{"id":3470,"date":"2019-05-07T12:23:11","date_gmt":"2019-05-07T03:23:11","guid":{"rendered":"http:\/\/163.180.4.222\/lab\/?p=3470"},"modified":"2019-05-07T12:23:11","modified_gmt":"2019-05-07T03:23:11","slug":"the-new-techniques-revealing-the-varied-shapes-of-chromatin","status":"publish","type":"post","link":"https:\/\/biochemistry.khu.ac.kr\/lab\/?p=3470","title":{"rendered":"The new techniques revealing the varied shapes of chromatin"},"content":{"rendered":"

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Researchers are realizing that the DNA\u2013protein complex doesn\u2019t just have one form but many.<\/h5>\n

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This multicoloured image of chromatin was created using multiplexed fluorescence\u00a0in situ<\/i>\u00a0hybridization and super-resolution microscopy.<\/span>Credit: Bogdan Bintu\/The Xiaowei Zhuang Laboratory\/The Alistair Boettiger Laboratory<\/p>\n<\/figcaption><\/figure>\n

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Molecular models suggest that chromosomes assemble in an ordered, hierarchical way: DNA wraps around proteins called histones to form nucleosomes, which fold into 30-nanometre fibres, then 120-nanometre \u2018chromonema\u2019, and further into larger chromatin structures until they reach their most tightly coiled form \u2014 the characteristic X-shaped bodies.<\/p>\n

Under the high-resolution microscopes of biophysicist Xiaowei Zhuang, these chromosomes resemble something from the mind of surrealist painter Salvador Dal\u00ed. Zhuang, who is at Harvard University in Cambridge, Massachusetts, is one of a growing number of researchers charting the topology of the genome to decode the relationship between chromatin structure and function. Using a highly multiplexed form of fluorescence\u00a0in situ\u00a0<\/i>hybridization (FISH) in combination with super-resolution microscopy, Zhuang\u2019s team mapped several million bases of human chromosome 21 at 30 kilobase resolution, tracing their shape like a dot-to-dot puzzle1<\/a><\/sup>. The resulting multicoloured image resembles one of the melting clocks in Dal\u00ed\u2019s 1931\u00a0The Persistence of Memory<\/i>.<\/p>\n

But that was in just one cell. In each cell that Zhuang\u2019s team looked at, the chromosome assumed a different shape \u2014 each one a different solution to some ineffable cellular calculation. \u201cThere is very strong cell-to-cell heterogeneity,\u201d Zhuang says.<\/p>\n

Ting Wu, a geneticist at Harvard Medical School in Boston, Massachusetts, who combined a similar super-resolution FISH approach with sequencing analysis to map a chunk of human chromosome 19 to 10 kilobase resolution in late 2018, observed similar heterogeneity2<\/a><\/sup>. The chromosomes in that study look more like space-filling protein models, and when the team overlaid markers of inactive and active chromatin, they observed distinct patterns. \u201cWe have never seen a structure of that 8.6-megabase region twice,\u201d says Wu. \u201cThe variability, which people had thought was there, and there are hints of, is truly astounding.\u201d Brian Beliveau, a genomic scientist at the University of Washington, Seattle, and a co-author of the paper, says bluntly: \u201cChromosomes are almost certainly like snowflakes.\u201d<\/p>\n

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A deeper look<\/strong><\/p>\n

In biology, function derives from form. It is shape, as a result of amino-acid sequence, that determines whether a given protein acts as a structural scaffold, signalling molecule or enzyme. The same is probably true of the genome. But until recently, there was no easy way for researchers to determine that structure.<\/p>\n

Using a sequencing-based method called Hi-C, which calculates the frequencies at which different chromosomal segments interact in space, researchers discovered that chromatin organizes into relatively stable structures called topologically associating domains (TADs), and larger domains called compartments. But Hi-C works by averaging chromosome conformation across millions of cells. To see chromatin forms as they exist in cells, researchers must study them individually.<\/p>\n

Wu and Zhuang focused on relatively small regions of the genome \u2014 a few million base pairs out of three billion. They had previously used a similar approach to map whole chromosomes at lower resolution3<\/a><\/sup>. Biophysical chemist Sunney Xie at Peking University in Beijing takes an even bigger-picture view.<\/p>\n

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A super-resolution microscopy system for imaging chromatin.<\/span>Credit: Bogdan Bintu\/The Xiaowei Zhuang Laboratory\/The Alistair Boettiger Laboratory<\/p>\n<\/figcaption><\/figure>\n

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Xie and his colleagues have used Dip-C \u2014 a sequencing method akin to single-cell Hi-C \u2014 to computationally map chromosomes at 20 kilobase resolution in individual cells with two sets of chromosomes4<\/a><\/sup>. The method\u2019s sensitivity is such that it picks up both intra- and interchromosomal contacts \u2014 about one million per cell \u2014 from which the team can infer the organization of the entire nucleus. It looks, in computer-generated renderings, like a multicoloured skein of yarn. \u201cWe know how that 6\u2009\u00d7\u2009109<\/sup>\u00a0bases are located in the nucleus,\u201d Xie says.<\/p>\n

And in olfactory neurons, they found5<\/a><\/sup>\u00a0that structure reflects cellular biology. Whereas most cells pack their thousand-odd olfactory-receptor genes at the periphery of the nucleus, olfactory neurons mostly pack them near the nuclear centre, where they are silenced \u2014 except, presumably, one that remains free to produce the neuron\u2019s olfactory receptor. \u201cChromatin structure determines cell function,\u201d Xie says.<\/p>\n

But unlike in proteins, that structure is highly variable. In one 2019 study6<\/a><\/sup>, Elizabeth Finn, a postdoctoral fellow in the laboratory of Tom Misteli, director of the Center for Cancer Research at the National Cancer Institute in Bethesda, Maryland, and her colleagues selected 125 pairs of contacts from Hi-C maps and used a high-throughput FISH platform to map the physical locations of contacts in human cells. In general, they found, regions that were strongly associated in Hi-C tended to be close together in space, whereas more weakly associated regions tended to co-localize less frequently.<\/p>\n

\u201cSo it correlates,\u201d Misteli says \u2014 but not completely. In many cells, no interaction was observed. That\u2019s not surprising, says Job Dekker, a chromatin biologist at the University of Massachusetts Medical School in Worcester, and a co-author of the study. Even a strong Hi-C signal might reflect just a handful of cells. And the genome is almost certainly dynamic, Dekker adds. A configuration that exists at one moment might disappear minutes later, as the cell samples the genomic landscape. \u201cWhat we do think is that most of these structures can happen in all cells, but occur transiently.\u201d<\/p>\n

Other mechanisms are probably also at play, says Misteli. Some loci, for instance, are characteristically spaced so far apart it would be difficult for them to interact through chromosome diffusion alone.<\/p>\n

What researchers are looking for, then, are the overarching patterns. At the Salk Institute for Biological Studies in La Jolla, California, molecular biologist Clodagh O\u2019Shea has developed a method for mapping chromatin structure in single cells with nanometre precision. The method involves coating cellular chromatin in a thin metal shell \u2014 as she puts it, like Han Solo encased in carbonite in the 1980 film\u00a0The Empire Strikes Back<\/i>. Working with Mark Ellisman at the University of California, San Diego, her team then uses 3D electron microscopy to make a CT scan of that metallic cast, and computer algorithms to track its tortuous path through the nucleus.<\/p>\n

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