Analysis of gene expression level changes (mRNA and protein) provide insights into regulatory influences and not necessarily mechanisms responsible for mediating those changes. To approach a mechanistic level it is imperative to map the physical linkages among proteins (protein-protein interactions) and proteins and DNA (protein-DNA interactions). These linkages form the "wiring" for gene regulatory circuits. The goal of this project is to use systems query tools to decipher physical linkages in gene regulatory networks in Halobacterium NRC-1 specified by two families of general transcription factors (GTFs include TATA binding proteins (TBPs) and transcription factor binding sites (TFBS)) in conjunction with ~120 transcription regulators and cis-regulatory elements. Together, these GTFs and regulators control transcription of ~2400 genes in the Halobacterium NRC-1 genome.

For deciphering protein-DNA interactions, we have developed a ChIP-chip* strategy to localize transcription factor binding sites (TFBSs) in the Halobacterium genome. Likewise, for protein-protein interactions we have developed a co-immuno-precipitation strategy to enrich protein complexes which are then analyzed by microcapillary liquid chromatography, electrospray ionization, and tandem mass spectrometry (μLC-ESI-MS/MS). To facilitate detection of conserved cis-regulatory elements, we have also recently completed the whole genome sequencing and annotation of a second archaeal halophile, Haloarcula marismortui, which is an inhabitant of the Dead Sea. Here, comparative genomic analysis is expected to shed insights into evolutionarily conserved regulatory circuits. Finally, we have designed plugins for Cytoscape to view the mass spectrometry data as protein-protein interaction graphs and to overlay TFBS onto the genome map of Halobacterium. Both of these specialized viewers are part of the Gaggle that enables exploration of interactions in the context of gene expression profiles, metabolic pathways, functional associations, etc.

Primary methodologies/approaches/strategies used to accomplish the goals

For deciphering protein-DNA interactions, we have developed a ChIP-chip* strategy to localize transcription factor binding sites in the Halobacterium genome. Likewise, for protein-protein interactions we have developed a co-immuno-precipitation strategy to enrich protein complexes which are then analyzed by microcapillary liquid chromatography, electrospray ionization, and tandem mass spectrometry (μLC-ESI-MS/MS). Finally, to facilitate detection of conserved cis-regulatory elements, we have also recently completed the whole genome sequencing and annotation of a second archaeal halophile, Haloarcula marismortui, which is an inhabitant of the Dead Sea.

*Chromatin Immunoprecipitation followed by microarray chip analysis. The procedure is used for experimentally mapping protein-DNA interactions.

Key results obtained for the project as of June 2005.

A comprehensive study of the general transcription regulatory network is currently being conducted. We have deciphered protein-DNA and protein-protein interactions among a large number of these factors to demonstrate a fascinating multi-tiered transcription regulatory network in Halobacterium. Using these interactions, we are now validating many of the key regulatory influences we have previously deciphered through analysis of large gene expression datasets with unsupervised statistical learning algorithms.

Future directions of this project.

Most of the interactions that we are currently studying have been under steady state and standard culture conditions. However, many of these interactions might occur under defined conditions with respect to both environmental and temporal space. A goal for future research is to map these dynamic interactions in time-series samples from environmentally perturbed cultures.

How does the project address the issues of predictive, preventive or personalized medicine?

This project aims to delineate experimental methodology and computational algorithms specifically for the purpose of modeling gene regulatory circuits. The long-term vision includes use of predictive mathematical models for gene regulatory networks in Halobacterium NRC-1, for engineering of designer circuits for a variety of biotechnological applications such as environmental clean-up projects. With respect to predictive, preventive and personalized medicine, the approaches delineated here will not only provide software and experimental technology but will also serve as a roadmap for designing similar strategies for constructing predictive models for complex diseases.

Group members involved with Project

Nitin Baliga (PI)
Marc Facciotti,
Amy Schmid,
Madhavi Vuthoori Amardeep Kaur,
Min Pan

Internal ISB groups/people involved with project.

Leroy Hood
Jeff Ranish

Representative publication(s):

Baliga, N.S., and DasSarma, S. (1999) Saturation mutagenesis of the TATA box and upstream activator sequence in the haloarchaeal bop gene promoter. J Bacteriol 181: 2513-2518.

Baliga, N.S., and Dassarma, S. (2000) Saturation mutagenesis of the haloarchaeal bop gene promoter: identification of DNA supercoiling sensitivity sites and absence of TFB recognition element and UAS enhancer activity. Mol Microbiol 36: 1175-1183.

Baliga, N.S., Goo, Y.A., Ng, W.V., Hood, L., Daniels, C.J., and DasSarma, S. (2000) Is gene expression in Halobacterium NRC-1 regulated by multiple TBP and TFB transcription factors? Mol Microbiol 36: 1184-1185.

Baliga, N.S., Pan, M., Goo, Y.A., Yi, E.C., Goodlett, D.R., Dimitrov, K., Shannon, P., Aebersold, R., Ng, W.V., and Hood, L. (2002) Coordinate regulation of energy transduction modules in Halobacterium sp. Analyzed by a global systems approach Proc Natl Acad Sci U S A 99: 14913-14918.

Baliga, N.S., Bonneau, R., Facciotti, M.T., Pan, M., Glusman, G., Deutsch, E.W., Shannon, P., Chiu, Y., Weng, R.S., Gan, R.R., Hung, P., Date, S.V., Marcotte, E., Hood, L., and Ng, W.V. (2004) Genome sequence of Haloarcula marismortui: A halophilic archaeon from the Dead Sea. Genome Res. 14: 2221-2234.