Mike Levine - Uc Berkeley - Part 1 - Transcriptional precision - Enhancers

Hi, I'm Mike Levine. I'm at the University of California at Berkeley. And I'm going to discuss gene regulation in the early Drosophila embryo and it's long been recognized that the precise control of gene activity is very important in animal development. And a very striking example of this can be seen on the first slide. Here you're looking at the heads of adult fruit flies, Drosophila melanogaster. On the left is a fly that's normal. It has a pair of eyes, small antennae and mouth parts. On the right is the head of an Antennapedia mutant. And here you see that in place of the normal antennae, there are perfectly sculpted middle legs. And this results from the misexpression of the Antennapedia gene in the wrong tissue. So normally Antennapedia is active in the middle thoracic regions of developing embryo where it controls the development of middle legs but in this Antennapedia mutant, the normal Antennapedia transcription unit is misexpressed in head tissues and this causes a transformation of antennae into legs. And so I'm going to discuss specifically gene regulation during early Drosophila embryogenesis. And this is summarized on the next slide. So here you're looking at a series of progressively older embryos beginning with the fertilization of the egg at stage 1. The two pronuclei fuse to form a zygotic diploid nucleus. And then this nucleus undergoes a series of mitoses indicated by the numbers 2, 3, and 4 and so forth. These are among the fastest mitoses known for any animal embryo system with a division every 6 to 8 minutes. After 13 rounds of division you wind up with a nuclear cleavage cycle 14 embryo which contains about 6000 nuclei forming a monolayer at the periphery of the egg; the cortex of the egg enclosing the central yolk. And the next slide shows some more details on the last two rounds of mitoses. You can see that the nuclei are dividing in a syncytium. This means that there are no cell membranes. The nuclei are in a common cytoplasm; the ooplasm of the egg. They're at the periphery of the egg and you see the last two division cycles leading to that nuclear cleavage cycle 14 embryo I discussed before. So at the nuclear cleavage cycle 14 stage, once again, you have 6000 nuclei forming a monolayer at the periphery of the egg and there are no further mitoses. Now, cell membranes are laid down between the individual nuclei over a one hour period of development. And this is the period I'm going to discuss. This is when localized patterns of gene expression establish the basic blaue plan or blueprint of the adult fly. Now this system is ideally suited for visualizing gene expression in development. First, the nuclei are at the periphery. There's no need to focus the objective of the microscope into the interior of the egg and embryo because all the action is at the surface. Secondly, you can see that the system is naturally synchronous. All these mitoses are occurring synchronously. All the cells, all the nuclei are at the same stage of the cell cycle. And this is a big advantage, as you'll see, for studying gene regulation. So, in the first part of my talk, I want to emphasize the importance of enhancers in controlling basic on-off patterns of gene expression. An example is shown here for a segmentation gene called Even-Skipped or Eve. Now, eve is expressed in a series of 7 stripes along the length of the embryo and these 7 stripes foreshadow the subdivision of an embryo into a repeating series of body segments. Although, segmentation isn't observed until several hours later in development. The eve gene is controlled by a series of separate enhancers located both upstream of the transcription start site (indicated by the arrow in the digram below) and three enhancers located downstream of the transcription start site. So, for example, a 500 base pair enhancer located about 1kb upstream of the eve transcription start site controls the expression of stripe number 2. A separate enhancer located about 3 kilobases upstream of the transcription start site controls the expressions of stripes 3 and 7. All together, the 5 enhancers located both upstream and downstream of the eve gene control the 7 stripes of eve expression. And the next slide shows more details for the regulation of one of these enhancers, the Eve-Striped 2 enhancer, which has been studied in the greatest detail. The Stripe 2 enhancer is diagrammed here. It is about 500 base pairs in length, located between 1 and 1.5kb upstream of the transcription start site, and you can see it contains a cluster of binding sites for different sequence specific transcription factors; both activators and repressors. And the distribution of these transcription factors relative to the Stripe 2 pattern is shown in the middle diagram. As I'll discuss a little later, the broad, maternal Bicoid gradient works together with Hunchback to define a broad domain in the entire anterior half of the embryo where the Stripe 2 enhancer could be switched on. Bicoid and Hunchback work as activators of the Stripe 2 pattern. But, the Stripe 2 enhancer also contains binding sites for sequence specific transcriptional repressors including Kruppel (the binding sites are indicated by Kr) and Giant (with binding sites indicated by Gnt). And these work as transcriptional repressors to delineate the Stripe 2 pattern, to form both the anterior and posterior orders. So the only place in the embryo where you have unfettered Bicoid and Hunchback activators that can bind to the activator binding sites within the Stripe 2 enhancer without impedance from the Giant and Kruppel repressors is right here, right within the limits of the Stripe 2 pattern. So all together the 500 base pair Stripe 2 enhancer integrates both positive and negative regulatory information to produce this sharp on-off stripe of transcription. And the picture below shows the relative expression pattern of the Eve stripes So you see the 7 Eve stripes in red and the Giant expression pattern in green. And you see that the anterior Giant pattern tightly straddles the anterior border of Eve Stripe 2. Giant binds to the Stripe 2 enhancer to form that stripe. Then the last point I want to make about transcriptional enhancers in development is that multiple enhancers can work independently of one another to produce an additive pattern of expression. So above you see the expression pattern produced by an enhancer called the NEE. And the NEE produces a lateral stripe of transcription. With expression off in the bottom of the embryo due to a localized repressor called Snail. Now, the embryo in the bottom panel shows the expression pattern of a synthetic transgene that contains the NEE enhancer placed next to the Eve Stripe 3 enhancer. And what you observe is an additive pattern of expression; a criss-cross of the NEE lateral stripe and Eve Stripe 3. And the reason why the two enhancers are working additively is because of short range transcriptional repression. The Snail repressor binds to the NEE as I mentioned to keep it off in the bottom regions of the embryo. But the binding of Snail to the NEE does not interfere with the neighboring Eve Stripe 3 enhancer which you can see is expressed just fine in the bottom regions of the embryo where there are high levels of the Snail repressor. So this property of short range repression... repressors only inhibit the activators to which they are bound. This permits the different enhancers to work independently of one another and give an additive pattern of expression; in this case a criss-cross of the NEE and Eve Stripe 3. So to summarize this introductory part of my talk: Enhancers are typically 500 base pairs in length. They contain binding sites for both activators and repressors and thereby produce sharp on-off patterns of gene expression. Multiple enhancers work in an additive pattern to produce complex patterns of expression due to short range transcriptional repression.