Worcester, MA, USA. Scientists now can perform structure function studies on chromosomes. The first high-resolution 3D structure of a bacterial genome generates higher-resolution and genome-wide insights, with implications for detailed examination of other organisms.

Co-author Job Dekker predicts that "This work will open an entirely new field of 3D genetics that analyzes spatial folding of genomes, and provides the first tantalizing evidence that researchers can now start to experimentally manipulate the folding of complete chromosomes and study the effect on biology!"

The current research follows up on previous work that mapped the 3D structure of genomes, including the work described in an earlier TS-Si report, Human Genome 3D Structure Optimal For DNA Packing (2009). Scientists then had deciphered the three-dimensional structure of the human genome, which paved the way for new insights into genomic function and expanded our understanding of how cellular DNA folds at scales that dwarf the double helix. The new technology, called Hi-C, helped answer how each of our cells stows some three billion base pairs of DNA while maintaining access to functionally crucial segments.

The 3D structure of the genome reflects and regulates fundamental DNA-based processes.

The team derived models of the entire genomic shape and structure in the bacterium Caulobacter crescentus.

Structure-function analyses revealed that a single cluster of DNA sequence elements involved in chromosome segregation defines the 3D organization.

The interdisciplinary study team included researchers from the University of Massachusetts Medical School, Harvard Medical School, Stanford University and the Prince Felipe Research Centre (CIPF; Spain).

Their study findings appear in the journal Molecular Cell.The approach combines Hi-C-like genome-wide analysis of genome folding with model building, imaging and genetics to present the first complete high-resolution 3D structure of a bacterial genome. Discovery of this structure led to the identification of DNA elements that determine the complete 3D organization of the entire genome. Moving these elements to different locations in the genome results in genome-wide changes in 3D structure in which the entire genome rotates in the cell.

Scientists know that the three-dimensional shape of a cell's chromosome plays a role in how genetic sequences and genes are regulated. However, technical challenges have limited genome-wide analysis of a chromosome's architecture that would allow for simultaneous identification of the elements involved in shaping it and analysis of specific features of the structure.

In this study, researchers used high-throughput chromatin interaction detection; next-generation DNA sequencing; computational modeling; and fluorescent microscopy to build the first 3D model of the architecture of the bacteria's chromosome and analyze the resulting structures. This new experimental approach revealed novel characteristics of a specific genetic sequence called the parS site, which helps to define the chromosome's shape.



Job Dekker and his colleagues originally developed Chromosome Conformation Capture (3C) to detect physical interactions between genomic elements (Dekker et al. Science, 2002).

Dekker's group later combined 3C, now widely used for genome regulation studies, with ultra-high-throughput DNA sequencing. This significanty increased the effective scale at which interactions between genomic loci can be studied.

The new 5C is essentially a high-throughput 3C version for large-scale mapping of chromatin interaction networks (Dostie et al. Genome Research 2006).

The team obtained detailed insights into the 3D arrangements of complete genomes at Kb resolution when they developed Hi-C technology.

It is a genome-wide and unbiased method that combines both 3C with deep sequencing (Lieberman-Aiden, van Berkum et al. Science 2009).Job Dekker, PhD, is a pioneer in chromosome interaction detection technologies, professor of biochemistry and molecular pharmacology at the University of Massachusetts Medical School, and a study co-author. "What we've shown," says Dekker, "is that it's possible to combine molecular biology with 3D modeling technology to perform studies that tell us novel things about how genomes fold and identify the genetic sequences that are responsible."

Dekker and his colleagues used 5C technology to map more than 28,700 contact points in the Caulobacter crescentus genome, using these contacts to approximate spatial distance in the folded chromosome. Plugged into a computational model, these contact points yielded a structural model of the bacterial chromosome which was strikingly beautiful: ellipsoidal in shape with arms helically arranged on either side.

The resulting 3D models of the Caulobacter crescentus genome, in conjunction with fluorescent microscopy, illustrate that the parS sequence, located in the pole of one arm of the chromosome, potentially served as an anchor for the genome and were instrumental in defining its overall structure.

Marc A. Marti-Renom, PhD, a computational biologist who leads the Structural Genomics Laboratory at the Prince Felipe Research Centre (CIPF; Spain), said "This work demonstrates that hybrid methods combining 5C maps with the Integrative Modeling Platform can produce genome-wide 3D models of unprecedented resolution, which for the first time allows for spatially pinpointing regulatory elements responsible of organizing the structure of a genome."

To unravel the role the parS site plays in the 3D organization of the chromosomal structure, Dekker and colleagues constructed mutant bacteria in which the parS site had been moved away from its normal position. Building 3D models of the shape of the mutated bacteria, they observed a change in the chromosome's structure; the entire genome had rotated clockwise.

Changing the position of the parS site had resulted in a large-scale reorganization of the chromosome's shape that repositioned these sites at the cell's poles. Mark Umbarger, a post doctoral fellow at Harvard Medical School and study author notes, "Strikingly, we found that moving sequence elements which are no larger than 500 base pairs, led to a change in the conformation of all of the 4 million base-pairs of the chromosome!"

"Our study is the first to test the effect of altering chromosome architecture. We were able to show that a very simple system, with a single anchor, can orient the whole chromosome inside of the cell." said Esteban Toro, PhD, one of the study authors and now a post doctoral fellow at the University of Pennsylvania. "These results suggest that the parS site in Caulobacter crescentus determines the orientation and global structure of the entire chromosome and are the only sequence elements that stably anchor the chromosome to the cell."

The ability for scientists to perform structure function studies on chromosomes has the potential to yield powerful new insights into the biology of genomes. "When we began this project, most scientists were assessing the positions of a handful of genomic loci and attempting to derive general conclusions about genome structure. We were unhappy with this approach and sought to develop an integrated experimental approach to generate higher-resolution, and genome-wide insights," Umbarger said.

"This isn't something we could have predicted from just looking at the DNA sequence," said Dekker. "This study illustrates how an investigation of 3D genomic structure can provide insights into how the complex relationships between genome sequence and structure can impact function. By studying genomic architecture we can potentially identify new classes of genomic sequences that are important in chromosome function and structure that we otherwise couldn't."

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