3D mapping of genome in combine FISH and RNAi
Larry H. Bernstein, MD, FCAP, Curator
LPBI
Cellular factors that shape the 3D landscape of the genome identified
http://www.nih.gov/news/health/aug2015/nci-13.htm
Researchers, using novel large-scale imaging technology, have mapped the spatial location of individual genes in the nucleus of human cells and identified 50 cellular factors required for the proper three-dimensional (3D) positioning of genes. These spatial locations play important roles in gene expression, DNA repair, genome stability, and other cellular activities. The study, by scientists at the National Cancer Institute (NCI), part of the National Institutes of Health, appeared August 13, 2015, in Cell.
One of the fundamental properties of the genomes of higher organisms is the non-random arrangement of DNA in the cell nucleus. Researchers have long known that most genes occupy preferred 3D positions in the nucleus and that the location of genes matters for their function, but it has been difficult to determine the molecular players and mechanisms that determine the positions. Although genes can be visualized routinely and their positions determined using fluorescence in situ hybridization, or FISH, this mapping method has traditionally been limited to the analysis of a few samples at a time and cannot be used for large-scale genome mapping.
NCI researchers, in close collaboration with NCI’s High-Throughput Imaging Facility, which was established earlier this decade, have developed a method called HIPMap (High-throughput Imaging Position Mapping) that makes the large-scale determination of 3D gene positions possible. This method uses an optimized FISH detection protocol, fully automated microscopy, and combines it with sophisticated computational image analysis that delivers high-precision gene mapping information for thousands of samples in a single experiment.
In the study, NCI researchers, led by Tom Misteli, Ph.D., associate director, NCI Center for Cancer Research, used HIPMap and a method known as RNA interference (RNAi) knockdown to screen nearly 700 proteins in the nucleus to identify those that are involved in the 3D positioning of several human genes. RNAi knockdown uses RNA molecules to block the production of specific proteins in cells.
By collecting data continuously from automated microscopes for 27 days and then analyzing more than three million data points, the scientists were able to identify 50 cellular factors that determine the location of genes in the cell nucleus. This list provides the basis for further investigation of the molecular mechanisms of genome organization.
“The importance of HIPMap is that it is a starting point for numerous applications, including cancer biology,” said Misteli. “In addition to addressing basic questions about the mechanisms of how genomes are organized in intact cells, the ability to map gene positions in a large number of samples and cells has already been used to detect very rare chromosome translocation events in cancer and to ask what cellular factors determine where chromosomes break.” During translocations, chromosomes break and reattach, which can cause the fusion of otherwise unconnected genes, resulting in hybrid genes whose protein products may contribute to the development of cancer.
As an example of the implications of HIPMap, Misteli pointed to a study from his lab published last month (Burman et al., Genes and Development. July 1, 2015). In that study researchers used a method derived from HIPMap to probe mechanisms that contribute to the susceptibility of chromosomes to break and form a cancer-causing translocation between the NPM1 gene and the ALK gene in a cancer known as anaplastic large cell lymphoma. Another possible application of HIPMap is in cancer diagnostics. The researchers have previously shown that some genes assume distinct positions in cancer. As a result, the 3D positions of genes could be used as diagnostic markers in diseases such as breast cancer and prostate cancer.
“HIPMap will be a powerful tool in many ongoing efforts to map the genome in 3D space and to translate the findings from these studies to cancer biology,” Misteli concluded.
About the National Institutes of Health (NIH): NIH, the nation’s medical research agency, includes 27 Institutes and Centers and is a component of the U.S. Department of Health and Human Services. NIH is the primary federal agency conducting and supporting basic, clinical, and translational medical research, and is investigating the causes, treatments, and cures for both common and rare diseases. For more information about NIH and its programs, visit www.nih.gov.
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Reference
Shachar S, Voss TC, Pegoraro G, Sciascia N, Misteli T. Identification of Gene Positioning Factors Using High-throughput Imaging Mapping. Cell. August 13, 2015. DOI: 10.1016/j.cell.2015.07.035.
Stephen J. Williams, PhD
It is interesting they were able to complete this work and develop this technology which I see has other applications than which they suggested. It has been shown how certain factors (HIRAII) accumulate in certain areas of the chromatin during earliest stages of transformation and facilitate massive chromatin remodeling. That genetic information is spatially regulated, particular specific genes, and their technology can map it in a way I feel which is more accurate than probe methodologies or common sequencing methodologies is exciting news. however it may be difficult to use this as an early detection platform, unless it is done post-biopsy. A proceedure, possibly sensor based would need to be developed as well an an invitral imaging methodology.
The particular interesting application I would find would be the detection of insertion sites of genetic therapy. Currently it requires a long procedure involving knowledge of flanking sequences but this mapping procedure would help greatly, especially as I see it in determining insertion sites in development of personalized CART therapy. I would be interested if this group have been able to transfect in a gene and use their HIMAP to determine the spatial insertion site. Will be nice to see this work evolve..
This is very insightful. There is no doubt that there is the bias you refer to. 42 years ago, when I was postdocing in biochemistry/enzymology before completing my residency in pathology, I knew that there were very influential mambers of the faculty, who also had large programs, and attracted exceptional students. My mentor, it was said (although he was a great writer), could draft a project on toilet paper and call the NIH. It can’t be true, but it was a time in our history preceding a great explosion. It is bizarre for me to read now about eNOS and iNOS, and about CaMKII-á, â, ã, ä – isoenzymes. They were overlooked during the search for the genome, so intermediary metabolism took a back seat. But the work on protein conformation, and on the mechanism of action of enzymes and ligand and coenzyme was just out there, and became more important with the research on signaling pathways. The work on the mechanism of pyridine nucleotide isoenzymes preceded the work by Burton Sobel on the MB isoenzyme in heart. The Vietnam War cut into the funding, and it has actually declined linearly since.
A few years later, I was an Associate Professor at a new Medical School and I submitted a proposal that was reviewed by the Chairman of Pharmacology, who was a former Director of NSF. He thought it was good enough. I was a pathologist and it went to a Biochemistry Review Committee. It was approved, but not funded. The verdict was that I would not be able to carry out the studies needed, and they would have approached it differently. A thousand young investigators are out there now with similar letters. I was told that the Department Chairmen have to build up their faculty. It’s harder now than then. So I filed for and received 3 patents based on my work at the suggestion of my brother-in-law. When I took it to Boehringer-Mannheim, they were actually clueless.