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Penn Epigenetics > Faculty > Institute Core Members

Glennis Logsdon, Ph.D.

Contributions to Science

Centromeres are specialized regions on each chromosome that mediate the segregation of sister chromatids during cell division. Errors in this process can cause aneuploidy, or an imbalance in chromosome number, which can result in cancer, infertility, and birth defects. Although centromeres are essential chromosomal regions, their sequence has remained unresolved in the human genome for the past two decades. The lack of complete centromeric sequences has limited our understanding of the role these regions play in essential cell biological processes required to maintain genome integrity and sustain life. During her postdoctoral training, Dr. Logsdon developed wet- and dry-lab methods to determine the first complete sequence of a human autosomal centromere (Logsdon et al., Nature, 2021). This work led to the complete sequence of all human centromeres (Altemose, Logsdon et al., Science, 2022) and, ultimately, the completion of the human genome (Nurk et al., Science, 2022).

The complete sequence of each human centromere provides an unprecedented opportunity to determine their variation and evolution for the first time. As such, the Logsdon Lab aims to uncover the genetic and epigenetic variation of centromeres among the human population and in diseased individuals, develop a model of human centromere variation, and use this model to study their basic biology and function. In addition, the Logsdon Lab plans to reconstruct the evolutionary history of centromeres over the last 25 million years using phylogenetic and comparative approaches with both human and non-human primate species. Finally, the Logsdon lab will apply our discoveries of centromeres to design and engineer new ones on human artificial chromosomes (HACs). This effort will build on Dr. Logsdon’s previous success in engineering HACs (Logsdon et al., Cell, 2019) and has the potential to revolutionize scientific research and medicine through the design of custom chromosomes and genomes. Together, our lab’s research will advance our understanding of the complex biology of human centromeres and will generate HACs that have the potential to fundamentally transform scientific research and medicine. Below, we provide an overview of each of these three research areas.

1. Centromere variation among the human population

With advances in long-read sequencing technologies and genome assembly algorithms, we are now in an era where the systematic assembly of centromeres is becoming a reality. The complete assembly of centromeres enables the study of their sequence and structural variation for the first time, and it allows for the precise mapping of histones and other centromeric proteins that were previously unmappable. As such, we are standing on the precipice of uncovering the complex biology of centromeres through the discovery of their genetic and epigenetic landscapes. The Logsdon Lab will lead the effort in this area by sequencing and assembling hundreds of human genomes from both healthy and diseased individuals, determining their centromeric genetic and epigenetic variation, and experimentally testing how this variation impacts centromere function. This work is foundational and will greatly advance our understanding of centromere biology and its role in chromosome segregation during cell division. This work will be done in close collaboration with the Human Pangenome Reference Consortium (HPRC) and the Human Genome Structural Variation Consortium (HGSVC).

a. Logsdon GA, Rozanski AN, Ryabov F, Potapova T, Shepelev VA, Catacchio CR, Porubsky D, Mao Y, Yoo D, Rautiainen M, Koren S, Nurk S, Lucas JK, Hoekzema K, Munson KM, Gerton JL, Phillippy AM, Ventura M, Alexandrov IA, Eichler EE. The variation and evolution of complete human centromeres. Accepted at Nature. Available on bioRxiv. doi: 10.1101/2023.05.30.542849

b. Logsdon GA, Vollger MR, Hsieh P, Mao Y, Liskovykh MA, Koren S, Nurk S, Mercuri L, Dishuck PC, Rhie A, …, Miga KH, Phillippy AM, Eichler EE. The structure, function and evolution of a complete human chromosome 8. Nature. 2021 April 7. doi: 10.1038/s41586-021-03420-7. PMCID: PMC7877196

c. Altemose N, Logsdon GA*, Bzikadze AV*, Sidhwani P*, Langley SA*, Caldas GV*, Hoyt SH, Uralsky L, Ryabov FD, Shew CJ, …, Eichler EE, Phillippy AM, Timp W, Dennis MY, O’Neill RJ, Schatz MC, Pevzner PA, Diekhans M, Langley CH, Alexandrov IA, Miga KH. Complete genomic and epigenetic maps of human centromeres. Science. 2022 April 1. doi: 10.1126/science.abl4178. PMCID: PMC9233505

*Authors contributed equally

d. Nurk S, Koren S, Rhie A, Rautiainen M, Bzikadze AV, Mikheenko A, Vollger MR, Altemose N, Uralsky L, Gershman A, Aganezov S, Hoyt SJ, Diekhans M, Logsdon GA, …, Eichler EE, Miga KH, Phillippy AM. The complete sequence of a human genome. Science. 2022 April 1. doi: 10.1126/science.abj6987. PMCID: PMC9186530

2. Centromere evolution among primate species

Centromeres are among the most rapidly evolving regions of the genome, with a mutation rate at least four-fold greater than the unique portions (Logsdon et al., Nature, 2021). This rapid evolution leads to variation in a‑satellite sequence and structure, and it contributes to the emergence of new a-satellite repeats. The forces that shape the evolution of human centromeres are not well understood, and this is largely due to a lack of complete sequence assemblies of centromeres from other primates. The Logsdon Lab will fill this gap in knowledge by sequencing and assembling centromeres from diverse primate species and using these assemblies to reconstruct the evolutionary history of centromeres over the last 25 million years. We will initially focus on the bonobo, chimpanzee, gorilla, orangutan, and macaque species but plan to expand to other primates that comprise the lesser apes and New World monkeys. This work will be done in close collaboration with the Telomere-to-Telomere (T2T) Consortium, which is planning to generate the first complete reference genomes for nearly all primates. We will also work with our long-standing collaborators who have expertise in primate centromere evolution and pangenomics.

a. Mao Y, Harvey WT, Porubsky D, Munson KM, Hoekzema K, Lewis AP, Audano PA, Rozanski A, Yang X, Zhang S, . . ., Logsdon GA, . . ., Eichler EE. Structurally divergent and recurrently mutated regions of primate genomes. Accepted at Cell. Available on bioRxiv. doi: 10.1101/2023.03.07.531415

b. Logsdon GA, Vollger MR, Hsieh P, Mao Y, Liskovykh MA, Koren S, Nurk S, Mercuri L, Dishuck PC, Rhie A, …, Miga KH, Phillippy AM, Eichler EE. The structure, function and evolution of a complete human chromosome 8. Nature. 2021 April 7. doi: 10.1038/s41586-021-03420-7. PMCID: PMC7877196

c. Sulovari A, Li R, Audano PA, Porubsky D, Vollger MR, Logsdon GA, Human Genome Structural Variation Consortium, Warren WC, Pollen AA, Chaisson M, Eichler EE. Human-specific tandem repeat expansion and differential gene expression during primate evolution. PNAS. 2019 October 28. doi: 10.1073/pnas.1912175116. PMCID: PMC6859368

3. Engineered centromeres on human artificial chromosomes

Human artificial chromosomes (HACs) have the potential to revolutionize scientific research and medicine through the development of numerous radical advancements, such as engineered viral immunity and cancer resistance in cell lines as well as cost-effective vaccine and pharmaceutical development. The Human Genome Project-Write is leading the way in this area by proposing to synthesize human chromosomes and genomes from scratch, building on previous successes in budding yeast. Among the many potential hurdles in translating success from yeast to human, perhaps the greatest is the centromere. Unlike yeast, human centromeres are comprised of hundreds of thousands of a-satellite repeats, which have been challenging to sequence and assemble for the past two decades. The lack of complete assemblies of these regions has hindered our ability to identify sequences that can form a centromere on a HAC, such as those associated with centromeric chromatin and the kinetochore. Because the Logsdon Lab will resolve the sequence of hundreds of human centromeres, we are in an ideal position to identify sequences that may be able to form a centromere on a HAC. Therefore, we plan to identify centromere-competent DNA sequences from natural human centromeres and test them for centromere formation and long-term stability on a HAC. This work will lay the groundwork for the construction of future synthetic human chromosomes and genomes that may fundamentally transform scientific research and medicine.

a. Gambogi CW, Mer E, Brown DM, Arora UP, Yankson G, Gavade JN, Logsdon GA, Heun P, Glass JI, Black BE. Efficient formation of single-copy human artificial chromosomes. Accepted at Science. Available on bioRxiv. doi: 10.1101/2023.06.30.547284

b. Gambogi CW, Dawicki-McKenna J, Logsdon GA, Black BE. The unique kind of human artificial chromosome: bypassing the requirement for repetitive centromere DNA. Exp Cell Res. 2020 April 1. doi: 10.1016/j.yexcr.2020.111978. PMCID: PMC7253334

c. Logsdon GA, Gambogi CW, Liskovykh MA, Barrey EJ, Larionov V, Miga KH, Heun P, Black BE. Human artificial chromosomes that bypass centromeric DNA. Cell. 2019 July 25. doi: 10.1016/j.cell.2019.06.006. PMCID: PMC6657561

Research Interest

Research in the Logsdon laboratory focuses on investigating the variation, evolution, and function of human centromeres. We use a combination of long-read sequencing technologies and synthetic biology approaches to determine how centromeres vary among humans and throughout evolution. We also design and engineer centromeres from scratch on human artificial chromosomes to better understand the human genome.

Yanxiang Deng

Yanxiang Deng, Ph.D.

Contributions to Science

  1. Spatially resolved profiling of histone modifications

To investigate the mechanisms underlying spatial organization of different cell types and functions in the tissue context, it is highly desired to examine not only gene expression but also epigenetic underpinnings in a spatially resolved manner to uncover the causative relationship determining what drives tissue organization and function. Despite recent advances in spatial transcriptomics to map gene expression, it has not been possible to determine the underlying epigenetic mechanisms controlling gene expression and tissue development with high spatial resolution. We developed a first-of-its-kind technology called spatial-CUT&Tag for genome-wide profiling of histone modifications pixel by pixel on a frozen tissue section without dissociation. This method resolved spatially distinct and cell-type-specific chromatin modifications in mouse embryonic organogenesis and postnatal brain development. Single-cell epigenomic profiles were derived from the tissue pixels containing single nuclei. Spatial-CUT&Tag adds a new dimension to spatial biology by enabling the mapping of epigenetic regulations broadly implicated in development and disease. In addition, epigenetic drugs are emerging now, so potentially we can develop drugs to target those epigenetic mechanisms. Having the tools to understand the epigenetic origin of different disease states could open up a whole new avenue of therapeutics.

  • Deng, Y., Bartosovic, M., Kukanja, P., Zhang, D., Liu, Y., Su, G., Enninful, A., Bai, Z., Castelo-Branco, G. and Fan, R. Spatial-CUT&Tag: spatially resolved chromatin modification profiling at the cellular level, Science, 375: 681-686 (2022).
  1. Spatially resolved profiling of chromatin accessibility

An ambitious global initiative has been undertaken to map cell types across all human organs. Single-cell sequencing has been critical to this effort, but it is hard to map the location of cell types to the original tissue environment. We developed spatial-ATAC-seq, which for the first time allows for directly observing cell types in a tissue as defined by global epigenetic state. Spatial-ATAC-seq allows us to identify which regions of the chromatin are accessible genome-wide in cells at specific locations in a tissue. This chromatin accessibility is required for genes to be activated, which then provides unique insights on the molecular status of any given cell. Combining the ability to analyze chromatin accessibility with the spatial location of cells is a breakthrough that can improve our understanding of cell identity, cell state and the underlying mechanisms that determine the expression of genes in the development of different tissues or diseases. Profiling mouse embryos using spatial-ATAC-seq delineated tissue-region-specific epigenetic landscapes and identified gene regulators involved in the development of the central nervous system. Mapping the accessible genome in the mouse and human brain revealed the intricate arealization of brain regions. Applying spatial-ATAC-seq to tonsil tissue resolved the spatially distinct organization of immune cell types and states in lymphoid follicles and extrafollicular zones. This technology progresses spatial biology by enabling spatially resolved chromatin accessibility profiling to improve our understanding of cell identity, cell state and cell fate decision in relation to epigenetic underpinnings in development and disease.

  • Deng, Y., Bartosovic, M., Ma, S., Zhang, D., Kukanja, P., Xiao, Y., Su, G., Liu, Y., Qin, X., Rosoklija, B.R, Dwork, A., Mann, J.J., Xu, M.L., Halene, S., Craft, J.E., Leong, W.K., Boldrini, M., Castelo-Branco, G. and Fan, R. Spatial profiling of chromatin accessibility in mouse and human tissues, Nature, 609: 375–383 (2022).
  1. Spatially resolved transcriptomics and proteomics

In multicellular systems, cells do not function in isolation but are strongly influenced by spatial location and surroundings. Spatial gene expression heterogeneity plays an essential role in a range of biological, physiological, and pathological processes. We developed a novel microfluidic platform (DBiT-seq) to deliver molecular barcodes to formaldehyde or FFPE fixed tissue sections in a spatially confined manner, enabling simultaneous barcoding of mRNAs and proteins, and construction of a high-spatial-resolution multi-omics atlas by NGS sequencing. The unique microfluidic in-tissue barcoding technique has enabled high-spatial-resolution mapping of whole transcriptome and tens of proteins at cellular level.

  • Liu, Y#, Yang, M#, Deng, Y#, Su, G, Enninful, A, Guo, C, Tebaldi, T, Zhang, D, Kim, D, Bai, Z, Norris, E, Pan, A, Li, J, Xiao, Y, Halene, S and Fan, R. “High-Spatial-Resolution Multi-Omics Sequencing via Deterministic Barcoding in Tissue”, Cell, 183: 1–17 (2020).

Research Interest

The Deng lab is developing novel technologies for spatial omics to solve challenging biological problems, including cancer and neurodegenerative diseases.

Kahlilia Blanco headshot

Kahlilia Morris-Blanco, Ph.D.

Research Interest

Dr. Morris-Blanco’s laboratory investigates epigenetic mechanisms involved in stroke pathophysiology by examining the interplay between spatial and temporal epigenetic dynamics, transcriptional regulation, and mitochondrial function in the post-stroke brain. Using both in vitro and in vivo experimental stroke models, they employ gene-specific and genome wide assessments of epigenomic organization, single-cell omics, metabolomics, and functional assessments of mitochondria and neuroprotection. Dr. Morris-Blanco is especially interested in using these mechanistic studies to develop novel treatment strategies, with the goal of translating epigenetic therapies to the clinic.

Aman Husbands, Ph.D.

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.

Mustafa A. Mir, Ph.D.

Contributions to Science

1. Label-free blood screening instruments (Masters work). Whole blood analysis is a critical and familiar part of clinical workflows. Widely used clinical blood cytometers are generally expensive and bulky, require proprietary reagents, and provide limited quantitative information on cell morphology. When these automated cytometers do find abnormalities, manual examination of a stained blood smear using light microscopy is required for further diagnosis. This process is costly, slow, and not quantitative. In my early graduate career, working Prof. Gabriel Popescu, I set out to address these issues by utilizing Quantitative Phase Imaging (QPI). In QPI the phase shift (optical delay) of light passing through a sample is measured with sub-nanometer accuracy using interferometry providing high resolution topographic and tomographic data (Mir et al, Prog. Optics, 2012). I started by building a new QPI instrument utilizing commercial CD-ROM technology (Mir et al., Opt. Express., 2009) with the goal of developing a low-cost platform for an automated point-of-care blood screening tool. A comparison of results on patient samples with those from a clinical cytometer shows excellent agreement (Mir et al., J. Biomed. Opt., 2010 & Biomed. Opt. Exp., 2011) for clinical parameters on red blood cells. Additionally, my approach provides quantitative single cell morphological data, uses 2 orders of magnitude lower input sample, requires no reagents other than whole blood, and significantly lowers instrument cost. Collectively, these studies established that QPI based technologies are more sensitive than currently used clinical whole blood analyzers for common parameters used to characterize erythrocytes and also provide additional information that could lead to earlier diagnosis of certain diseases. 

a) M. Mir, B. Bhaduri, R. Wang, R. Zhu, G. Popescu, Chapter 3 – Quantitative Phase Imaging, Progress in Optics, Ed: W. Emil, Elsevier, Volume 57, pp. 133-217 (2012) 

b) M. Mir, Z. Wang, K. Tangella and G. Popescu, Diffraction Phase Cytometry: Blood on a CD-ROM, Optics Express, 17 (4), 2579-2585 (2009) 

c) M. Mir, H. Ding, Z. Wang, J. Reedy, K. Tangella and G. Popescu, Blood Screening using Diffraction Phase Cytometry, Journal of Biomedical Optics, 15 (2), 027016 (2010) 

d) M. Mir, K. Tangella and G. Popescu, Blood Testing at the Single Cell Level using Quantitative Phase and Amplitude Microscopy, Biomedical Optics Express, 2 (12), 3259-3266. (2011) 

2. Non-perturbative, quantitative, and label-free live imaging spanning broad spatio-temporal scales (PhD work). A major challenge in the live imaging of cells and tissues is the need to non-destructively capture data at a broad range of temporal and spatial scales. High temporal and spatial resolution data is required to measure dynamics and morphological changes at the sub-cellular level, at sub-micron and millisecond scales, while at the same time placing this data in the context of the population (e.g. whole tissue) at scales of millimeters and days. In Prof. Gabriel Popescu’s lab we developed new quantitative phase imaging (QPI) hardware and analytical tools which provide quantitative information on morphology, mass density, and dynamics while spanning this broad range of spatial-temporal scales. Critically, these new microscopes use low-power white-light illumination and are sensitive to the endogenous optical properties of the sample, circumventing challenges associated with fluorescence microscopy such as photobleaching and phototoxicity (at the expense of molecular specificity). One of our instruments, known as the Spatial Light Interference Microscope (Wang et al, Opt. Express, 2011), for which I developed control hardware and software, has also been successfully commercialized (phioptics.com). I used these new tools to 1) Characterize single cell mass growth in a cell-cycle dependent manner, overturning widely used models of constant exponential growth (Mir et al, PNAS, 2011); 2) Measure the effects of estrogen and estrogen agonists on the growth and dynamics of estrogen sensitive breast cancer cells, showing that estrogen blockers lead to slower growing cells with lower cell masses (Mir et al, PLoS One, 2014); 3) Quantify neuronal network formation in human stem cell derived neurons and showed correlations between single cell mass growth, transport within the forming network, and the emergence of self-organization of the developing network (Mir et al, Sci. Rep., 2014). These studies have had a major impact on the rapid growth of the field of Quantitative Phase Imaging and its adoption by life scientists which is best illustrated by the increasing presence of dedicated QPI instruments in core facilities and laboratories around the world. 

a) Z. Wang, L. Millet, M. Mir, H. Ding, S. Unarunotai, J. Rogers, M. Gillette, and G. Popescu, Spatial Light Interference Microscopy, Optics Express, 19 (2), 1016-1026 (2011) 

b) M. Mir, Z. Wang, Z. Shen, M. Bednarz, R. Bashir, I. Golding and G. Popescu*, Optical Measurement of Cell Cycle Dependent Growth, Proceedings of the National Academies of Sciences (PNAS), 108 (32), 13124-13129 (2011) 

c) M. Mir, A. Bergamaschi, B.S. Katzenellenbogen and G. Popescu, Highly sensitive quantitative imaging for single cancer cell growth kinetics and drug response, PLoS ONE, 9 (2), e89000 (2014) 

d) M. Mir, T. Kim, A. Majumder, M. Xiang, R. Wang, S. C. Liu, M. U. Gillette, S. Stice and G. Popescu, Label-Free Characterization of Emerging Human Neuronal Networks” Scientific Reports, 4, 4434 (2014) 

3. Computational Imaging (PhD and Postdoc work). Live-cell microscopy involves harsh tradeoffs between spatial resolution, temporal resolution, and sensitivity. In all optical microscopes spatial resolution is limited by the diffraction limit of light. Additionally, in the case of fluorescence microscopy, photo-bleaching and photo-toxicity present significant barriers for high-speed 3D imaging. Over the past two decades the field of compressed sensing has emerged to take advantage of an intrinsic property of natural images known as sparsity. Sparsity can be exploited to reconstruct high resolution images from lower resolution data (time or space) provided this data is acquired in an appropriate manner. Such computational imaging approaches, integrate hardware design and computational algorithms to boost spatial and temporal imaging resolution. During my graduate work, I developed a 3D deconvolution method (with Dr. D. Babacan) that exploits the sparsity of quantitative phase images to provide super-resolution 3D tomograms of living cells in a completely label-free manner (Mir et al., PLoS ONE, 2012). Using this approach I was able to resolve and characterize coiled and helical sub-cellular structures in E. coli which were previously only visible using fluorescence based super-resolution methods. We then further refined this approach by including a near-complete physical model of our optical system in a technique known as White-Light Diffraction Tomography (Kim et al., Nat. Photonics, 2014) which provides quantitative 3D tomograms of live cells with sub-cellular resolution. During my post-doctoral work, I led a project (senior author) to develop a generalizable compressed sensing scheme which provides a 5-10 fold reduction of light exposure and acquisition time in 3D fluorescence microscopy (Woringer et al., Opt. Express 2017). Collectively these works have demonstrated novel computational imaging approaches to improve the temporal and spatial resolution of light-microscopes and decrease photo-bleaching and photo-toxicity in 3D fluorescence imaging. 

a) M. Mir, D. Babacan, M. Bednarz, I. Golding and G. Popescu, Three Dimensional Deconvolution Spatial Light Interference Tomography for studying Subcellular Structure in E. coli , PLoS ONE, 7 (6), e38916 (2012) 

b) T. Kim, R. Zhou, M. Mir, S. D. Babacan, P. S. Carney, L. L. Goddard, and G. Popescu, White-light diffraction tomography of unlabelled live cells, Nature Photonics, (2014) 

c) M. Woringer, X. Darzacq, C. Zimmer, and M. Mir, Faster and less phototoxic 3D fluorescence microscopy using a versatile compressed sensing scheme, Optics Express, 25(12), 13668-13683, (2017) 

4. Single molecule tracking and high-resolution 4D imaging in live embryos (Postdoc work). The application of single-molecule tracking to study transcription factor dynamics has shed significant insight on their search and DNA-binding kinetics over the past decade in cell culture models. At the start of my postdoc the technology to perform such measurements in live embryos did not exist. I overcame this problem by building a customized lattice light-sheet microscope to acquire the first data on single-molecule kinetics of proteins within the nuclei of living animal embryos (demonstrated in Mice, C. elegans, and Drosophila, Mir et al., Methods Mol Biol., 2018). In addition to enabling single molecule tracking in embryos, lattice light-sheet microscopy also provides high resolution 4D imaging over large fields-of-views and extended periods of times. These unique imaging capabilities provided opportunities for several collaborative efforts centered on questions of nuclear organization and transcription regulation (these collaborative works are in addition to my primary focus on studying transcription factor binding in Drosophila embryos as described below). Notably, these efforts include 1) Revealing the role of phase separation in the formation of heterochromatin domains in collaboration with Gary Karpen’s group (Strom et al, Nature 2017), 2) Characterizing the role of intrinsically disordered regions in replication initiation factor loading in collaboration with Michael Botchan’s group (Parker et al., eLife 2019), 3) Developing capabilities to label non-repetitive genomic loci with Cas9 in collaboration with Ahmet Yildiz’s group (Qin et al, 2017) and 4) Using single molecule-imaging in C. elegans embryos to study the regulation of RNA polymerase II recruitment to chromatin during X-chromosome dosage compensation in collaboration with Barbara Meyer’s group (in preparation). These collaborative efforts have provided unique biophysical insights on nuclear organization, chromatin biology, and transcription regulation and have helped mature the technologies of single molecule imaging in live embryos and software to analyze large high-dimensional imaging datasets. 

a) M. Mir, A. Reimer, M. Stadler, A. Tangara, A. S. Hansen, D. Hockemeyer, M. B. Eisen, H. Garcia, X. Darzacq*, Chapter 32 – Single Molecule Imaging in Live Embryos using Lattice Light-Sheet Microscopy, Methods in Molecular Biology, Nanoscale Imaging: Methods and Protocols, Volume 1814, Ed: Y. L. Lyubchenko, Springer, pp 541-559 (2018) 

b) A. R. Strom, A. V. Emelyanov, M. Mir, D. V. Fyodorov, X. Darzacq, and G. Karpen, Phase separation drives heterochromatin domain formation, Nature, doi:10.1038/nature22989 (2017) 

c) M. W. Parker, M. Bell, M. Mir, J. A. Kao, X. Darzacq, M. R. Botchan, J. M. Berger, A new class of disordered elements controls DNA replication through initiator self-assembly, eLife 8:e48562, (2019) Download PDF 

d) P. Qin, M. Parlak, C. Kuscu, J. Bandaria, M. Mir, K. Szlachta, R. Singh, X. Darzacq, A. Yildiz, and M. Adli, Live cell imaging of low- and non- repetitive chromosome loci using CRISPR-Cas9, Nature Communications, 8, 14725, (2017) 

5. High concentration multi-factor hubs accentuate transcription factor binding and gene activation (Postdoc work). During my postdoc I became fascinated with linking transcription regulation at the molecular scale to cell-fate patterning during embryonic development. Central to this question is the biophysical problem of how transcription factors find their specific genomic targets in the crowded nuclear milieu. I utilized single molecule imaging to study the anteroposterior morphogen gradient formed by the transcription factor Bicoid in Drosophila melanogaster embryos. Bicoid has long been a model for classical cooperative binding, in which protein-protein interactions stabilize its interaction with target sites. However, when I analyzed Bicoid dynamics in nuclei along its anterior to posterior gradient, I saw no evidence for the concentration-dependent binding off-rates this model predicts. My data instead revealed that Bicoid binding occurs in high-local concentration hubs, which are dependent on the presence of a maternally deposited pioneer factor, Zelda. These multi-factor hubs increase the frequency of Bicoid binding events and thus result in higher Bicoid time-averaged occupancy without directly stabilizing its interactions with DNA (Mir et al, Genes Dev. 2017). Remarkably, these hubs themselves are highly dynamic, forming and melting on the order of seconds. By combining single-molecule tracking, high-speed 3D imaging, and live-visualization of nascent transcription I showed that Zelda-Bicoid hubs drive enrichment of transcription factors at their target loci through transient but preferential interactions (Mir et al, eLife, 2018) which then result in bursts of transcriptional activity (unpublished). Recently I have shown that hub formation is dependent on interactions between the disordered protein domains of hub components and independent of DNA binding (unpublished). These discoveries have come at a time when there is great interest in the role of disordered proteins in driving nuclear organization, particularly in the context of liquid-liquid phase separation. However, my work illustrates that disordered protein interactions can lead to dynamic compartmentalization distinct from phase separation, emphasizing that we have barely begun to understand the myriad biophysical forces that are at play (Mir et al, Development, 2019; McSwiggen et al., Genes Dev 2019). 

a) M. Mir, A. Reimer, J. E. Haines, X.Y. Li, M. Stadler, H. Garcia, M. B. Eisen, X. Darzacq, Dense Bicoid hubs accentuate binding along the morphogen gradient, Genes Dev., 31, 1784-1794 (2017) 

b) M. Mir, M. R. Stadler, S.A. Ortiz, X. Darzacq, M. B. Eisen, Dynamic Multifactor hubs transiently interact with sites of active transcription in Drosophila embryos, eLife, 7:e40497, (2018) 

c) M. Mir, W. Bickmore, E. E. M. Furlong, G. J. Narlikar, Chromatin topology, condensates, and gene regulation: shifting paradigms or just a phase? Development, 146,dev182766 (2019) 

d) D. T. McSwiggen, M. Mir, X. Darzacq, R. Tjian, Evaluating phase separation in live cells: diagnosis, caveats, and functional consequences, Genes Dev, (2019) 

Research Interest

Gene expression is regulated by a complex choreography of highly dynamic events, including the binding of transcription factors to non-coding regulatory regions of the genome, regulation of chromatin topology, and the assembly of large macromolecular complexes, all of which occur in the crowded nuclear environment. Our understanding of these dynamic processes has largely been driven by approaches that provide population averaged and static snapshots which have delivered remarkable insights, but are inherently ill suited for elucidating processes that vary greatly in space and time.  Comprehending the mechanisms that regulate gene expression, and the role of nuclear organization in this regulation, requires technological and theoretical approaches that bridge spatial scales from molecular to organismal and temporal scales from milliseconds to days. We develop and utilize high-resolution microscopy methods which allows us to probe this vast range of spatial and temporal scales within living embryos. For example, we acquire high-speed volumetric data to quantify chromatin dynamics, multi-color datasets  to study the interaction and distribution of protein domains associated with gene activation or repression as cell fates are determined in young embryos, and use single-molecule localization techniques to quantify the kinetics of individual transcription factors as they whizz around the nucleoplasm searching for and binding to their genomic targets. The goal of our lab is to use these advanced imaging technologies in combination with biophysical modelling, genomics, and gene editing to comprehend and manipulate the interplay between nuclear organization, transcription regulation, and gene expression patterns during cell-fate determination.

Richard Phillips, M.D., Ph.D.

Contributions to Science

  1. Identification of oncogenic pathways as therapeutic targets in epigenetically driven gliomas

I investigated the mechanism of action of a menin-inhibitor, MI-2, in H3K27M gliomas following up on studies which identified a menin-inhibitor (MI-2) as the top ‘hit’ in a chemical screen in a new model of this disease (Funato et. al, 2014, Science). Menin is an epigenetic regulator which we hypothesized may be specifically required in the setting of H3K27M induced epigenetic dysregulation. I demonstrated that MI-2 exhibits anti-glioma activity in H3K27M mutant and H3 wild-type glioma subtypes and showed menin is not the relevant molecular target for this drug in gliomas. Instead, using an integrated approach employing genetic, biochemical and metabolomic methods, I discovered the direct molecular target of MI-2 in glioma as lanosterol synthase, a cholesterol biosynthesis enzyme; revealing a novel metabolic vulnerability in glioma and more broadly implicating cholesterol homeostasis as an attractive pathway to target in this malignancy. 

Phillips RE, Yang Y, Smith R, Thompson B, Yamasaki T, Soto-Feliciano Y, Funato K, Liang Y, Garcia-Bermudez J, Wang X, Garcia B, Yamasaki K, McDonald J, Birsoy K, Tabar V, Allis CD. Target identification reveals lanosterol synthase as a vulnerability in glioma. Proc Natl Acad Sci U S A. 2019 Apr 16;116(16):7957-7962. PMCID: PMC6475387

Phillips RE, Soshnev AA, Allis CD. Epigenomic Reprogramming as a Driver of Malignant Glioma. Cancer Cell. 2020 Aug 31:S1535-6108(20)30419-0.

  1. Development of novel therapeutics in glioma targeting epigenetic mechanisms

We identified the chromatin regulator EZH2 as a context-specific dependency in H3K27M gliomas and through computational modeling of existing, non-brain penetrant EZH2 inhibitor scaffolds, and I led a collaboration which devised a chemical strategy resulting in the discovery of the first brain-penetrant small molecule targeting EZH2 for brain tumors.

A Chemical Strategy toward Novel Brain-Penetrant EZH2 Inhibitors. Liang R, Tomita D, Sasaki Y, Ginn J, Michino M, Huggins DJ, Baxt L, Kargman S, Shahid M, Aso K, Duggan M, Stamford AW, DeStanchina E, Liverton N, Meinke PT, Foley MA, Phillips RE. ACS Med Chem Lett. 2022 Feb 10;13(3):377-387. PMCID: PMC4981478

  1. Treatment approaches for management of brain tumors

Medulloblastoma is the most common primary brain tumor in children and management of extra-neural recurrence is a controversial and difficult-to-treat clinical scenario in Neuro-Oncology. We demonstrated efficacy of a combination standard-dose chemotherapy regimen which obviated the need for high-dose chemotherapy and stem cell transplantation (which both have significant potential side-effects) to induce long-term remission in this clinical entity.

Phillips RE, Curran KJ, Khakoo Y. Management of late extra-neural recurrence of medulloblastoma without high-dose chemotherapy. J Neurooncol. 2015 Sep;124(3):523-4. PMCID: PMC4981478

RESEARCH INTEREST

Coordinated epigenetic regulation enables cells to adopt specific gene expression programs to orchestrate normal differentiation and maintain cell fate. Gliomas are the most common type of brain cancer and exome-sequencing data has identified mutations in epigenetic regulators as a major driver of these tumors. However, it remains incompletely understood how these epigenetic drivers rewire the chromatin landscape and how this epigenetic dysregulation alters cellular phenotypes such as differentiation and immune evasion. In the Phillips Lab, we employ a number of cutting-edge techniques – from the development of forward genetics tools (i.e. CRISPR-Cas9 screening technology), epigenomic profiling, using neural stem cell models, and patient-derived models of glioma – to elucidate how epigenetic mechanisms contribute to gliomagenesis. Our long term research goal is to understand the how epigenetic pathways are rewired in brain cancer during tumorigenesis, therapy, and evasion of immunity.

Melike Lakadamyali, Ph.D.

Contributions to Science

  1. Developed versatile methods for quantitative, multiplexed imaging of molecular complexes using super-resolution microscopy:Proteins in cells assemble into nanoscale complexes in order to carry out a specific function. The spatial organization and stoichiometry of proteins within these nanoscopic functional units is highly important for maintaining a cell’s healthy physiology. Changes in nanoscale organization of protein complexes, for example a change from monomeric to oligomeric stoichiometry, often triggers disease states. However, visualizing many proteins simultaneously and quantifying protein copy number with high spatial resolution is highly challenging. Super-resolution methods hold promise for overcoming this hurdle, however, the complex photophysics of fluorophores limits both multi-color imaging capabilities and the ability to extract quantitative information. Lakadamyali lab has been developing new methods to overcome these challenges. To address the limitations in simultaneous, high-throughput, multi-color super-resolution imaging, we recently combined DNA-Paint with excitation multiplexing to demonstrate the initial proof of concept for fm-DNA-Paint. Further, to address the challenges with quantification of protein copy number at the nanoscale level, we built calibration standards based on DNA origami. Overall, these methods begin to overcome some of the main challenges associated to super-resolution microscopy making it a multiplexed and quantitative tool.

 

  1. “Quantifying protein copy number in super-resolution using an imaging invariant calibration”,F.C. Zanacchi, C. Manzo, R. Magrassi, N.D. Derr,M. Lakadamyali, Biophysical Journal, 116, 2195-2203 (2019)
  2. “Excitation-multiplexed multicolor super-resolution imaging with fm-STORM and fm-DNA-Paint” P.A. Gómez-García, E.T. Garbacik, M.F. Garcia-Parajo,M. Lakadamyali,PNAS, 115, 12991-12996 (2018)
  3. “DNA Origami: Versatile super-resolution calibration standard for quantifying protein copy number” F. Cella Zanacchi, C. Manzo, A. Sandoval Alvarez, N, Derr,M. Lakadamyali,Nature Methods, doi:10.1038/nmeth.4342 (2017)
  4. “Single molecule evaluation of fluorescent protein photoactivation efficiency using anin vivonanotemplate”, N. Durisic, L. L. Cuervo, A. S. Álvarez, J.Borbely, M. Lakadamyali, Nature Methods, 11, 156-162 (2014)
  1. Determined a novel nanoscale organization of chromatin:An important goal of the Lakadamyali lab is to reconcile the epigenomic and microscopic views of chromatin organization and determine how chromatin structure regulates gene function. Nuclear organization of the chromatin fiber spans many length-scales and while the nanoscale level organization plays a key role in regulating gene expression, this organization is impossible to visualize using conventional microscopy methods. Lakadamyali lab has been using and further developing quantitative super-resolution microscopy methods to overcome this limitation. We visualized and estimated the number of nucleosomes along the chromatin fiber of different cells at nanoscale resolution. We discovered that nucleosomes are assembled in heterogeneous groups of varying sizes, which we termed “clutches”. Remarkably, the median number of nucleosomes and their packing density inside clutches highly correlated with cellular state, such that clutch size correlates with gene expression and pluripotency grade of iPSCs. Additionally, nanoscale chromatin organization is aberrantly remodeled in disease states.
  2. “Aberrant chromatin reorganization in cells from diseased fibrous connective tissue in response to altered chemomechanical cues”. S. Heo, S. Thakur, X. Chen, C. Loebel, B. Xia, R. McBeath, J.A. Burdick, V.B. Shenoy, R.L. Mauck, M. Lakadamyali, Nature Biomedical Engineering, in press (2022)

b.     “Two-Parameter mobility assessments discriminate diverse regulatory factor behaviors in chromatin”, J. Lerner, P.A. Gomez-Garcia, R. McCarthy, Z. Liu, M. Lakadamyali, K. S. Zaret, Molecular Cell, doi: https://doi.org/10.1016/j.molcel.2020.05.036, (2020)

  1. “Super-resolution microscopy reveals how histone tail acetylation affects DNA compaction within nucleosomes in vivo”, J. Otterstrom, A.C. Garcia, C. Vicario, P.A. Gomez-Garcia, M.P. Cosma, M. Lakadamyali, Nucleic Acids Research,  doi: 10.1093/nar/gkz593 (2019)
  2. “Chromatin fibers are formed by heterogeneous groups of nucleosomes in vivo”, M. A. Ricci, C. Manzo, M. Garcia-Parajo, M. Lakadamyali†, M. P. Cosma†, (†co-senior authors), Cell, 160, 1145-1158 (2015)

3. Developed cutting-edge methods for studying intracellular trafficking: One of the overarching goals of Lakadamyali lab is to determine how molecular motors coordinate to transport vesicles and organelles in the complex cellular environment. To achieve this goal, we developed an “all-optical correlative imaging method” that combines single particle tracking in living cells with super-resolution microscopy of the same cell after fixation. Super-resolution microscopy can reveal the details of cellular architecture with exquisite spatial resolution; however, the current methods cannot capture fast dynamic processes (millisecond time scale) due to limited temporal resolution. With the correlative approach, we overcame this limitation and for the first time related cargo transport dynamics to the organization of the microtubule cytoskeleton in the cellular context. By mapping cargo trajectories to individual microtubules, we studied how the local cytoskeletal architecture regulates vesicle trafficking. We found that the cytoskeleton acts as a selective filter to vesicles that are comparable in size to or larger than the mesh size of the microtubule network leading to their pausing. We further showed that lysosomal and autophagosomal compartments are selectively enriched on specific microtubule tracks defined by post-translational modifications in order to enhance their fusion and regulate autophagy. Overall, we have developed powerful methods that reveal how the 3D local cytoskeletal architecture and the microtubule post-translational modifications combine to regulate, opening a new window into studying vesicle-cytoskeleton interactions in the physiological as well as disease contexts. We have recently expanded these studies to visualizing the distribution of microtubule associated proteins like tau under physiological and pathological conditions.

  1. “Tau forms oligomeric complexes on microtubules that are distinct from pathological oligomers”, M.T. Gyparaki, A. Arab, E.M. Sorokina, A.N. Santiago-Ruiz, C.H. Bohrer, J. Xiao, M. Lakadamyali, PNAS, 118 (19) e2021461118 (2021)
  2.  “Detyrosinated microtubules spatially constrain lysosomes facilitating lysosome-autophagosome fusion” N. Mohan, I. V. Verdeny, A. S. Alvarez, E. Sorokina, M. Lakadamyali, Journal of Cell Biology, doi: 10.1083/jcb.201807124, (2018)
  3. “3D Motion of Vesicles Along Microtubules Helps Them to Circumvent Obstacles in Cells”, I. V. Vilanova, F. Wehnekamp, N. Mohan, A. S. Alvarez, J. S. Borbely, J. Otterstrom, D. Lamb, M. Lakadamyali, Journal of Cell Science, doi: 10.1242/jcs.201178, (2017)
  4. “Correlative live-cell and superresolution microscopy reveals cargo transport dynamics at microtubule intersections”, Š. Bálint, I. V. Verdeny, A. S. Álvarez, M. Lakadamyali, PNAS, 110, 3375-3380 (2013) (cover page illustration)

Research Interest

The overarching goal of the Lakadamyali lab is to understand the molecular mechanisms that regulate sub-cellular organization and the significance of this organization on cell function. Cells are highly compartmentalized: the sub-cellular positioning of organelles, nucleic acids and proteins are spatially and temporally coordinated to ensure that biochemical reactions take place at the right place and time. Dr. Lakadamyali’s program has three major focus areas that seek to advance our understanding of sub-cellular organization. First, she develops advanced microscopy methods that provide the technological advances necessary to visualize the spatial organization of cellular machinery with near molecular spatial resolution, including quantitative tools for measuring protein stoichiometry and multiplexed, high-throughput imaging methods. Second, she seeks to determine how the microtubule cytoskeleton and motors regulate transport and positioning of organelles within the cytoplasm and the functional consequences of disrupting proper organelle organization. As part of this work, her lab has recently expanded their focus to studying microtubule associated protein tau and its aggregation in neurological diseases. Third, she seeks to understand how the spatial organization of chromatin within the nucleus regulates gene activity. These three areas integrate synergistically to move forward our understanding of how sub-cellular organization emerges and impacts cell physiology and pathology.

George Burslem, Ph.D.

Contributions to Science

Targeted Protein Degradation
Approximately 75% of the proteome is considered undruggable by traditional small molecule inhibition approaches. As an LLS postdoctoral fellow at Yale, I contributed to the development and application of Proteolysis Targeting Chimera (PROTACs) – heterobifunctional small molecules which recruit an E3 ligase to a protein target, resulting in target ubiquitination and subsequent degradation via the proteasome. This enables small molecule induced degradation of disease relevant proteins in native systems including in vivo. One of my contributions was to expand this approach to transmembrane proteins (specifically receptor tyrosine kinases) which are not endogenously degraded via the proteasome but can be degraded in this manner under small molecule control. The PROTAC approach has also been applied to crucial targets (BCR-Abl and FLT-3 ITD) in hematological malignancies demonstrating the power of the PROTAC approach for drug discovery and revealing previously unknown scaffolding roles of these kinases.
– Proteolysis-Targeting Chimeras as Therapeutics and Tools for Biological Discovery, G.M. Burslem and C.M. Crews, Cell, 2020, 181, 102. PMCID: PMC7319047
– Targeting BCR-ABL1 in Chronic Myeloid Leukemia by PROTAC-mediated Targeted Protein Degradation, G.M. Burslem, A. Reister-Schultz, D.P. Bondeson, C. Eide, S. Savage, B. Druker and C.M. Crews, Cancer Research, 2019, 79. 4744. PMCID: PMC6893872
– Enhancing Antiproliferative Activity and Selectivity of a FLT-3 Inhibitor by Proteolysis Targeting Chimera Conversion, G.M. Burslem, J. Song, X. Chen, J. Hines and C.M. Crews, J. Am. Chem. Soc., 2018, 140, 16428. PMID: 30427680
– The Advantages of Targeted Protein Degradation over Inhibition: an RTK Case Study, G.M. Burslem, B.E. Smith, A. Lai, S. Jaime-Figueroa, D. McQuaid, D.P. Bondeson, M. Toure, H. Dong, Y. Qian, J. Wang, A.P. Crew, J. Hines and C. M. Crews, Cell Chemical Biology, 2018, 25, 67. PMCID: PMC5777153

HIF-1α/p300 Inhibition
Mammalian cells have developed an elaborate pathway for oxygen sensing with a key player in this pathway being hypoxia inducible factor 1α (HIF-1α). Under hypoxic conditions, HIF-1α accumulates, heterodimerizes and translocated to the nucleus where it forms a protein-protein interaction with p300. This complex is transcriptionally active resulting in the hypoxic response and resupply of oxygen to the hypoxic tissue. This pathway is crucial for growth and development but is also exploited by solid tumors to enable growth to continue after exhaustion of their oxygen supply. As a graduate student, I focused on elucidating the molecular recognition between HIF-1α and p300 using biophysical and biochemical approaches before applying that knowledge to develop the first biophysically characterized inhibitors of this protein-protein interaction. Furthermore, this project led to the identification of novel peptide and protein aptamer inhibitors of the HIF-1α/p300 interaction via phage display and a novel class of chemical probes which incorporates both natural and unnatural recognition elements to provide enhanced selectivity and potency.
– Hypoxia Inducible Factor as a Model for Studying Inhibition of Protein-Protein Interactions, G.M. Burslem, H.F. Kyle, A.S. Nelson, T.A. Edwards, A.J. Wilson, Chemical Science, 2017, 8, 4188. PMCID: PMC5576430
– Towards “Bionic” Proteins: Replacement of Continuous Sequences from HIF-1α with Proteomimetics to Create Functional p300 Binding HIF-1α Mimics, G.M. Burslem, H.F. Kyle, A. L. Breeze, T.A. Edwards, S.L. Warriner, A. S. Nelson and A.J. Wilson, Chem. Commun., 2016, 52, 5421. PMCID: PMC4843846
– Small molecule proteomimetic inhibitors of the HIF-1α/p300 protein-protein interaction, G.M. Burslem, H. Kyle, A. Breeze, T.A. Edwards, A. Nelson, S.L. Warriner and A.J. Wilson, ChemBioChem, 2014, 15, 1083. PMCID: PMC4159589
– Exploration of the HIF-1α/p300 binding interface using peptide and adhiron phage display technologies to locate binding hot-spots for inhibitor development, H. F. Kyle, K. F. Wickson, J. Stott, G. M. Burslem, A. L. Breeze, D. C. Tomlinson, S. L. Warriner, A. Nelson, A. J. Wilson and T. A. Edwards, Mol. Biosyst., 2015, 11, 2738. PMID: 26135796

Modulating Protein-Protein Interactions
There are an estimated 650,000 pairwise protein-protein interactions in the human interactome and these interactions are implicated in all biological pathways. As such, the ability to modulate protein-protein interactions with chemical probes can provide unique insights in the functional roles of proteins and their binding partners. Over my career I have developed chemical biology approaches to both inhibit and induce protein-protein interactions as tool compounds and therapeutic approaches. An attractive approach to the inhibition of protein-protein interactions is to develop molecules which mimic a portion of one of protein binding partners and thus preferentially occupy the binding site on the other. Peptides are capable of doing this but must pay a significant entropic penalty to adopt the required conformation due to their inherent flexibility. One approach to combat this is to pre-organize the peptide into the desired conformation by chemically “stapling” it. Another approach is to develop small molecule mimetics capable of recapitulating the recognition elements of a protein/peptide but with less conformational flexibility. I have applied both of these techniques to generate inhibitors of a variety of protein-protein interactions including p53/hDM2, Bcl-XL/BID and RNase S-peptide/S-protein. Furthermore, protein-protein interactions can be induced by small molecules known as molecular glues which we have employed to stabilize interactions between Cereblon and IKZF1. We have also demonstrated the ability to induce protein-protein interactions between various proteins using heterobifunctional compounds.
– Double Quick, Double “Click” Reversible Peptide “Stapling”, C.M. Grison, G.M. Burslem, J.A. Miles, L. Pilsl, D.J. Yeo, S.L. Warriner, M. E. Webb and A. J. Wilson, Chemical Science, 2017, 8, 5166. PMCID: PMC5618791
– Synthesis of Highly Functionalized Oligobenzamide Proteomimetic Foldamers by Late-Stage Introduction of Sensitive Groups, G.M. Burslem, H.F. Kyle, P. Prabhakaran, A. L. Breeze, T.A. Edwards, S.L. Warriner, A. Nelson and A.J. Wilson, Org. Biomol. Chem. 2016, 14, 3782. PMCID: PMC4839272
– Efficient Synthesis of Immunomodulatory Drug Analogues Enables Exploration of Structure Degradation Relationships. G.M. Burslem*, P. Ottis, S. Jaime-Figueroa, A. Morgan, P.M. Cromm, M. Toure and C.M. Crews*, ChemMedChem, 2018, 12, 1508 (*Co-corresponding authors). PMCID: PMC6291207
– Lessons on Selective Degradation with a Promiscuous Warhead: Informing PROTAC Design, D.P. Bondeson, B.E. Smith, G.M. Burslem, A.D. Buhimschi, J. Hines, S. Jaime-Figueroa, J. Wang, B. Hamman, A. Ishchenko, C.M. Crews, Cell Chemical Biology, 2018, 25, 78. PMCID: PMC5777153

Epigenetic Chemical Biology
Epigenetic drug discovery provides a wealth of opportunities for the discovery of new therapeutics but has been hampered by low hit rates, frequent identification of false-positives, and poor synthetic tractability. Since establishing my laboratory at the University of Pennsylvania, we have endeavored to remedy the low hit rate in drug discovery efforts against epigenetic targets, by careful chemo-informatic analysis of active compounds and screening libraries. We have used the information gathered in these analysis to inform the design and synthesis of privileged compound collections for epigenetic chemical biology and probe discovery.
– Photochemical Synthesis of an Epigenetic Focused Tetrahydroquinoline Library, A.I. Green and G.M. Burslem, RSC Medicinal Chemistry, 2021, DOI: 10.1039/D1MD00193K
– Focused Libraries for Epigenetic Drug Discovery: The Importance of Isosteres, A.I. Green and G.M. Burslem, J. Med. Chem, 2021, 64, 7231-7240. PMID: 34042449
– Advances and Opportunities in Epigenetic Chemical Biology, J. Beyer, N. Raniszewski and G.M. Burslem, ChemBioChem, 2021, 22, 17-42. PMID: 32786101

Research Interest

The Burslem lab is interested in developing chemical tools to understand and modulate lysine post-translational modifications, specifically acetylation and ubiquitination. The laboratory is particularly interested in novel pharmacological approaches to modulate post-translational modifications which regulate gene expression and protein stability.

Colin Conine, Ph.D.

Contributions to Science

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

Liling-Wan

Liling Wan, Ph.D.

Contribution to Science

Molecular link between histone acetylation and oncogenic gene activation Histone acetylation is a chromatin mark generally associated with gene activation, yet the molecular mechanisms underlying this correlative relationship remain incompletely understood. I led a collaborative study in which we identified a novel ‘reader’ for histone acetylation named ENL. We showed in leukemia cells that ENL interacts with histone acetylation via the well-conserved YEATS domain, and in so doing, helps to recruit and stabilize its associated transcriptional machinery to drive transcription of leukemogenic genes. By determining the structure of ENL in complex with an acetylated histone peptide, we and our collaborators demonstrated that disrupting the reader function reduced chromatin recruitment of ENL-associated transcriptional machinery and resulted in suppression of oncogenic programs. Furthermore, blocking the functionality of ENL sensitized leukaemia cells to inhibitors that target another distinct class of histone acetylation readers, the BET proteins, thus highlighting the crosstalk between epigenetic readers and potential benefit of combinatorial therapies. Our work established ENL as a missing molecular link between histone acetylation and gene activation critical for leukemia malignant state, and has inspired following studies investigating other YEATS domain-containing proteins as a new class of chromatin ‘readers’ in a broad range of human cancers. In addition to bringing novel insights into our basic understanding of chromatin regulation, this work also provides mechanistic guidance and structural basis for ongoing drug development to target chromatin reading activity of ENL in aggressive leukemias.

Wan L#, Wen H#, Li Y#, Lyu J, Xi Y, Hoshii T, Joseph JK, Wang X, Loh YE, Erb MA, Souza AL, Bradner JE, Shen L, Li W, Li H*, Allis CD*, Armstrong SA*, Shi X*. ENL Links Histone Acetylation to Oncogenic Gene Activation in Leukemias. Nature 2017 Mar 9;543(7644):265-269. (#Equal contribution). PMC5372383
Li Y*, Sabari BR*, Panchenko T*, Wen H, Zhao D, Guan H, Wan L, Huang H, Tang Z, Zhao Y, Roeder RG, Shi X, Allis CD, Li H. Molecular Coupling of Histone Crotonylation and Active Transcription by AF9 YEATS Domain. Mol Cell2016 Apr 21;62(2):181-93. (*Equal contribution) PMC4841940

New type of gain-of-function mutations in chromatin readers Recognition of modified histones by ‘reader’ proteins constitutes a key mechanism mediating the function of histone modifications, yet the mechanisms by which their dysregulation contributes to diseases remain poorly understood. Recurrent, hotspot mutations in the acetylation-reading domain (YEATS domain) of ENL were recently found in Wilms’ tumor, the most common type of pediatric kidney cancer. Whether and how these mutations cause the disease remained unknown. Our current work shows that these mutations confer ENL gain of function in driving abnormal gene expression implicated in cancer. Unexpectedly, these mutations promoted ENL self-association, resulting in the formation of discrete nuclear puncta that are characteristic of biomolecular condensates, a newly recognized form of protein assembly that often involves weak, multivalent molecular interactions and commonly underlies the formation of membrane-less organelles. We demonstrated that such a property drives ‘self-reinforced’ chromatin targeting of mutant ENL protein and associated transcriptional machinery, thus enforcing active transcription from target loci. Aberrant gene control driven by ENL mutations, in turn, perturbs developmental programs and derails normal cell fate to a path towards tumorigenesis. This work is a remarkable demonstration of how mistakes in chromatin reader-mediated process can act as a driving force for tumor formation. These mutations represent a new class of oncogenic mutations which impair cell fate through promoting self-association and reinforcing chromatin targeting.

Wan L#, Chong S, Fan X, Liang A, Cui X, Gates L, Carroll TS, Li Y, Feng L, Chen G, Wang S, Ortiz MV, Daley S, Wang X, Xuan H, Kentsis A, Muir TW, Roeder RG, Li H, Li W, Tjian R, Wen H#, Allis CD#. Impaired Cell Fate through Gain-of-function Mutations in a Chromatin Reader. Nature 2019 in press (#co-corresponding)

Targeting chromatin readers as cancer therapies My postdoctoral work suggested that the displacement of histone acetylation reader ENL from chromatin may be a promising epigenetic therapy, alone or in combination with BET inhibitors, for aggressive leukemia. I have contributed to the development of peptidomimetic and small molecule inhibitors targeting the YEATS domain protein family. The ultimate goal of these and other ongoing efforts is to develop chemical probes targeting the ‘reading’ activity of ENL and other family members as valuable research tools and potential therapeutic agents.

Li X*, Li XM*, Jiang Y, Liu Z, Cui Y, Fung K, van der Beelen S, Tian G, Wan L, Shi X, Allis CD, Li H, Li Y#, Li X#. Structure-guided Development of YEATS Domain Inhibitors by Targeting π-π-π Stacking. Nat Chem Biol. 2018 Dec;14(12):1140-1149. (*Equal contribution) PMC6503841

Molecular mechanisms of cancer metastasis Metastasis accounts for > 90% cancer-related deaths and yet is the most poorly understood aspect of cancer biology. I have contributed to studies in which we identified and characterized new molecular mechanisms for cancer metastasis. My graduate work focused on Metadherin (MTDH), a novel cancer gene identified by our group to be prevalently amplified in breast cancer and strongly associated with a high risk of metastasis and poor prognosis. What drives the strong selection of MTDH in primary tumors was unclear. By generating genetically engineered mouse models, we provided first evidence supporting an essential role of MTDH in the initiation and metastasis of diverse subtypes of breast cancer. We further showed that MTDH regulates the expansion and activity of cancer stem cells through working with its binding partner SND1. By determining the atomic structure of the complex via collaboration, we demonstrated that disrupting the complex impairs breast cancer development and metastasis in vivo. Our work establishes MTDH and SND1 as critical regulators of cancer development and provides mechanistic guidance for ongoing drug development efforts to target this complex as cancer therapy. More broadly, this work provides crucial experimental support for the emerging concept that metastatic potential could be conferred by early oncogenic events that possess additional metastasis-promoting function and advances our understanding of the origin of metastatic traits.

Wan L, Lu X, Yuan S, Wei Y, Guo F, Shen M, Yuan M, Chakrabarti R, Hua Y, Smith HA, Blanco MA, Chekmareva M, Wu H, Bronson RT, Haffty BG, Xing Y, Kang Y. MTDH-SND1 Interaction Is Crucial for Expansion and Activity of Tumor-Initiating Cells in Diverse Oncogene- and Carcinogen-Induced Mammary Tumors. Cancer Cell 2014 Jul 14;26(1):92-105. PMC4101059
Wan L, Pantel K, Kang Y. Tumor Metastasis: Moving New Biological Insights into the Clinic. Nature Medicine 2013 Nov;19(11):1450-64. PMID: 24202397
Guo F#, Wan L#, Zheng A, Stanevich V, Wei Y, Satyshur KA, Shen M, Lee W, Kang Y, Xing Y. Structural Insights into the Tumor-Promoting Function of the MTDH-SND1 Complex. Cell Reports 2014 Sep 25;8(6):1704-13.  (#Equal contribution). PMC4309369
Wan L, Hu G, Wei Y, Yuan M, Bronson RT, Yang Q, Siddiqui J, Pienta KJ, Kang Y. Genetic Ablation of Metadherin Inhibits Autochthonous Prostate Cancer Progression and Metastasis. Cancer Research 2014 Sep 15;74(18):5336-47. PMC4167565
Kang Y, Xing Y, Wan L, Guo F. Use of peptides that block metadherin-SND1 interaction as treatment for cancer. (U.S. Patent No. 10,357,539 B2).

Research Interest

The research interests in our laboratory lie in the intersection of cancer biology and epigenetics. We focus on chromatin – the complex of DNA and histone proteins – and its regulatory network. Cancer genome studies revealed that at least 50% of human cancers harbor mutations in genes encoding chromatin-associated factors, suggesting widespread roles of chromatin misregulation in cancer. We strive to understand chromatin function and its dysregulation in human cancer, with a focus on addressing how chromatin-based mechanisms regulate cellular fate transition and plasticity that endow cancer cells with tumor-promoting potentials. We use a host of different approaches in genetics, epigenetics, biochemistry, genome-wide sequencing, bioinformatics and functional genomics to address these questions. We are also interested in leveraging our basic mechanistic discoveries for therapeutics development.

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