Environmental Factor, April 2009, National Institute of Environmental Health Sciences
Transcriptional Control During Embryogenesis
By Robin Arnette
Studies with the common fruit fly (Drosophila melanogaster) and sea squirt (Ciona intestinalis) are helping to unravel the connection between transcriptional networks and cellular behavior. Michael Levine, Ph.D., a specialist in the genetic control of dorsal-ventral patterning and heart cell migration, gave a seminar at NIEHS that featured some of his latest findings. “Transcriptional Precision in the Drosophila Embryo” took place on March 10 and was hosted by Karen Adelman, Ph.D.(http://www.niehs.nih.gov/research/atniehs/labs/lmc/tre/index.cfm), a principal investigator in the Laboratory of Molecular Carcinogenesis.
Levine(http://mcb.berkeley.edu/index.php?option=com_mcbfaculty&name=levinem) said that the genetic control of dorsal-ventral patterning in Drosophila is controlled by a sequence-specific transcription factor called dorsal (Dl), which is related to mammalian NF-κB. In a two-hour fly embryo, Dl is distributed in a broad nuclear gradient with peak levels present in ventral regions — the future belly of the adult fly — and progressively lower levels in lateral and more dorsal regions. This Dl gradient controls dorsal-ventral patterning by regulating 60-70 target genes in a concentrated-dependent fashion.
“To understand how the Dl gradient generates these distinct threshold readouts of gene activity, we've isolated and analyzed about 35 Dl target enhancers,” he said. "The Dl gradient generates six distinct patterns of gene expression across the dorsal-ventral axis of the embryo, and the protein works in a highly combinatorial fashion with other regulatory factors to produce these different threshold readouts.”
Levine explained that the Dl gradient activates the target gene Twist (Twi) and the two work synergistically to regulate most of the Dl target genes. Because the binding sites for these two proteins are very close and oriented toward each other on the DNA template, they are inextricably linked. Levine said, “If the Twi binding site points away from Dl, it disrupts cooperative interactions between the two and leads to a severe reduction in expression. This is an example of how [developmental] grammar — a fixed arrangement of binding sites — is absolutely essential for enhancer function.”
Levine's group, along with a collaborator at Massachusetts Institute of Technology, used chromatin immunoprecipitation techniques, ChIP-chip or ChIP-seq assays, to look for Dl/Twi binding sites throughout the Drosophila genome. The surprising finding is that nearly half of the 60-70 Dl target sites that contain a 5' primary enhancer also contain a duplicate enhancer for a single pattern of gene expression. Levine calls these secondary enhancers “shadow” enhancers and, in some cases, they are located within the intron of neighboring genes. Further ChIP-chip studies using the distantly-related Anopheles mosquito proved that the intronic enhancer was actually the primary one.
Additional ChIP-chip assays identified stalled RNA Polymerase II (Pol II) in the promoter regions of most Dl target genes prior to their activation in the early embryo. Quantitative confocal imaging suggests that genes containing stalled Pol II are activated in a synchronous manner throughout the field of cells where they are expressed.
Levine spent the last few minutes of his talk discussing the other organism that his lab studies— the sea squirt. The larval stage of this simple chordate looks like a tadpole and is composed of about 1000 cells, but the sea squirt heart arises from the 110 cell stage from a pair of blastomeres — undifferentiated cells formed by the cleavage of a fertilized egg. During this stage, several transcription factors such as Mesp, Xp2 and Fox4, along with the fibroblast growth factor (FGF) signaling pathway allow these procardio myocytes to migrate from the anterior tail region to the head region to form the heart. According to Levine, if FGF signaling is blocked, these tail cells don't receive the signal and remain in the tail to become muscle.
Levine explained why his sea squirt studies are important. “Recent molecular phylogenetic studies suggest that this chordate is the closest living relative of the vertebrates, so whatever we can learn from this system will be directly applicable to far more complex vertebrates, which isn't always the case in Drosophila.”