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mnC1 homozygote and WT worm
The Gronostajski Lab:
The Nuclear Factor I (NFI) family of transcription/replication proteins

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Brief Description:

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) family 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 have constructed and are annotating the NFIRegulome database which contains all genes for which there is published evidence for regulation by NFI 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.

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.  We're also determining the specific cell type in the lung in which Nfib is required for normal lung maturation.

Most recently, the NFI-X knockout mouse we've made (Nfix-) has an ENLARGED brain and abnormal cells that contain markers of neural stem cells within the normally empty ventricles.  We're characterizing these cells and how they relate to the increased brain size.  These animals also have defects in intestine morphogenesis and physiology that we are examining.

(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.  We've shown that worms lacking nfi-1 are viable, but have several interesting phenotypes including a shortened lifespan.  We've demonstrated the first cell-autonomous function of NFI by showing that expression of the protein specifically in pharyngeal muscle cells rescues the pharyngeal pumping defect and shortened lifespan of nfi-1 deficient animals.  We're also recently published the mapping the in vivo binding sites of NFI-1 in whole worms, the first whole genome analysis of in vivo NFI binding sites in any organism.

(4) We have recently created the NFIRegulome database, which contains all genes for which there is published evidence that NFI transcription factors regulate their expression.  This database is a work in progress with several dozens of genes being annotated with a few hundred to go.  We will soon be able to query this database for tissue- and cell-type specific genes regulated by known transcription factors that cooperate with NFI proteins.

Above is a picture of NFI expression in the developing mouse embryo. These in situs were produced through a collaboration with Dr. Gary Lyons. For a closer look click here!(800kB,beware)	NFI-C expression in P15 mouse molar.  Note expression in Odontoblasts and Preodontoblasts.  Picture is courtesy of ASM and appears as the cover of the April, 2003 Mol. Cell Biol.

A Nuclear Factor I binding Site on DNA For those structurally minded people, here's a molecular model of an NFI binding site (TTGGCNNNNNGCCAA is the consensus sequence on duplex DNA). The green balls represent nitrogens in methyl groups of thymidine residues in the major groove while the red balls represent nitrogens in guanosine residues in the major groove protected from methylation by NFI. We don't know what the protein looks like sitting on DNA, but we're working on it!

Expression of an nfi-1-lacZ transgene      Above are pictures of C. elegans embryos expressing an nfi-1-lacZ transgene.  Expression occurs only during embryogenesis and not in the adult worm.  Expression starts in a few cells and appears to spread throughout most of the embryo.  We are currently identifying the cell types in which nfi-1 is expressed.

Members of the Lab:

• Christine E. Campbell, Ph.D. Research Assistant Professor
  
• Jason Osinski, B.Sc.
  
• Yu-Chih (Jay) Hsu, M.Sc.
• Bryan Winograd, undergrad.
• Sean Boland, undergrad.
• Sanel Sadibasic, undergrad.

Gronostajski, R.M. Roles of the NFI/CTF gene family in transcription and development. Gene 249: 31-45, 2000.

Behrens, M., G. Venkatraman, R.M. Gronostajski, R.R. Reed and F.L. Margolis. NFI in the development of the olfactory neuroepithelium and the regulation of olfactory marker protein gene expression. Eur. J. Neurosci. 12:1372-84, 2000.

Baumeister, H., R.M. Gronostajski, G.E. Lyons and F.L. Margolis. Identification of NFI-binding sites and cloning of NFI-cDNAs suggest a regulatory role for NFI transcription factors in olfactory neuron gene expression. Mol. Brain. Res. 72: 65-79, 1999.

das Neves, L., C. Duchala, F. Godinho, M. Haxhiu, C. Colmenares, W. Macklin, C.E. Campbell, K. Butz and R.M. Gronostajski.  Disruption of the murine Nuclear Factor I-A gene (Nfia) results in perinatal lethality, hydrocephalus and agenesis of the corpus callosum. Proc. Natl. Acad. Sci. (USA) 96: 11946-51, 1999.

Fletcher, C.F., N.A. Jenkins, N.G. Copeland, A.Z. Chaudhry and R.M. Gronostajski.  Exon structure of the Nuclear Factor I DNA-binding domain from C. elegans to mammals.  Mammalian Genome 10: 390-396, 1999.

Leahy, P., D.R. Crawford, G. Grossman, R.M. Gronostajski and R.W. Hanson.  CREB binding protetin coordinates the function of multiple transcription factors including Nuclear Factor I to regulate phosphoenolpyruvate carboxykinage (GTP) gene transcription. J. Biol. Chem. 274: 8813-8822, 1999.

Chaudhry, A.Z., A. Vitullo and R.M. Gronostajski. Nuclear Factor I-mediated repression of the Mouse Mammary Tumor Virus promoter is abrogated by the coactivators p300/CBP and SRC-1. J. Biol. Chem. 274: 7072-7081, 1999.

Chaudhry, A.Z, A. Vitullo and R.M. Gronostajski. Nuclear Factor I (NFI) isoforms differentially activate simple versus complex NFI-responsive promoters. J. Biol. Chem. 273: 18538-18546, 1998.

Crawford, D., P. Leahy, C. Hu, A. Chaudhry, R. Gronostajski, G. Grossman, J. Woods, P. Hakimi, W. Roesler and R.W. Hanson. Nuclear Factor I regulates expression of the gene for phoshoenolpyruvate carboxykinase (GTP). J. Biol. Chem. 273: 13387-13390, 1998.

Bandyopadhyay, S., D.W. Starke, J.J. Mieyal and R.M. Gronostajski. Thioltransferase (Glutaredoxin) Reactivates the DNA-binding Activity of Oxidation-inactivated Nuclear Factor I. J. Biol. Chem. 273: 392-397, 1998.

Chaudhry, A.Z., G.E. Lyons and R.M. Gronostajski.  Expression patterns of the four Nuclear Factor I genes during mouse embryogenesis indicate a potential role in development. Developmental Dynamics 208: 313-325, 1997.

Golden Oldies:

Gronostajski, R. M., K. Nagata and J. Hurwitz. Isolation of human DNA sequences that bind to nuclear factor I, a host protein involved in adenovirus DNA replication. Proc. Natl. Acad. Sci. (USA) 81:4013-7, 1984.

Hi, I'm Rich Gronostajski   (requires audio)   The State University of New York at Buffalo Dept. of Biochemistry, Developmental Genomics Group New York State Center of Excellence in Bioinformatics and Life Sciences 701 Ellicott St. Buffalo, New York 14203  Telephone : (716) 829-3471 e-mail: rgron@buffalo.edu

This page last updated Oct. 10th, 2009.

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