Glennis Logsdon, Ph.D.

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, Ph.D.

  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 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.

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.

  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.

  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. LakadamyaliNature 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 resolutionWe 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 iPSCsAdditionally, 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. LakadamyaliNature 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. LakadamyaliNucleic 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), Cell160, 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. LakadamyaliJournal 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. LakadamyaliPNAS1103375-3380 (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.

Maya Capelson, Ph.D.

Contact Information

The Perelman School of Medicine at the University of Pennsylvania
Department of Cell and Developmental Biology
9-101 Smilow Center for Translational Research
3400 Civic Center Blvd
Philadelphia, PA 19104-6059
Office: 215-898-0550
Lab: 215-573-7548
capelson@pennmedicine.upenn.edu

Robert Babak Faryabi, Ph.D.

Deciphering Mechanisms of Genome Mis-folding In Cancer

Our lab deploys data-rich experimental techniques to elucidate the role of genome mis-folding in controlling oncogenic gene expression programs. Specifically, we are interested in moving beyond the status quo to understand how oncogenic subversion of lineage-determining transcription factors set topology of cancer genome. To tackle this question, we combine genomics and super-resolution imaging and focus on investigating molecular mechanisms of genome mis-folding in breast and blood cancers. These mechanistic studies aim to identify precise epigenetic vulnerabilities of cancer cells and guide treatments disrupting cancer cells’ transcriptional addiction.

Representative Publication:

Oncogenic Notch Promotes Long-Range Regulatory Interactions Within Hyperconnected 3D Cliques. Petrovic J*, Zhou Y*, Fasolino M, Goldman N, Schwartz GW, Mumbach MR, Nguyen SC, Rome KS, Sela Y, Zapataro Z, Blacklow SC, Kruhlak MJ, Shi J, Aster JC, Joyce EF, Little SC, Vahedi G, Pear WS, Faryabi RB Molecular Cell. 2019;73(6):1174-90 e12

Determining Epigenetic Mechanisms Of Resistance To Targeted Therapies

Targeting oncogenic drivers of cancers commonly leads to drug resistance. Mechanisms of acquiring resistance to oncology drugs mostly remain unknown, partly due to the limitations of population-based assays in elucidating heterogeneity of drug-naive and complexity of drug-induced tumor evolution. Using single-cell genomics and imaging, we study how heterogeneity and plasticity of transcriptional dependencies confer resistance to targeted therapeutics such as Notch inhibitors.

Representative Publication

TooManyCells Identifies And Visualizes Relationships Of Single-cell Clades. Schwartz GW, Zhou Y, Petrovic J, Fasolino M, Xu L, Shaffer SM, Pear WS, Vahedi G, Faryabi RB Nature Methods, 2020; 17: 405-413

Innovating Computational Methods To Enable Cancer Discovery

Our lab innovates statistical and machine learning approaches to accelerate discovery of novel therapeutics and biomarkers by elucidating complexity and heterogeneity of tumors. Recently, we have developed a computational ecosystem for mapping molecular and spatial heterogeneity in tumors. As part of the Center for Personalized Diagnostics, we also mine cancer patient genotypic/phenotypic data to improve patient health. patient health.

Representative Publication:

Classes of ITD Predict Outcomes in AML Patients Treated With FLT3 Inhibitors. Schwartz GW, Manning B, Zhou Y, Velu P, Bigdeli A, Astles R, Lehman AW, Morrissette JJD, Perl AE, Li M, Carroll M, Faryabi RB Clinical Cancer Research. 2019;25(2):573-83

Research Interest

Cancer is typically considered a genetic disease. However, recent progress in our understanding of epigenetic aberrations in cancer has challenged this view. Overarching goal of our lab is to understand epigenetic mechanisms of transcriptional addiction in cancer and exploit this information to advance cancer therapeutics.

To pursue this objective, we use cutting-edge chromatin conformation capture, high-content imaging, single-cell epigenomics, functional genomics, and combine these technologies with our expertise in computational sciences to systematically explore: i) how epigenetic control of gene expression is disrupted in cancer, ii) why transcriptional addiction can develop, and iii) how heterogeneity and plasticity of transcriptional dependencies enable drug resistance.

Kathryn E. Wellen, Ph.D.

1. Role of STAMP2 (STEAP4) in modulating inflammatory and metabolic responses in adipocytes: My interest in metabolism began while I was a graduate student in the laboratory of Gökhan S. Hotamisligil. My work in the Hotamisligil lab specifically focused on the role of six-transmembrane protein of prostate 2 (STAMP2; also known at STEAP4). We because interested in STAMP2 from a gene expression study that I performed at the start of my graduate training, in which we found that STAMP2 expression was induced by the inflammatory cytokine TNFα and suppressed by thiazolidinediones (Endocrinology, 2004). During the rest of my graduate training years, I worked to elucidate the function of STAMP2 in adipocytes, using both cell culture and mouse models. We found that STAMP2 acts to prevent inappropriate activation of inflammatory pathways in adipocytes, thereby contributing to the maintenance of systemic insulin sensitivity (Cell, 2007). We also published an influential review article that has been cited over 4000 times to date.
Wellen KE, Uysal KT, Wiesbrock S, Yang Q, Chen H, Hotamisligil GS. Interaction of tumor necrosis factor-alpha- and thiazolidinedione-regulated pathways in obesity. Endocrinology. 2004 May;145(5):2214-20. PubMed PMID: 14764635.
Wellen KE, Hotamisligil GS. Inflammation, stress, and diabetes. J Clin Invest. 2005 May;115(5):1111-9. PubMed PMID: 15864338; PubMed Central PMCID: PMC1087185.
Wellen KE, Fucho R, Gregor MF, Furuhashi M, Morgan C, Lindstad T, Vaillancourt E, Gorgun CZ, Saatcioglu F, Hotamisligil GS. Coordinated regulation of nutrient and inflammatory responses by STAMP2 is essential for metabolic homeostasis. Cell. 2007 May 4;129(3):537-48. PubMed PMID: 17482547; PubMed Central PMCID: PMC2408881.
The hexosamine biosynthetic pathway in coordination of metabolic and signaling pathways: After obtaining my PhD, I joined Craig B. Thompson’s laboratory, to further develop my expertise in cellular metabolism and gain training in the re-emerging field of cancer metabolism. I sought to understand how cells gauge nutrient availability and, to this end, investigated the role of the hexosamine biosynthetic pathway, which generates the glycosyl donor UDP-GlcNAc. We found that glucose utilization in the hexosamine pathway impacts growth factor receptor N-glycosylation and surface presentation, and that this serves as a mechanism to coordinate glucose and glutamine metabolism to support proliferation in hematopoietic cells (Genes and Development, 2010). More recently, we have reexamined the regulation of the hexosamine pathway under conditions of nutrient deprivation, identifying that pancreatic cancer cells employ a little studied hexosamine salvage pathway in response to glutamine deprivation to feed UDP-GlcNAc pools (eLife, 2021).
Wellen KE, Lu C, Mancuso A, Lemons JM, Ryczko M, Dennis JW, Rabinowitz JD, Coller HA, Thompson CB. The hexosamine biosynthetic pathway couples growth factor-induced glutamine uptake to glucose metabolism. Genes Dev. 2010 Dec 15;24(24):2784-99. PubMed PMID: 21106670; PubMed Central PMCID: PMC3003197.
Wellen KE, Thompson CB. A two-way street: reciprocal regulation of metabolism and signalling. Nat Rev Mol Cell Biol. 2012 Mar 7;13(4):270-6. PubMed PMID: 22395772.
Campbell SL and Wellen KE. Metabolic Signaling to the Nucleus in Cancer. Mol Cell, 2018 Aug 2; 71(3): 398-408. PMID: 30075141.
Campbell SL, Mesaros C, Izzo L, Affronti H, Noji M, Schaffer, BE, Tsang T,  Sun K,  Trefely S, Kruijning S, Blenis J, Blair IA, Wellen KE. Glutamine deprivation triggers NAGK-dependent hexosamine salvage, eLife, 2021
Acetyl-CoA at the interface of lipid metabolism and epigenetics: roles in cellular and organismal physiology: My interest in metabolic regulation of the epigenome, which constitutes a major current focus on my lab, also developed during my postdoctoral work in the Thompson lab. When I joined the Thompson lab in 2006, the lab had been studying acetyl-CoA metabolism and its role in supporting tumor growth through de novo lipid synthesis. Whether acetyl-CoA levels are also regulatory for lysine acetylation had been speculated and evidence for this had emerged in yeast, but little to no evidence for this possibly existed in mammalian cells. We found that acetyl-CoA production by ATP-citrate lyase (ACLY) is critical for maintaining overall levels of histone acetylation in multiple mammalian cell types, including adipocytes. This initial study, published in Science, was one of the first papers demonstrating metabolic control of the epigenome in mammalian cells. Since starting my own laboratory in 2011, we have extensively investigated the role of acetyl-CoA metabolism in regulation of lipid metabolism and the epigenome. We reported the development of Aclyf/f mice and MEF cell lines, as well as Adiponectin-Cre;Aclyf/f (adipocyte-specific KO) mice (Cell Reports, 2016). Using these reagents, we demonstrate that upregulation of ACSS2 and engagement of acetate metabolism is a key mechanism of compensation to supply acetyl-CoA for histone acetylation and lipid synthesis in the absence of ACLY, in vitro and in vivo. We further found that fructose metabolism to acetate produced by gut microbiota represents a key source of acetyl-CoA for hepatic lipogenesis (Nature, 2020). We also demonstrated that ACLY is crucial for sucrose-induced activation of ChREBP in adipocytes and in sustaining systemic metabolic homeostasis during carbohydrate feeding (Cell Reports, 2019).
Wellen KE, Hatzivassiliou G, Sachdeva UM, Bui TV, Cross JR, Thompson CB. ATP-citrate lyase links cellular metabolism to histone acetylation. Science. 2009 May 22;324(5930):1076-80. PubMed PMID: 19461003; PubMed Central PMCID: PMC2746744.
Zhao S, Torres AM, Henry R, Trefely T, Wallace M, Lee JV, Carrer A, Sengupta A, Kuo YM, Frey AJ, Meurs N, Viola JM, Blair IA, Weljie A, Snyder NW, Andrews AJ, Wellen KE. ATP-citrate lyase controls a glucose-to-acetate metabolic switch, Cell Rep, 2016, Oct 18;17(4):1037-1052. Pubmed PMID: 27760311; PubMed Central PMCID: PMC5175409
Fernandez S, Viola JM, Torres A, Wallace M, Trefely S, Zhao S, Affronti HC, Gengatharan JM, Guertin DA, Snyder NW, Metallo CM, Wellen KE. Adipocyte ACLY facilitates dietary carbohydrate handling to maintain metabolic homeostasis in females. Cell Rep, 2019, May 28;27(9):2772-2784. PubMed PMID:31141698; PubMed Central PMCID: PMC6608748
Zhao S, Jang C, Liu J, Uehara K, Gilbert M, Izzo L, Zeng X, Trefely S, Fernandez S, Carrer A, Miller KD, Schug ZT, Snyder NW, Gade TP, Titchenell PM, Rabinowitz JD, Wellen KE. Dietary fructose feeds hepatic lipogenesis via microbiota-derived acetate, Nature. 2020, Mar;579(7800):586-591. PubMed PMID: 32214246.
Acetyl-CoA at the interface of lipid metabolism and epigenetics: roles in tumor development and progression. A major emphasis of my laboratory has been to investigate the role of acetyl-CoA metabolism and metabolic control of the epigenome in tumor development and progression. One of the key questions we have sought to answer is whether oncogene-mediated metabolic rewiring impacts acetyl-CoA pools in such a way as to modulate the tumor epigenome. We found that oncogenic activation of the PI3K-AKT pathway promotes nuclear-cytosolic acetyl-CoA production and histone acetylation via ATP-citrate lyase (ACLY). Consistently, in human tumors, we identified a significant positive correlation between pAKT-S473 and histone acetylation levels. This study was published in Cell Metabolism and was one of the first demonstrations that oncogenic metabolic reprogramming contributes to alterations in the tumor epigenome independent of mutations in genes encoding metabolic enzymes. Following up on this study, we have identified a role for ACLY-S455 phosphorylation within the nucleus in providing acetyl-CoA for histone acetylation near sites of DNA double strand breaks, which facilitates BRCA1 recruitment and DNA repair by homologous recombination (Molecular Cell, 2017). We have also found that ACLY is crucial for KRASG12D-driven histone acetylation in pancreatic acinar cells and plays a distinct role in supporting acinar-to-ductal metaplasia in early pancreatic tumorigenesis. Once tumors form, however, ACSS2 is highly expressed and tumors can grow even in the absence of ACLY. Despite this metabolic flexibility, we found that targeting of downstream acetyl-CoA producing processes, specifically the mevalonate pathway and the reading of acetyl-lysine, can suppress tumor growth (Cancer Discovery, 2019). Most recently, in collaborative work with Nathaniel Snyder, we have developed methodology for compartmentalized acyl-CoA analysis termed SILEC-SF and leveraged this approach to discover that propionyl-CoA is enriched in the nucleus and that isoleucine catabolism feeds nuclear propionyl-CoA pools and histone lysine propionylation (Trefely et al, Mol Cell, 2022).

Lee JV, Carrer A, Shah S, Snyder NW, Wei S, Venneti S, Worth AJ, Yuan ZF, Lim HW, Liu S, Jackson E, Aiello NM, Haas NB, Rebbeck TR, Judkins A, Won KJ, Chodosh LA, Garcia BA, Stanger BZ, Feldman MD, Blair IA, Wellen KE. Akt-dependent metabolic reprogramming regulates tumor cell histone acetylation. Cell Metab. 2014 Aug 5;20(2):306-19. PubMed PMID: 24998913; PubMed Central PMCID: PMC4151270.
Sivanand S, Rhoades S, Jiang Q, Viney I, Zhang J, Tang J, Benci J, Yuan S, Zhao S, Carrer A, Bennett MJ, Minn AJ, Weljie AM, Greenberg RA, Wellen KE. Nuclear acetyl-CoA production by ACLY promotes homologous recombination, Mol Cell, 2017 Jul 20 Jul 20;67(2):252-265. PubMed PMID: 28689661; PubMed Central PMCID: PMC5580398
Carrer A, Trefely S, Zhao S, Campbell SL, Norgard RJ, Schultz KC, Sidoli S, Parris JLD, Affronti HC, Sivanand S, Egolf S, Sela Y, Trizzino M, Gardini A, Garcia BA, Snyder NW, Stanger BZ, Wellen KE. Acetyl-CoA metabolism supports multi-step pancreatic tumorigenesis. Cancer Discov. 2019 Mar;9(3):416-435. PubMed PMID: 30626590; PubMed Central PMCID: PMC6608748
Trefely S, Huber K, Liu J, Noji M, Stransky S, Singh J,  Doan MT, Lovell CD, von Krusenstiern E, Jiang H, Bostwick A, Pepper HL, Izzo L, Zhao Z, Xu JP, Bedi Jr KC, Rame JE,  Sidoli S, Bogner-Strauss J, Mesaros C, Wellen KE*, Snyder NW*. Quantitative sub-cellular acyl-CoA analysis reveals distinct nuclear metabolism and isoleucine-dependent histone propionylation, Mol Cell, 2022 (*co-corresponding authors)

Research Interest

cancer metabolism, metabolic regulation of the epigenome, metabolic signaling

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