Intellectulal Merit. Expression of eukaryotic genes is regulated at the transcriptional level by sequence-specific DNA-binding regulators that control the assembly and activity of the general/basal RNA polymerase II transcription machinery at the "core promoter". The core promoter encompasses the transcription initiation site of the gene and is composed of various core promoter DNA elements including the TATA box, the Initiator and other elements that are variably present in different genes. These DNA elements control not only the strength of the core promoter but also the selective response of the core promoter to specific enhancers and transcription regulators and therefore play a critical role in the regulation of differential gene expression. Recent analyses have revealed the conservation and prevalence of the Initiator element in eukaryotic gene core promoters from yeast to humans. However, the functions of the Initiator and the mechanisms of TATA-Initiator synergy, have remained poorly understood. This project will characterize the mechanisms of Initiator function and TATA-Initiator synergy and the roles of transcription factors and recently identified cofactors in Initiator-dependent gene transcription in vitro and in vivo. Mechanistic analyses will be performed in reconstituted transcription reactions in vitro, and gene-selective transcription functions in vivo will be analyzed by a number of approaches. The results promise to transform our understanding of the one of the most fundamental processes involved in regulation of gene expression in eukaryotes.
Broader Impacts. This project will enhance the infrastructure of research and training through the continued instruction of undergraduate and graduate students and mentoring of postdoctoral researchers. The research integrates into the PI's long-term interest in mammalian gene regulation and will be synergistic with his teaching responsibilities. Results and generated tools will be communicated and made available to the scientific community via research publications and through discussions in scientific meetings, and to broader audiences via the internet. The PI is committed to increase the participation of underrepresented minorities in science and will continue to engage minority students in research through established minority access programs at the University of California at Riverside.
Scientific outcomes. The project has transformed our understanding of one of the most fundamental processes in living organisms: regulation of gene expression. The project investigated the molecular mechanisms in volved in regulation of gene expression in mammalian cells, which are of broad relevance to most eukaryotic organisms, including animals and plants. The project has extended our understanding the Central Dogmal of Molecular Biology by identifying novel molecular mechanisms for regulation of eukaryotic gene expression at the transcriptional level. How eukaryotic genes, and especially the more complex mammalian genes, are regulated has been an intensive area of study by many laboratories for over 40 years. However, most studies have concentrated on gene regulation by distal regulatory DNA elements and cognate regulators. This NSF-supported project has focused on the poorly understood functions and regulatory roles of the core promoter region (i.e., the transcription initiation region) of genes. The results of the project have extended the paradigm of combinatorial control of specific gene expression by different assortments of distal DNA-binding regulators to also include combinations of DNA sequence elements within the core promoter region. This project uncovered some of the previously unknown protein factors and mechanisms involved in specific gene regulation by the combinatorial effects of core promoter DNA elements and distal regulatory DNA elements. The project also uncovered that the stereo-specific arrangement of regulatory DNA elements relative to core promoter DNA elements is also important to achieve proper gene-specific regulation. The information gathered has led to a better understanding in molecular terms of how specificity in gene regulation is achieved in mammalian cells. Specific products of the research project included one research article published in the prestigious journal "Genes and Development", one Methods article published in "Methods in Molecular Biology", and two review articles published in the journals "Cell Cycle" and "Transcription". The project generated additional results that are being prepared for publication: one review article commissioned by the journal "Transcription" and one research article that describes the most recent results uncovering for the first time a combinatorial and stero-specific function of regulatory DNA elements in conjunction with core promoter elements, including the possible mechanisms involved. Broader outcomes. The project outcomes impact broad disciplines. Given the general and fundamental nature of the research project which investigates phyllogenetically conserved factors and basic mechanisms involved in regulation of eukaryotic gene expression, the results obtained (briefly described above) are likely to influence a broad range of disciplines including medicine, plant and agricultural sciences, and environmental and bioengineering sciences, which all exploit or benefit from knowledge of the molecular mechanisms that regulate the expression of eukaryotic genomes. Results and generated reagents/tools were communicated and made available to the scientific community via research publications and through discussions in scientific meetings, and to broader audiences via the internet. The project impacted human resources and minorities. This project introduced five undergraduate students to research in a laboratory and has fostered the development of research, teaching, and mentoring skills of two graduate students and two postdocs. One graduate student obtained his PhD degree for his work, which was entirely supported by this project. This project also provided development opportunities to minorities in the sciences, which included six women and four undergraduate students of Hispanic origins who contributed to different aspects of the project. This research project enhanced the educational activities of the PI as a Professor teaching in the field of gene regulation and allowed the PI to increase the participation of underrepresented minorities in the sciences. The PI of this project served as a Mentor and the Director of the Minority Access to Research Careers for Undergraduates (MARCU) Program and participated in other minority programs such as the California Alliance for Minority Participation (CAMP) Program at the University of California at Riverside, an officially classified Minority Institution. Impacts on society. The teaching and mentoring supported by this project has allowed students and postdocs to realize, or better appreciate, the values of hard work, team work and collaborative effort, professionalism, ethical conduct, and considerate non-sicriminative behavior in an ethnically diverse work environment. It also taught the value of outreach to communities and the importance of communicating with the general public about the essential role of fundamental research in the continued development of our society. The project also helped the economy, especially of the southern California Inland empire region, by employing personnel and by purchasing research materials and services.
