Understanding the language encrypted in the gene regulatory regions of the human genome is a challenging goal for the genomic era. Although customary extrapolations from steady-state mRNA levels have been effective, deciphering these regulatory codes will require additional empirical data sets that more closely reflect the dynamic progression of molecular events responsible for inducible transcription. We describe an approach using chromatin immunoprecipitation to profile the kinetic occupancy of the transcriptional coactivator and histone acetyltransferase p300 at numerous mitogen-induced genes in activated T cells. Comparison of these profiles reveals a class of promoters that share common patterns of inducible expression, p300 recruitment, dependence on selective p300 domains, and sensitivity to histone deacetylase inhibitors. Remarkably, this class also shares an evolutionarily conserved promoter composition and structure that accurately predicts additional human genes with similar functional attributes. This ''reverse genomic'' approach will have broad application for the genome-wide classification of promoter structure and function.

Using a novel cell-based assay to profile transcriptional pathway targeting, we have identified a new functional class of thalidomide analogs with distinct and selective antileukemic activity. These agents activate nuclear factor of activated T cells (NFAT) transcriptional pathways while simultaneously repressing nuclear factor-B (NF-B) via a rapid intracellular amplification of reactive oxygen species (ROS). The elevated ROS is associated with increased intracellular free calcium, rapid dissipation of the mitochondrial membrane potential, disrupted mitochondrial structure, and caspase-independent cell death. This cytotoxicity is highly selective for transformed lymphoid cells, is reversed by free radical scavengers, synergizes with the antileukemic activity of other redox-directed compounds, and preferentially targets cells in the S phase of the cell cycle. Live-cell imaging reveals a rapid drug-induced burst of ROS originating in the endoplasmic reticulum and associated mitochondria just prior to spreading throughout the cell. As members of a novel functional class of "redoxreactive" thalidomides, these compounds provide a new tool through which selective cellular properties of redox status and intracellular bioactivation can be leveraged by rational combinatorial therapeutic strategies and appropriate drug design to exploit cell-specific vulnerabilities for maximum drug efficacy. IntroductionThalidomide is a synthetic glutamic acid derivative originally marketed as a sedative and antiemetic in 1954. 1,2 However, in 1961 it was quickly withdrawn from distribution when its teratogenic properties were discovered. 1,2 Several years later the serendipitous finding that thalidomide could allay the symptoms of erythema nodosum led to its re-emergence as a treatment for various proinflammatory and autoimmune conditions. 3 Although many of the anti-inflammatory properties of thalidomide have been linked to its ability to repress tumor necrosis factor-␣ (TNF-␣) expression, 4 the mechanisms underlying most of its therapeutic effects, including its ability to costimulate T cells, 5 remained a mystery. In 1994, speculation that thalidomide teratogenicity is linked to the repression of angiogenesis 6 spawned a new wave of clinical investigations that expanded the use of thalidomide for the treatment of various malignancies, including multiple myeloma, melanoma, renal-cell carcinoma, and prostate cancer. 1,2 The therapeutic promise of thalidomide became a motivation to develop more effective derivatives with reduced toxicity. Several chemical classes of compounds were subsequently developed. One group, referred to as immunomodulatory drugs (IMiDs), 2 was identified because of its potential to promote T-cell costimulatory activity. A second group, referred to as the selective cytokine inhibitory drugs (SelCIDs), were found to be potent phosphodiesterase 4 (PDE4) inhibitors. 7 Both groups repress TNF-␣ expression. IMiDs are currently in phase 2 and phase 3 clinical trials for multiple myeloma, metastatic melanoma, and prosta...

The blueprint for cellular diversity and response to environmental change is encoded in the cis-acting regulatory sequences of most genes. Deciphering this 'cis-regulatory code' requires multivariate data sets that examine how these regions coordinate transcription in response to diverse environmental stimuli and therapeutic treatments. We describe a transcriptional approach that profiles the activation of multiple transcriptional targets against combinatorial arrays of therapeutic and signal transducing agents. Application of this approach demonstrates how cis-element composition and promoter context combine to influence transcription downstream of mitogeninduced signaling networks. Computational dissection of these transcriptional profiles in activated T cells uncovers a novel regulatory synergy between IGF-1 and CD28 costimulation that modulates NF-kB and AP1 pathways through signaling cascades sensitive to cyclosporin A and wortmannin. This approach provides a broader view of the hierarchical signal integration governing gene expression and will facilitate a practical design of combinatorial therapeutic strategies for exploiting critical control points in transcriptional regulation.

Burkholderia cenocepacia is a significant problem in individuals with cystic fibrosis and is a member of the B. cepacia complex of closely related antibiotic resistant bacteria. A salicylate-regulated antibiotic efflux operon has been identified in B. cenocepacia and one of its four genes, llpE, is without parallel in previously reported efflux operons. PCR amplification and sequencing of llpE from B. cepacia complex isolates demonstrated the highest prevalence in B. cenocepacia with a high degree of sequence conservation. While at least one non-synonymous mutation was identified between isolates from different genomovars, only synonymous differences were identified within the IIIA and IIIB sub-groups of B. cenocepacia. Structural modeling suggests that LlpE is a member of the alpha/beta hydrolase enzyme family. Identification of strong structural homology to hydrolases and a high degree of conservation in B. cenocepacia suggests an enzymatic function for LlpE, benefiting survival in the cystic fibrosis lung.