DNA Mambo in the Nucleus

Some organizational principles for nuclear DNA to organize genes for local regulation.

There has been a long and productive line of research on the mechanisms of transcription from DNA to RNA- the process that reads the genome and translates its code into a running stream of instructions going out to the cell through development and all through life. This search has generally gone from the core of the process outwards to its regulatory apparatus. The opening of DNA by simple RNA polymerases was one of the first topics of study, followed by how the polymerase is positioned at the start site by "promoter" DNA sequences, with ever more ornate and distant surrounding machinery coming under scrutiny over time, as researchers climbed the evolutionary trajectory of life, from viruses and bacteria to mammals. 

But how this process fits into the larger structure of the nucleus, and how it is globally organized eukaryotes has long been an intriguing question, and tools are finally available to bring this level of organization into focus. For example, genes are known to be activated by direct contact with "enhancer" elements located thousands, even many tens of thousands, of basepairs away on the DNA- so why can't those enhancers activate other genes elsewhere in the nucleus, rather than the genes they are nearest to on the one-dimensional DNA? The nucleus is a small place with a lot of DNA. Roughly 1/100 of its physical space is taken up by DNA, and it is highly likely that such enhancers could be closer in 3-D space to other genes than the ones they are supposed to regulate, if everything were arranged randomly. Similarly, how do such enhancer elements find their proper targets, amid the welter of other DNA and proteins? A hundred thousand base pairs is long enough to traverse the entire nucleus.

So there has to be some organization, and new techniques have come along to illuminate it. These are crosslinking methods where the cells are treated with a chemical to crosslink / freeze a fraction of protein and DNA interactions in place, then enzymes are introduced to chop everything up, to various degrees of completeness. What is left are little clumps of DNA and protein that hopefully include distant cross-links, between enhancers and promoters, between key organizational sites and the genes they interact with, etc. Then comes the sequencing magic. These clumped stray DNAs are diluted and ligated together (only to local ends), amplified and sequenced, generating a slew of DNA sequences. Those hybrid sequences can be interpreted, (given the known sequence of the reference genome), to say whether some genomic location X got tangled up with some other location Y, reflecting their 3-D interaction in the cell when it was originally treated.

A recent paper pushed this method forward a bit, with finer-grained enzymatic digestion and deeper sequencing, to come up with the most detailed look ever at a drosophila genome, and at some particular genes that have long held interest as key regulators of development. This refined detail, plus some experiments mutating some of the key DNA sites involved, allowed them to come up with a new class of organizing elements and a theory of how the nuclear tangle works.

Long range contacts in the Antennapedia locus of flies. Micro-C refers to the crosslinking and sequencing method that maps long-range DNA contacts mediated by proteins. Pyramids in the top diagram map binary location-to-location contacts. Local contacts generally predominate over distant ones, but a few distant connections are visible, such as between the ends of the ftz gene. TAD stands for topologically associating domain, mapping out the connections seen above between pink sites. This line also lists the genes residing in each zone (Deformed, micro RNA 10, Sex combs reduced, fushi terazu, and Antennapedia promoters P1 and P2). The contacts track shows where the authors map specific sites where organizing factors (including Trl (trithorax-like) and CP190 (centrosomal protein of 190 kDa)) bind. The overall idea is that there are two kinds of contacts, boundaries and tethers. Boundaries insulate one region from the next, preventing regulatory spill-over to the wrong gene. Tethers serve as pro-regulatory staging points, helping enhancers contact their proper promoter targets, even though the tether complex does not itself promote RNA transcription.

Insulator elements have been recognized for some time. These are locations that seem to block regulatory interactions across them, thus defining, between two such sites, a topologically associated domain, (TAD). How they work is not entirely clear, but they may stitch themselves to the nuclear membrane. They are thought to interact with a DNA pump called cohesin to extrude a loop of DNA between two insulator sites, thereby keeping that DNA clear of other interactions, at least temporarily, and locally clumped. The authors claim to find a new element called a distal tethering element (DTE), which works like an enhancer in promoting interaction between distant activating regulatory sites and genes, but doesn't actually activate. They just structure the region so that when a signal comes, the gene is ready to be activated efficiently. 

One theory of how insulator elements work. The insulator sites "CTCF motif" are marked on the DNA with dark blue arrow heads. They control the boundaries of action by the protein complex cohesin, which forms dimeric doughnuts around DNA and can pump DNA. Cohesins are central to the mechanisms of meiosis and mitosis. The net effect is to produce a segregated region of DNA as portrayed at the bottom, which should have a much higher rate of local interactions (as seen in the Micro-C method) than distant interactions.

At the largest scale, these authors claim that there are, in the whole fly genome and at this particular (early) point in development, 2034 insulator locations (TADs) and 620 tethering elements (TEs or DTEs). They show that DTEs in the locus they study closely play an active role in turning the nearby genes on at early times in development, and in directing activation from enhancers near the DTE, rather than ones farther away. What binds to the DTEs? So-called "pioneer" regulatory factors(such as Zelda) that have the power to make way through nucleosomes and other chromatin proteins to bind their target DNA. The authors say that these tether sites, once set up, are then stable on a permanent basis, through all developmental stages, even though the genes they assist may only be active transiently. 

The "poised" nature of some genes had been observed long ago, so it is not entirely surprising to see this mechanism get fleshed out a little, as a structural connection that is made between genes and their regulatory sites in advance of the actual activator proteins arriving at the associated enhancers and turning them on.

 

Final model: the normal case around the Antennapedia locus is shown at top, with insulator sites shown in pink, and tethering sites shown in teal. If one of the tethering elements is removed (middle), then the enhancer EE has less effect on the gene Scr, whose expression is reduced. If an insulator is removed (bottom), the re-organized domain allows the ftz gene's regulators, including the enhancer AE1, to affect Scr expression, altering its timing and location of expression.

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