The T-cell Transcription Regulation Group applies transcriptional approaches to understanding how molecular signaling influences gene regulation in mammalian cells. We use T-cells and the process of T-cell activation as a model system. The work and progress in the group over the past year can be defined in 3 major categories: I. Profiling of Transcriptional Targets II. Defining kinetic profiles of factor occupancy in vivo using Promoter Arrays III. Characterization of hierarchical complex assembly and short and long range chromatin structure at the Interleukin 2 (IL-2) promoter. I. Profiling of Transcriptional Targets This year we have a developed a transcriptional approach termed Profiling of Transcriptional Targets (PTT) to help better define how molecular signaling events are integrated in the nucleus to influence gene transcription. The first element of this approach is a high-throughput transcriptional reporter assay where reporter constructs containing various promoters or other gene regulatory elements are inserted into human T-cells and then challenged with a variety (16 or more) of combinations of mitogenic agents in the presence or absence of numerous (8 or more) pharmacological agents with known or presumed immuno-modulatory activity. As a result, multiple gene regulatory region of specific genes can be assessed under over one hundred (128) conditions. Since this process generates a large volume of """"""""high dimensional"""""""" or multivariate data, we have effectively applied the use of several computational methods well suited to the type and volume of this data. These methods include unsupervised neural networks, hierarchical clustering and principal component analysis. By this method, we have been able to determine relationships between specific gene regulatory elements and promoter in terms of how they respond to different mitogens and pharmacological agents. This approach has enabled us to identify an operational signaling pathway in activated T-cells that responds to the addition of IGF-1 by up-regulating signaling through a PI3-kinase dependent pathway to exert reciprocal control over NF-kappa B and AP-1 transcriptional activity. In order to retrieve transcriptional information that more closely resembles the in vivo regulation of target genes we have adapted PTT to be used with nuclear run-on experiments. This approach combines the molecular specificity of the nuclear run-on technique with micro-array. This process allows the simultaneous assessment of nuclear pre-mRNA populations in comparison to the steady state pool of cytoplasmic RNA. Thus changes in gene expression can be examined at both the level of transcriptional regulation and transcript stabilization. II. Kinetic Profiling of Promoter Occupancy in in vivo We have adapted the chromatin immunoprecipitation assay (ChIP) for highthroughput analysis by combining it with microarray technology (ChIP to chip). This allows us to profile the kinetic association of transcription factors and other co-regulatory elements with tens to potentially thousands of different promoter regions simultaneously. In collaboration with the NCI microarray facility and using a microarray printed with genomic PCR fragments containing the promoter regions of known genes, we have been analyzing the kinetic association of the co-activator protein p300 with multiple gene promoter regions in activated T-cells. Measurements are taken over 15 minute intervals following stimulation of the T-cells. The resulting data is a series of kinetic occupancy curves or retentions times that profile the changing interactions of p300 with each gene on the array. The use of these genomic """"""""Promoter Arrays"""""""" reveals that genes and their promoters can be classified by the manner in which p300 associates with them under a particular stimulus. Comparing the promoter regions of these classes using bioinformatics software reveals patterns of sequences composition and structure that has predictive value in determining their functional requirements for p300. We have currently organized a multi-institutional pilot promoter array study (pPROMP) under the supervision of the Cellular, Molecular and Developmental Biology Faculty of CCR. The study is a collaboration between CCR, the Cancer Genotype Facility, CGAP, National Institute on Aging, the Bioinformatic Group at Ohio State University to provide an 1000+ genomic promoter array for ChIP to Chip studies. Primer sets designed and used for the production of PCR fragments for printing onto arrays at NCI and NIA will be provided to CGF/CGAP for screening regulatory SNPs in several populations. This array will then become a source of the production of several orthologous data sets that will provide basic, clinical and population scientists access to empirically correlated information about promoter structure and function. III. Characterization of hierarchical complex assembly and short and long range chromatin structure at the Interleukin 2 (IL-2) promoter. The model system that we studying in T-cells is the interleukin 2 promoter. This year we have determined that there is a dynamic and ordered assembly of transcriptional complexes at the IL-2 promoter that has a defined hierarchy. Moreover we have identified that there is an epigenetic marking of the IL-2 promoter that allows for a constitutive binding of NF-kappa B factors to the IL-2 promoter that is lineage specific. Associated with these kappa B factors at the IL-2 promoter in resting cells is the ubiquitous and multifunctional mediator complex. Continued studies are aimed at characterizing the kinetic of the association of other factors at the promoter to generate a detailed picture of the dynamics of the factor assembly at IL-2.
