DNA Loop-the-Loops

A new full-genome map indicates how DNA is folded within the nuclei of human cells.

By | December 11, 2014

Physics simulation of 5 megabases of DNA forming loops and domainsADRIAN SANBORN, EREZ LIEBERMAN AIDEN Researchers have created the highest-resolution map to date of how the human genome folds within the nucleus, according to a study published today (December 11) in Cell. The work illuminates basic facts about the genome’s 3-D structure, including that it forms around 10,000 loops. It also sheds light on how genome structure influences gene expression, as looping DNA brings promoters and enhancers into close proximity. The work covers one mouse and eight human cell types.

“This is indeed a standard-setting paper,” said Bing Ren, a professor of cellular and molecular medicine at the University of California, San Diego, who was not involved in the study. “It's a landmark in the field of genome architecture.” Ren’s lab published its own 3-D map of genome structure last year, but according to Ren, this latest version has five to 10 times better resolution.

“This huge dataset will be used as a highly valuable resource for many researchers to mine and address all sorts of questions related to the functioning of our genome,” Wouter de Laat, who studies DNA architecture at the Hubrecht Institute in the Netherlands, wrote in an e-mail to The Scientist.

The work was led by Erez Lieberman Aiden, director of the Center for Genome Architecture at Baylor College of Medicine and Rice University in Houston, Texas, and Eric Lander of the Broad Institute in Cambridge, Massachusetts.

The researchers created their map using an updated version of a previously published method called Hi-C. (See “Nuclear Cartography,” The Scientist, October 2014.) First, they crosslinked the DNA in cells with formaldehyde to preserve its 3-D structure. Next, they treated the DNA with restriction enzymes, cutting it into tiny pieces. They then added biotin markers to the ends of the cut DNA, followed by ligase, which binds together any free DNA ends that are in close proximity. The idea is that DNA will form small circles near where strands were bound together. Finally, the researchers sheared the DNA and pulled down biotin-marked fragments, which they sequenced.

The slight difference between the new “in situ” Hi-C and the original Hi-C is that, through the new approach, the DNA remains in the nucleus during ligation, rather than being released into a solution. This reduces the potential for accidental binding of DNA fragments to other fragments that were not actually their neighbors in the nucleus.

Hi-C yielded a dizzying array of contacts between different regions of the genome. The researchers used algorithms to determine the genome’s structure. Defining loops as regions of DNA demarcated by two loci that were more frequently in contact with one another than with other loci in between them, the researchers broke the genome down the into contact domains—regions of the genome in which loci tend to interact with each more than with other genomic regions. In many cases, contact domains are contained within loops.

“The contents of the loop tends to interact with itself,” explained study coauthor Suhas Rao, a researcher at the Center for Genome Architecture. Loci within contact domains also tend to have relatively uniform histone modifications. Finally, the researchers found that the genome can be divided into at least six subcompartments, or regions that segregate into similar sectors of the nucleus.

Altogether, the team identified around 10,000 loops—far fewer than previously estimated. “I was shocked, frankly speaking, when I saw that number,” said Ren, who himself once estimated that there were more than 1 million loops in the human genome.

“I think much depends on how you define a loop,” said Giacomo Cavalli, who studies chromatin and cell biology at the Institute of Human Genetics in Montpellier, France, and was not involved in the study.

The researchers also found that the protein CTCF often binds to DNA at the point where it forms a loop. Moreover, they found that the two CTCFs, each binding one piece of DNA, usually face each other.

The researchers further attempted to understand the function of the loops. They confirmed that loops often bring together distant enhancers and promoters and that these pairings often lead to changes in gene expression. Thirty percent of the loops found in a lymphoblastoid cell line (GM12878) were formed by promoters and enhancers coming together, the researchers found.

And many loops were conserved among cell types, and even between mice and humans. But others seemed to drive cell type-specific gene expression patterns. The researchers said that their map will help scientists to understand the functional effects of mutations to non-coding DNA regions.

“Detailed 3-D genome maps are crucial for the interpretation of disease-associated variants in the genome,” said de Laat. Genome-wide association studies “often identify risk variants located in non-coding intergenic sequences, making it difficult to understand how they drive disease,” he noted, adding that 3-D genome maps could help researchers to find the targets of regulatory elements.

“We’ve associated the genes with the distal regions that control them,” said Rao. “[Now] we can actually start to make sense of all the mutations that we weren’t able to before.”

Finally, the researchers compared looping in diploid chromosome pairs. They found that homologous chromosomes do show slightly different looping patterns, possibly reflecting imprinting that only turns one copy of a gene at a time. Predictably, they found more pronounced differences in looping between active and inactive X chromosomes in female cells. The inactive X chromosomes had superdomains and superloops much larger than those found in other chromosomes.

The researchers said they hope other scientists will explore their data using their downloadable tool, Juicebox. “It allows anybody, us and other users, to look at any regions that we’re interested in,” said study coauthor Miriam Huntley, a PhD student at Harvard University. “Any user who [is] interested in a particular gene or  . . . a particular enhancer or a particular mutation. They can . . . zoom into a particular region, and they can try to see what’s happening there. How is the genome folding in this area that I care about?”

“There’s a lot we can learn not just by reading the paper, but by going through the data and comparing them to other works,” said Cavalli.

S.S.P. Rao et al., “A 3D map of the human genome at kilobase resolution reveals principles of chromatin looping,” Cell, doi:10.1016/j.cell.2014.11.021, 2014.

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Avatar of: James V. Kohl

James V. Kohl

Posts: 384

December 12, 2014

Nutrient-dependent/pheromone-controlled adaptive evolution: a model. Excerpt: "A form of GnRH associated with sexual orientation in yeasts links control of the feedback loops and developmental processes required for nutrient acquisition, movement, reproduction, and the diversification of species from microbes to man [via RNA-directed DNA methylation and RNA-mediated amino acid substitutions that stabilize protein folding in the DNA of organized genomes].

I think this report on how DNA is folded within the nuclei of human cells makes it obvious that nutrient-dependent pheromone-controlled RNA-mediated amino acid substitutions, protein folding, and feedback loops link the epigenetic landscape to the physical landscape of DNA via what is currently known about the bio-physically constrained chemistry of protein folding and the conserved molecular mechanisms of cell type differentiation.

For cross species examples that include the honeybee model organism, see my 2012 and 2013 reviews, which are based on what we (T.B.) included in the molecular epigenetics section of our 1996 Hormones and Behavior review: From Fertilization to Adult Sexual Behavior  and on extension of RNA-mediated cell type differentiation to insects in Organizational and activational effects of hormones on insect behavior and to the life history transitions of honeybees in Honey bees as a model for understanding mechanisms of life history transitions.

Avatar of: James V. Kohl

James V. Kohl

Posts: 384

Replied to a comment from James V. Kohl made on December 12, 2014

December 15, 2014

I think the lack of response here can be attributed to what is now known about protein folding and cell type differentiation. It can be compared to the claims of theorists that "...genomic conservation and constraint-breaking mutation is the ultimate source of all biological innovations and the enormous amount of biodiversity in this world. In this view of evolution there is no need of considering teleological elements." (p. 199) Nei (2013)

Two additional video representations of what is currently known about physical and chemical constraints on the conserved molecular mechanisms of nutrient-dependent protein folding can now be compared.

One links thermodynamic cycles of protein biosynthesis and degradation to cell type differentiation via amino acid substitutions in species from microbes to man. ISHE Summer Institute 2013

One links mutations to cell type differentiation in a mouse to human model. 2 Cell Studies Reveal Genetic Variation Driving Human Evolution

Taken together with the video that reveals the priciples of chromatin looping,  less than 25 minutes of video representations could be used as a basis for conclusions about cell type differentiation that would move science forward -- minimally, into the current century.  Is that a problem?

Avatar of: James V. Kohl

James V. Kohl

Posts: 384

Replied to a comment from James V. Kohl made on December 15, 2014

December 17, 2014

I realize that most of the biologically uniformed are already concerned that they were taught to believe in a ridiculous theory based on the pseudoscientific nonsense of definitions and assumptions. However, I cannot help but add another level of examination that evolutionary theorists seem to have ignored as they touted their ridiculous theories.

What about The ribosome as a missing link in the evolution of life

In theory, it links nutrient-dependent RNA-mediated events to cell type differentiation across all genera, and that takes us back to this claim:

"We cannot conceive of a global external factor that could cause, during this time, parallel evolution of amino acid compositions of proteins in 15 diverse taxa that represent all three domains of life and span a wide range of lifestyles and environments. Thus, currently, the most plausible hypothesis is that we are observing a universal, intrinsic trend that emerged before the last universal common ancestor of all extant organisms." -- A universal trend of amino acid gain and loss in protein evolution

The problems with use of de Vries definition of 'mutation' and assumptions about how cell type differentiation occurs in species from microbes to man is the lack of pattern recognition AND the the lack of experimental evidence of biologically-based cause and effect that links the epigenetic landscape to the physical landscape of DNA in organized genomes of species from microbes to man. That problem has led to claims about constraint-breaking mutation that defy any common sense approach to cell type differentiation in any organism that eats or reproduces.

Coelacanths and birds eat and reproduce. GnRH and GnRH receptors link their nutrient-dependent physiology of pheromone-controlled reproduction to the honeybee model organism via its hormone-organized and hormone-activated behaviors. Pattern recognition suggests that dinosaurs did not evolve into birds because nutrient-dependent pheromone-controlled amino acid substitutions link coelacanths to mammals via conserved molecular mechanisms of cell type differentiation.

Is anyone who plans to continue touting theories about mutations and evolution willing to address the concerns that others may have about the end of that pseudoscientific nonsense as others begin to examine the biological basis of cause and effect?

Avatar of: Pombe Researcher

Pombe Researcher

Posts: 2

August 1, 2015

Recent cancer biology papers make so much more sense after reading this paper, along with Madabhushi's paper on Top2b-mediated DSBs which may resolve topological constraints formed by chromatin loops.  It's like "Oh, so that's why all these c-myc translocations with enhancers occur during lymphoma and myeloma development.  They were brought into close proximity by Top2b-mediated DSBs, but errors in the DSB repair machinery led to a translocation, causing constitutive upregulation of this oncogene."

The findings and tools from this paper will help many scientists develop their hypotheses and experiments.

Avatar of: Pombe Researcher

Pombe Researcher

Posts: 2

August 1, 2015

Recent cancer biology papers make so much more sense after reading this paper, along with Madabhushi's paper on Top2b-mediated DSBs which may resolve topological constraints formed by chromatin loops.  It's like "Oh, so that's why all these c-myc translocations with enhancers occur during lymphoma and myeloma development.  They were brought into close proximity through changes in chromatin loops by Top2b-mediated DSBs, but errors in the DSB repair machinery led to a translocation, causing constitutive upregulation of this oncogene."

The findings and tools from this paper will help many scientists develop their hypotheses and experiments.

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