Our laboratory is interested in the organizing principles of the eukaryotic genome and the epigenetic functions in development and disease. We are focused on two types of related regulatory DNA elements that have seemingly opposite functions: (1) insulators, also known as chromatin boundary elements, which are DNA elements that block unwanted gene activation by enhancers from neighboring genes and (2) Promoter Targeting Sequences, PTS, a novel type of regulatory element which selectively facilitates long-range enhancer-promoter interactions. Both of these elements were found in the Drosophila homeotic gene complex, a conserved cluster of Hox genes, which control cellular identities throughout the animal kingdom. Mutations in these genes and their regulatory regions result in developmental defects in flies and in a range of diseases including birth defects and cancer in humans.
One of the most important questions of the functional eukaryotic genome is how large-scale genome structure and epigenetic function are organized and regulated. One class of specialized DNA regions called insulators, or chromatin boundary elements, have been identified from yeast to humans. These elements are believed to organize the genome into distinct areas called functional loop domains, in order to restrict regulatory activities locally by preventing inappropriate mis-regulation.
Recently, we and others identified the Drosophila CTCF and demonstrated that this protein mediates the insulator function of the Fab-8 element from the Abd-B locus. To understand the role of CTCF in large-scale organization of chromosome structure, we conducted a Chromatin Immunoprecipitation followed by genome-wide tiling array analysis on CTCF-binding sites. Our analysis reveals that CTCF binds to many known domain boundaries within the Abd-B gene of the BX-C including previously characterized Fab-8 and MCP insulators, and the Fab-6 region. We also found that dCTCF-binding sites are often situated between closely positioned gene promoters, consistent with the role of CTCF as an insulator protein. Importantly, CTCF tends to bind gene promoters at the upstream of transcription start sites, in contrast to the predicted binding sites of the insulator protein Su(Hw). These findings suggest that CTCF plays a more active role in regulating gene activity and that it functions differently from other insulator proteins in the genome.
Currently we are analyzing how CTCF binding relates to other genomic features, epigenetic marks, and chromosomal proteins in collaboration with Dr. Ramana Davuluri’s group also from the Wistar Institute. Preliminary results suggest that CTCF tends to associate with Polycomb group silencers and the cohesin/condensing family of proteins. In contrast, the insulator protein SuHw does not exhibit such a trend. This observation and the study discussed above suggest that SuHw and CTCF have different organizing roles in the genome.
The impending release of genome-wide binding profiles for approximately 150 chromosomal proteins and histone modifications by the ENCODE project offers an exciting opportunity to discover specific chromosomal proteins or epigenetic marks associated with CTCF and to subsequently examine how they are related to the insulator function of CTCF. A specific example to be tested is the H4K3me3 modification, which has been shown to be associated with CTCF in both human and fly genomes. We are currently investigating the role of H3K4me3 and related histone modifying enzymes in CTCF insulator function.
Finally, to gain a thorough mechanistic understanding of insulators, we need to identify the additional proteins factors necessary for insulator function. For this purpose, we are developing an RNAi screen to identify proteins or transcripts necessary for CTCF insulator function.
My earlier study identified a novel regulatory DNA element from the bithorax gene complex, the Promoter Targeting Sequence (PTS). PTS has anti-insulator activity as it allows an enhancer to activate a promoter despite intervening insulators. It also has a "promoter-targeting" function, restricting the enhancer activity to a single promoter even when additional promoters are located nearby. Our recent studies showed that the PTS facilitates the activity of a distant enhancer and its targeting activity is "memorized" in following generations. Further study of this epigenetic element using biochemical, molecular and genetic approaches is essential to understanding this unique regulatory element and will thus shed light on higher order chromosome organization and epigenetic inheritance. Currently, my lab is working on two projects related to the PTS element:
Chromatin structure and PTS function. Because regulatory activities of gene expression ultimately converge on the chromatin through histone modifications, nucleosome remodeling, and modulations of the transcription machinery, the anti-insulator and promoter targeting activities of the PTS will affect aspects of chromatin structure. Our main research goal is to investigate the role of chromatin in PTS-mediated anti-insulator and promoter targeting activities. Specifically, we are testing how local chromatin structures near transgene insertion sites affect PTS activities. Secondly, we are examining how PTS activities affect histone modifications at the PTS, the targeted promoter, the insulator, and the enhancer involved. Thirdly, we are studying how the anti-insulator function of PTS affects CTCF binding to insulators, and Pol II-binding to the target promoter. Through these studies, we will provide mechanistic insights into the PTS functions.
Identification of proteins that function through the PTS. To study the mechanism of PTS activities, we are also in the process of identifying and characterizing PTS-interacting proteins by DNA affinity purifications and Yeast One-Hybrid screen using a minimal 27 bp PTS element. We will characterize the identified proteins by EMSA, antibody staining, and ChIP to demonstrate their interaction with PTS, both in vitro and in vivo. Finally, we will generate point mutations and dsRNA knockdowns to study the genetic functions of the potential PTS genes. In the future, we will test whether these proteins mediate PTS function by affecting chromatin structures.
In addition to studying higher order genome organization in model genetic organisms, we are also interested in well-characterized animal disease models. Human herpes simplex virus (HSV) is the main cause of cold sores and genital sores. Herpes outbreaks can overload the immune system, reducing its ability to defend the body against other viral infections. The HSV-1 genome contains 152 kilobases of double-stranded DNA encoding close to 100 transcripts. During latent infections, the viral genome is maintained in sensory neurons with the transcription of only one large transcript, the Latency Associated Transcript (LAT). How this active chromatin is kept separate from the repressed chromatin in the nearby ICP0 region remains crucial to the understanding of the HSV life cycle. Recently, we showed that the LAT intron region contains an 800 bp insulator, which interacts with CTCF both in vitro using EMSA and in vivo by ChIP assay. The deletion of CTCF binding sites impaired insulator activity in human K562 cells and Drosophila cells. These results suggest that the LAT insulator works as a chromatin boundary during latency to separate active chromatin associated with the LAT promoter region from repressed chromatin in the ICP0 gene.
More recent studies from our lab identified a second insulator at the 5’ of the LAT promoter, thus the LAT region is protected by two insulators to prevent LAT from being silenced during latent infections. Currently, we are making mutations to the two insulators to examine their in vivo function. More importantly, we are mapping the HSV genome-wide interaction sites for CTCF using ChIP-chip. This study is not only aimed at gaining detailed insights into how insulators regulate other genes within the HSV genome, but also to provide a potential connection between the HSV genome organization and the higher order structure of the host genome.