Molecular Recognition in Biological Systems and Bioinformatics

Transcription regulation; developmental biology; mouse knockouts; genetics; biochemistry.

The goal of our laboratory is to gain a better understanding of how proteins that interact with DNA regulate RNA transcription, DNA replication and metazoan development. Our focus is on the structure and function of the Nuclear Factor I (NFI) and T-box families of site-specific DNA binding proteins. In vertebrates, NFI family members function in both the replication of viral DNA and the transcription of viral and cellular genes. We are currently analyzing the role of the NFI gene family in both vertebrate and C. elegans development. Studies on mouse NFI genes can be divided into two major themes: (1) biochemical analysis of NFI protein structure and function and (2) molecular genetic studies on NFI's role in cell growth, differentiation and development. We are also assessing the function of the single C. elegans NFI gene (nfi-1, (3)) and the function of T-box transcriptional activation domains in the in vivo target gene selectivity of T-box transcription factors (4).

(1) The DNA-binding domain of NFI differs from those found in other well characterized DNA-binding proteins. Four major questions being addressed in the laboratory are: What is the structure of the NFI DNA-binding domain? How does NFI recognize and interact with DNA? Does NFI change the structure of DNA when it binds? What proteins interact with NFI to stimulate RNA transcription and/or DNA replication? We are asking these questions both in our laboratory and in collaboration with a number of talented investigators.

We have shown that the NFI-C protein represses the glucocorticoid-dependent expression of the MMTV promoter. This repression can be overcome by overexpression of the co-activator proteins CBP, p300 or SRC-1, suggesting a role of these co-activators in MMTV expression. Surprisingly, NFI-C doesn't repress progesterone stimulation of MMTV. We are currently working out the biochemical mechanism for this repression by NFI-C and the roles of co-activators, histone acetylase activity and chromatin remodeling activity in the process.

(2) We've been generating targeted mutations in mouse NFI genes to determine the roles of the different NFI family members in development.

The NFI-A deficient mouse we generated (Nfia-) has major neurological defects including agenesis of the corpus callosum, hydrocephalus and defects, in the generation of specific midline glial cell populations. We're now studying the biochemical pathways leading to these developmental defects with the goal of determining how loss of a single transcription factor results in major neuroanatomical changes. We're focusing on whether loss of NFI-A causes changes in: 1) cell proliferation or death, 2) cell migration or differentiation, 3) axonal outgrowth, 4) axonal pathfinding, 5) glial cell differentiation and 6) patterns of neuronal or glial cell gene expression.

The NFI-C deficient mouse we generated (Nfic-) has novel defects in tooth development. Although NFI-C was one of the first transcription factors cloned and is expressed in many embryonic and adult tissues, the only defect seen in mice lacking Nfic is that the molar roots fail to develop and the incisors are dysmorphic and poorly developed. This defect is severe enough that most mutant mice die within a few months if fed a standard lab chow, but have a normal lifespan and are fertile if fed a soft dough diet. Since this is the first mutation that affects primarily tooth root formation, it should allow us to determine the molecular pathways needed for this important postnatal developmental process.

The NFI-B deficient mouse we recently made (Nfib-) has both major neuroanatomical defects and defects in lung maturation. The brain defects are more extensive then seen in the Nfia- mouse above and include agenesis of the corpus callosum, loss of the basilar pons, and hippocampal defects. The lung defects are of interest since lung immaturity is a major problem in premature newborns. We are determining the biochemical and genetic pathways by which Nfib regulates lung maturation.

(3) While all vertebrates examined contain 4 highly conserved NFI genes (NFI-A, -B, -C and -X), the nematode Caenorhabditis elegans has only a single NFI gene (nfi-1). Unlike the case in vertebrates, where all 4 NFI genes are expressed in many tissues during both embryogenesis and throughout adult life, the C. elegans nfi-1 gene is expressed primarily during embryogenesis. In collaboration with Yuji Kohara we've identified where nfi-1 mRNA is expressed in C. elegans and are assessing the phenotype of worms deficient in the nfi-1 gene product. By comparing the function of NFI in worms and mice, we are asking how NFI-dependent developmental pathways have been conserved through over 500M years of evolution.

(4) Recently we've begun to examine the functions of the T-box family of transcription factors in the regulation of gene expression. With this family we are asking how genes with similar DNA-binding domains can regulate the activation of different endogenous target genes in vivo. We now have data indicating that the region of T-box genes often referred to as the "transactivation domain" can influence both the degree of transcriptional activation seen on a transfected reporter gene and also, surprisingly, the selectivity of endogenous in vivo targets of the T-box family member, TBX2. These data suggest that the in vivo target gene selectivity of a transcription factor is determined by both the specificity of its DNA-binding domain, and also by specific interactions of its transactivation/repression domain with other components of the transcriptional machinery. This added level of complexity of transcriptional regulation of endogenous genes in vivo has often been suggested, and we now have strong evidence for it in the T-box gene family.

• Mouse
• Human
• C. elegans
• Worms

• NFI
• t-bet
• Tbx-1
• Nuclear Factor I

Lung maturation, callosal agenesis, kidney formation, brain development.

• Gene targeting
• Microarrays
• Transient and stable transfection
• QPCR

DAPI

• HeLa
• 293
• Embryonal stem cells
• Neural stem cells
• ES cells

Congenital birth defects, Crohn's disease, RDS, COPD, spina bifida, callosal agenesis, hydrocephalus, neurodegeneration.

Lung, brain, kidney, intestine.

http://elegans.swmed.edu/Worm_labs/Gronostajski/