Dana Silverbush, PhD

Research Interest

A major obstacle to effective cancer therapy is the co-existence of multiple cell states with distinct molecular and phenotypical profiles within a tumor (intra-tumoral heterogeneity). The ability of tumor cells to transition between these states (tumor plasticity) has been shown to mediate treatment resistance and disease progression. As a result, malignancies with high rates of heterogeneity and plasticity—such as glioma, acute myeloid leukemia, pancreatic, and lung cancers—remain highly treatment-refractory. Notably, intra-tumoral heterogeneity encompasses genetic, epigenetic, transcriptional, and phenotypic components, and is increasingly thought to be driven by an interplay of genetic and epigenetic factors. The aim of the Silverbush lab is to map the genetic and epigenetic patterns driving transcriptomic heterogeneity and tumor plasticity, leveraging these patterns to enhance diagnostic and prognostic accuracy. To achieve this, we develop single-cell multi-omic assays and corresponding analysis solutions, employing them to explore intra-tumoral heterogeneity and tumor plasticity. The lab consists of both a wetlab component and a computational component, and we welcome students with diverse backgrounds and interested in either or both.

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.

Vikram Paralkar, MD

Research Interest

Research in the Paralkar Lab spans the spectrum from human patient sample studies and mouse models to cutting-edge molecular biology tools, high-throughput sequencing approaches, and novel computational algorithms, all with the goal of gaining insight into how the transcription of coding genes and noncoding ribosomal DNA genes is regulated in hematopoietic stem cells, myeloid progenitors, and in leukemia.

rRNA Transcription in Hematopoiesis and Leukemia

Ribosomal RNA (rRNA) forms the majority of cellular RNA, and its transcription in the nucleolus by RNA Polymerase I from ribosomal DNA (rDNA) repeats accounts for the bulk of all transcription. rRNA transcription rates vary dramatically between different normal cell types in the hematopoietic tree, and leukemic cells have characteristic prominent nucleoli, indicating robust ribosome synthesis.

The rate of ribosome production has far-reaching influence on the fate of the cell, and dictates its size, proliferation, and ability to translate global or specific mRNAs. Little is known however about how rRNA transcription is regulated and fine-tuned across normal and malignant tissues, and whether this regulation can be targeted for leukemia treatment.

The Paralkar Lab has identified that key hematopoietic and leukemic transcription factors bind to rDNA and regulate rRNA transcription, and we are interested in understanding how the binding of cell-type-specific transcription factors regulates the activity of Polymerase I and the transcription of rRNA in normal hematopoiesis, and how this regulation is co-opted in leukemia to drive abundant ribosome biogenesis.

Stemness and Differentiation in Hematopoiesis and Leukemia

Normal hematopoiesis requires an intricate balance in the bone marrow between the ability of stem cells to maintain themselves for decades of life while producing billions of mature blood cells every day. This balance is maintained by the combinatorial activity of transcription factors and chromatin proteins that dictate the transcription of coding gene networks instructing fate choice decisions. Several of the critical factors involved in these decisions are mutated in acute and chronic leukemias, and their mutations tip the equilibrium in the bone marrow towards accumulation of aberrant progenitor populations.

The Paralkar Lab is interested in gaining a detailed mechanistic understanding of how chromatin proteins regulate the stemness-differentiation balance, and how mutations in them produce malignancy.

Bioinformatic Pipelines for Genetics and Epigenetics

Current bioinformatic pipelines for high-throughput studies like whole genome sequencing, RNA-seq, ChIP-seq, and single cell RNA-seq are limited in their ability to map repetitive elements of the genome like ribosomal DNA. Such loci therefore tend to be ignored in genome wide analyses. Given that rRNA accounts for the bulk of the transcriptional output of the cell, the inability to map datasets to rDNA has historically been a major limitation, and has created a significant knowledge gap in our understanding of the most abundant RNA in the cell.

The Paralkar Lab has developed customized genomes and computational pipelines to map datasets to rDNA, and we are interested in developing advanced tools to map and interpret the genetic and epigenetic profiles of rDNA in normal and malignant cells.

Selected Publications

George SS, Pimkin M, Paralkar VR: Customized genomes for human and mouse ribosomal DNA mapping. BioRxiv Nov 2022.

Antony C, George SS, Blum J, Somers P, Thorsheim CL, Wu-Corts DJ, Ai Y, Gao L, Lv K, Tremblay MG, Moss T, Tan K, Wilusz JE, Ganley ARD, Pimkin M, Paralkar VR: Control of ribosomal RNA synthesis by hematopoietic transcription factors. Molecular Cell 82(20): 3826-3839, Oct 2022.

Lv K, Gong C, Antony C, Han X, Ren J, Donaghy R, Cheng Y, Pellegrino S, Warren AJ, Paralkar VR, Tong W: HectD1 controls hematopoietic stem cell regeneration by coordinating ribosome assembly and protein synthesis. Cell Stem Cell 28: 1-16, Jul 2021.

Xu P, Palmer LE, Lechauve C, Zhao G, Yao Y, Luan J, Vourekas A, Tan H, Peng J, Scheutz JD, Mourelatos Z, Wu G, Weiss MJ, Paralkar VR: Regulation of gene expression by miR-144/451 during mouse erythropoiesis. Blood 133(23): 2518-2528, Jun 2019.

Traxler EA, Thom CS, Yao Y, Paralkar V, Weiss MJ: Non-specific inhibition of erythropoiesis by short hairpin RNAs. Blood 131(24): 2733-2736, Jun 2018.

Paralkar VR, Taborda CC, Huang P, Yao Y, Kossenkov AV, Prasad R, Luan J, Davies JO, Hughes JR, Hardison RC, Blobel GA, Weiss MJ: Unlinking an lncRNA from Its Associated cis Element. Molecular Cell 62(1): 104-10, Apr 2016.

Paralkar VR, Mishra T, Luan J, Yao Y, Kossenkov AV, Anderson SM, Dunagin M, Pimkin M, Gore M, Sun D, Konuthula N, Raj A, An X, Mohandas N, Bodine DM, Hardison RC, Weiss MJ: Lineage and species-specific long noncoding RNAs during erythro-megakaryocytic development. Blood 123(12): 1927-37, Mar 2014.

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.

Shuo Zhang, Ph.D.

Research interest/work responsibility
My main role at the Penn Epigenetics Institute is to provide bioinformatic services to the Institute’s core members. I am interested in analyzing next-generation sequencing data to gain biological insights. In addition, I am interested in training and applying machine learning models to tackle biological questions.

Skills

  • Whole genome bisulfite sequencing, DNA methylation ChIP array
  • Bulk/single-cell RNA-seq, small RNA-seq (piRNA)
  • ChIP-seq, CUT&RUN, ATAC-seq
  • HiC

Background

I have ten years of hands-on experience in analyzing a variety of next-generation sequencing data. During my PhD research in genomics, I developed a novel computational pipeline to annotate transposable elements (DNA parasites accounting for ~45% of the human genome) from a terabyte-scale whole genome re-sequencing D. melanogaster strains. During my first postdoctoral training, I developed computational pipelines that used HiC data to identify three-dimensional chromatin changes, including split/merge of topologically associated domains (TADs) and changes in chromatin stripes. During my postdoctoral training under the mentorship of Dr. Elizabeth A Heller, I expanded my expertise towards computational analysis of neuronal epigenetic regulation. This training involved analysis and mining of CUT&RUN, ChIP-seq and RNA-seq data. I have also expanded my expertise into machine learning models, such as PLIER, to interrogate cocaine-regulated gene expression in preclinical models of addiction.

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