The mammalian genome employs some 3000 transcription factors to achieve specific patterns of gene expression. Because these patterns are critical for successful development and signal response, aberrations in transcription factor function frequently underlie human disorders such as cancer and immune dysfunction. Our laboratory employs biochemical, genetic and genomic approaches to determine transcription factor function in tumorigenesis, lymphocyte development/function, and to elucidate specific gene regulatory circuits in normal and diseased cells. Our efforts focus on gene regulation both from the perspective of mechanisms of action and biological effects of specific transcription factors, as well as the means of coordinate and reciprocal regulation of specific groups of genes.

1. We are investigating the properties and biological functions of an unusual class of homeodomain-related transcription factors: Oct-1, Oct-2, and Oct-4. The prototypic member of this class is Oct-1, which is widely expressed in adult cells. Oct-2 expression is largely confined to B lymphocytes and the central nervous system. Oct-4 expression is confined to early embryonic development and is critical for the maintenance of embryonic stem cell identity. Tying several lines of evidence together, we have found that octamer-binding proteins act as signal integrators—coupling cellular metabolic, oxidative and genome integrity inputs to transcriptional output. For example, Oct-1 deficient fibroblasts and B cells are hypersensitive to DNA damaging and oxidative stress agents. DNA-PK phosphorylation sites in the Oct-1 N-terminus are critical for the ability of Oct-1 to correct sensitivity to DNA damaging agents. Gene chip analyses reveal that Oct-1 deficient cells differentially express genes involved in DNA damage and oxidative stress response. Oct-1 deficient fibroblasts and B cells also have elevated levels of reactive oxygen species (ROS). A conserved but non-essential cysteine residue within the DNA binding domain of Oct-1 modulates ROS levels. Experiments are ongoing to determine whether this cysteine is post-translationally modified. We have also recently determined that loss of Oct-1 induces a major metabolic shift away from glycolytic function and towards oxidative utilization of amino acids as a carbon source. These changes are exactly opposite those frequently encountered in tumor cells. We are identifying stress-dependent Oct-1 co-factors and post-translational modifications that mediate these effects. Consistent with these phenotypes, we have found that Oct-1 deficiency strongly alters tumor onset and spectrum in mouse models and transformation efficiency in vitro. Oct-1 expression is also altered in a number of human tumors. The DNA sequence which interacts with these factors is present many times in the major Immunoglobulin (Ig) loci, as well as in viruses such as human immunodeficiency virus-1 (HIV-1), and a number of cell-cycle, inflammatory, cytokine, and leukocyte-specific genes. In the case of Oct-4 where relatively few targets are known, we have identified new interacting sequences within enhancer and promoter regions of putative target genes using a high-throughput genomic oligonucleotide library approach. The ultimate goals of this project are to understand the mechanisms transcriptional regulation by octamer-binding proteins and their biological consequences.

2. We are studying the signaling networks and transcription factors that regulate gene expression in B cells. We are interested in a group of genes including the immunoglobulins (Ig), which encode the primary effector molecules of B cells, as well as genes encoding the surrogate Ig molecules, co-receptors, and the molecular machinery that shapes Ig structure. These genes follow a program of coordinate and reciprocal regulation during B cell development, and later during B cell activation and the generation of an immune response, yet the mechanism is unknown. Using statistical and molecular approaches, we have found that the TFII-I family of transcriptional regulatory proteins can interact with Ig regulatory sequences and can modulate Ig expression. TFII-I has been previously identified in a signaling cascade emanating from the B cell receptor (surface Ig). This result suggests the presence of a regulatory feedback loop. We are investigating whether TFII-I also controls the ancillary genes that shape B cell development and activity, and whether it interacts with Oct proteins. The ultimate goal of this work is to determine the mechanisms by which these genes are regulated in B cells.