CONTROL OF GENE EXPRESSION DURING DEVELOPMENT
Judith A. Kassis, PhD, Head, Section on Gene Expression
J. Lesley Brown, PhD, Staff Scientist
Yuzhong Cheng, PhD, Senior Research Technician1
Melissa Durant, PhD, Postdoctoral Fellow
Deborah Kwon, BS, Postbaccalaureate Fellow2
Catherine Stefaniuk, BS, Postbaccalaureate Fellow2
During development and differentiation, genes either become competent to be expressed or are stably silenced in an epigenetically heritable manner. This selective activation/repression of genes leads to differentiation of tissue types. Recent evidence suggests that modifications of histones in chromatin contribute substantially to determining whether a gene will or will not be expressed. Our group is interested in understanding how chromatin-modifying protein complexes are recruited to the DNA. In Drosophila, two groups of genes, the Polycomb group (PcG) and the Trithorax group (TrxG), are important for inheritance of the silenced and active chromatin state, respectively. Regulatory elements called Polycomb group response elements (PREs) are cis-acting sequences required for the recruitment of chromatin-modifying PcG protein complexes. Recently, it has been suggested that TrxG proteins act through either the same or overlapping cis-acting sequences. Our group is working on understanding how PcG and TrxG proteins are recruited to the DNA.
DNA sequences that constitute a PRE
PREs are DNA elements through which the PcG protein transcriptional repressors act. Many of the PcG proteins are associated in two protein complexes that repress gene expression by modifying chromatin. Both complexes specifically associate with PREs in vivo. However, it is not known how they are recruited or held at the PRE. PREs are complex elements made up of binding sites for many proteins. Our laboratory has been working to define all the sequences and DNA-binding proteins required for the activity of a 181-bp PRE from the Drosophila engrailed gene. At least nine binding sites are present within this 181-bp PRE. Two of the sites are for the PcG proteins Pleiohomeotic (Pho) and Pleiohomeotic-like (Phol). Binding sites for the proteins GAGA factor, Pipsqueak, Zeste, and Dsp1 also are present within the engrailed PRE. Proteins that bind to the other three sites were not identified. We found that one of the unidentified sites necessary for PRE activity is bound by members of the Sp1/KLF family of zinc-finger proteins. This family of proteins encodes transcription factors and has been extensively studied in mammals, revealing 20 Sp1/KLF family members. Drosophila accounts for 10 Sp1/KLF family members, of which 9 bind to the engrailed PRE. We derived a consensus-binding site for the Sp1/KLF Drosophila family members and showed that the consensus sequence is present in most of the molecularly characterized PREs. The data suggest that one or more Sp1/KLF family members play a role in PRE function in Drosophila.
We have been working to determine which of the Sp1/KLF family members in Drosophila may be involved in PcG function. We made antibodies to the three most likely candidates, and our analysis now focuses on one candidate ubiquitously expressed in embryos. We are generating a mutant in the gene that encodes Sp1/KLF to determine if it plays a role in PcG repression.
Although much is known about the protein-binding sites required for PRE function, we are not able to predict the location of a PRE based on the presence of binding sites alone. To help us identify either other protein-binding sites required for PRE function or other important characteristics of PREs (such as the number or spacing of binding sites), we have begun analyzing other PREs from the engrailed region of the genome. Our work should lead to a better understanding of the protein-binding sites required for PRE function.
Brown JL, Grau DJ, DeVido SK, Kassis JA. An Sp1/KLF binding site is important for the activity of a Polycomb group response element from the Drosophila engrailed gene. Nucleic Acids Res 2005;33:5181-9.
Müller J, Kassis JA. Polycomb response elements and targeting of Polycomb group proteins in Drosophila. Curr Opin Genet Dev 2006;16:476-84.
Understanding the role of PREs and flanking sequences at the engrailed gene
The Drosophila engrailed gene encodes a homeodomain protein that plays an important role in the development of many different parts of the embryo, including the formation of the segments, nervous system, head, and gut. It also plays an important role in the development of the adult, specifying the posterior compartment of each imaginal disk. Accordingly, engrailed is expressed in a highly specific and complex manner in the developing organism. The 181-bp engrailed PRE is located near the engrailed promoter from −576 to −395 upstream of the transcription start site. We were interested in determining the role of this PRE in the control of engrailed expression and learned, for example, that it is redundant with other flanking PREs in the endogenous engrailed gene; another strong PRE is located from −1100 to −1500, and other weak PREs are probably located nearby. In fact, when we examined the location of Ph and Pho proteins on engrailed DNA by chromatin immunoprecipitation (ChIP), we found the proteins bound to a 2.5-kb region extending from the engrailed promoter to about −2.5kb upstream. Therefore, it is perhaps not too surprising that a 500-bp deletion that includes the 181-bp PRE and flanking sequence did not lead to ectopic engrailed expression. The remaining PREs were apparently sufficient to recruit PcG proteins. However, we were surprised that loss of this DNA led to a loss-of-function phenotype, suggesting that the DNA must also play a positive role in the expression of engrailed. Recent experiments suggest several positive elements either overlapping or coincident with the PREs. We are determining whether the positive and negative sequences can be separated.
The regulatory sequences for the engrailed gene extend over a 70-kb region. We used reporter constructs to find sequences important for expression in stripes, the nervous system, the head, and so forth and identified discrete regulatory elements located throughout the 70-kb region. We also found at least seven additional potential PREs located throughout the region. PcG protein complexes have been shown in vitro to bring together DNA fragments, and the complexes possibly cause looping in vivo. We are interested in learning whether the additional PREs are involved in mediating interactions between distant enhancers and the engrailed promoter.
A genetic screen identifies new members of the Trithorax and Polycomb groups
We performed a genetic screen to identify new members of the TrxG and PcG. When making transgenic Drosophila, we used the eye color gene white to detect transgenic Drosophila. Eye color is dependent on the expression level of the white gene; more expression of white causes a darker eye color, and less expression a lighter eye color. PREs linked to white (a PRE–white transgene) cause less white expression, leading to a lighter eye color. We performed a genetic screen to identify mutations that would darken the eye color of transgenic flies with a PRE–white transgene. We reasoned that mutation of a PcG gene, which encodes a repressor, might lead to a darkening of the eye color. Increasing the activity of an activator protein (a potential TrxG gene) might also darken the eye color by competing with the PcG repressors. We screened over 60,000 flies and obtained 9 mutants. We have now characterized 2 of those mutants. Interestingly, one has a PcG phenotype and the other a TrxG phenotype. We are currently completing our analyses of these two genes and beginning characterization of other mutants isolated in the screen. We hope to understand the molecular function of these newly identified PcG and TrxG proteins.
1 Joined the laboratory during the reporting period.
2 Left the laboratory during the reporting period.
3 Sarah DeVido, MS, former Technician
4 Stefanie Kremer, BS, former Postbaccalaureate Fellow
5 Alayne Brown, BS, former Postbaccalaureate Fellow
6 Amanda Gordon, BS, former Postbaccalaureate Fellow
COLLABORATORS
James A. Kennison, PhD, Program in Genomics of Differentiation, NICHD, Bethesda, MD
For further information, contactjkassis@mail.nih.gov.
