin-producing beta cells lead to loss of beta-cell mass and a dependence on exogenous insulin administration for survival. As a drastic departure from the status quo, our laboratory discovered misfolding of DNA at megabase-pair diabetes-susceptibility regions, leading to reorganization of large transcriptionally coordinated regions in a mouse model of T1D as a result of sequence variation associated with diabetes development (Fasolino and Goldman et al, Immunity, 2020). Remarkably, we demonstrated the relevance of these findings to human T1D, thanks to our team efforts in the Human Pancreas Analysis Program (HPAP) at the University of Pennsylvania. Our ongoing work built on transcriptional profiling of beta cells in human T1D (Fasolino, et al, Nature Metabolism, 2022), assessing if and how the genome is misfolded in primary immune cells from pancreatic tissues of individuals with T1D, will not only enable us to devise molecular and optical strategies to detect T1D at critical time points where interventions can delay progression to clinical diagnosis, but is also key for understanding the molecular etiology of this disease. Due to the unprecedented opportunity to work with primary immune cells in pancreatic tissues of more than 100 human organ donors and our ability to work with the mouse model of T1D, our laboratory is at an exemplary position to describe the cause and effect relationships between genetics, nuclear architecture, and gene regulation in T1D.
To achieve our lab’s central goal, which is to better understand the chromatin biology of T cells in health and disease, we also innovate computational techniques to fully understand the complexity of multidimensional epigenomic datasets in T cells. We devised a computational workflow to rigorously detect architectural stripes using computer vision (Yoon, et al, Nature Communications, 2022).
Research Interest
Our protection against microorganisms such as viruses, bacteria, and fungi is achieved by the orchestrated interactions among a multitude of distinct and specific cells of the innate and adaptive immune responses. Among many players in this system, the white blood cells called T lymphocytes possess the most powerful ability to recognize and target the pathogenic microorganisms. The overarching goal of the Vahedi laboratory is to understand the molecular mechanisms through which genomic information is interpreted in normal development of T cells and further dissect how common genetic variation can lead to misinterpretation of the genetic material in T mediated diseases such as autoimmune disorders. The multidisciplinary nature of our laboratory allows us to exploit cutting-edge computational and experimental approaches and generate unbiased maps of genome organization in primary immune cells in humans and mice. We further follow our hypothesis-generating yet unbiased efforts with experiments dissecting the mechanisms of our predictions using genome editing in mice or cell lines which provides us with an unparalleled opportunity to rigorously define the link between genetics, chromatin organization, and immune cell functions.
While studying sperm small RNA in mice, I discovered that RNAs are shipped from the epididymis to maturing sperm via extracellular vesicles, establishing a novel soma-to-germline transfer of RNA in mammals. This transfer of RNAs from epididymis to sperm is important for embryonic development as embryos fertilized by early epididymal sperm exhibit altered embryonic gene expression and fail to develop to term. Remarkably, both the molecular gene expression and embryonic viability phenotypes are rescued when early epididymal sperm embryos are injected with miRNAs acquired as sperm transit the epididymis.
Conine CC, Sun F, Song L, Rivera-Pérez JA, Rando OJ. MicroRNAs Absent in Caput Sperm Are Required for Normal Embryonic Development. Dev Cell. 2019 Jul 1;50(1):7-8. PubMed PMID: 31265813.
Conine CC, Sun F, Song L, Rivera-Pérez JA, Rando OJ. Small RNAs Gained during Epididymal Transit of Sperm Are Essential for Embryonic Development in Mice. Dev Cell. 2018 Aug 20;46(4):470-480.e3. PubMed Central PMCID: PMC6103825.
Lee G, Conine CC. The Transmission of Intergenerational Epigenetic Information by Sperm microRNAs. Epigenomes. 2022 April 07; 6(2):12-20. PubMed PMID: 35466187.
Research Interest
The functions of noncoding RNAs in fertility, epigenetic inheritance, and development
R-loop biology.
R-loops are RNA containing chromatin structures that have the potential to be powerful regulators of epigenetic gene expression. Many questions about where R-loops form, what protein factors function to modulate these structures in vivo, and their mechanisms in gene regulation remain unanswered. As an independent investigator, I drew on my considerable expertise in protein and RNA biochemistry to initiate projects to understand the function and mechanisms of R-loops. We developed a novel technique that we named, “MapR”, that combines the specificity of RNase H for DNA:RNA hybrids with the sensitivity, speed, and convenience of the CUT&RUN approach, whereby targeted genomic regions are released from the nucleus by micrococcal nuclease (MNase) and sequenced directly, without the need for affinity purification. MapR is an antibody independent, recombinant protein-based technology that is fast, sensitive, and easy to implement (Yan et al, 2019). Also, we have recently improved MapR to increase resolution and confer strand specificity (Wulfridge and Sarma, 2021) and have used proximity proteomics to identify the R-loop proteome (Yan et al, 2022). We discovered a previously unappreciated role for zinc finger and homeodomain proteins in R-loop regulation and identified ADNP, a high confidence autism spectrum disorder gene, as an R-loop resolver.
Yan Q*, Shields, EJ*, Bonasio R†, Sarma K†. 2019. Mapping native R-loops genome-wide using a targeted nuclease approach. Cell Rep, 29(5):1369-1380. PMC6870988.
Yan Q, Sarma K. 2020. MapR: A method for identifying native R-loops genome-wide. Curr Protoc Mol Biol. 130(1):e113. PMC6986773.
Wulfridge P, and Sarma K. 2021. A nuclease- and bisulfite-based strategy captures strand-specific R-loops genome-wide. eLife 10:e65146. PMC7901872.
Yan Q*, Wulfridge P*, Doherty J, Fernandez-Luna JL, Real PJ, Tang HY, Sarma K. 2021. Proximity labeling identifies a repertoire of site-specific R-loop modulators. Nature Commun. 13(1):53. PMCID: PMC8748879. ‘*’- equal contribution.
Non-coding RNA regulation.
As a post-doctoral fellow, I applied my expertise in biochemistry to investigate the significance of interactions between proteins and long non-coding RNAs (lncRNAs) in the context of X chromosome inactivation. I discovered a novel use for locked nucleic acids (LNAs) in the study of lncRNA function. The mechanism of Xist RNA spreading had remained an open question in the field since its discovery in the 1990s. Using LNAs, I found that Xist RNA utilizes both repetitive and unique elements within itself to spread uniformly over the entire X chromosome to silence it (Sarma et al, 2010). Furthermore, combining the use of LNAs with a high throughput sequencing approach (CHART) allowed us to identify regions of the genome that were bound by Xist RNA (Simon et al, 2013). The use of LNAs was key in discovering that Xist RNA utilizes distinct mechanisms to coat the inactive X chromosome at different developmental stages. This technology has been patented and commercialized and is now being used to functionally characterize several different non-coding RNAs that play important roles in cellular homeostasis. During this time, I also contributed to examining the interactions between the PRC2 complex and Xist RNA (Cifuentes-Rojas et al, 2014), and discovered ATRX to be a high affinity RNA binding protein that interacts with Xist RNA to regulate PRC2 enrichment on the inactive X chromosome (Sarma et al, 2014).
Sarma K, Levasseur P, Aristarkhov A, Lee JT. 2010. Locked nucleic acids (LNAs) reveal sequence requirements and kinetics of Xist RNA localization to the X chromosome. Proc Natl Acad Sci U S A 107:22196-22201. PMCID: PMC3009817.
Simon MD*, Pinter SF*, Fang R*, Sarma K, Rutenberg-Schoenberg M, Bowman SK, Kesner BA, Maier VK, Kingston RE, Lee JT. 2013. High-resolution Xist binding maps reveal two-step spreading during X-chromosome inactivation. Nature 504:465-469. PMCID: PMC3904790.
Cifuentes-Rojas C, Hernandez AJ, Sarma K, Lee JT. 2014. Regulatory interactions between RNA and polycomb repressive complex 2. Mol Cell 55:171-185. PMCID: PMC4107928.
Sarma K, Cifuentes-Rojas C, Ergun A, Del Rosario A, Jeon Y, White F, Sadreyev R, Lee JT. 2014. ATRX regulates binding of PRC2 to Xist RNA and Polycomb targets. Cell 159:1228. PMID: 28898627.
Histone methyltransferases and Polycomb proteins.
I have spent a large part of my career working toward understanding the role of various chromatin factors in epigenetic gene regulation. As a graduate student, I purified and characterized some of the first reported histone lysine methyltransferases (HKMTs) such as PR-Set7, Set9, and the Polycomb Repressive Complex 2 (PRC2). During this time, I also developed and characterized reagents and tools that were critical to advance research in this field. My discovery that the mammalian homolog of the drosophila polycomblike protein (PHF1), a PRC2 accessory factor, facilitates the activity of the PRC2 complex provided first evidence of the role of accessory proteins in the function of the PRC2 complex (Sarma et al, 2008). Recent publications by several independent groups underscore the importance of PHF1 and other polycomblike proteins in PRC2 function. As a post-doctoral fellow, I discovered that the chromatin remodeler ATRX regulates PRC2 localization genome-wide (Sarma et al, 2014). In my own lab, we showed that ATRX-RNA interactions specify PRC2 targeting to a subset of polycomb targets (Ren et al, 2020). We also discovered that ATRX mutations in the histone binding or the helicase domains have distinct effects on PRC2 localization and neuronal differentiation (Bieluszewska et al, 2022).
Sarma K, Margueron R, Ivanov A, Pirrotta V, Reinberg D. 2008. Ezh2 requires PHF1 to efficiently catalyze H3 lysine 27 trimethylation in vivo. Mol Cell Biol 28:2718-2731. PMCID: PMC2293112.
Sarma K, Cifuentes-Rojas C, Ergun A, Del Rosario A, Jeon Y, White F, Sadreyev R, Lee JT. 2014. ATRX regulates binding of PRC2 to Xist RNA and Polycomb targets. Cell 159:1228. PMID: 28898627.
Ren W*, Medeiros N*, Warneford-Thomson R, Wulfridge P, Yan Q, Bian J, Sidoli S, Garcia BA, Skordalakes E, Joyce E, Bonasio R, Sarma K. 2020. Disruption of ATRX-RNA interactions uncovers roles in ATRX localization and PRC2 function. Nat Commun. 6;11(1):2219. PMID: 32376827
Bieluszewska A, Wulfridge P, Doherty J, Ren W, Sarma K. (2022). ATRX histone binding and helicase activities have distinct roles in neuronal differentiation. Nucleic Acids Res. PMC in progress
Research Interest
The Sarma lab focuses on elucidating the molecular mechanisms of RNA mediated epigenetic gene regulation in eukaryotes. Using various cell culture models including mouse embryonic stem cells, neuronal differentiation systems, and cancer models, we employ biochemical and various -omics tools to examine the mechanisms of RNAs and their interactions with chromatin and epigenetic modifiers in gene regulation. We have become increasingly interested in RNA containing chromatin structures called R-loops and how they are misregulated in neurodevelopmental disorders and cancers and their impact on genomic processes that can drive disease. Most recently, we developed high throughput sequencing methods to profile native R-loops and identified R-loop interactors using a proximal proteomic approach.