Gronostajski Lab Homepage

The Former Gronostajski Lab:

The Nuclear Factor I (NFI) family of transcription/replication proteins

I have now retired and am not keeping this page up but leave it around for anyone who is interested. Thanks to all of my former lab members for their great work.

Role of NFI proteins in mammalian and nematode development.

Mechanisms of transcriptional modulation by NFI proteins.

Structure and Function of NFI DNA-binding domain.

Link to NFI expression vector page.

Quicktime movie of worm development

Requires Quicktime: 4-cell to adult (size 2Mb)

In vivo target gene selectivity of T-box transcription factors

New! Link to NFI-Regulome Database.

Real-time PCR primers for mouse NFI genes.

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 NFI-Regulome 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.

(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. 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 cerebral ventricles. We're characterizing these cells and how they relate to the increased brain size. We've also shown that NFI-X functions in both the switch between quiescence and activation of adult neural stem cells and in the decision made by neural stem cells to proceed down the neural, astrocyte and oligodendrocyte lineages. Since we've also shown that NFI-A and NFI-B affect lineage determination we are examining the interaction of these 3 genes in neural stem cell homeostasis.

(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 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 created the NFI-Regulome 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.

Above are images of Wild Type (A-C) and nfi-1 null C. elegans (D-F). Single young adults were spotted in the center of fresh plates and left for 10 min. (A, D) Photographs of N2 worms and nfi-1 mutants; (B, E) Track patterns of N2 worms and nfi-1 mutants; (C, F) Track patterns of N2 worms and nfi-1 mutants with higher magnification. Note less regular tracks in nfi-1 mutant vs. N2 worms.

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:

Selected Lab References: (Click on those with links for PDF file download)

Grabowska, M. M., S. M. Kelly, A. L. Reese, J. M. Cates, T. C. Case, J. Zhang, D. J. DeGraff, D. W. Strand, N. L. Miller, P. E. Clark, S. W. Hayward, R. M. Gronostajski, P. D. Anderson, and R. J. Matusik. Nfib Regulates Transcriptional Networks That Control the Development of Prostatic Hyperplasia. Endocrinol. 157 (2016) 1094-109. PMID:26677878 PMC:PMC4769366

Ding, B., J. W. Cave, P. R. Dobner, D. Mullikin-Kilpatrick, M. Bartzokis, H. Zhu, C. W. Chow, R. M. Gronostajski, and D. L. Kilpatrick. Reciprocal autoregulation by NFI occupancy and ETV1 promotes the developmental expression of dendrite-synapse genes in cerebellar granule neurons. Mol. Biol. Cell 27 (2016) 1488-99. PMID:26941328 PMC:PMC4850036

Zhou, B., J. M. Osinski, J. L. Mateo, B. Martynoga, F. J. Sim, C. E. Campbell, F. Guillemot, M. Piper, and R. M. Gronostajski. Loss of NFIX Transcription Factor Biases Postnatal Neural Stem/Progenitor Cells Toward Oligodendrogenesis. Stem Cells Dev 24 (2015) 2114-26. PMID:26083238

Vidovic, D., L. Harris, T. J. Harvey, Y. H. Evelyn Heng, A. G. Smith, J. Osinski, J. Hughes, P. Thomas, R. M. Gronostajski, T. L. Bailey, and M. Piper. Expansion of the lateral ventricles and ependymal deficits underlie the hydrocephalus evident in mice lacking the transcription factor NFIX. Brain Res. 1616 (2015) 71-87. PMID:25960350

O'Connor, C., J. Campos, J. M. Osinski, R. M. Gronostajski, A. M. Michie, and K. Keeshan. Nfix expression critically modulates early B lymphopoiesis and myelopoiesis. PLoS One 10 (2015) e0120102. PMID:25780920 PMC:4363787

Mellas, R. E., H. Kim, J. Osinski, S. Sadibasic, R. M. Gronostajski, M. Cho, and O. J. Baker. NFIB Regulates Embryonic Development of Submandibular Glands. J. Dent. Res. 94 (2015) 312-9. PMID:25403566

Heng, Y. H., B. Zhou, L. Harris, T. Harvey, A. Smith, E. Horne, B. Martynoga, J. Andersen, A. Achimastou, K. Cato, L. J. Richards, R. M. Gronostajski, G. S. Yeo, F. Guillemot, T. L. Bailey, and M. Piper. NFIX Regulates Proliferation and Migration Within the Murine SVZ Neurogenic Niche. Cereb. Cortex 25 (2015) 3758-78. PMID:25331604 PMC:PMC4585514

Harris, L., L. A. Genovesi, R. M. Gronostajski, B. J. Wainwright, and M. Piper. Nuclear factor one transcription factors: Divergent functions in developmental versus adult stem cell populations. Dev Dyn 244 (2015) 227-38. PMID:25156673

Grabowska, M. M., S. M. Kelly, A. L. Reese, J. M. Cates, T. C. Case, J. Zhang, D. J. DeGraff, D. W. Strand, N. L. Miller, P. E. Clark, S. W. Hayward, R. M. Gronostajski, P. D. Anderson, and R. J. Matusik. Nfib regulates transcriptional networks that control the development of prostatic hyperplasia. Endocrinol. (2015) en20151312. PMID:26677878

Robinson, G. W., K. Kang, K. H. Yoo, Y. Tang, B. M. Zhu, D. Yamaji, V. Colditz, S. J. Jang, R. M. Gronostajski, and L. Hennighausen. Coregulation of Genetic Programs by the Transcription Factors NFIB and STAT5. Mol. Endocrinol. 28 (2014) 758-67. PMID:24678731 PMC:4004779

Piper, M., G. Barry, T. J. Harvey, R. McLeay, A. G. Smith, L. Harris, S. Mason, B. W. Stringer, B. W. Day, N. R. Wray, R. M. Gronostajski, T. L. Bailey, A. W. Boyd, and L. J. Richards. NFIB-Mediated Repression of the Epigenetic Factor Ezh2 Regulates Cortical Development. J. Neurosci. 34 (2014) 2921-30. PMID:24553933

Lee, D. S., H. W. Choung, H. J. Kim, R. M. Gronostajski, Y. I. Yang, H. M. Ryoo, Z. H. Lee, H. H. Kim, E. S. Cho, and J. C. Park. NFI-C Regulates Osteoblast Differentiation via Control of Osterix Expression. Stem Cells 32 (2014) 2467-79. PMID:24801901

Lajoie, M., Y. C. Hsu, R. M. Gronostajski, and T. L. Bailey. An overlapping set of genes is regulated by both NFIB and the glucocorticoid receptor during lung maturation. BMC genomics 15 (2014) 231. PMID:24661679 PMC:4023408

Heng, Y. H., R. C. McLeay, T. J. Harvey, A. G. Smith, G. Barry, K. Cato, C. Plachez, E. Little, S. Mason, C. Dixon, R. M. Gronostajski, T. L. Bailey, L. J. Richards, and M. Piper. NFIX regulates neural progenitor cell differentiation during hippocampal morphogenesis. Cereb. Cortex 24 (2014) 261-79. PMID:23042739 PMC:PMC3862270

Grabowska, M. M., A. D. Elliott, D. J. DeGraff, P. D. Anderson, G. Anumanthan, H. Yamashita, Q. Sun, D. B. Friedman, D. L. Hachey, X. Yu, J. H. Sheehan, J. M. Ahn, G. V. Raj, D. W. Piston, R. M. Gronostajski, and R. J. Matusik. NFI transcription factors interact with FOXA1 to regulate prostate-specific gene expression. Mol. Endocrinol. 28 (2014) 949-64. PMID:24801505 PMC:4042066

Martynoga, B., J. L. Mateo, B. Zhou, J. Andersen, A. Achimastou, N. Urban, D. van den Berg, D. Georgopoulou, S. Hadjur, J. Wittbrodt, L. Ettwiller, M. Piper, R. M. Gronostajski, and F. Guillemot. Epigenomic enhancer annotation reveals a key role for NFIX in neural stem cell quiescence. Genes Dev. 27 (2013) 1769-86. PMID:23964093 PMC:3759694

Harris, L., C. Dixon, K. Cato, Y. H. Heng, N. D. Kurniawan, J. F. Ullmann, A. L. Janke, R. M. Gronostajski, L. J. Richards, T. H. Burne, and M. Piper. Heterozygosity for nuclear factor one x affects hippocampal-dependent behaviour in mice. PLoS One 8 (2013) e65478. PMID:23776487, PMC:3679126

Dixon, C., T. J. Harvey, A. G. Smith, R. M. Gronostajski, T. L. Bailey, and M. Piper. Nuclear Factor One X Regulates Bobby Sox During Development of the Mouse Forebrain. Cell Mol. Neurobiol, (2013) PMID:23852417,

Ding, B., W. Wang, T. Selvakumar, H. S. Xi, H. Zhu, C. W. Chow, J. D. Horton, R. M. Gronostajski, and D. L. Kilpatrick. Temporal regulation of nuclear factor one occupancy by calcineurin/NFAT governs a voltage-sensitive developmental switch in late maturing neurons. J. Neurosci. 33 (2013) 2860-72. PMID:23407945,

Chang, C. Y., H. A. Pasolli, E. G. Giannopoulou, G. Guasch, R. M. Gronostajski, O. Elemento, and E. Fuchs. NFIB is a governor of epithelial-melanocyte stem cell behaviour in a shared niche. Nature 495 (2013) 98-102. PMID:23389444, PMC:3635831

Yusuf, D., S. L. Butland, M. I. Swanson, E. Bolotin, A. Ticoll, W. A. Cheung, X. Y. Zhang, C. T. Dickman, D. L. Fulton, J. S. Lim, J. M. Schnabl, O. H. Ramos, M. Vasseur-Cognet, C. N. de Leeuw, E. M. Simpson, G. U. Ryffel, E. W. Lam, R. Kist, M. S. Wilson, R. Marco-Ferreres, J. J. Brosens, L. L. Beccari, P. Bovolenta, B. A. Benayoun, L. J. Monteiro, H. D. Schwenen, L. Grontved, E. Wederell, S. Mandrup, R. A. Veitia, H. Chakravarthy, P. A. Hoodless, M. M. Mancarelli, B. E. Torbett, A. H. Banham, S. P. Reddy, R. L. Cullum, M. Liedtke, M. P. Tschan, M. Vaz, A. Rizzino, M. Zannini, S. Frietze, P. J. Farnham, A. Eijkelenboom, P. J. Brown, D. Laperriere, D. Leprince, T. de Cristofaro, K. L. Prince, M. Putker, L. del Peso, G. Camenisch, R. H. Wenger, M. Mikula, M. Rozendaal, S. Mader, J. Ostrowski, S. J. Rhodes, C. Van Rechem, G. Boulay, S. W. Olechnowicz, M. B. Breslin, M. S. Lan, K. K. Nanan, M. Wegner, J. Hou, R. D. Mullen, S. C. Colvin, P. J. Noy, C. F. Webb, M. E. Witek, S. Ferrell, J. M. Daniel, J. Park, S. A. Waldman, D. J. Peet, M. Taggart, P. S. Jayaraman, J. J. Karrich, B. Blom, F. Vesuna, H. O'Geen, Y. Sun, R. M. Gronostajski, M. W. Woodcroft, M. R. Hough, E. Chen, G. N. Europe-Finner, M. Karolczak-Bayatti, J. Bailey, O. Hankinson, V. Raman, D. P. LeBrun, S. Biswal, C. J. Harvey, J. P. DeBruyne, J. B. Hogenesch, R. F. Hevner, C. Heligon, et al. The transcription factor encyclopedia. Genome Biol. 13 (2012) R24. PMID:22458515,

Plachez, C., K. Cato, R. C. McLeay, Y. H. Heng, T. L. Bailey, R. M. Gronostajski, L. J. Richards, A. C. Puche, and M. Piper. Expression of nuclear factor one A and -B in the olfactory bulb. J. Comp. Neurol. 520 (2012) 3135-49. PMID:22886731,

Kilpatrick, D. L., W. Wang, R. Gronostajski, and E. D. Litwack. Nuclear factor I and cerebellar granule neuron development: an intrinsic-extrinsic interplay. Cerebellum 11 (2012) 41-9. PMID:22548229, PMC:3175246

Kang, P., H. K. Lee, S. M. Glasgow, M. Finley, T. Donti, Z. B. Gaber, B. H. Graham, A. E. Foster, B. G. Novitch, R. M. Gronostajski, and B. Deneen. Sox9 and NFIA coordinate a transcriptional regulatory cascade during the initiation of gliogenesis. Neuron 74 (2012) 79-94. PMID:22500632,

Heng, Y. H., R. C. McLeay, T. J. Harvey, A. G. Smith, G. Barry, K. Cato, C. Plachez, E. Little, S. Mason, C. Dixon, R. M. Gronostajski, T. L. Bailey, L. J. Richards, and M. Piper. NFIX Regulates Neural Progenitor Cell Differentiation During Hippocampal Morphogenesis. Cereb. Cortex (2012) PMID:23042739,

Gronostajski, R. M., J. Guaneri, D. H. Lee, and S. M. Gallo. The NFI-Regulome Database: A tool for annotation and analysis of control regions of genes regulated by Nuclear Factor I transcription factors. Journal of clinical bioinformatics 1 (2011) 4. http://www.ncbi.nlm.nih.gov/pubmed/21884625

Piper, M., L. Harris, G. Barry, Y. H. Heng, C. Plachez, R. M. Gronostajski, and L. J. Richards. Nuclear factor one X regulates the development of multiple cellular populations in the postnatal cerebellum. J. Comp. Neurol. 519 (2011) 3532-48. http://www.ncbi.nlm.nih.gov/pubmed/21800304

Subramanian, L., A. Sarkar, A. S. Shetty, B. Muralidharan, H. Padmanabhan, M. Piper, E. S. Monuki, I. Bach, R. M. Gronostajski, L. J. Richards, and S. Tole. Transcription factor Lhx2 is necessary and sufficient to suppress astrogliogenesis and promote neurogenesis in the developing hippocampus. Proc. Natl. Acad. Sci. (USA) 108 (2011) E265-74. http://www.ncbi.nlm.nih.gov/pubmed/21690374

Meng, F., T. M. Suchyna, E. Lazakovitch, R. M. Gronostajski, and F. Sachs. Real Time FRET Based Detection of Mechanical Stress in Cytoskeletal and Extracellular Matrix Proteins. Cell Mol Bioeng 4 (2011) 148-59 . http://www.ncbi.nlm.nih.gov/pubmed/21625401

Muthusamy, N., H. C. Chen, G. Rajgolikar, K. G. Butz, F. W. Frissora, and R. M. Gronostajski. Recombination activation gene-2-deficient blastocyst complementation analysis reveals an essential role for nuclear factor I-A transcription factor in T-cell activation. Int. Immunol. 23 (2011) 385-90. http://www.ncbi.nlm.nih.gov/pubmed/21602176

Hsu, Y. C., J. Osinski, C. E. Campbell, E. D. Litwack, D. Wang, S. Liu, C. J. Bachurski, and R. M. Gronostajski. Mesenchymal nuclear factor I B regulates cell proliferation and epithelial differentiation during lung maturation. Dev. Biol. 354 (2011) 242-52. http://www.ncbi.nlm.nih.gov/pubmed/21513708

Piper, M., G. Barry, J. Hawkins, S. Mason, C. Lindwall, E. Little, A. Sarkar, A. G. Smith, R. X. Moldrich, G. M. Boyle, S. Tole, R. M. Gronostajski, T. L. Bailey, and L. J. Richards. NFIA controls telencephalic progenitor cell differentiation through repression of the Notch effector Hes1. J. Neurosci. 30 (2010) 9127-39. http://www.ncbi.nlm.nih.gov/pubmed/20610746

Zheng, S., S. M. Eacker, S. J. Hong, R. M. Gronostajski, T. M. Dawson, and V. L. Dawson. NMDA-induced neuronal survival is mediated through nuclear factor I-A in mice. J. Clin. Invest. 120 (2010) 2446-56.

Wang, W., J. E. Crandall, E. D. Litwack, R. M. Gronostajski, and D. L. Kilpatrick. Targets of the nuclear factor I regulon involved in early and late development of postmitotic cerebellar granule neurons. J. Neurosci. Res. 88 (2010) 258-65.