Research


Research in the Wu Lab

Our laboratory studies how chromosome behavior and positioning influence genome function and evolution, with implications for gene regulation, genome stability, and disease. Using genetic, molecular biological, computational, and imaging tools, we approach these topics from a variety of angles. We also develop technologies. For example, in the area of microscopy, we are continuing to work with Oligopaints1,2, an oligo-based strategy for fluorescent in situ hybridization (FISH) that has enabled homolog-specific probes (HOPs)3 as well as methods for the in situ super-resolution imaging of chromosomal DNA (OligoSTORM and OligoDNA-PAINT)3,4. Our laboratory is also involved in the Consortium for Space Genetics, which addresses the application of genetic technologies to long-term exploration of Space and, most recently, climate change on Earth. In addition, we are the home for the Personal Genetics Education Project (pgEd.org), which promotes awareness of, and dialog about, personal genetics in our communities.

Haplotype-resolved Hi-C reveals somatic homolog pairing in early Drosophila embryos. Adapted from ref. 12.

In tight pairing, the frequency of trans-homolog (thom) interactions between two loci representing 1 kb of genomic separation is equivalent to that of cis interactions between two loci across 5 kb of genomic separation (top, left). In contrast, the frequency of thom interactions for two loci representing 1 kb of genomic separation in a loosely paired region is equivalent to that of cis interactions across 30 kb of genomic separation (top, right). Tightly and loosely paired regions also produce different patterns in haplotype-resolved Hi-C maps (bottom). Adapted from ref. 13.

Homolog pairing in somatic cells

Localized as well as telomere-to-telomere pairing of homologous chromosomal regions in somatic cells has been implicated in the regulation of gene expression across a wide variety of species (e.g., Drosophila, mammals, plants, and fungi) and in phenomena as diverse as transvection, pairing-sensitive silencing, repeat-induced point mutation, parental imprinting, meiotic silencing of unpaired DNA, X-inactivation, VDJ recombination, allelic skewing, Oct4 behavior during stem cell differentiation, misexpression of human chromosome 19 in renal oncocytoma cells, and sequence ultraconservation (reviewed in 5; also refs. 6, 7). Using a method we developed for high-throughput FISH (Hi-FISH), we have conducted a whole-genome screen for genes that control pairing, uncovering candidates that promote pairing as well as those that inhibit pairing8,9, even revealing a pairing-based signature that distinguishes germ line from soma in early Drosophila embryogenesis10. These studies have also brought us face-to-face with sister chromatid cohesion11. Most recently, we developed and then applied a haplotype-resolved Hi-C approach (Ohm) to elucidate the structure of homologously paired somatic chromosomes in early Drosophila embryos and cultured Drosophila cells, observing at least two types of pairing (tight and loose) and a correlation between pairing and gene expression12,13.

Genome visualization with Oligopaints

We develop technologies for visualizing chromosomes in situ in order to study the 3D organization of the genome. One such technology is Oligopaints, an oligo-based approach for FISH that can be used to image single copy regions as small as 3 kb or less and as large as tens of megabases1-4,14.

The maternal and paternal 2.6 Mb regions containing the X-inactivation centers (XICs) in tetraploid 129xCAST hybrid mouse cells can be distinguished using 129-specific and CAST-specific HOPs probes. Simultaneous RNA FISH showed 100% co-localization of Xist RNA with the paternal 129 XICs, as would be expected in these cells. Adapted from ref. 3.

HOPs, a strategy for distinguishing homologs:

We have harnessed single nucleotide polymorphisms (SNPs) to enable an Oligopaint-based method for visually distinguishing the maternal and paternal homologous chromosomes in Drosophila and human systems3,14. Most recently, we combined HOPs with super-resolution imaging to observe that the maternal and paternal homologs of at least one region of human chromosome 19 differ more from each other in terms of ellipticity when they are present in the same nucleus than would be expected at random14.

OligoSTORM can reveal details of chromosome structure and organization that are not accessible via conventional microscopy. The heatmap (left) depicts the bithorax complex (BX-C) in Drosophila cell (cartoon) as imaged with conventional widefield FISH. OligoSTORM images (right) reveals loops and aggregates.

OligoSTORM and OligoDNA-PAINT, two single-molecule super-resolution strategies for visulaizing the genome:

We have developed two methods, called OligoSTORM and OligoDNA-PAINT, for the in situ super-resolution imaging of chromosomal DNA3,4,14. Application of these technologies has enabled us to visualize genomic regions as small as a 2.9 kb differentially methylated region (DMR) and the 59.5 kb DNMT1 gene and as large as an 8.16 Mb region on human chromosome 19. As the latter was imaged in sequential steps, it revealed a variety of 3D organizational features, including a loop (290 kb), contact domains/topologically associating domains, or TADs (90 – 350 kb), and compartmental segments (360 kb – 1 Mb)14. We found that active (A-type) or inactive (B-type) compartments, as defined by Hi-C interaction frequencies, correspond to 3D organizational structures and, because OligoSTORM can address the physical parameters of distance, surface area, volume, sphericity, and density, it was able to reveal both the apposition as well as intermingling of compartmental segments. Furthermore, the integration of Hi-C and OligoSTORM via integrative modeling of genomic regions (IMGR) produced a 10-kb resolution model of chromosome folding across an 8.16 Mb region. We are now examining the structures of paired homologs, centromeres, developmentally regulated chromosomal domains, and highly condensed chromosomes.

OligoSTORM image of 8.16 Mbs of human chromosome 19 in 9 steps ranging in size from 360 kb to 1.8 Mb. Adapted from ref. 14.

OligoSTORM produces space-filling images, wherein each signal (sphere) represents a single blink of a single fluorophore. This image shows a 290 kb loop of human chromosome 19, including its two 10 kb anchors. Adapted from ref. 14.

Distribution of 896 UCEs representing human-mouse-rat (HMR), human-mouse-dog (HMD), and human-chicken (HC) sequences. Adapted from ref. 15.

Ultraconserved elements (UCEs) and genome integrity

UCEs con­sist of up to thousands of DNA sequences, scattered across the genome, that have been main­tained essen­tial­ly unchanged for hundreds of millions of years, appearing today exactly as they were when birds, rep­tiles, and mammals diverged. Because no known function requires such con­serva­tion, the mere exist­ence of UCEs is considered one of the most profound mys­ter­ies to emerge from the genome era. Breaking rank from other models, we have proposed that ultraconser­va­tion can be explained if the two allelic copies of each UCE physically pair up and then undergo sequence compari­son such that dis­ruptions in sequence, copy number, or allelic pairing lead to loss-of-fitness via disease or reduced fertility15. In this way, disruptions of UCEs would ulti­mately be lost from the popu­la­tion, explaining how UCEs sur­vive unchanged through time. If true, this mechanism would contribute to the main­te­nance of genome integrity. We are testing this model using computational and wet bench strategies to reveal the relationship between UCEs, copy number variants (CNVs), balanced rearrangements, selection pressure, and disease15-19.

X-inactivation

Our overarching interest in how diploid genomes respond to the presence of homologous chromosomal regions has led us to consider mechanisms that can cause a diploid cell to be functionally hemizygous at specific loci or across an entire chromosome. These mechanisms include random mononallelism, parental imprinting, X-inactivation, and loss-of-heterozygosity through mitotic recombination. Here, we have ventured to publish a number of speculative models20-22.

Biomedicine in space

We are interested in addressing issues of human health in Space via two lines of investigation. The first stems from our proposal that UCEs (above) may contribute to the maintenance of genome integrity by removing cells that have acquired deleterious rearrangements15-19. In this way, UCEs could offer a strategy for protecting humans and other organisms from radiation damage during long-term travel in space. The second avenue aims to assess the impact of extreme environments on the genome. In particular, we aim to apply Oligopaint technologies1-4, including OligoSTORM and OligoDNA-PAINT, to determine whether the conditions of Space, such as absence of gravity, can affect 3D genome organization.

references

1. Beliveau BJ*, Joyce ER*, Apostolopoulos N, Yilmaz F, Fonseka CY, McCole RB, Chang Y, Li JB, Senaratne TN, Williams BR, Rouillard J-M, Wu CT. Versatile design and synthesis platform for visualizing genomes with Oligopaint FISH probes. Proc Natl Acad Sci USA 2012; 109(52):21301-6. PMCID: PMC3535588.

2. Beliveau BJ, Apostolopoulos N, Wu CT. Visualizing genomes with Oligopaint FISH probes. Curr Protoc Mol Biol 2014; 105:Unit 14.23 doi: 10.1002/0471142727.mb1423s105. PMCID: PMC3928790.

3. Beliveau BJ, Boettiger AN, Avendaño MS, Jungmann R, McCole RB, Joyce EF, Kim-Kiselak C, Bantignies F, Fonseka CY, Erceg J, Hannan MA, Hoang HG, Colognori D, Lee JT, Shih WM, Yin P, Zhuang X, Wu CT. Single-molecule super-resolution imaging of chromosomes and in situ haplotype visualization using Oligopaint FISH probes. Nat Commun 2015; 6:7147. PMCID: PMC4430122.

4. Beliveau BJ*, Boettiger AN*, Nir G*, Bintu B*, Yin P, Zhuang X, Wu CT. In situ super-resolution imaging of genomic DNA with OligoSTORM and OligoDNA-PAINT. Methods Mol Biol 2017; 1663:231-252. PMCID: PMC5919218.

5. Joyce EF, Erceg J, Wu CT. Pairing and anti-pairing: a balancing act in the diploid genome. Curr Opin Genet Dev 2016; 37:119-128. PMCID: PMC4939289.

6. Selker EU, Cambareri EB, Jensen BC, Haack KR. Rearrangement of duplicated DNA in specialized cells of Neurospora. Cell 1987; 51:741-52. PMID: 2960455.

7. Shiu PK, Raju NB, Zickler D, Metzenberg RL. Meiotic silencing by unpaired DNA. Cell 2001; 107:905-16. PMID: 11779466.

8. Joyce EF, Williams BR, Xie T, Wu CT. Identification of genes that promote or antagonize somatic homolog pairing using a high-throughput FISH-based screen. PLoS Genetics 2012; 8:e1002667. PMCID: PMC3349724.

9. Joyce EF. Toward high-throughput and multiplexed imaging of genome organization. Assay Drug Dev Technol 2017; 15:11-14. doi: 10.1089/adt.2016.770. PMID: 28092459.

10. Joyce EF, Apostolopoulos N, Beliveau BJ, Wu CT. Germline progenitors escape the widespread phenomenon of homolog pairing during Drosophila development. PLoS Genetics 2013; 9:e1004013. PMCID: PMC3868550.

11. Senaratne TN, Joyce EF, Nguyen SC, Wu CT. Investigating the interplay between sister chromatid cohesion and homolog pairing in Drosophila nuclei. PLoS Genetics 2016; 12:e1006169. PMCID: PMC4991795.

12. Erceg J*, AlHaj Abed J*, Goloborodko A*, Lajoie BR, Fudenberg G, Abdennur N, Imakaev M, McCole RB, Nguyen SC, Saylor W, Joyce EF, Senaratne TN, Hannan MA, Nir G, Dekker J, Mirny LA, Wu CT. The genome-wide, multi-layered architecture of chromosome pairing in early Drosophila embryos. Nat Commun 2019; 10:4486. PMCID: PMC6776651.