Posts classified under: Developmental Epigenetics Interest Group

Penn Epigenetics > Faculty > Developmental Epigenetics Interest Group

Rajan Jain, M.D.

Contributions to Science

Our work is broadly characterized by three major themes.

First, we are defining the molecular factors regulating 3D chromatin spatial positioning. We have shown that lamina-chromatin interactions (lamina-associated domains or LADs) mediate cardiomyocyte differentiation during cardiac development, establishing physiologic relevance of spatial positioning. More recently, we have shown that pathogenic LMNA variants associated with cardiomyopathy compromise genome organization at the nuclear lamina in cardiomyocytes but not in hepatocytes or adipocytes. The disruption in lamina-chromatin interactions is targeted and results in the misexpression of non-myocyte genes in mutant cardiomyocytes. Finally, we have generated an atlas of lamina-chromatin interactions across multiple human cell types and defined a new, intermediate LAD state. Collectively, my work has shown that 3D spatial positioning regulates cardiac myocyte cellular identity and contributes to phenotypes observed in human laminopathies. We are now working to reveal the molecular mechanisms underlying spatial positioning, using genome-wide screens, mouse and human iPSC models, high resolution imaging and genomics. We work closely with the Joyce and Lakadamyali laboratories. This work has been supported by my NIH New Innovator award (DP2HL147123) and expanded by our interactions with the 4D Nucleome Consortium (U01DA052715).

A second major area of investigation in the laboratory is understanding how Bromodomain and Extraterminal Domain (BET) proteins regulate cardiac cell fate and state. I first became interested in BET proteins as an undergraduate in the Tjian laboratory (PMID: 12620225). In my program, we are examining the cell type specific roles of individual BET proteins in cardiac cell types, with a particular focus on BRD4. We have shown that BRD4 gains locus-specificity in adult cardiac myocytes by interacting with GATA4. Most recently, my group discovered a novel role for BRD4 in genome folding by regulating stability of cohesin on chromatin. Loss of BRD4 in neural crest results in phenotypes resembling human syndromes associated with mutations in cohesin and associated proteins which we mechanistically attribute to the role of BRD4 in genome folding. Our data is amongst the first demonstrating the functional relevance of genome folding to cellular identity. We are extending this work to identify other regulators of genome folding, their mechanism of action and relevance to development and disease. The work is supported by an NHLBI R01 (HL139783).

Our work is further advanced by a third theme in longstanding collaborative work with the Raj lab to elucidate determinants of cellular plasticity, which may inform our understanding of transdifferentiation. My group has shown demonstrated the lineage relationships in various tissues and organs, both in vivo and in vitro. Taken together, our studies suggest that lineage plasticity exists and helps maintain tissue homeostasis. This is funded through a Transformative Research Award (R01GM137425).

My lab strives to explain our science to the lay public:

https://www.youtube.com/watch?v=fthq4chhrGo&list=LL&index=1&t=1s

https://www.youtube.com/watch?v=t2uOlbohsNo&list=LL&index=2&t=271s

Research Interest

Our research program is driven by the central hypothesis that three-dimensional (3D) genome organization orchestrates the establishment and maintenance of cardiac cellular identity. Uncovering the molecular mechanisms that guide nuclear architecture will transform our understanding of how coordinated gene expression is regulated and cellular identity is achieved. Progenitor cells progressively restrict their lineage potential during cardiac development. The mechanisms that establish and maintain cellular identity through mitosis and over a lifespan underlie health. Compromised differentiation and identity have been linked to several diseases. Models underlying fate determination and identity often focus on transcription factors and/or niche signals. Current paradigms fail to reconcile how the interplay between a finite number of morphogens and lineage specific transcription factors result in 200+ cell types with distinct and stable identities. We posit that nuclear architecture represents a critical mechanism for achieving coordinated regulation of hundreds of genes underlying cellular identity by governing their accessibility or availability. My interdisciplinary training and research program in developmental biology and epigenetics, background as a practicing cardiologist, collaborative network have allowed me to build a strong body of work demonstrating that nuclear architecture regulates cellular identity in development and disease. We aim to decipher the rules governing spatial positioning of loci and genome folding, and to reveal their importance to cardiac development and disease.

Klaus Kaestner, Ph.D., M.S.

Research Interest

The Kaestner lab employs modern mouse genetic approaches, such as gene targeting, tissue-specific and inducible gene ablation, to understand the molecular mechanisms of organogenesis and physiology of the liver, pancreas and gastrointestinal tract. We also employ next-generation sequencing explore the differences between the transcriptome and epigenome of normal vs diseased tissues.
The prevalence of Diabetes Mellitus has reached epidemic proportions world-wide, and is predicted to increase rapidly in the years to come, putting a tremendous strain on health care budgets in both developed and developing countries. There are two major forms of diabetes and both are associated with decreased beta-cell mass. No treatments have been devised that increase beta-cell mass in vivo in humans, and transplantation of beta-cells is extremely limited due to lack of appropriate donors. For these reasons, increasing functional beta-cell mass in vitro, or in vivo prior to or after transplantation, has become a “Holy Grail” of diabetes research. Our previous studies clearly show that adult human beta-cells can be induced to replicate, and – importantly – that cells can maintain normal glucose responsiveness after cell division. However, the replication rate achieved was still low, likely due in part to the known age-related decline in the ability of the beta-cell to replicate. We propose to build on our previous findings and to develop more efficacious methods to increase functional beta-cell mass by inducing replication of adult beta-cells, and by restoring juvenile functional properties to aged beta-cells. We will focus on mechanisms derived from studies of non-neoplastic human disease as well as age-related phenotypic changes in human beta-cells.
We are determining  the mechanisms of age-related decline in beta-cell function and replicative capacity, by mapping the changes in the beta-cell epigenome that occur with age. Selected genes will then be targeted using cutting-edge and emerging technologies such as Crispr-activation and inhibition systems that are already established or are being developed in our laboratories. The research team combines clinical experience with expertise in molecular biology and extensive experience in genomic modification aimed at enhancing beta-cell replication. By basing interventions on changes found in human disease and normal aging, this approach will increase the chances that discoveries made can be translated more rapidly into clinically relevant protocols.

Kavitha Sarma, Ph.D.

Contributions to Science

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.

Montserrat Anguera, Ph.D.

Contributions to Science

1. Functional characterization of long noncoding RNAs that regulate gene expression during development and in adulthood. As a postdoctoral fellow, I began investigating epigenetic mechanisms and modeling these mechanisms of gene expression using conditional knockout mouse models. I demonstrated that the Tsx gene does not encode for a protein but instead is transcribed as a long noncoding RNA, similar to other neighboring genes. I generated a conditional knockout mouse for Tsx and discovered that Tsx RNA has functions beyond the germline, affecting stem cell growth, expression of neighboring genes that regulate X-chromosome inactivation, and also behavior in a gender-specific fashion. This work provided a conceptual foundation for experimentally testing for coding potential of noncoding RNAs and for their functional characterization. As an associate professor, my lab continues to study epigenetic mechanisms of gene expression involving long noncoding RNAs from the X chromosome. We have also investigated sex-specific gene expression profiles during human placental development using in vitro pluripotent stem cell derived model systems. In addition, my lab continues to have a long-standing interest in functional characterization of novel X-linked long noncoding RNAs. We discovered LNCRHOXF1 which functions during early placental development.

  • Anguera, M.C., Ma, W., Clift, D., Namekawa, S., Kelleher, R.J, and Lee, J.T. (2011). “Tsx produces a long noncoding RNA and has general functions in the germline, stem cells, and brain.” PLoS Genet. 7 (9):1-14. PMCID: PMC3164691
  • Ian Penkala, Camille Syrett, Jianle Wang, and Montserrat C. Anguera. (2016). “LNCRHOXF1: a long noncoding RNA from the X-chromosome that suppresses viral response genes during development of the early human placenta”. Mol Cell Biol. Apr 11, 2016, PMID: 27066803
  • Camille Syrett, Isabel Sierra, Corbett Berry, Daniel Beiting, and Montserrat C. Anguera. (2018) “Sex-specific gene expression during differentiation of human pluripotent stem cells to trophoblast progenitors”. Stem Cells Dev. July 11; PMID: 29993333

2. Defining abnormal X-Chromosome Inactivation maintenance in human pluripotent stem cells. During my postdoc, I also investigated human X-Chromosome Inactivation (XCI) in the pluripotent state to specifically determine whether reprogramming human female fibroblasts would reactive the inactive X chromosome. I discovered that the reprogramming process generated a heterogeneous mixture of X-Inactivation cells, distinguishable by their expression pattern for the long noncoding RNA XIST. I made the critical observation that loss of XIST RNA expression in human pluripotent stem cells results in the acquisition of cancer-like phenotypes. This work was the first to demonstrate that human female pluripotent stem cells are potentially dangerous in a clinical setting. These experiments are the basis for my current research program investigating how abnormal expression from the inactive X, resulting from aberrant XCI maintenance, is involved in female-biased autoimmunity, where X-silencing is compromised.

  • Anguera, M.C., Sadreyev, R., Zhang, Z., Szanto, A., Sheridan, S., Haggerty, S., Jaenisch, R., Gimbelbrandt, A., Mitalipova, M., and Lee, J.T. (2012). “Molecular signatures and epigenetic stability of human induced pluripotent stem cells.” Cell Stem Cell 11(1):75-90. PMCID: PMC3587778
  • Lessing, D., Anguera, M.C., and Lee, J.T. (2013). “X Chromosome Inactivation and Epigenetic Responses to Cellular Reprogramming.” Annu Rev Genomics Hum Genet. 14:85-110.

3. Mechanisms of X-Chromosome Inactivation in lymphocytes and other immune cells. As an associate professor at Penn, I have continued investigating the molecular mechanisms of X-Chromosome Inactivation, and how altered dosage of X-linked genes contributes to sex-biased disease. As auto-antigen driven activation of lymphocytes drives autoimmune disease we first investigated the epigenetic status of the inactive X in female lymphocytes from humans and mice, and made the remarkable discovery that these cells do not maintain X-Chromosome Inactivation in the same way as other female somatic cells. We were the first to discover that the inactive X has euchromatic features in female lymphocytes, which may underlie the female-bias in autoimmune disorders including lupus. Our research also demonstrated that T and B cells, upon antigen-mediated stimulation, exhibit relocalization of Xist RNA and heterochromatic marks to the inactive X chromosome, which we have termed ‘dynamic XCI maintenance’. My lab is investigating the molecular details of dynamic XCI maintenance in T and B cells, and has identified protein factors that are required for localization of epigenetic modifications to the inactive X.

  • Wang, J., Syrett, C.M., Kramer, M. Basu, A., Atchison, M. & Anguera, M.C. (2016). “Unusual maintenance of X-chromosome Inactivation predisposes female lymphocytes for increased expression from the inactive X”. Proc Natl Acad Sci, Mar. 21, 2016. PMCID: PMC4833277
  • Syrett, C.M., Sindhava, V., Hodawadekar, S., Myles, A., Liang, G., Zhang, Y., Nandi, S., Cancro, M., Atchison, M., & Anguera, M.C. (2017) “Loss of Xist RNA from the inactive X during B cell development is restored in a dynamic YY1-dependent two-step process in activated B cells”. Plos Genetics, Oct 9; 13 (10). PMCID: PMC5648283
  • Camille Syrett, Vishal Sindhava, Isabel Sierra, Aimee Dubin, Michael Atchison, and Montserrat C. Anguera (2018). “Diversity of Epigenetic Features of the inactive X-chromosome in NK cells, dendritic cells, and macrophages”. Frontiers in Immunology. Dec 14. PMCID: PMC6331414.
  • Sierra I, Anguera MC. (2019). “Enjoy the silence: X-chromosome inactivation diversity in somatic cells.” Curr Opin Genet Dev. 2019 Apr;55:26-31. Epub 2019 May 17. Review. PubMed PMID: 31108425; PubMed Central PMCID: PMC6759402.

4. Impact of autoimmune disease on X-Chromosome Inactivation mechanisms
We continue to investigate diseases exhibiting a female bias that also have abnormal increased expression of X-linked genes. My lab has focused on the autoimmune disorder systemic lupus erythematosus (SLE), which has a strong female-bias and exhibits aberrant over-expression of the X-linked genes CXCR3, CD40LG, TLR7, and FOXP3 in lymphocytes. We made the remarkable discovery that female SLE patients and mouse models with lupus-like disease have perturbations with markers of the inactive X, most notably XIST/Xist RNA localization and heterochromatic modifications H3K27me3 and H2AK119ubiquitin. Our work also found female-specific X-linked gene signatures of SLE patients. We are actively continuing this work in an effort to understand how transcription from the inactive X contributes to autoimmune disease susceptibility and disease severity, and also investigate mouse models of spontaneous lupus disease to examine how disease onset impacts XCI maintenance and X-linked gene expression.

  • Syrett CM, Paneru B, Sandoval-Heglund D, Wang J, Banerjee S, Sindhava V, Behrens EM, Atchison M, & Anguera M.C. (2019) “Altered X-chromosome Inactivation in T cells may promote sex-biased autoimmune diseases”. JCI Insight, Apr 4;4(7). PMCID: PMC6483655.
  • Syrett CM, Sierra I, Beethem ZT, Dubin AH, Anguera MC. “Loss of epigenetic modifications on the inactive X chromosome and sex-biased gene expression profiles in B cells from NZB/W F1 mice with lupus-like disease.” J Autoimmun. (2020) Feb;107:102357. Epub 2019 Nov 25. PubMed PMID: 31780316; PubMed Central PMCID: PMC7237307.
  • Pyfrom S, Paneru B, Knoxx, J, Posso S, Buckner J, Cancro M, Anguera MC. “The dynamic epigenetic regulation of the inactive X chromosome in healthy human B cells is dysregulated in lupus patients”. Proc Natl Acad Sci. (2021) June;Vol.118.
  • Jiwrajka N, Anguera MC. “The X in SeX-Biased Immunity and Autoimmune Rheumatic Disease.” J Exp Med. (2022). Review. doi 10.1084/jem.20211487.

Research Interest

My research program focuses on epigenetic gene regulation that underlies sex differences in development and disease. In particular, I am interested in understanding how gene expression from the X-chromosome is regulated to ensure dosage compensation between males and females, and how these mechanisms become altered in diseases exhibiting a sex-bias, such as autoimmunity. We investigate the female-specific epigenetic process of X-chromosome Inactivation (XCI). The X-chromosome contains genes necessary for cell proliferation, metabolism, cognition, reproduction, and has the highest density of immunity-related genes. Thus, gene expression from the X-chromosome is critical for cellular identity, and XCI ensures proper dosage of these genes in females. Abnormal expression of X-linked genes is a feature of many diseases exhibiting a sex-bias, including cancer, cardiovascular disease, and autoimmunity. I have a broad background in epigenetics, spanning the metabolism of one-carbon units that generate methyl groups that alter gene expression, to the epigenetic regulatory mechanisms involved in XCI. My independent research program at the University of Pennsylvania continues to investigate the epigenetic mechanisms that regulate X-linked gene expression, and how alterations result in female-biased disease. My laboratory has undertaken and completed studies establishing a new field of research, defining novel epigenetic pathways involving the X-chromosome that impact human development, immune responses during development, and lymphocyte function. We routinely use RNA and DNA fluorescence in situ hybridization, immunofluorescence experiment, and allele-specific RNA sequencing, which enables single-cell resolution of the epigenetic characteristics of the inactive X and transcriptional activity. Our long-term goal is to understand how dosage compensation of X-linked genes is maintained in lymphocytes, how perturbations with this silencing contribute to female-biased autoimmune disease, and how XCI maintenance mechanisms can be targeted to correct aberrant X-linked gene expression.

Ongoing Projects:
R01 AI134834
Anguera (PI)
6/01/2018-5/31/2023
Gene regulation from the inactive X in activated B cells

Lupus Mechanisms and Targets Award
Anguera (PI)
6/1/2020-5/30/2023
Targeting the inactive X for correcting dosage imbalances in lupus

Roberto Bonasio, Ph.D.

Contributions to Science

1. Chromatin complexes and noncoding RNAs
The maintenance of epigenetic memory critically relies on proper targeting of complexes that modify chromatin structure. A key question is how are these complexes targeted to the proper genomic regions in different lineages. Using protein biochemistry and next generation sequencing, I discovered that multiple proteins of the Polycomb group, critical for epigenetic repression, bind to RNA and that their RNA binding activity contributes both to chromatin localization and to regulation of PRC2 activity at specific genes (Kaneko 2013). More recently, I have adapted my protein–RNA interaction mapping technique to identify additional regions of PRC2 that interact RNA and found the protein surface likely responsible for the RNA-mediated inhibition of its activity (Zhang 2019). Because we identified RNA-binding sites on both “flavors” of PRC2 (PRC2.1 and PRC2.2) we also investigated their function, discovering distinct contribution of these two complexes to the epigenetic changes that occur during neural differentiation (Petacovici 2021). We also discovered the RNA-binding activity of another chromatin modifier, TET2, and found that this enzyme regulates levels of small noncoding RNAs derived from tRNAs (He 2020). Our studies on Polyomb
Kaneko S, Son J, Shen SS, Reinberg D†, Bonasio R†. PRC2 binds active promoters and contacts nascent RNA in embryonic stem cells. Nature Structural and Molecular Biology 2013;20:1258–64. PMID: 24141703; PMCID: PMC3839660.
Zhang Q*, McKenzie NJ*, Warneford-Thomson R*, Gail EH, Flanigan SF, Owen BM, Lauman R, Levina V, Garcia BA, Schittenhelm RB, Bonasio R†, Davidovich C†. RNA exploits an exposed regulatory site to inhibit the enzymatic activity of PRC2. Nature Structural and Molecular Biology. 2019;26:297. PMID: 30833789.
He C†, Bozler J, Janssen KA, Wilusz JE, Garcia BA, Schorn AJ, Bonasio R†. TET2 chemically modifies tRNAs and regulates tRNA fragment levels. Nature Structural and Molecular Biology 2020; doi:10.1038/s41594-020-00526-w. PMID: 33230319.
Petracovici A, Bonasio R. Distinct PRC2 subunits regulate maintenance and establishment of Polycomb repression during differentiation. Molecular Cell 2021; doi:10.1016/j.molcel.2021.03.038. PMID: 33887196.

2. Epigenetics of social behavior in ants
My ultimate research goal is to understand how epigenetic processes of gene regulation impact complex organism-level phenomena such as brain function and behavior. To this end, since my postdoctoral work I have led efforts to develop ants as experimental organisms for neuroepigenetic research. Ant workers and queens exhibit dramatically different morphologies, lifespans, and behaviors, which are encoded by the same genome and, therefore, must be specified at the epigenetic level. With our high-quality assemblies of the ant genomes and annotation of coding and non-coding genes (Shields 2018), as well as the recent demonstration of feasibility of reverse genetics by genome editing, we have contributed to establish Harpegnathos saltator as a molecular model system for neuroepigenetic research. Our transcriptomic analyses combined with genetic and social manipulations revealed key roles for neuropeptides (Gospocic 2017) and steroid hormones (Gospocic 2021) in establishing and maintaining distinct neurotranscriptomes in the different castes. We also discovered that social reprogramming in Harpegnathos causes extensive cellular plasticity in the brain, including the expansion of a neuroprotective glia population (Sheng 2020), which has spurred my interest in a new direction for my research: aging and neurodegeneration.
Gospocic J, Shields EJ, Glastad KM, Lin Y, Penick CA, Yan H, Mikheyev AS, Linksvayer TA, Garcia BA, Berger SL, Liebig J, Reinberg D, Bonasio R. The neuropeptide corazonin controls social behavior and caste identity in ants. Cell 2017;170:748. PMID 28802044; PMCID: PMC5564227.
Shields EJ, Sheng L, Weiner AK, Garcia BA, Bonasio R. High-quality genome assemblies reveal long non-coding RNAs expressed in ant brains. Cell Reports 2018;23:3078. PMID 29874592; PMCID: PMC6023404.
Sheng L*, Shields EJ*, Gospocic J, Glastad KM, Ratchasanmuang P, Raj A, Little S, Bonasio R. Social reprogramming in ants induces longevity-associated glia remodeling. Sci Adv 2020;6: eaba9869. PMID 32875108; PMCID: PMC7438095.
Gospocic J*, Glastad KM*, Sheng L, Shields EJ, Berger SL‡, Bonasio R‡. Kr-h1 maintains distinct caste-specific neurotranscriptomes in response to socially regulated hormones. Cell 2021;184:5807. PMID: 34739833.

3. Technology development
Throughout my career I have been actively engaged in developing new technologies. Some of them became part of the studies described above and below, others were reported on their own. The first primary publications originating from my own lab were a novel method to detect protein–RNA interactions based on their proximity and not their affinity (Beck 2014) and a mass spectrometry screen to map the RNA-binding sites of hundreds of known and novel RNA-binding proteins in the nucleus of embryonic stem cells (He 2016). My lab was also an early adopter of high-throughput single-cell RNA sequencing techniques (e.g. Drop-seq) and helped other laboratories on campus to obtain access to this technology (Nicetto 2019). Most recently, we developed a new technique to map R-loops (Yan 2019).
Beck D*, Narendra V*, Drury WJ 3rd, Casey R, Jansen PW, Yuan ZF, Garcia BA, Vermeulen M, Bonasio R. In vivo proximity labeling for the detection of protein–protein and protein–RNA interactions. Journal of Proteome Research 2014;13:6135. PMID: 25311790; PMCID: PMC4261942.
He C, Sidoli S, Warneford-Thomson R, Tatomer DC, Wilusz JE, Garcia BA, Bonasio R. High-resolution mapping of RNA-binding regions in the nuclear proteome of embryonic stem cells. Molecular Cell 2016;64:416. PMID: 27768875; PMCID: PMC5222606.
Nicetto D, Donahue G, Jain T, Peng T, Sidoli S, Sheng L, Montavon T, Becker JS, Grindheim JM, Blahnik K, Garcia BA, Tan K, Bonasio R, Jenuwein T, Zaret KS. Loss of H3K9me3 heterochromatin at protein coding genes enables developmental lineage specification. Science 2019;363:294. PMID: 30606806; PMCID: PMC6664818.
Yan Q*, Shields EJ*, Bonasio R†, Sarma K†. Mapping native R-loops genome-wide with a targeted nuclease approach. Cell Reports 2019;179:953. PMID 31665646; PMCID PMC6870988.

4. Migratory routes of dendritic cells
In my graduate studies in immunology, I discovered that dendritic cells sample antigens in the periphery and, rather than migrating to primary lymphoid tissues, reenter the circulation to reach distant sites of action (Cavanagh 2005, Bonasio 2006). These discoveries challenged the long-held view that dendritic cells only traverse a unidirectional route from the blood to the peripheral tissues and from here to lymphoid organs, and suggested entirely new mechanisms by which central tolerance could be achieved and immunological memory reactivated. I also developed a new technique to covalently label T lymphocytes with quantum dots for in vivo visualization by intravital multiphoton microscopy (Bonasio 2007). Although less relevant for my current area of research, my studies in immunology demonstrate the breadth of my training and provide me with a valuable skill set in imaging, mouse genetics, and in vivo approaches that is typically lacking in more conventionally trained researchers in biochemistry and functional genomics.
Cavanagh LL*, Bonasio R*, Mazo IB, Halin C, Cheng G, van der Velden AW, Cariappa A, Chase C, Russell P, Starnbach MN, Koni PA, Pillai S, Weninger W, von Andrian UH. Activation of bone marrow-resident memory T cells by circulating, antigen-bearing dendritic cells. Nature Immunology 2005;6:1029–37. PMID: 16155571; PMCID: PMC1780273.
Bonasio R, Scimone ML, Schaerli P, Grabie N, Lichtman AH, von Andrian UH. Clonal deletion of autoreactive thymocytes by circulating dendritic cells homing to the thymus. Nature Immunology 2006;7:1092–100. PMID: 16951687.
Bonasio R, Carman CV, Kim E, Sage PT, Love KR, Mempel TR, Springer TA, von Andrian UH. Specific and covalent labeling of a membrane protein with organic fluorochromes and quantum dots. Proceedings of the National Academy of Sciences 2007;104:14753. PMID: 17785425; PMC1976196.

*Equal contributions
†Co-corresponding authors

Research Interest

The Bonasio Lab investigates the contribution of epigenetic gene regulation to brain function, with a focus on the role of noncoding RNAs. We study these fundamental biological processes at a mechanistic level in mouse stem cells and neurons and at a functional level the brain of traditional and less traditional model organisms, such as ants, fruit flies, and planarians.

Elizabeth Heller, Ph.D.

Contribution to Science

Proteomic characterization of inhibitory synapses. During doctoral training at The Rockefeller University under the mentorship of Dr. Nathaniel Heintz, I aimed to genetically tag and purify individual synapse types in the mammalian brain, in order to characterize their protein content using an innovative biochemical enrichment strategy coupled with high throughput proteomic analysis. In pursuit of this goal, I developed the first protocol for the specific biochemical isolation and characterization of the elusive inhibitory synapse.  We made a remarkable discovery, namely, that inhibitory synapses consist of structural proteins and ion channels, yet are completely lacking in the signaling molecules that comprise the major component of excitatory synapses.

Selimi F, Cristea IM, Heller E, Chait BT, Heintz N. Proteomic studies of a single CNS synapse type: the parallel fiber/purkinje cell synapse. PLoS Biol. 2009 Apr 14;7(4):e83. PubMed PMID: 19402746; PubMed Central PMCID: PMC2672601.
Heller EA, Zhang W, Selimi F, Earnheart JC, Ślimak MA, Santos-Torres J, Ibañez-Tallon I, Aoki C, Chait BT, Heintz N. The biochemical anatomy of cortical inhibitory synapses. PLoS One. 2012;7(6):e39572. PubMed PMID: 22768092; PubMed Central PMCID: PMC3387162.

Identification of critical period for sleep-consolidated spatial memory. During my undergraduate training I conducted an independent study under Dr. Ted Abel, aimed at elucidating the time course of sleep-induced memory formation in mice by examining memory deficits that result from sleep deprivation during discrete times following learning. We found that fear conditioning is blocked by sleep deprivation during a time period 5-10 hours post training, but unaffected by sleep deprivation for five hours immediately following training. This finding provided critical insights into the time-course of sleep-induced memory consolidation.

Graves LA, Heller EA, Pack AI, Abel T. Sleep deprivation selectively impairs memory consolidation for contextual fear conditioning. Learn Mem. 2003 May-Jun;10(3):168-76. PubMed PMID: 12773581; PubMed Central PMCID: PMC202307.

Locus-specific epigenetic editing for the study of addiction and depression. My postdoctoral research aimed to investigate the causal molecular mechanisms by which chromatin modifications contribute to reward-related pathology in the mammalian brain. There is a preponderance of compelling evidence implicating epigenetic modifications in the pathology of addiction and depression, in both human patients and animal models, yet previous studies have been unable to distinguish between the mere presence and the functional relevance of epigenetic modifications at relevant loci. To elucidate the molecular function of epigenetic regulation relevant to reward pathology, I have developed the use of engineered transcription factors to deliver histone modifications to a specific gene of interest in reward-related regions of the mammalian brain (major publications listed in Personal Statement).
Identification of cellular and molecular mechanisms underlying addiction and stress. In addition to pursuing my main postdoctoral research project, described above, I have also worked with others both inside and outside of the Nestler lab to investigate the molecular basis of drug addiction. For example, I have studied the role of serum- and glucocorticoid-inducible kinase 1 (SGK1) in regulating morphine and cocaine reward, and found that while its transcription and activity are upregulated in vivo by morphine and cocaine, exogenous SGK1 overexpression causes opposite behavioral responses to these two drugs. I have also contributed to several additional studies on the epigenetics of addiction, such as the role of nucleosome remodeling and the Sirtuin family of histone deacetylase.

Ferguson D, Koo JW, Feng J, Heller E, Rabkin J, Heshmati M, Renthal W, Neve R, Liu X, Shao N, Sartorelli V, Shen L, Nestler EJ. Essential role of SIRT1 signaling in the nucleus accumbens in cocaine and morphine action. J Neurosci. 2013 Oct 9;33(41):16088-98. PubMed PMID: 24107942; PubMed Central PMCID: PMC3792451.
Cates HM, Thibault M, Pfau M, Heller E, Eagle A, Gajewski P, Bagot R, Colangelo C, Abbott T, Rudenko G, Neve R, Nestler EJ, Robison AJ. Threonine 149 phosphorylation enhances ΔFosB transcriptional activity to control psychomotor responses to cocaine. J Neurosci. 2014 Aug 20;34(34):11461-9. PubMed PMID: 25143625; PubMed Central PMCID: PMC4138349.
Koo JW, Lobo MK, Chaudhury D, Labonté B, Friedman A, Heller E, Peña CJ, Han MH, Nestler EJ. Loss of BDNF signaling in D1R-expressing NAc neurons enhances morphine reward by reducing GABA inhibition. Neuropsychopharmacology. 2014 Oct;39(11):2646-53. PubMed PMID: 24853771; PubMed Central PMCID: PMC4207344.
Heller EA, Kaska S, Fallon B, Ferguson D, Kennedy PJ, Neve RL, Nestler EJ, Mazei-Robison MS. Morphine and cocaine increase serum- and glucocorticoid-inducible kinase 1 activity in the ventral tegmental area. J Neurochem. 2015 Jan;132(2):243-53. PubMed PMID: 25099208; PubMed Central PMCID: PMC4302038.

Research Interest

The Heller Lab studies the mechanisms by which remodeling of the epigenome leads to aberrant neuronal gene function and behavior.  To approach this problem, we directly manipulate histone and DNA modifications at specific genes in vivo, using viral delivery of epigenetic editing tools.  We focus on uncovering the mechanisms by which chromatin modifications interact with the transcriptional machinery following exposure to psychostimulants, such as drugs of abuse and stress. Because the behavioral disease traits of addiction and depression persist long after cessation of the harmful experience,  stable epigenetic remodeling is an attractive mechanism for such long-lasting effects and presents an intriguing target for therapeutic intervention.

Mia Levine, PhD

Mia Levine, Ph.D.

Contribution to Science

The evolution of young genes via de novo- and duplication- based mechanisms
Evolutionary mechanisms of innovation at the molecular level are numerous. Codons diverge, regulatory elements arise and degenerate, and new genes are born. These signatures of adaptive evolutionary change are frequently species-restricted. My PhD research identified very young genes that harbor no homology to exons in related genomes. In contrast to classic mechanisms of novel gene formation like gene duplication, these de novo genes arise instead from fortuitous sequence evolution at noncoding DNA. These genes exhibited testis-biased expression and signatures of adaptive evolution, implicating male germline processes as potent agents of selection of these rare mutations. This publication was the first to describe such de novo genes that have since been documented in a wide array of taxa, including humans. My postdoctoral research focused instead on gene duplication as a potent mechanism of adaptive diversification. A shared domain structure between parent and daughter proteins facilitated my goal to identify lineage-specific innovations in proteins that package DNA. Prior to my research, the Heterochromatin Protein 1 (HP1) gene family was thought to encode between 2 and 5 members across eukaryotes. I discovered 22 new HP1 members in Drosophila. These 22 paralogs were all born less than 20 million years ago. Nevertheless, the number of HP1 genes per species remains relatively constant. This revolving door of gene replacement implicates conserved, currently undefined chromatin functions encoded by unconserved components recurrently generated by gene duplication.

Levine, M.T., C. D. Jones, A. D. Kern, H. A. Lindfors, and Begun, D.J. (2006) Novel genes derived from noncoding DNA in Drosophila melanogaster are frequently X-linked and exhibit testis-biased expression.Proceedings of the National Academy of Sciences 103: 9935-9939. PMCID: PMC1502557.
Levine, M.T., McCoy, C. Vermaak. D., LeeY.C.G, Hiatt, M.A., Matsen, F.A., and H.S. Malik (2012) Phylogenomic analysis reveals dynamic evolutionary history of the Drosophila Heterochromatin Protein 1 (HP1) gene family.  PLOS Genetics8(6): e1002729. PMCID: PMC3380853.

Adaption to novel environments at DNA packaging proteins

melanogaster, like humans, evolved in Africa but more recently invaded the New World and established populations from tropical to temperate climates. These geographically structured populations are panmictic (randomly mating), so any genotypic or phenotypic differences observed are likely the product of fitness variation across environments, i.e., natural selection. This spatially-structured system therefore presents a unique opportunity to elucidate the molecular basis of adaptation. I have taken both whole genome- and single locus- approaches. In one report, genomic DNA fromD. melanogastertropical and temperate populations from both Australia and the US were hybridized to whole-genome tiling arrays from which we inferred geographic sequence divergence (allele frequency variation) based on geographically structured differences in probe intensities. Our analysis demonstrated that a remarkably large fraction of the D. melanogaster genome has been targeted by spatially-varying positive selection. Our whole-genome analysis also uncovered many previously unsuspected biological functions associated with adaptation to novel environments. One of the most intriguing of these functions was chromatin binding. In light of the extensive data on the environment sensitivity of chromatin dynamics, I was especially interested in the unexplored role that chromatin-remodeling factors play in adaptation to novel habitat. Under one model, chromatin-remodeling factors evolve to maintain chromatin structure that is perturbed by environmental fluctuations. I focused primarily on the Polycomb Group genechameau. I discovered a linear relationship between latitude and allele frequency at several SNPs in both the US and Australian populations, which represent independent colonization events. Moreover, an amino acid-changing SNP predicted variation in tolerance to freezing temperatures. These data strongly implicated the action of natural selection and introduced chromatin-remodeling factors as a potentially rich source of adaptive genetic variation. Inspired by the observation that chromatin-based gene regulation can span more than one promoter, I also tested the hypothesis that adaptive expression variation across latitudinal gradients spans physically linked genes. I found that gene “neighborhoods” (of up to 15 genes), rather than single genes, exhibit adaptive transcriptional profiles, consistent with the notion that chromatin factors regulate adaptive expression variation across space.
Turner, L.T., Levine, M.T., and Begun, D.J. (2008). Genomic analysis of adaptive differentiation in Drosophila melanogaster.Genetics 179: 475-485. PMCID: PMC2390623.
Levine, M.T. and Begun, D.J. (2008). Evidence of spatially varying selection at four chromatin-remodeling loci inDrosophila melanogaster. Genetics 179: 455-473. PMCID: PMC2390624.
Levine, M.T., Eckert, M., and D.J. Begun (2011) Whole genome expression plasticity across tropical and temperateDrosophila melanogaster populations from eastern Australia. Molecular  Biology and Evolution 28: 249–256. PMCID: PMC3002243.

Evolutionary and functional diversification of essential DNA packaging proteins
Conserved nuclear proteins support conserved nuclear processes. Yeast and humans, for example, share essential, homologous chromatin proteins that package eukaryotic DNA and support shared, essential functions like chromosome segregation and telomere integrity. These cellular processes, however, also rely on unconserved molecular machinery. A surprisingly large fraction of essential genes that encode chromatin proteins evolve rapidly. My dissertation documented early evidence of this paradoxical phenomenon. The Dosage Compensation Complex (DCC) is responsible for equalizing X-linked gene dosage via chromatin remodeling of the single male X chromosome.  Loss of function at DCC genes is lethal. I discovered population genetic evidence of positive selection at four of the five DCC complex components. Continuing this theme during my postdoctoral research, I uncovered the essential function of the Heterochromatin Protein 1 paralog, HP1E. HP1E is required for faithful segregation of paternal DNA during the first embryonic mitosis. Nevertheless, a subset of Drosophila species apparently persists without HP1E. I discovered that in D. melanogaster not all paternal chromosomes are equally vulnerable to chromatin bridging during the first embryonic mitosis—the heterochromatin-rich sex chromosomes are more likely to mis-segregate than the large autosomes. Intriguingly, over evolutionary time major rearrangements of these same sex chromosomes co-occur with the pseudogenization of HP1E in the obscura group of Drosophila. These data support a model under which karyotype evolution rendered dispensable a once-essential gene. My findings thus provided a neat hypothesis to resolve the apparent paradox of HP1E’s essentiality in D. melanogaster together with its loss in related species.

Levine, M.T., Holloway, A.K., Arshad, U., and Begun, D.J. (2007) Pervasive and largely lineage-specific adaptive protein evolution in the dosage compensation complex of Drosophila melanogaster.Genetics 177: 1959–1962. PMCID: PMC2147993.
Levine, M.T. and H.S. Malik (2013) A rapidly evolving genomic toolkit of Drosophila heterochromatin.Fly 7: 137-141. PMCID: PMC4049844.
Levine, M.T., Vander Wende, H., and H.S. Malik (2015) Mitotic fidelity requires transgenerational action of a testis-restricted HP1.eLife 4: e07378. PMCID: PMC4491702

Research Interest

Chromatin proteins package our genomic DNA. Essential, highly conserved cellular processes rely on this genome compartmentalization, yet many chromatin proteins are wildly unconserved over evolutionary time. We study the biological forces that drive chromatin protein evolution and the functional consequences for chromosome segregation, telomere integrity, and genome defense.

Arjun-Raj

Arjun Raj, Ph.D.

Contribution to Science

We have contributed to the understanding of mechanisms that create and control cell-to-cell variability in gene expression. In particular, our work was amongst the first to use quantitative single molecule RNA detection techniques to describe the phenomenon of transcriptional bursts, in which we found that transcription is a pulsatile process consisting of pulses of activity interspersed with periods when the gene is completely inactive (Raj et al. PLOS Bio 2006, Leveque and Raj Nat Meth 2013a). We also contributed to the mathematical modeling of this field (Raj et al. PLOS Bio 2006). We have now shown how these pulses relate to homeostatic mechanisms that maintain transcript concentration despite changes in cell volume and DNA content (Padovan-Merhar et al. Mol Cell 2015). We have also shown that variability can be used as a tool for dissecting mechanisms of transcriptional control of molecules such as long non-coding RNA (Maamar et al. Genes and Dev. 2013).

Raj A, Peskin CS, Tranchina D, Vargas DY, Tyagi S. Stochastic mRNA synthesis in mammalian cells.PLoS Biol. 2006 Oct;4(10):e309. PubMed PMID: 17048983; PubMed Central PMCID: PMC1563489.
Levesque MJ, Raj A. Single-chromosome transcriptional profiling reveals chromosomal gene expression regulation. Nat Methods. 2013 Mar;10(3):246-8. doi: 10.1038/nmeth.2372. Epub 2013 Feb 17. Erratum in: Nat Methods. 2013 May;10(5):445. PubMed PMID: 23416756; PubMed Central PMCID: PMC4131260.
Maamar H, Cabili MN, Rinn J, Raj A. linc-HOXA1 is a noncoding RNA that represses Hoxa1 transcription in cis. Genes Dev. 2013 Jun 1;27(11):1260-71. doi: 10.1101/gad.217018.113. Epub 2013 May 30. PubMed PMID: 23723417; PubMed Central PMCID: PMC3690399.
Padovan-Merhar O, Nair GP, Biaesch AG, Mayer A, Scarfone S, Foley SW, Wu AR, Churchman LS, Singh A, Raj A. Single Mammalian Cells Compensate for Differences in Cellular Volume and DNA Copy Number through Independent Global Transcriptional Mechanisms. Mol Cell. 2015 Apr 16;58(2):339-52. doi: 10.1016/j.molcel.2015.03.005. Epub 2015 Apr 9. PubMed PMID: 25866248; PubMed Central PMCID: PMC4402149.

We have contributed to the understanding of how cell-to-cell variability can lead to phenotypic consequences. Specifically, we showed that variability in transcription can lead to random cell fate decisions in bacteria (Maamar and Raj et al. Science 2008), and that variability in gene expression can lead to phenotypic variability in metazoan development (Raj and Rifkin et al. Nature 2010). More recently, we have linked gene expression variability to single cell non-genetic resistance mechanisms in melanoma (Shaffer et al. Nature, in press).

Maamar H, Raj A, Dubnau D. Noise in gene expression determines cell fate in Bacillus subtilis. Science. 2007 Jul 27;317(5837):526-9. Epub 2007 Jun 14. PubMed PMID: 17569828; PubMed Central PMCID: PMC3828679.
Raj A, Rifkin SA, Andersen E, van Oudenaarden A. Variability in gene expression underlies incomplete penetrance. Nature. 2010 Feb 18;463(7283):913-8. doi: 10.1038/nature08781. PubMed PMID: 20164922; PubMed Central PMCID: PMC2836165.
Shaffer SM, Dunagin M, Torborg S, Torre EA, Emert T, Krepler C, Beqiri M, Sproesser K, Brafford P, Xiao M, Eggan E, Anastopoulos IN, Vargas-Garcia CA, Singh A, Nathanson K, Heryn M, Raj A. Rare cell variability and drug-induced reprogramming as a mode of cancer drug resistance. Nature, in press.

We have contributed to methodological approaches to measuring expression and transcription in single cells via RNA fluorescence in situ hybridization (RNA FISH). First, we developed a method that greatly simplifies the detection of individual RNA molecules by RNA FISH (Raj et al. Nat Meth 2008). We have since pushed the method to high multiplexing in the detection of chromosome structure and gene expression simultaneously (Levesque and Raj, Nat Meth 2013a). We also have enabled the detection of single nucleotide variants on individual RNA molecules (Levesque et al. Nat Meth 2013b), which allows for mutation detection and measurements of allele-specific expression. Further, we have developed an ultra-fast variant of RNA FISH that enables use in diagnostic and point of care settings (Shaffer et al. PLOS ONE 2013).

Raj A, van den Bogaard P, Rifkin SA, van Oudenaarden A, Tyagi S. Imaging individual mRNA molecules using multiple singly labeled probes. Nat Methods. 2008 Oct;5(10):877-9. doi: 10.1038/nmeth.1253. Epub 2008 Sep 21. PubMed PMID: 18806792; PubMed Central PMCID: PMC3126653.
Levesque MJ, Raj A. Single-chromosome transcriptional profiling reveals chromosomal gene expression regulation. Nat Methods. 2013 Mar;10(3):246-8. doi: 10.1038/nmeth.2372. Epub 2013 Feb 17. Erratum in: Nat Methods. 2013 May;10(5):445. PubMed PMID: 23416756; PubMed Central PMCID: PMC4131260.
Levesque MJ, Ginart P, Wei Y, Raj A. Visualizing SNVs to quantify allele-specific expression in single cells. Nat Methods. 2013 Sep;10(9):865-7. doi: 10.1038/nmeth.2589. Epub 2013 Aug 4. PubMed PMID: 23913259; PubMed Central PMCID: PMC3771873.
Shaffer SM, Wu MT, Levesque MJ, Raj A. Turbo FISH: a method for rapid single molecule RNA FISH. PLoS One. 2013 Sep 16;8(9):e75120. doi: 10.1371/journal.pone.0075120. eCollection 2013. PubMed PMID: 24066168; PubMed Central PMCID: PMC3774626.

Research Interest

Our lab aims to develop a quantitative understanding of the molecular biology of the cell. Interests include chromosome structure and gene expression, non-coding RNA, and global regulation of gene expression. Applications include genetics, cancer and stem cells.

Doris Wagner, Ph.D.

Contribution to Science

Topics of study
The role and Regulation of SWI/SNF chromatin remodeling complexes in plants.
ATP-dependent chromatin remodeling can change the chromatin state by using the energy derived from ATP hydrolysis to alter histone/DNA interactions. We uncovered key roles for plant SWI/SNF subfamily remodelers in stem cell maintenance and in overcoming Polycomb repression for induction of the floral homeotic genes during flower patterning. Finally, activity of one of the SWI/SNF remodelers, BRM, is modulated directly by signaling components of the stress hormone ABA for drought tolerance.
Kwon, C.S., Chen, C., and Wagner, D. (2005). WUSCHEL is a primary target for transcriptional regulation by SPLAYED in dynamic control of stem cell fate in Arabidopsis. Genes Development 19, 992-1003.
Bezhani, S., Winter, C., Hershman, S., Wagner, J.D., Kennedy, J.F., Kwon, C.S., Pfluger, J., Su, Y., and Wagner, D. (2007). Unique, Shared, and Redundant Roles for the Arabidopsis SWI/SNF Chromatin Remodeling ATPases BRAHMA and SPLAYED. Plant Cell 19, 403-416.
Han, S.K., Sang, Y., Rodrigues, A., BIOL425F2010, B., Rodriquez, P.L. and Wagner, D. (2012) The SWI2/SNF2 chromatin remodeling ATPase BRAHMA represses Abscisic Acid Responses in the Absence of the Stress Stimulus in Arabidopsis, Plant Cell 24 4892-4906.
Wu, M.F., Sang, Y., Bezhani, S., Yamaguchi, N., Han, S.K., Li, Z., Su, Y., Slewinski, T.L., and Wagner, D. (2012). SWI2/SNF2 chromatin remodeling ATPases overcome polycomb repression and control floral organ identity with the LEAFY and SEPALLATA3 transcription factors. Proceedings of the National Academy of Sciences of the United States of America 109, 3576-3581. http://www.eurekalert.org/pub_releases/2015-10/uop-ph101315.php
Peirats-Llobet, M., Han, S.K., Gonzalez-Guzman, M., Jeong, C.W., Rodriguez, L., Belda-Palazon, B., Wagner, D*., and Rodriguez, P.L*. (2016). A Direct Link between Abscisic Acid Sensing and the Chromatin-Remodeling ATPase BRAHMA via Core ABA Signaling Pathway Components. Molecular Plant 9, 136-147. * corresponding authors
The switch to flower formation, a major developmental switch critical for reproductive success.
Plants generate different types of lateral organs (leaves, then branches and finally flowers) post-embryonically from stem cell descendants at the shoot apex. When flowers form is critical for plant reproductive success. Our research has established that the plant specific helix-turn-helix transcription factor LEAFY is a key regulator of the onset of flower formation. We showed that the regulatory network downstream of LFY is comprised of a set of interlocking feed-forward loops that together control the timing of the upregulation of the direct LFY target APETALA1, a commitment factor of floral fate. We further found that LFY directly alters the hormone environment in newly formed primordia to promote floral fate.
Yamaguchi, A., Wu, M.F., Yang, L., Wu, G., Poethig, R.S., and Wagner, D. (2009). The microRNA-regulated SBP-Box transcription factor SPL3 is a direct upstream activator of LEAFY, FRUITFULL, and APETALA1. Developmental Cell 17, 268-278. (faculty 1000 recommended)
Winter, C.M., Austin, R.S., Blanvillain-Baufume, S., Reback, M.A., Monniaux, M., Wu, M.F., Sang, Y., Yamaguchi, A., Yamaguchi, N., Parker, J.E., J.E., Parcy, F., Jensen, S.T., Li, H., Wagner, D. (2011). LEAFY Target Genes Reveal Floral Regulatory Logic, cis Motifs, and a Link to Biotic Stimulus Response. Developmental Cell 20, 430-443.
Yamaguchi, N., Winter, C., Wu, M-F., Kanno, Y., Yamaguchi, A., Seo, M., and Wagner, D. (2014) Gibberellin acts positively then negatively to control onset of flower formation in Arabidopsis. Science 344, 638-41. https://stke.sciencemag.org/content/7/325/ec127); http://www.eurekalert.org/pub_releases/2014-05/uop-phh050814.php
Zhu, Y., Klasfeld, S., Jeong, C.W., Jin, R., Goto, K., Yamaguchi, N., and Wagner, D. TERMINAL FLOWER 1-FD complex target genes and competition with FLOWERING LOCUS T. Nature Communications, 11, 5118. https://www.eurekalert.org/pub_releases/2020-10/uop-dpg100920.php
Jin, R., Klasfeld, S., Garcia, M.F., Xiao, J., Han, S.K., Konkol, A., Zhu, Y., and Wagner, D.. LEAFY is a pioneer transcription factor and licenses cell reprogramming to floral fate. 2021  Nature Communications, 12(1):626. https://www.eurekalert.org/pub_releases/2021-01/uop-fe012721.php
Organogenesis and differentiation in response to hormonal cues
We have recently become interested in the question how hormone responses control organogenesis and differentiation. We have identified key targets of a master transcriptional regulator of the auxin hormone response, AUXIN RESPONSE FACTOR5/MONOPTEROS during organogenesis (flower primordium initiation) and uncovered an auxin hormone triggered chromatin state switch. In addition, we have elucidated how BRM together with leaf maturation transcription factors triggers leaf differentiation by lowering response to the hormone cytokinin.
Yamaguchi, N., Wu, M.-F., Winter, C., Berns, M., Nole-Wilson, S., Yamaguchi, A., Coupland, G., Krizek, B., and Wagner, D. (2013) Auxin-mediated Initiation of the Flower Primordium. Developmental Cell 24, 1–12. (faculty 1000 recommended)
Efroni, I., Han, S.K., Kim, H.Y., Wu, M.F., Sang, Y., Hong, J.C., Eshed, Y*., and Wagner, D*. (2013). Regulation of leaf maturation by chromatin-mediated modulation of hormonal responses. Developmental Cell. 24, 438-445. *corresponding authors
Wu, M-F, Yamaguchi, N., Xiao J., Bargmann, B. Estelle, M., Sang, Y. and Wagner, D. (2015) Auxin-regulated chromatin switch directs acquisition of flower primordium founder fate. eLife;4:e09269 http://www.eurekalert.org/pub_releases/2015-10/uop-ph101315.php
Chung, Y., Zhu, Y., Wu,M.-F., Simonini, S., Armenta-Medina, A.,  Jin, R.,  Østergaard, L.,  Gillmor, C.S., and Wagner, D. (2019) Auxin Response factors promote organogenesis by chromatin-mediated repression of the pluripotency gene SHOOTMERISTEMLESS. Nature Communications 10, 886.
Polycomb silencing and recruitment in plants
Cell identity depends on silencing of unnecessary or detrimental gene expression programs. To understand the regulation of epigenetic gene silencing we have elucidated how Polycomb Repressive Complexes are targeted to developmental genes in plants. We have uncovered a genome-encoded recruitment mechanism very similar to that previously described in the fruitfly. Our studies pave the way to future epigenetic manipulation of desirable plant traits.
Bossi, F., Fan, J., Xiao, J., Chandra, L., Shen, M., Dorone, Y., Wagner, D., and Rhee, S.Y. (2017). Systematic discovery of novel eukaryotic transcriptional regulators using sequence homology independent prediction. BMC Genomics 18, 480.
Xiao, J., Jin, R., Yu, X., Shen, M., Wagner, J.D., Pai, A., Song, C., Zhuang, M., Klasfeld, S., He, C., Santos, A. M., Helliwell, C., Pruneda-Paz, J. L., Kay, S. A., Lin, X., Cui, S., Garcia, M. F., Clarenz, O., Goodrich, J., Zhang, X., Austin, R. S., Bonasio, R., Wagner, D. (2017). Cis and trans determinants of epigenetic silencing by Polycomb repressive complex 2 in Arabidopsis. Nat Genet. 49 (10) 1546-1552  preview in NG. https://www.eurekalert.org/pub_releases/2017-08/uop-pbs082117.php
Sun, B., Zhou, Y., Cai, J., Shang, E., Yamaguchi, N., Xiao, J., Looi, L.S., Wee, W.Y., Gao, X., Wagner, D. and Ito, T. (2019). Integration of transcriptional repression and Polycomb-mediated silencing of WUSCHEL in floral meristems. Plant Cell.
Bieluszewski, T., Xiao, J., Yang, Y., and Wagner, D. (2021). PRC2 activity, recruitment, and silencing: a comparative perspective. Trends Plant Sci 26, 1186-1198.
Lee, U.S., Bieluszewski, T., Xiao, J., Yamaguchi, A., and Wagner, D. (2022). H2A.Z contributes to trithorax activity at the AGAMOUS locus. Mol Plant 15, 207-210.

Research Interest

(Re)programming Cell Identity and Function in the Context of Chromatin

Our research focuses on the reprogramming of cell identity and function during developmental transitions and in response to environmental inputs in plants. Reprogramming offers a window of opportunity to unravel the regulatory logic that underlies cell fate and function as existing programs are shut down and new one’s are put in place. Plants are an excellent experimental system to address this question as they tailor their final form and cell function to a changing environment to optimize growth and survival. We have shown that master transcriptional regulators, hormone response and chromatin state together orchestrate cell fate reprogramming in plants.

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