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Our work focuses on elucidation of the biological role and mechanism of action of the TOX family of proteins. TOX is the founding member of a small subfamily of highly evolutionarily conserved HMG-box proteins. The HMG-box protein superfamily is defined by a ~80 amino acid DNA-binding domain, and individual members function in regulation of gene expression, chromatin remodeling, genomic stability, DNA repair, and other DNA-dependent cellular processes. The sequence of TOX places it clearly in the class of DNA sequence-independent but structure-dependent HMG-box factors. However, TOX has a single HMG-box domain and a developmentally regulated and cell-type specific pattern of expression that makes it stand out among this subfamily of chromatin-interacting proteins.
Crystal structure of the TOX DNA-binding domain, in collaboration with Ramachandran Murali, PhD.
Nuclear Factor TOX, A Key Regulator of Immune System Development and Function
Mature T lymphocytes develop, in a complex multistep process, from bone marrow-derived progenitors that migrate to the thymus. The end result is production of T cells that are largely self-tolerant (i.e. do not cause autoimmunity) but collectively, have the ability to mount an immune response against a wide range of pathogens. Moreover, this thymic “education” is a lineage diversification process, resulting in development of CD4 helper/inducer T cells, CD8 cytolytic T cells, T regulatory cells (Treg) and natural killer T cells (NKT) from common precursors. Our data has demonstrated the importance of TOX in regulating gene expression during this process, as well as other aspects of development of both the adaptive and innate immune systems. Mice that lack TOX have profound deficiencies in development of CD4-lineage T cells (including Treg cells) and NKT cells in the thymus, a block in NK cell development in the bone marrow, and a lack of fetal lymphoid tissue inducer cells. The latter deficit causes failure of lymph node organogenesis in these animals. We are continuing to work to understand the molecular mechanism of action of the protein in these various contexts.
TOX-deficient mice lack lymph nodes.
A reporter mouse allows visualization of CD4 T cells (green) that express TOX (red) during an immune response.
TOX also appears to play an important role during immune responses. We have created “knock-in” fluorescent reporter mice that allow us to track expression of TOX, during the development of the various cell types indicated above, in mature T cells, as well as other non-immune cell types. This ongoing work has led to identification of the protein in a subset of T lymphocytes that regulates antibody production by B cells. Using conditional knockout mice, we are working to define the specific molecular role of TOX in this, and other, T lymphocyte subsets.
The HMG-box protein domain, including that in TOX, folds into a concave L-shaped structure that binds the minor groove of DNA. Our lab, in collaboration with Murali’s Laboratory, has recently determined the crystal structure of the DNA-binding domain of TOX. Modeling studies have suggested that this protein domain may undergo conformational change upon interaction with DNA, opening an approach for development of small molecule inhibitors or activators of the TOX family of proteins.
Since TOX is also expressed in the liver (and the brain), we have a collaborative study that point to a role for the protein as an important regulator of liver metabolism.
TOX3 in Breast Cancer
Some breast tumors highly express the TOX3 nuclear protein.
There are four TOX protein family members, defined by a near identical DNA-binding domain and a related N-terminal domain with transactivation ability. Genome-wide association studies first demonstrated that variations in a sequence upstream of the TOX3 locus were associated within creased breast cancer risk. This has recently been attributed to an increased binding affinity of the FOXA1 transcriptional regulator to an enhancer region that controls TOX3 expression. We produced a rabbit monoclonal anti-TOX3 antibody that allowed us to detect high-level expression of TOX3 in a subset of breast tumors, as well as a subset of normal ER+ mammary epithelial cells. The identity of the latter subpopulation is under investigation, as are the signals that may control TOX3 expression. We also created a conditional transgenic strain of mouse to study the consequences of over expression of TOX3in vivo. Expression of the protein in a breast cancer cell line indicates that TOX3 is primarily an activator of transcription, and influences expression of many genes implicated in cell cycle or tumor migration. Our ongoing work is aimed at revealing the biological role of TOX3 as a regulator of gene expression in breast cancer cells, and whether, similar to TOX in the immune system, TOX3 may regulate normal mammary epithelial cell development and/or function. In this context, small molecules that can modulate TOX3 activity may prove useful both as probes and as a foundation for development of future therapeutics.