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Penn Epigenetics > Faculty > Institute Core Members
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.

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.

Golnaz-Vahedi

Golnaz Vahedi, Ph.D.

Contribution to Science

Our lab demonstrated for the first time that a transcription factor called TCF-1 has an exceptional property to unwrap DNA from nucleosomes and create open chromatin, establishing the epigenetic identity of T cells (Johnson et al., Immunity, 2018). Strikingly, we found that the ectopic expression of TCF-1 in fibroblasts can unwrap DNA from nucleosomes even at stretches with the most refractory chromatin states, leading to gene expression. Most recently, we extended the role of this transcription factor to the three-dimensional (3D) genome organization (Wang et al, Nature Immunology, 2022). Using high-resolution molecular and optical mapping of the 3D genome, we found that TCF-1 is linked to changes in the structure of topologically associating domains in T cell progenitors that lead to interactions between previously insulated regulatory elements and target genes at late stages of T cell development. To the best of our knowledge, this is the first report describing the role of a transcription factor in dismantling topologically associating domains during a developmental trajectory. Whether other lineage-determining transcription factors act in a way similar to TCF-1 to enable interactions between regulatory elements and the genes required for various developmental pathways remains to be studied. Our ongoing focus on this transcription factor aims to elucidate the precise epigenetic mechanisms through which TCF-1 interacts with chromatin remodeling enzymes and the specific domain of this protein which is required to unwrap DNA from nucleosomes and bend DNA for genome folding, thus leading to a highly orchestrated cascade of gene expression events that drive T cell development and function. 

 

Although the main function of T cells is to protect us from infectious agents, many medically important diseases are associated with abnormal T cell responses directed against proteins produced by our own body’s tissues. This broad category of immune-mediated diseases is referred to as autoimmune disorders and includes diseases such as type 1 diabetes (T1D), inflammatory bowel disease, rheumatoid arthritis, and multiple sclerosis. In the case of T1D, interactions between T lymphocytes and insulin-producing beta cells lead to loss of beta-cell mass and a dependence on exogenous insulin administration for survival. As a drastic departure from the status quo, our laboratory discovered misfolding of DNA at megabase-pair diabetes-susceptibility regions, leading to reorganization of large transcriptionally coordinated regions in a mouse model of T1D as a result of sequence variation associated with diabetes development (Fasolino and Goldman et al, Immunity, 2020). Remarkably, we demonstrated the relevance of these findings to human T1D, thanks to our team efforts in the Human Pancreas Analysis Program (HPAP) at the University of Pennsylvania. Our ongoing work built on transcriptional profiling of beta cells in human T1D (Fasolino, et al, Nature Metabolism, 2022), assessing if and how the genome is misfolded in primary immune cells from pancreatic tissues of individuals with T1D, will not only enable us to devise molecular and optical strategies to detect T1D at critical time points where interventions can delay progression to clinical diagnosis, but is also key for understanding the molecular etiology of this disease. Due to the unprecedented opportunity to work with primary immune cells in pancreatic tissues of more than 100 human organ donors and our ability to work with the mouse model of T1D, our laboratory is at an exemplary position to describe the cause and effect relationships between genetics, nuclear architecture, and gene regulation in T1D.

 

To achieve our lab’s central goal, which is to better understand the chromatin biology of T cells in health and disease, we also innovate computational techniques to fully understand the complexity of multidimensional epigenomic datasets in T cells. We devised a computational workflow to rigorously detect architectural stripes using computer vision (Yoon, et al, Nature Communications, 2022).

Research Interest

Our protection against microorganisms such as viruses, bacteria, and fungi is achieved by the orchestrated interactions among a multitude of distinct and specific cells of the innate and adaptive immune responses. Among many players in this system, the white blood cells called T lymphocytes possess the most powerful ability to recognize and target the pathogenic microorganisms. The overarching goal of the Vahedi laboratory is to understand the molecular mechanisms through which genomic information is interpreted in normal development of T cells and further dissect how common genetic variation can lead to misinterpretation of the genetic material in T mediated diseases such as autoimmune disorders. The multidisciplinary nature of our laboratory allows us to exploit cutting-edge computational and experimental approaches and generate unbiased maps of genome organization in primary immune cells in humans and mice. We further follow our hypothesis-generating yet unbiased efforts with experiments dissecting the mechanisms of our predictions using genome editing in mice or cell lines which provides us with an unparalleled opportunity to rigorously define the link between genetics, chromatin organization, and immune cell functions.

Gerd Blobel, M.D., Ph.D.

Contribution to Science

1) We are pursuing questions of how chromatin in organized in the nucleus, specifically, how enhancer-promoter contacts are formed or constrained. We found that the hematopoietic transcription factor GATA1 and its co-factor FOG1 are essential to juxtapose the enhancer of the b-globin locus with the promoter (Vakoc, Mol. Cell 2005). This study was among the first to define any nuclear factor in chromatin looping. We discovered that chromatin looping is highly dynamic and can occur even at repressed genes (Jing, Mol. Cell 2008). Using a novel approach of tethering the “looping” factor Ldb1 to an endogenous gene, we were able for the first time to generate an enhancer-promoter chromatin loop at a native endogenous gene locus and thus discovered that chromatin looping causally underlies gene expression (Deng et al., Cell 2012). We adapted this approach to reprogram the murine and human b-globin to reactivate the dormant embryonic and fetal globin genes, respectively (Deng, Cell 2014). We are advancing this strategy towards a clinical application in the setting of sickle cell anemia and thalassemia.

We discovered a novel developmental stage specific chromatin architectural element that constrains the functional range of the b-globin enhancer (Huang, Genes Dev. 2017). This element is a potential target for therapeutic genome editing.

By examining the chromosomal architecture during transcription elongation in erythroid cells we discovered that at some genes, instead of the RNA polymerase tracking down the gene, the gene is reeled alongside a polymerase complex that is stabilized by enhancer promoter loops (Lee et al., Genes Dev. 2015). This discovery modifies long-standing views of how transcription progresses in the nucleus.

  • Deng W, Lee J, Wang H, Reik A, Gregory PD, Dean A, and Blobel GA(2012) Controlling long-range chromosomal interactions at a native locus by targeted tethering of a looping factor. Cell149:1233-44. (Featured in a Cell video, and selected by Faculty of 1000) [PMCID: PMC3600827]
  • Deng W, Rupon JW, Krivega, I, Breda L, Motta, I, Jahn KS, Reik A, Gregory PD, Rivella S, Dean A, Blobel GA(2014) Reactivation of developmentally silenced globin gene expression by forced chromatin looping. Cell, 158:849-60. Selected by Faculty of 1000, and highlighted in Nature Genetics [PMCID: PMC4134511]
  • Lee K, Hsiung CC-S, Huang P, Raj A and Blobel GA(2015) Dynamic enhancer-gene body contacts during transcription elongation. Genes Dev., 29:1992-1997.
  • Huang P, Keller CA, Giardine B, Grevet, JD, Davies JOJ, Hughes JR, Kurita R, Nakamura Y, Hardison RC, and Blobel GA(2017) Comparative analysis of 3-dimensional chromosomal architecture identifies novel fetal hemoglobin regulatory element. Genes Dev. 31:1704-1713. [PMCID: PMC5647940]

2) A key question in the establishment and maintenance of cellular lineages is how transcriptional programs are stably maintained throughout the cell cycle to preserve lineage identity. This question is intimately related to how transcription factors interact with the appropriate gene-specific elements within chromatin and how these interactions are controlled throughout the cell division cycle. During mitosis chromosomes condense and transcription is silenced globally as a result of eviction of most nuclear factors from chromatin. Our studies have been aimed at understanding how the cell epigenetically “remembers” to restore appropriate transcription patterns upon G1 entry. By studying the tri-thorax protein MLL and the hematopoietic transcription factor GATA1 we have gained important insights into mitotic “bookmarking” mechanisms, including the first genome wide location analyses of transcription factors in pure mitotic populations (Blobel, Mol. Cell. 2009; Kadauke, Cell 2012) and functional insights into post-mitotic reactivation of bookmarked vs non-bookmarked genes. In the process we developed key reagents that are being used by many others in the field, including a new method to purify mitotic cells to virtual homogeneity and new tools to degrade proteins of interest specifically in mitosis. We have carried out the first genome wide survey of chromatin accessibility in pure mitotic chromatin and found that remarkably, that chromosome retain most of their “openness” with enhancers being more susceptible to partial loss of accessibility that promoters (Hsiung, Genome Res. 2015). We have examined for the first time on a global scale how the genome is transcriptionally reawakened following mitosis and discovered a window in time at which the genome is hyperactive and at which cell to cell variation in transcription patterns is established (Hsiung, Genes Dev. 2016). We also describe how genome architecture is hierarchically re-built when cells exit mitosis and re-enter the G-1 phase of the cell cycle. (Zhang, Nature, 2019)

  • Blobel GA, Kadauke S, Wang E, Lau AW, Zuber J, Chou MM, and Vakoc CR (2009). A Reconfigured Pattern of MLL Occupancy within Mitotic Chromatin Promotes Rapid Transcriptional Reactivation Following Mitotic Exit. Molecular Cell, 36:970-983. [PMCID: PMC2818742] (Preview in Developmental Cell 18:4, 2010).
  • Kadauke S, Udugama M., Pawlicki JM, Achtman JC, Jain DP, Cheng Y, Hardison RC, and Blobel GA(2012) Tissue-specific Mitotic Bookmarking by Hematopoietic Transcription Factor GATA1. Cell 150:725-737. [PMCID: PMC3425057]
  • Hsiung CC-S, Morrissey C, Udugama M, Frank CL, Keller CA, Baek S, Giardine B, Crawford GE, Sung M-H, Hardison RC, Blobel GA(2015) Genome accessibility is widely preserved and locally modulated during mitosis. Genome Research, 2:213-25 [PMCID: PMC4315295]
  • Hsiung CC-S, Bartman C, Huang P, Ginart P, Stonestrom AJ, Keller CA, Face C, Jahn KS, Evans JP, Sankaranarayanan L, Giardine B, Hardison RC, Raj A, Blobel GA(2016) A hyperactive transcriptional state marks genome reactivation upon mitotic exit. Genes Dev. 30:1423-39. [PMCID: PMC 4926865]

3) We are interested in the factors that establish higher order chromatin organization and how long range chromatin interactions impact on transcription. We used forced chromatin looping in combination with single molecule RNA-FISH to understand mechanistically how enhancer-promoter contacts impact on transcription output and to define the dynamics of chromatin looping (Bartman, Mol. Cell 2016, Bartman, Mol. Cell 2019).

We examine how architectural factors such CTCF and cohesins function in organizing the genome and how the impact enhancer promoter communication and gene expression. We discovered that the epigenetic “reader” BET protein BRD2 and the architectural transcription factor CTCF and found that BRD2 contributes to the formation of chromatin boundaries that insulate enhancers from contacting and activating inappropriate genes (Hsu, Molecular Cell 2017). We profiled CTCF and cohesin during the cell cycle and linked these molecules to hierarchical genome folding when cells exit mitosis and enter the G1 phase of the cell cycle (Zhang, Nature 2019). We are examine chromatin contacts and domain boundaries via gain-of-function perturbative studies (Zhang, Nature Genetics 2020).

  • Bartman CR, Hsiung CC-S, Raj A, Blobel GA(2016) Enhancer regulation of transcriptional bursting parameters revealed by forced chromatin looping. Molecular Cell 62:237-247 [PMCID: PMC4842148]
  • Hsu SC, Bartman CR, Gilgenast TG, Edwards, CR, Stonestrom AJ, Huang P, Emerson DJ, Evans P, Werner MT, Keller CA, Giardine ., Hardison RC, Raj A, Phillips-Cremins JE and Blobel GA (2017) The BET protein BRD2 cooperates with CTCF to enforce architectural and transcriptional boundaries. Molecular Cell  66:102-116 (Selected by Faculty of 1000) [PMCID: PMC5393350]
  • Zhang H, Emerson DJ, Gilgenast TG, Titus KR, Lan Y, Huang P, Zhang D, Wang H, Keller CA, Giardine B. Hardison RC, Phillips-Cremins JE, and Blobel GA(2019) Chromatin Structure Dynamics During the Mitosis to G1-Phase Transition. Nature, 576:158-162 [PMCID: PMC6895436]
  • Zhang D, Huang P, Keller CA, Giardine B, Zhang H, Gilgenast TG, Phillips-Cremins JE, Hardison RC, and Blobel GA(2020) Alteration of genome folding via engineered boundary insertion. Nature Genetics, 52:1076-1087 (featured in News and Views) [PMCID: PMC7541666]

4) We are aim to find new modalities to raise fetal hemoglobin production to benefit patients with sickle cell disease and thalassemia. Recently our focus has been to identify molecules involved in fetal hemoglobin regulation that might be druggable. We improved CRISPR-Cas9 technology as a screening tool and employed it to discover an erythroid specific protein kinase HRI as regulator of fetal hemoglobin (Grevet, Science 2018). We followed up with mechanistic studies elucidating the pathway leading from HRI to fetal globin silencing (Huang et al. 2020). Using this CRISPR screening approach we discovered a new zinc finger transcription factor, ZNF410, that is potentially targetable via a PROTAC that silences fetal hemoglobin transcription via a single target gene, the NuRD subunit CHD4 (Lan, Molecular Cell, 2020). Deep mechanistic studies include the clinically relevant question as to why some cells respond to fetal hemoglobin induction while others do not (Khandros, Blood 2020).

  • Grevet JD, Lan X, Hamagami N, Edwards CR, Sankaranarayanan L, Ji X, Bhardwaj SK, Face CJ, Posocco DF, Abdulmalik O, Keller CA, Giardine B, Sidoli S, Garcia BA, Chou ST, Liebhaber SA, Hardison RC, Shi J, and Blobel GA(2018) Domain-focused CRISPR screen identifies HRI as a fetal hemoglobin regulator in human erythroid cells. Science 361:285-290 (Preview in the New England Journal of Medicine 379:17,2018) [PMCID:PMC6257981]
  • Huang P, Peslak SA, Lan X, Khandros E, Yano JA, Sharma M, Keller CA, Giardine B, Qin K, Abdulmalik O, Hardison RC, Shi J  and Blobel GA(2020). HRI activates ATF4 to promote BCL11A transcription and fetal hemoglobin silencing. Blood, 135:2121-2132 (Plenary paper, featured in Preview) [PMCID: PMC7290097]
  • Lan X. Ren R., Feng R., Ly L.C., Lan Y., Zhang Z., Aboreden N., Qin K., Horton J.R., Grevet J.D.,Mayuranathan T.,Abdulmalik O., Keller C.A., Giardine B., Hardison R.C., Crossley M., Weiss M.J., Cheng X., Shi J., Blobel G.A. (2020) ZNF410 uniquely activates the NuRD component CHD4 to silence fetal hemoglobin expression. Molecular Cell, in press
  • Khandros E., Huang P., Peslak S.A., Sharma, M., Abdulmalik O., Giardine B., Zhang Z., Keller C.A., Hardison R.C., and Blobel G.A. (2020). Understanding Heterogeneity of Fetal Hemoglobin Induction through Comparative Analysis of F- and A-erythroblasts. Blood135;1957-1968 (featured in Preview) [PMCID: PMC7256358]

 

Complete List of Published Work in MyBibliography 

https://www.ncbi.nlm.nih.gov/myncbi/1-Q-j5i56qQAo/bibliography/public/

Research Interest

We study how genetic regulatory elements are organized spatially in the nucleus and how transcription programs and chromatin architecture are organized throughout the cell cycle to maintain lineage identity. A major effort in the lab is directed towards understanding the regulation of globin gene expression and developing approaches to perturb globin gene expression to ameliorate sickle cell disease. Our work bridges basic science with preclinical studies. For our studies we combine molecular, genomic, biochemical, and imaging approaches with studies in normal and gene targeted mice.

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

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