Jumin Zhou, Ph.D.

Assistant Professor
Gene Expression and Regulation Program
215-898-3988, Office
zhouj@wistar.org

Introduction

The Zhou laboratory is studying certain specialized DNA elements involved in regulating the genes responsible for the proper development of the body. The laboratory is using the fruit fly as an experimental model, but the genes being investigated appear throughout the animal kingdom, and mutations in these genes or the DNA elements that help to regulate them often result in cancer, birth defects, and other disorders.

Research Summary

The Zhou laboratory is interested in the mechanisms that control how genes are turned on and off, using the fruit fly as an experimental model. This special class of genes, the homeotic genes, controls the body plan of the entire animal kingdom. Homeotic genes turn on transcription of groups of genes to make structures such as legs, wings, and antennae develop properly. Mutations in these genes and their regulatory regions often result in developmental defects, cancer, and other diseases. Proper regulation of homeotic genes requires specialized DNA elements. The Zhou research team is specifically interested in regions of DNA that either facilitate or disrupt gene transcription. From fruit flies to humans, large gene clusters or single gene loci with complex regulatory regions are necessary to orchestrate the intricate expression patterns of proteins during developmental and physiological processes. The right protein has to be expressed at the right time. The Hox gene cluster and the b-globin locus are excellent examples. A central question for genes with a large and complex regulatory region is how to establish specific enhancer-promoter interactions (that is, how genes are turned on to produce a specific protein) whereby enhancer-interacting activator proteins recognize and activate specific, but often distantly located, gene promoters, and how to prevent inappropriate interactions, and thereby mistakes in development. A class of special DNA regions called insulators, or chromatin boundary elements, have been identified from yeast to man. These elements are believed to organize genomes into distinct areas called functional loop domains to restrict regulatory activity locally, thus preventing inappropriate mis-regulations. Insulators are believed to exist between neighboring genes to prevent enhancers from activating the wrong promoters. A few years ago the Zhou team identified a regulatory element, the Promoter Targeting Sequence (PTS), which has an anti-insulator activity. This permits an enhancer to activate a promoter despite an intervening insulator. It also has a "promoter targeting" function, restricting the enhancer activity to a single promoter even when more than one promoter is available. Recent studies also showed that the PTS facilitates the activity of a distant enhancer, and its targeting activity is "memorized" in all successive generations and is even maintained following transgene transposition. The Zhou lab is now engaged in determining the basic biological properties of PTS and identifying the genes and proteins that mediate its activity.

Recent Scientific Advances

The Zhou laboratory is interested in the mechanisms controlling long-distance gene activation in the fruit fly. The research team is specifically interested in regulatory DNA elements that either facilitate or disrupt gene transcription by distant enhancers such as insulators and Promoter Targeting Sequences (PTS). In higher metazoans from fruit flies to man, large gene clusters or single gene loci with complex regulatory regions are necessary to orchestrate the intricate expression patterns of proteins during developmental or physiological processes. The Hox gene cluster and the b-globin locus are excellent examples. A central question for genes with a large and complex regulatory region is how to establish specific enhancer-promoter interactions, whereby enhancer-interacting activator proteins recognize and activate specific, but often distantly located, gene promoters, and how to prevent inappropriate interactions. A class of special DNA regions called insulators, or chromatin boundary elements, have been identified from yeast to man. These elements are believed to organize genomes into distinct areas called functional loop domains to restrict regulatory activity locally, thus preventing inappropriate mis-regulations. Insulators are believed to exist between neighboring genes to prevent enhancers from activating the wrong promoters.

Paradoxically, the recently identified Frontabdominal (Fab)-7 and Fab-8 insulator elements from the Abdominal-B (Abd-B) locus of the Drosophila Bithorax gene complex (BX-C) are located between enhancers and their promoter. The Abd-B gene consists of several abdominal specific enhancer domains called infraabdominal (iab). The Fab elements are required to separate these domains to prevent inappropriate developmental consequences such as duplication of an abdominal segment. However, they apparently do not block enhancer-promoter interactions in Abd-B. This suggests that there must be a mechanism in place to help enhancers in Abd-B to bypass the insulators and activate their promoters. This hypothesis has led to the recent identification of a novel cis-regulatory element, the Promoter Targeting Sequence (PTS) (Zhou and Levine, 1999). The PTS has an anti-insulator activity, permitting an enhancer to activate a promoter despite an intervening insulator. It also has a "promoter targeting" function, restricting the enhancer activity to a single promoter even when more than one promoter is available. Recent studies also showed that the PTS facilitates the activity of a distant enhancer (Lin et al., 2003), and its function is genetically stable in that the PTS-mediated promoter targeting activity is "memorized" in all successive generations and is even maintained following transgene transposition. Mutations in the PTS region disrupt enhancer-promoter interactions in the Abd-B locus, resulting in corresponding homeotic transformations, whereby the posterior abdominal segment resemble more anterior segment. These properties offer significant insights into the mechanism of PTS function and provide a new window to examine the fundamental questions about enhancer-promoter interaction and the mechanism of insulator function. Given the unique activities of the PTS and the overall conservation among the Hox clusters, similar PTS elements are likely to be found in other Hox clusters or other large complex loci. Current research interests are to determine the basic mechanism of PTS function and to identify genes whose product function through this element. Long-term goals are to determine the molecular mechanism of PTS function and to test whether a similar mechanism is employed to regulate vertebrate genes.

Determining the basic properties of PTS using transgenic embryos: The research team plans to determine whether the PTS possesses promoter specificity and if so, what dictates such specificity by analyzing the Abd-B promoter and its proximal region in transgenic embryos of fruit flies. Second, the investigators will test if the chromosomal location determines whether or not the PTS can mediate promoter targeting at a specific insertion site, and if so, whether it also determines which promoter the PTS targets an enhancer to. They will use the gene conversion technique to place different PTS-containing P-elements at the same chromosomal location and examine the promoter targeting activity of the PTS from different transgenes.

Lastly, they will test the stable-looping model by characterizing the epigenetic maintenance property of the PTS. The working model is that the PTS forms a stable loop between the enhancer and the promoter regions. This interaction is independent of activator or repressor proteins that interact with the enhancer. This model could explain how the PTS bypasses an insulator and why the PTS restricts an enhancer to only a single promoter. A key prediction of this model is that these activities will be at least to a certain degree independent of which enhancers and insulators are present. They will test the prediction that formation of a heritable, stable enhancer-promoter interaction is independent of enhancer identity and enhancer-binding proteins, as well as test whether there is direct physical association between the chromatin around the enhancer and that at the promoter using the Chromosome Conformation Capture (CCC) and Chromatin Immunoprecipitation (ChIP) techniques.

Determining the role of histone modifications in PTS mediated functions: Promoter targeting results in the selective activation of only one of the two closely positioned (about 600 bp apart) w and lacZ promoters in transgenic constructs. One possible molecular mechanism of how this is achieved is by differential modifications of the histones at the promoters. One can imagine that PTS-interacting proteins recognize the target promoter, and recruit histone-modifying enzymes, which either phosphorylate or acetylate histones at the promoter to create an activator-accessible conformation. Equally possible, the PTS may require a repression mechanism to keep the nearby, non-targeted promoter silent. Thus, the PTS might recruit histone deacetylase or methylase, which create silenced, activator-inaccessible structure.

To investigate whether the promoter-targeting activity of PTS involves promoter-specific histone modification, the investigators will specifically examine whether histone H3 at the targeted and the non-targeted promoters is differentially modified using the ChIP assay in collaboration with Dr. Shelley Berger's laboratory (also at The Wistar Institute). The colleagues will begin by selecting a transgenic strain where PTS targets IAB8 to the lacZ promoter, but not to the w promoter, and compare Ser-10 phosphorylation, which activates transcription, and Lys-14 acetylation, which also activates transcription, at these two promoters. They will also examine Lys-9 methylation, which represses chromatin and transcription, at these promoters to detect whether there is a correlation with the silencing of the w promoter. A control transgene without the PTS and the insulator will also be included. If the analysis proves to be successful, they will examine similar modifications in the endogenous Abd-B locus, as well as determine whether histone modification changes of the Abd-B promoter in PTS deletion mutants. This involves labeling Abd-B expressing cells with GFP, followed by cell sorting before ChIP analysis. These studies may provide crucial pieces of evidence of the molecular mechanism of long-range enhancer-promoter interactions in the Drosophila Hox gene cluster.

Identifying genes and proteins that mediate PTS activities: Identifying proteins that mediate PTS activities is essential for determining the molecular mechanisms of these activities and will guide future studies on how enhancer-promoter interactions are regulated in large gene complexes. In order to identify the genes and proteins that function through the PTS, the lab will conduct a genetic screen for mutants that inactivate or alter PTS activity. Because it is likely that the proteins encoded by these genes will be essential, they will conduct a comprehensive F1 Flp-FRT screen that is specifically designed for recovering recessive lethal mutations as wells as other types of mutations that can be isolated by traditional screens. Mutations that modify PTS activity will be mapped and modifier-encoded genes will be cloned using standard genetic and molecular procedures. The involvement of the cloned genes in PTS function will be tested by introducing transgenes into mutant backgrounds for these genes. Mutant phenotypes will be carefully studied with emphasis on how they affect Abd-B function.

Selected Publications

Moon, H., Filippova, G., Loukinov, D., Pugacheva, E., Chen, Q., Smith, S.T., Munhall, A., Grewe, B., Bartkuhn, M., Arnold, R., Burke, L.J., Renkawitz-Pohl, R., Ohlsson, R., Zhou, J., Renkawitz, R., Lobanenkov, V.: CTCF is conserved from Drosophila to humans and confers enhancer blocking of the Fab-8 insulator. EMBO Rep, 2005.

Zhou, J., Berger, S.L.: Good fences make good neighbors: barrier elements and genomic regulation. Mol Cell 16:500-502, 2004.

Lin, Q., Chen, Q., Lin, L., Zhou, J.: The Promoter Targeting Sequence mediates epigenetically heritable transcription memory. Genes Dev 18:2639-2651, 2004.

Lin. Q., Chen, Q., Wu, Di., and Zhou, J., A chromatin insulator is essential for initiating promoter targeting in the Drosophila embryo. Submitted.

Lin, Q., Di Wu., and Zhou, J. (2003). The PTS element facilitates and restricts a distant enhancer to a single promoter in the transgenic Drosophila embryos. Development. 130, 519-526.

Zhou, J., and Levine, M. (1999). A novel cis-regulatory element, the PTS, mediates an anti-insulator activity in the Drosophila embryo. Cell. 99, 567-575.

Zhou, J., Ashe, H., Burks, C., and Levine, M. (1999). Characterization of the transvection mediating region of the Abdominal-B locus in Drosophila. Development. 126, 3057-3065.

Zhou, J., Zwicker, J., Szymanski, P., Levine, M., and Tjian, R. (1998). TAFII mutations disrupt Dorsal activation in the Drosophila embryo. PNAS. 95, 13483-13488.

Zhou, J., Cai, H. N., Ohtsuki, S, and Levine, M. (1997). The regulation of enhancer-promoter interactions in the Drosophila embryo. Cold Spring Harbor Symp. Quant. Biol. LXII: 307.

Zhou, J., Barolo, S., Szymansky, P. and Levine, M. (1996). The Fab-7 element of the bithorax complex attenuates enhancer-promoter interactions in the Drosophila embryo. Genes Dev. 10: 3195-3201.