(6jb) Integrated single-cell genomics: Combined epigenome and transcriptome sequencing of single cells to understand cellular differentiation

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: s.dey@hubrecht.eu.
Website: http://siddharthdey.com

Education

·  
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).

Research Appointments

·  
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.

Research Interests

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.

Research Experience

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.

Future Directions

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.

Publications

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).