HAINI N. CAI
Associate Professor
Ph.D., 1988
University of Cincinnati
RESEARCH
Transcriptional regulation underlies crucial aspects of development, physiological responses, and many human diseases including cancer. Study of pattern formation during Drosophila development has established the importance of complex enhancers in integrating regulatory information and directing localized gene expression. One goal of my research is to understand how enhancers specifically interact with genes in complex genetic loci in the context of the chromosomal environment. We have used a stripe expression assay in transgenic Drosophila embryos to analyze how enhancers are specified for two neighboring genes, ftz and Scr, in Antennapedia Complex (ANT-C), a Drosophila homeotic gene complex. Our study revealed that specific gene activation is influenced by two complementary mechanisms: the function of a special class of regulatory DNA called chromatin boundaries (or insulators), as well as competition among neighboring gene promoters for enhancers (Development 2001, 128:4339).
More recent studies of insulator mechanism and function in my laboratory focus on the following areas:
1. Testing insulator interactions: To probe whether insulator interaction represents a general mechanism of insulator activity, we tested enhancer-blocking activity of twelve selected pairs of Drosophila insulators including suHw, scs, Fab-7, SF1 and ftz-MAR. We found that, unlike suHw, whose activity is abolished by homologous paring, the heterologous and even homologous pairing of other Drosophila insulators, such as Fab7, SF1, scs, and ftz-MAR, do not abolish their insulator activity. Rather, pairing of these boundary elements generally resulted in an enhancement of their enhancer-blocking activity. These results suggest that diverse mechanisms may underlie insulator function, and that selective interactions between different classes of boundary elements may exist (PNAS, 2003, 100:5223).
2. Study of insulator anchor sites in the nucleus: We also examined the functional sites of boundary elements in the nucleus. Studies from the Corce�s group indicate that the Gypsy retrotransposon, which harbors the suHw insulator, is preferentially associated with the nuclear envelope, suggesting that this association may be required for its boundary function. We have probed the nuclear localization of transgenes containing either functional or non-functional arrangements of the 340 bp suHw insulator. Our results indicate that, unlike the full-length Gypsy, the functional suHw insulator does not locate preferentially to the nuclear periphery, suggesting that the suHw boundary may require local interactions, such as those between neighboring insulators, or anchorage to structures that are extensive in nucleus (submitted).
3. Identification of SF1, a novel boundary in ANT-C: We have recently identified a novel chromatin boundary, SF1, between the Drosophila ftz and Scr genes in ANT-C. SF1 exhibits a strong enhancer-blocking activity that is orientation- and enhancer-independent and persists throughout the fly life cycle. SF1 contains clusters of GAGA sites that are critical for its boundary activity, a common feature shared with boundaries from the Bithorax complex, such as Fab7 and Mcp1. We show that SF1 can block a ftz enhancer, ftz-distal, in transgenic assays. The genomic position and function of SF1 lead us suggest that boundary may interacts with like elements downstream of ftz to �loop-out� the ftz domain to ensure independent Scr regulation. Our results indicate that conserved boundary function may regulate gene activity in both homeotic complexes in Drosophila (EMBO J., 2003, 22:(12)).
4. Separation of insulator and barrier function in SF1: Our study of the SF1 boundary further indicates that it can effectively protect the mini-white transgene against chromosomal position effect (CPE), an indicator of �chromatin barrier� function. Functional mapping of the SF1 boundary has revealed that the insulator and barrier activities are mediated by distinct DNA elements within the 2.3 kb boundary. For example, SF1b, a 675 bp fragment that exhibits the strongest enhancer-blocking activity among SF1 sub-fragments, is ineffective in protecting a mini-white transgene against CPE, whereas SF1c, which showed no enhancer-blocking activity, contains a strong barrier activity. Another indication of the separation of the insulator and the barrier function are their distinct sequence requirements: SF1b insulator activity depends on the GAGA sites whereas the barrier function of the SF1c appears to be GAGA-independent. Our results provided the first example in an animal model that insulator and barrier activity associated with complex boundary elements may be mediated by different cis and trans factors, and possibly through distinct mechanisms, a phenomenon originally described by the Felsenfeld group. The regulatory function of complex boundaries, such as SF1, may depend on the composition and placement of these distinct activities (Manuscript in preparation).
We are currently using molecular, biochemical, and genetic approaches to dissect the SF1 boundary and to further understand its in vivo function. In particular, we are trying to identify cis and trans factors associated with the insulator as well as the barrier activities. We are searching for other specialized DNAs, such as other boundaries or PRE elements downstream of ftz, that could potentially interact with SF1 to form the looped ftz domain. We are also in the process of disrupting SF1 in vivo in order to test its function in gene regulation in the ftz-Scr genomic region.
Chromatin boundaries are DNA-protein complexes that have been shown to play an important role in gene regulation from yeast to humans. They are operationally defined by their ability to block enhancer-promoter interactions and to protect transgenes from the influences of neighboring chromatin. In spite of their wide presence, the mechanism of insulator activity is not known. Previous studies of suHw, an insulator from the Drosophila Gypsy retrotransposon, showed that the boundary activity of suHw depends on its cis-arrangement, including copy number and position. Specifically, the enhancer-blocking activity of the insulator is abolished by the tandem pairing of insulator elements. These results suggest that insulators may interact with like elements elsewhere in the genome or anchor sites in the nucleus to partition chromatin into distinct domains or loops, resulting in the separation of enhancers from promoters (Science 2001, 291:493).
Test for insulator-nuclear envelope association
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| CONTACT INFORMATION |
| (706) 542-3329, hcai@uga.edu |
| SEE ALSO |
| Biomedical & Health Sciences Institute |
GRANT SUPPORT
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| Kevin Jiayang Liu | |
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| Sharmila Roy | |
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 Polytene Chromosome from Drosophilia |
Cai, H. and M. Levine. Modulation of enhancer-promoter interactions by insulators in the Drosophila embryo Nature 1995. 376(6540): 533-6.Cai, H. N., D. N. Arnosti, and M. Levine. Long-range repression in the Drosophila embryo. Proc Natl Acad Sci U S A 93 1996. (18): 9309-14.
Cai, H. N., and M. Levine The gypsy insulator can function as a promoter-specific silencer in the Drosophila embryo. EMBO J 1997. 16(7): 1732-41.
Ohtsuki S., M. Levine and H. N. Cai. Different core promoters possess distinct regulatory activities in the Drosophila embryo. Genes Dev 12 1998: 547-556.
Cai, HN, Shen P. Effects of cis arrangement of chromatin insulators on enhancer-blocking activity. Science. 2001 ;291(5503):493-5.
Cai, HN, Zhang, Z, Adams, JR and Shen, P Integrated transcriptional regulation by enhancers, insulators and neighboring genes Development 2001, 128 :4339-4347
Majumder P and Cai HN The functional analysis of insulator interactions in the Drosophila embryo Proc Natl Acad Sci U S A 2003; 100:5223-5228
Belozerov VE, Majumder P, Shen P and Cai HN A novel boundary element may facilitate independent gene regulation in the Antennapedia Complex of Drosophila EMBO J. 2003, 22 (12) 3113-3121
Xu QH, Li M , Adams J and Cai HN.?Study of insulator function and nuclear localization. J Cell Sci 2004; 117 (7) :1025-1032
Majumder P, Belozerov VE,Bosu D, Roy S and Cai HN. Distinct mechanisms of transcriptional regulation revealed by non-overlapping activities within a compound chromatin boundary. Submitted.