Jun 25, 2018 in Science

Genomes in Three Dimensions

Studying the chromosome structure is a good way of understanding the mechanism of the genome. The focus in study of genomics has significantly switched to the study of the three dimensional structure of the chromosomes which is coiled in the nucleus. One of the biggest challenges in the study of structure of chromosome is the lack of knowledge on the dynamics of the chromosomes in relations to the cells change.

Knowledge of the DNA double helix structure has been known by scientists for many years. The DNA double helix is twisted about the proteins known as ‘histones’ to form chromatin strands that form the chromosomes. The chromosomes associate with each other in the nucleus all the time. For instance, a genome biologist, Peter Fraser in 2002, identified “long-range interactions that bring gene sequences into physical contact with far-off regulatory elements”. The three dimension arrangement of the chromosomes is very important for the functioning of the cell. This is emphasized by the mess that results when this arrangement is altered. For instance, several types of cancers have been associated with mutations that affect the spatial arrangement of the chromatin. Burkitt’s syndrome is a cancer of the lymphatic that results when a portion of chromosome 8 gets to chromosome 14 and vice versa.

The histones and DNA sequences are marked with ‘chemical modifications’ that turn genes on and off. These modifications are revealed by the three dimensional structure of chromatin. Researchers are therefore accepting that gene activity is not only determined by the chemical nature of chromosomes but also on the spatial arrangements of these chromosomes. However the dynamic and non-deterministic nature of chromosomes movements still remains a big challenge.

Techniques Used (Microscopy and chromosome capture)

For a long time researchers have relied on microscopy to study the arrangement of chromosomes. However, this could only reveal how the two loci are close to each other but not whether they come into contact with each other. Today, scientists use a technique known as “chromosome conformation capture” which was developed by Job Decker In 2002. The technique involves creation of hybrid molecules by combining free DNA strands. This is achieved by soaking cells with formaldehyde to stick DNA to its associated proteins and proteins to each other. While using this technique it is important to choose the restriction enzyme carefully. This is because various restriction enzymes cut varied fragment sizes.

In order to prepare a library of ligation products, one requires several reagents including formaldehyde, buffers and enzymes that cut and join back DNA. Most of these reagents can be purchased locally. Various techniques have been developed to detect various interactions. For instance 3C detects interaction between loci using quantitative PCR; 4C detects one locus and the rest of the genome using sequencing or microarrays while 5C detects multiple selected loci using sequencing.

Biologists have been able to detect various interacting loci that were not known before. For instance, a mediator protein complex is bound to enhancer sequences and “core promoters of genes transcribed in embryonic stem cells”. Another protein known as cohesion is bound along with mediator protein and connects two DNA segments. The challenges in mapping these loci include lack of powerful techniques that can match regulatory elements and genes across the genome. Another challenge is a signal to noise problem.

In order to learn how interactions occur labeled cells are counted under a microscope. However, it is tedious and labor intensive. An effective technique known as FISH, sixed cell technique fluorescence in situ. In this technique, nuclei are treated with formaldehyde and then denatured in order to allow “entry of DNA probes that fluorescently label certain sequences”. Interactions are only observed in one in ten cells. These lower rates don’t indicate that the interactions do not occur but it shows the dynamic nature of chromosome arrangement.

An advancement of the stand alone microscope is the opera instrument which examines loci in hundreds of cells per minute. This technique is faster and can be used by non-experts. Another technique known as cryoFISH technique produces fewer artifacts and better resolution than conventional FISH. Electron microscopy also produces better resolution although staining and imaging of cells may take long.

The microscopic techniques cannot differentiate between loci that are near each other and those that are in contact since all microscopic techniques are coarse detection techniques. Even sequencing techniques cannot show which interactions will occur together. The solution to this problem is a technique that incorporates DNA FISH and chromosome conformation capture.

The whole genome model

Researchers, led by Dekker and Marc Marti-Renom researched and published a three dimensional structure model of the 500 kilo base of the human chromosome 16. Dekker and her team used interaction-frequency maps to generate chromatin models for the cells. The model predicts that chromatin structures exist in active genes. In cells where both sets of genes are active, only a single globule forms.

In order to construct genome wide models at higher resolution, one starts with smaller genomes. This approach was used to obtain a high resolution of a model wide genome of the Schizosaccharomyces pombe, fission yeast which contains about 14 million base pairs and 5000 genes. Researchers also developed a model of the budding yeast, Saccharomyces cerevisiae.

In future, three dimensional genome models will represent more “dynamic, semi-random movements of chromosomes,” However; present versions will still be valuable in showing overall tendencies. According to Dekker, “By imaging you highlight the variability, by chromosome capture, you highlight the commonalities.

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