In early embryos it is not possible to distinguish between arms and legs: their developmental precursors are virtually identical. Dynamic changes in the 3D structure of the chromatin in the leg precursor cells bring the Pen enhancer into spatial proximity to the Pitx1 gene, thus enabling the activation of this critical gene specifically in the future leg. As a result, forelimb and hindlimb buds subsequently differentiate from each other and produce arms and legs, respectively. This surprising molecular mechanism has been discovered by scientists around Stefan Mundlos and Guillaume Andrey from the Max Planck Institute for Molecular Genetics and the Charitè-Universitätsmedizin Berlin.
In order to develop normal organs and tissue, the activity of genes needs to be tightly orchestrated in each cell during the development of an embryo. In particular, each gene must be activated and de-activated at specific moments in particular tissues, so that the stepwise morphological processes that construct the body can be executed. For this purpose, the DNA surrounding the approximately 20,000 human genes has control areas consisting of promoters and other regulatory elements, the so-called enhancers.
Enhancers are segments of DNA, which precisely control the activity of genes at the defined places and moments they are required. They constitute an important fraction of the genome and are positioned between or within genes. However, in order to control the activity of genes, they need to be in close physical proximity with them.
Pitx1 and Pen
For example, an enhancer, namely Pen, regulates a gene called Pitx1. During the development and outgrowth of undifferentiated hindlimb buds, Pitx1 is highly expressed to produce a leg with a thigh, knee, and foot. Appropriately the Pen enhancer is active in the cell of the future leg. Yet, the researchers have found that it is also active in presumptive arm cells.
This was surprising, as the expression of Pitx1 in forelimbs would lead to the partial transformation of arms into legs, with, for example, an elbow that looks like a knee. So, how does the body insure that the upper body develops an arm and not a leg from its forelimb bud? How does it prevent that Pen activates Pitx1 in this tissue?
Folding and winding of chromatin
"The basis for the activation of Pitx1 by Pen is the dynamic three-dimensional folding of the chromatin," says Stefan Mundlos, summarizing the latest study results of his team, which cooperated with several international partners in this research project. Chromatin consists of the DNA and its associated proteins.
It folds and winds into a highly organized three-dimensional structure in order to fit into the compact cell nucleus. The resulting 3D structure causes a close proximity of DNA regions that are actually far apart from each other on the linear DNA thread. Accordingly, this contact in the 3D space of the cell nucleus enables enhancers and genes to find each other and regulate themselves.
Prevention of chromatin interaction
Now the team around Andrey and Mundlos shows how the dynamics of alternating chromatin 3D folding controls the formation of the extremities. The Pitx1 gene locus, i.e. the position in the genome where the Pitx1 gene and its regulators are located the DNA strand, can take on different spatial and functional shapes depending on the folding.
These different structures were computationally modeled in 3D by Mario Nicodemi from the University of Naples with a new methodology based on the Berlin data. In the hindlimb bud of the later leg, an "active configuration" of the chromatin ensures that Pen reaches spatial vicinity to Pitx1, thus enabling its activation.
In this case, the limb bud becomes a leg. In contrast, in the forelimb buds of the later arms, an "inactive configuration" of chromatin folding allows that an adjacent inactive gene folds up between Pen and Pitx1 and prevents their interaction. Due to the blocked interaction, Pitx1 is not activated and arms can develop.
"The system can be compared to a lamp that can be switched on or off using a light switch," explains Andrey. "The lamp is constantly connected to the electric network, just as the gene and its enhancer are present in both forelimb and hindlimb buds. However, the current only flows after the light switch has been flipped. Similarly, it is only by altering the 3D structure of the chromatin that the molecular interaction between enhancer and gene becomes possible".
From arms to legs
However, so-called structural mutations in the genome can influence the folding of chromatin. The scientists have detected such mutations in patients with the so-called Liebenberg syndrome. They further analyzed the mutations in experiments with mice, in which the genetic modification was reconstructed. In this case, the chromatin also folds in the presumptive arm limb buds into a structure that brings Pen and Pitx1 together so that the gene is activated. In this way, arms with leg structures develop.
"Our results indicate that the 3D conformations of the chromatin directly influence the regulation of gene activity," says Mundlos. While the "lamp" normally shines only in the lower limb buds and is switched off in the upper ones, it seems to “burn” permanently in all four limb buds in patients with Liebenberg syndrome. According to the findings of the research group, this structural gene control is partly based on Hox genes - master genes of embryonic development.
Regulation of Pitx1 activity in the evolution of limbs
The Max Planck scientists also note that this particular mechanism of gene regulation in the developing limb is quite "circuitous". They speculate that this might have evolutionary reasons. When vertebrates left the water for a terrestrial life about 400 million years ago, "the anterior and posterior extremities were morphologically equal and Pitx1 and Pen were probably equally active in fore as well as hind limbs. Only in the further course of evolution, a distinction was made between arms and legs.
To achieve this, evolution had two choices, either change Pen to a hind limb-only activity, or selectively restrict Pen activity to the hind limbs and inactivate it in the forelimb. The latter was apparently the solution of choice: gene regulation via the chromatin structure was probably the most effective method.
Prof. Dr. Stefan Mundlos
Max Planck Institute for Molecular Genetics
Bjørt K. Kragesteen, Malte Spielmann, Christina Paliou, Verena Heinrich, Robert Schöpflin, Andrea Esposito, Carlo Annunziatella, Simona Bianco, Andrea M. Chiariello, Ivana Jerković, Izabela Harabula, Philine Guckelberger, Michael Pechstein, Lars Wittler, Wing-Lee Chan, Martin Franke, Darío G. Lupiáñez, Katerina Kraft, Bernd Timmermann, Martin Vingron, Axel Visel, Mario Nicodemi, Stefan Mundlos & Guillaume Andrey
Dynamic 3D Chromatin Architecture Contributes to Enhancer Specificity and Limb Morphogenesis.
Nature Genetics 2018
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