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Tower of Babel: Next Generation Sequencing Provides New Insights on Chromosome Construction

Biologists have long appreciated the complexity of genome organization, but until recently lacked the tools to discern the intricacies of this puzzle. Now, thanks to some handy cross-linking, careful amplification, and (of course!) next generation sequencing, teams from Massachusetts are taking us down the rabbit hole, with some surprising findings from Wonderland.

Bend Over Backwards

Eukaryotic genomes are packed pretty tight- each human cell harbors about two meters of genome. But for decades, the fine details of this packaging were a mystery. At interphase, chromatin (DNA strands and their associated proteins) adopts a somewhat ‘relaxed’ state. Whilst genes are accessible to transcriptional machinery, the entire genome is not open for business. So-called ‘silent’ heterochromatin and structural elements like centromeres and telomeres are largely intact and compact at interphase, though not visible.

Everything changes during cell division. In mitosis, chromatin of all types condenses rapidly into visible chromosomes. But the mechanisms of this condensation were beyond the technological grasp of generations of molecular biologists and cytogeneticists.

Abandon All Hope, Ye Who Enter Here

Traditional attempts to understand the complexities of the three-dimensional arrangement of the genome raised more questions than they answered. Researchers had only a paucity of tools available to address this issue, and could only look on either the extremely small scale (thousands of nucleotides) or the extremely large scale (whole chromosomes). On the small scale, protein and epigenetic markers partitioned gene-rich euchromatin from gene-poor heterochromatin. On the large scale, cytogeneticists nit-picked the intricate features of metaphase chromosomes. Early fluorescent staining techniques revealed that chromosomes occupied discreet (albeit loose) nuclear regions. More recently, reports surfaced that discrete chromatin regions were associated with different regions of the nucleus (such as the nuclear envelope or nucleolus), and that these regions varied by cell type and age. But the details? Good luck with that!

Won’t You Be My Neighbor?

Chromosome Conformation Capture (3C) technology began to revolutionize the realm of chromatin organization just over a decade ago. The principle is simple: cross-link chromatin using a chemical like formaldehyde, attaching an individual DNA strand to its 3-D neighbors (whether they be from the gene next door or another chromosome entirely). While original 3C used locus-specific PCR to reveal interactions between specific genomic loci, new 5C and Hi-C protocols utilize ligation-specific amplification steps to generate libraries of genomic interactions, which are then funneled into next-generation sequencing platforms.

New Ground

Studies using these new approaches, which are named 3C, 4C (I skipped that, but you can Google it), 5C, and Hi-C, have all shed new light on the complexities of genome organization in the nucleus (or cell, for prokaryotes). These have included studies comparing the interphase chromatin organization of different cell types and disease states. But, most of these endeavors have not shed definitive light on the chromatin organization of interphase chromosomes, or how interphase chromosomes compact and organize themselves into the tight, ordered chromosomes of metaphase.

Let’s Get Together

In November 2013, a joint team from Harvard, MIT, and the University of Massachusetts, published a paper in Science, where they sought answers in HeLa cells. They used 5C and Hi-C analyses on the Illumina platform to reveal striking differences between interphase and mitotic chromatin states. In all stages of interphase (G1, S, and G2), interactions among loci appeared stable. In interphase, a given locus is more likely to associate with nearby stretches of DNA. These so-called Topologically Associated Domains (TADs) were previously known, and confirmed in this paper with specific focus on human chromosome 21. Other interactions (on loci far apart within and between chromosomes) are possible, but less likely based on these data. TADs ranged in size from 100,000 to 10,000,000 bases in length. Critically, TADs varied in size and composition between HeLa cells and two other human cell types, implying that the cell and tissue differentiation process established unique local and global genome interactions.

The Tie That Binds

During mitosis, 5C and Hi-C data clearly show that TADs break down amid a flurry of chromosome condensation. For a given locus, interactions that spanned ten million bases in interphase shrink to 10,000 bases during mitosis. This breakdown wasn’t unique to HeLa cells. All cell types tested adopted strikingly similar compact mitotic interactions. So, although differences in gene expression and chromatin organization define distinct cell types during interphase, mitosis appears to bring them together again. The need for accurate and rapid chromatin compaction might be the tie that binds all cell types together.

Higher and Higher

The 5C and Hi-C mitotic interactions imply formation of local chromatin ‘loops’ during chromosome condensation, possibly by proteins already implicated in these processes (such as the aptly-named ‘condensin complex’).  But what about more higher-ordered chromosome structures? Well, the picture gets fuzzier! The authors took the novel approach of simulating Hi-C results for different chromosome condensation hypotheses and compared these hypothetical results to their real Hi-C data.  Their models tended to support ‘compaction’ of the main chromatin fiber along its axis, bringing local ‘loops’ together to form the typical metaphase chromosome. Still, caveats abound, and additional studies must probe the details with more certainty.

Circular Logic

In a separate study (also published in Science) an MIT-based team performed Hi-C analysis using an Illumina platform on the single circular chromosome of a bacterium called CaulobacterCaulobacter loci showed many local interactions of identical size to the TADs of human cells. Since prokaryotes and eukaryotes share some chromosome-associated proteins, this similar spacing may point to a baseline chromatin-organization structure for all life. In addition, this study demonstrated that Hi-C is an effective tool to assay the effect of antibiotics on chromosome structure and replication, an essential tool in the age of shrinking antimicrobial therapies.

Curiouser and Curiouser

Thus, with a little help from NGS, molecular biologists are finally treading into the realm of higher-order chromosome structure. Their ultimate findings could resolve the geometric puzzle of the two-meter mystery, or leave us more perplexed than before. But, if these recent studies in Science are any indication, this is one tower we can decipher.

 

Image Credit: Thomas Thomas

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