Integrated single-cell genomics: Combined epigenome and transcriptome sequencing of single cells to understand cellular differentiation
Siddharth S. Dey, Ph.D.
Alexander van Oudenaarden's group, Hubrecht Institute, The Netherlands.
Email: firstname.lastname@example.org. Website: http://siddharthdey.com
· University of California, Berkeley, USA. Ph.D., Chemical and Biomolecular Engineering (May 2012).
· Institute of Chemical Technology, Mumbai, India. B.S., Chemical Engineering (May 2006).
· Post-doctoral researcher (11/2012 - Present).
Quantitative Biology of Development & Stem Cells, Hubrecht Institute, The Netherlands.
Advisor: Alexander van Oudenaarden.
Project: Developing novel single-cell genomics methods to quantify cell-to-cell heterogeneity in cancers and stem cells.
· Graduate Research Assistant (08/2006 - 10/2012).
Department of Chemical and Biomolecular Engineering, University of California, Berkeley, USA.
Advisor: David V. Schaffer.
Ph.D. Dissertation: A computational and experimental approach to understanding HIV-1 evolution and latency for the design of improved antiviral therapies.
· Undergraduate Research Assistant (05/2004 - 07/2004).
Department of Organic Chemistry, Indian Institute of Science, Bangalore, India.
Advisor: Goverdhan Mehta.
Project: Total synthesis of cyclitols and investigation of their crystal structures.
The genome within all cell-types of a multicellular organism is identical, yet different cell-types display varied functions within an organism due to differences in other factors, collectively termed as the epigenome. Similarly, in specific cases, such as cancers, viral infections and in certain cell-types during normal development, mutations and other structural variations within the genome also influences cellular functions. Thus, a central question in biology is to understand how the genome or epigenome influences cellular phenotypes. From an engineering perspective, gene expression regulation can be viewed as the output of a network of complex chemical and physical processes, and understanding how these processes interact and integrate to govern cellular phenotypes has been a major focus of my graduate and postdoctoral research. More specifically, regenerative medicine has recently emerged as a promising therapeutic strategy for treating degenerating tissues using different types of stem cells. However, the full promise of regenerative medicine has been difficult to achieve so far, partly due to our incomplete understanding of the genetic and epigenetic mechanisms regulating differentiation of stem cells to specific lineages and tissues.
To understand how the genome and epigenome regulates cellular function, development of high-throughput sequencing methods, known as next-generation sequencing, are beginning to unravel genome-wide correlations between the genome, epigenome and transcriptome within a large population of cells or tissues. However, as these measurements are made from a bulk population, they only provide an average description of the system. Because tissues are composed of several cell-types and even cells within the same cell-type have been shown to display dramatic cell-to-cell variability in gene expression, bulk measurements obscure quantification of how genetic or epigenetic features directly influence the function of individual cells. To overcome this limitation, recent advances in molecular biology have enabled genome-wide single-cell measurements of the transcriptome, genome or certain epigenetic marks that capture this cell-to-cell heterogeneity. Thus, my research interests are to develop novel single-cell genomics methods to better understand how changes in the epigenetic landscape during normal development regulates cellular differentiation, information that is critical towards realizing the full potential of regenerative medicine.
My graduate and postdoctoral research has focused on investigating how the genome and epigenome regulates the dynamics of gene expression in viral and mammalian systems8. During my graduate studies, I used a systems biology approach to demonstrate that chromatin environments at different genomic loci decouple transcription factor mediated initiation of gene expression from subsequent gene activation6. Further, while cell-to-cell heterogeneity in gene expression has been shown to drive dramatic phenotypic variations, the upstream epigenetic mechanisms regulating this heterogeneity remain largely unknown8. I used DNA accessibility assays and single-molecule mRNA FISH (smFISH) to discover that repressed chromatin is associated with increased gene expression noise3. This project sparked my interest in single-cell biology. However, traditional single-cell techniques, such as smFISH, allowed quantification of only a handful of genes concurrently while next-generation sequencing techniques only enabled ensemble averaged genome-wide measurements starting from a bulk population. Therefore, as a post-doctoral researcher, I was interested in developing genome-wide quantification techniques in single cells. In particular, I developed the first genome-wide technology that enables sequencing both genomic DNA and mRNA from the same cell4. I found that low genomic copy numbers drive increased gene expression variability between tumor cells. Next, to understand the extent of cell-to-cell heterogeneity in the epigenome, I have been involved in several projects. First, we developed a method to map the 3D organization of the genome in single cells2. Similarly, I developed another technique to quantify the epigenetic modification, 5-hydroxymethylcytosine in single mouse embryonic stem cells1.
These studies have shown that a major unanswered question is to precisely understand how upstream epigenetic mechanisms regulate cell-to-cell heterogeneity in gene expression. This question has been difficult to study and the mechanisms of regulation are poorly understood because current technologies are limited to quantifying either the genome, epigenome or transcriptome from a single cell. To unambiguously understand how a particular gene expression program in a cell is regulated will require direct measurement of both the transcriptome together with the epigenome from the same cell. Historically, development of new technologies is closely followed by giant leaps in science and therefore in my laboratory, we will develop novel integrated technologies that enable simultaneous genome-wide measurements of the epigenome and transcriptome from the same cell to gain insights into early mammalian development and mechanisms contributing to maintenance and regeneration of adult tissues.
Specifically, we propose to quantify multiple epigenetic marks, such as DNA methylation, DNA hydroxymethylation, DNA accessibility and transcription factor occupancy together with the transcriptome of single cells, innovations that will require advances in protein engineering, molecular biology and robotics. These integrated single-cell sequencing techniques will provide a unique opportunity to explore differentiation of pluripotent stem cells into the three germ layers of the developing mouse embryo to address fundamental questions in the field and lay foundations for the design of better cell-based therapies in regenerative medicine.
1. Mooijman D, Dey SS (co-first author), Boisset JC, Crosetto N, van Oudenaarden A. Single-cell 5hmC sequencing reveals extensive chromosome-wide epigenetic heterogeneity. Nature Biotechnology (Manuscript under review).
2. Kind J, Pagie L, de Vries S, Azar LN, Dey SS, Bienko M, Zhan Y, Lajoie B, de Graaf CA, Amendola M, Imakaev M, Fudenberg G, Mirny L, Jalink K, Dekker J, van Oudenaarden A, van Steensel B (2015). Genome-wide maps of nuclear lamina interactions in single human cells. Cell (Accepted, in press).
3. Dey SS, Foley JE, Limsirichai P, Schaffer DV, Arkin AP (2015). Orthogonal control of expression mean and variance by epigenetic features at different genomic loci. Molecular Systems Biology 11:806.
o Study highlighted in: Tyagi S (2015). Tuning noise in gene expression. Molecular Systems Biology 11:805.
4. Dey SS, Kester L, Spanjaard B, Bienko M, van Oudenaarden A (2015). Integrated genome and transcriptome sequencing from the same cell. Nature Biotechnology 33:285-289.
o Study highlighted in: The genome and transcriptome of a single cell (2015). Nature Methods 12:173.
o Study highlighted in: One cell at a time (2015). Cell 161: 1479.
5. Wong VC, Fong LE (co-first author), Adams NM, Xue Q, Dey SS, Miller-Jensen K (2014). Quantitative evaluation and optimization of co-drugging to improve anti-HIV latency therapy. Cellular and Molecular Bioengineering 7:320-333.
6. Miller-Jensen K, Dey SS (co-first author), Pham N, Foley JE, Arkin AP, Schaffer DV (2012). Chromatin accessibility at the HIV-1 LTR promoter sets a threshold for NF-κB mediated viral gene expression. Integrative Biology 4:661-671.
7. Dey SS, Xue Y, Joachimiak MP, Friedland GD, Burnett JC, Zhou Q, Arkin AP, Schaffer DV (2012). Mutual information analysis reveals coevolving residues in Tat that compensate for two distinct functions in HIV-1 gene expression. Journal of Biological Chemistry 287:7945-7955.
8. Miller-Jensen K, Dey SS (co-first author), Schaffer DV, Arkin AP (2011). Varying Virulence: Epigenetic control of expression noise and disease processes. Trends in Biotechnology 29:517-525.
9. Dey SS, Prausnitz JM (2011). Opportunities for chemical engineering thermodynamics in biotechnology: Some examples. Industrial & Engineering Chemistry Research 50:3-15.
10. Mehta G, Sen S, Dey SS (2005). (1S*,2S*,4S*,5S*)-Cyclohexane-1,2,4,5-tetrol monohydrate. Acta Crystallographica Section C 61 (Pt 6):o358-360.
11. Mehta G, Sen S, Dey SS (2005). (1R*,2S*,4S*,5S*)-Cyclohexane-1,2,4,5-tetrol. Acta Crystallographica Section E 61 (Pt 4):o920-922.
Teaching and Mentoring Experience
· Mentored a Masters student, Department of Molecular and Cellular Life Sciences, Utrecht University (01/2014 - 08/2014).
· Mentored two Graduate students, Department of Plant and Microbial Biology, University of California, Berkeley (11/2011 - 05/2012).
· Mentored two Graduate students, Department of Bioengineering, University of California, Berkeley (01/2009 - 05/2009 & 08/2011 - 12/2011).
· Teaching assistant, Chemical engineering thermodynamics, University of California, Berkeley (01/2009 - 05/2009).
· Teaching assistant, Chemical process design, University of California, Berkeley (01/2008 - 05/2008).
Awards and Honors
· Keystone Symposia Scholarship: Conference Travel Scholarship (2011).
· JRD Tata Trust Scholarship: All round academic excellence (2004 - 2005).
· Sir Ratan Tata Trust Scholarship: All round academic excellence (2003 - 2004).
· Gujarat Ambuja Cement Award: First rank in Semester I of Chemical Engineering (2002 - 2003).
· Certificate of Honor from Maharashtra State Board: Ninth rank out of 200,000 students in High School (2001 - 2002).
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