Three dimensional (3D) genomics Siyuan (Steven) Wang Lab, Department of Genetics, YSM, http://campuspress.yale.edu/wanglab/ Three dimensional (3D) genomics DNA Nucleosomes Topologically Associating Domains (TADs) A-B compartments Chromosomal territories My lab is very interested in the three dimensional genomics or the spatial organization of chromatin particularly in mammalian cell nucleus, and how the spatial organization of chromatin impacts cellular states. We are interested in this topic first because how DNA is packaged in a nucleus is intrinsically a very interesting and fundamental biophysical question. In each of our cells, the total length of the genomic DNA is usually about 2 meters. Now how do cells fit 2 meters of DNA into a nucleus of about 10 um in diameter, and let it go through replication, cell cycle and other biological processes without messing up. That’s a daunting task if you ask me! We know for quite a while that DNA wraps around histones to form nucleosomes. And on large length scales during the interphase of cell cycle, individual chromosomes occupy distinct nuclear space to form chromosome territories. And only in recent years, thanks to the development of the new technique called Hi-C, people were to able to discover two new intermediate structures called topologically associating domains and the A-B compartment organization. These structures were shown to have intriguing dynamics during different biological processes such as cell cycle, differentiation of embryonic stem cell, gamete-to-zygote transition, cellular aging, and diseases such as cancer. But there are still many key gaps in our understanding of the 3D genome. One of them, a decades-old question that people have not been able to answer, is: What is the 3D folding path of chromatin at all length scales above the nucleosomes? From TADs to compartments to chromosomes territories, these are essentially larger and larger chromatin blobs. In structural studies of protein, we get to know the 3D folding path of peptide chains. How can we get the same kind of information for chromatin organization? To address this need, we developed a new technology to directly trace the spatial organization of numerous genomic regions in individual chromosomes in single cells. Fundamental cell biology and biophysics Intriguing dynamics during biological processes Functional consequences Misteli, Cell, 2007 Gorkin, Leung and Ren, Cell Stem Cell, 2014 Levine, Cattoglio and Tjian, Cell 2014 Dekker and Mirny, Cell 2016 Bickmore and van Steensel, Cell 2013 Cremer and Cremer, Cold Spring Harb. Perspect. Biol., 2010 What is the 3D folding path of chromatin at length scales above the nucleosomes?

Siyuan (Steven) Wang Lab, Department of Genetics, YSM, http://campuspress.yale.edu/wanglab/ The idea of tracing The idea behind the tracing is that we hope to fluorescently label numerous genomic loci and image their spatial positions. However, one major challenge is that with one fluorescent color, we cannot distinguish the genomic identities of these loci, so it’s going to be hard to figure out which locus connects with which locus in a image. Also, we showed here that if many loci in the same chromosome are simultaneously labeled with the same color, in the image their fluorescence are all connected to each other, so one cannot even resolve the different genomic loci. Traditionally, people have used different fluorescent colors to distinguish different genomic loci, but there are a only a few colors available for fluorescence microscopy, not enough to distinguish so many loci. To tackle this challenge, we developed a sequential imaging strategy. Wang et al, Science, 2016

Siyuan (Steven) Wang Lab, Department of Genetics, YSM, http://campuspress.yale.edu/wanglab/ The idea of tracing Multiplexed sequential fluorescence in situ hybridization (FISH) In this strategy, we first image only one genomic locus, and record its position in 3D. We then bleach the fluorescence, and label the next locus on the genomic map with the same fluorescence color. We can then connect the 3D positions of these loci because we know they are next to each other on the genomic map. We can repeat this bleach-label-connect procedure over and over again, until all the genomic loci are imaged and connected, and a chromatin trace is revealed. In this work we introduced this invention, which is a multiplexed sequential version of fluorescence in situ hybridization, or FISH. With this technique, we traced the spatial organization of chromatin in individual human chromosomes at the Mb-to-whole chromosome length scale in a common cell line, and already discovered a series of unexpected structural features. FISH, if you know, were traditionally applied to detect both DNA and RNA. Our multiplexed sequential FISH can surely be applied to RNA as well. In this work, …. Wang et al, Science, 2016

Siyuan (Steven) Wang Lab, Department of Genetics, YSM, http://campuspress.yale.edu/wanglab/ Multiplexed imaging of transcriptome Imaging 100-1000 distinct RNA species in single cells we developed a similar multiplexed FISH technology to image 100-1000 distinct RNA species in single cells. Here we used a combinatorial barcoding scheme and an error-robust encoding scheme borrowed from telecommunication to label 100-1000 different RNA species, and distinguish them with merely 16 rounds of imaging. For each RNA species we can count its copy number and get the single molecule localizations, and all these labeled RNA species can be profiled in the same cell. So we are essentially getting single cell transcriptome information with molecular and cellular spatial information at the same time. Chen et al, Science, 2015

Siyuan (Steven) Wang Lab, Department of Genetics, YSM, http://campuspress.yale.edu/wanglab/ All these work are made possible with our self built automated imaging and flow system. Here at yale my system has been fully up and running since last December, and we are generating exciting in situ DNA tracing the RNA profiling data.

Acknowledgements Yale: Prof. Yasuko Iwakiri Prof. Sherman Weissman Prof. Samuel Katz Prof. Joerg Bewersdorf Prof. David Baddeley Prof. In-Hyun Park Prof. Andrew Xiao Prof. Valentina Greco Prof. Jun Lu Prof. Stefania Nicoli Prof. Joan Steitz Prof. Antonio Giraldez Miao Liu MCGD 2nd year Yanbo Chen BQBS 2nd year Bing Yang MCGD 2nd year Yanfang Lu Postgrad research associate Finally I would like to thank my lab members for their contributions, and also thank my collaborators and former colleagues. Harvard: Prof. Xiaowei Zhuang Jun-Han Su Bogdan Bintu Jeffrey Moffitt Kok Hao Chen Alistair Boettiger Prof. Chao-ting Wu Brian Beliveau Angela Su Visiting student UConn Mark Pownall MCGD rotation Mengwei Hu MCGD rotation Jonathan Radda MCGD rotation