Richard M. Gronostajski

Roles of the NFI/CTF gene family in transcription and development

The Nuclear Factor I (NFI) family of site-specific DNA-binding proteins (also known as CTF or CAAT box transcription factor) functions both in viral DNA replication and in the regulation of gene expression. The classes of genes whose expression is modulated by NFI include those that are ubiquitously expressed, as well as those that are hormonally, nutritionally, and developmentally regulated. The NFI family is composed of four members in vertebrates (NFI-A, NFI-B, NFI-C and NFI-X), and the four NFI genes are expressed in unique, but overlapping, patterns during mouse embryogenesis and in the adult. Transcripts of each NFI gene are differentially spliced, yielding as many as nine distinct proteins from a single gene. Products of the four NFI genes differ in their abilities to either activate or repress transcription, likely through fundamentally different mechanisms. Here, we will review the properties of the NFI genes and proteins and their known functions in gene expression and development.

The Transcription Factor NFIA Controls the Onset of Gliogenesis in the Developing Spinal Cord

The mechanisms controlling the transition from neurogenesis to gliogenesis in the vertebrate CNS are incompletely understood. We identified a family of transcription factors, called NFI genes, which are induced throughout the spinal cord ventricular zone (VZ) concomitantly with the induction of GLAST, an early marker of gliogenesis. NFIA is both necessary and sufficient for GLAST induction in the VZ. Unexpectedly, NFIA is also essential for the continued inhibition of neurogenesis in VZ progenitors. This function is mediated by the requirement of NFIA for the expression of HES5, a Notch effector. However, Notch effectors are unable to promote glial-fate specification in the absence of NFIA. Thus, NFIA links the abrogation of neurogenesis to a generic program of gliogenesis, in both astrocyte and oligodendrocyte VZ progenitors. At later stages, NFIA promotes migration and differentiation of astrocyte precursors, a function that is antagonized in oligodendrocyte precursors by Olig2.

The Transcription Factor Gene Nfib Is Essential for both Lung Maturation and Brain Development

ABSTRACT The phylogenetically conserved nuclear factor I (NFI) gene family encodes site-specific transcription factors essential for the development of a number of organ systems. We showed previously that Nfia-deficient mice exhibit agenesis of the corpus callosum and other forebrain defects, whereas Nfic-deficient mice have agenesis of molar tooth roots and severe incisor defects. Here we show that Nfib-deficient mice possess unique defects in lung maturation and exhibit callosal agenesis and forebrain defects that are similar to, but more severe than, those seen in Nfia-deficient animals. In addition, loss of Nfib results in defects in basilar pons formation and hippocampus development that are not seen in Nfia-deficient mice. Heterozygous Nfib-deficient animals also exhibit callosal agenesis and delayed lung maturation, indicating haploinsufficiency at the Nfib locus. The similarity in brain defects in Nfia- and Nfib-deficient animals suggests that these two genes may cooperate in late fetal forebrain development, while Nfib is essential for late fetal lung maturation and development of the pons.

Abnormal Development of Forebrain Midline Glia and Commissural Projections in Nfia Knock-Out Mice

Nuclear factor I (NFI) genes are expressed in multiple organs throughout development (Chaudhry et al., 1997; for review, seeGronostajski, 2000). All four NFI genes are expressed in embryonic mouse brain, with Nfia, Nfib, andNfix being expressed highly in developing cortex (Chaudhry et al., 1997). Disruption of the Nfia gene causes agenesis of the corpus callosum (ACC), hydrocephalus, and reduced GFAP expression (das Neves et al., 1999). Three midline structures, the glial wedge, glia within the indusium griseum, and the glial sling are involved in development of the corpus callosum (Silver et al., 1982; Silver and Ogawa, 1983; Shu and Richards, 2001). BecauseNfia−/−mice show glial abnormalities and ACC, we asked whether defects in midline glial structures occur inNfia− / − mice. NFI-A protein is expressed in all three midline populations. InNfia− / − , mice sling cells are generated but migrate abnormally into the septum and do not form a sling. Glia within the indusium griseum and the glial wedge are greatly reduced or absent and consequently Slit2 expression is also reduced. Although callosal axons approach the midline, they fail to cross and extend aberrantly into the septum. The hippocampal commissure is absent or reduced, whereas the ipsilaterally projecting perforating axons (Hankin and Silver, 1988; Shu et al., 2001) appear relatively normal. These results support an essential role for midline glia in callosum development and a role for Nfia in the formation of midline glial structures.

Expression Patterns of the Four Nuclear Factor I Genes During Mouse Embryogenesis Indicate a Potential Role in Development

The nuclear factor I (NFI) family of site‐specific DNA‐binding proteins is required for both the cell‐type specific transcription of many viral and cellular genes and for the replication of adenovirus DNA. Although binding sites for NFI proteins within the promoters of several tissue‐specific genes have been shown to be essential for their expression, it is unclear which NFI gene products function in specific tissues during development. We have isolated cDNAs from all four murine NFI genes (gene designations Nfia, Nfib, Nfic, and Nfix), assessed the embryonic and postnatal expression patterns of the NFI genes, and determined the ability of specific NFI proteins to activate transcription from the NFI‐dependent mouse mammary tumor virus (MMTV) promoter. In adult mice, all four NFI genes are most highly expressed in lung, liver, heart, and other tissues but only weakly expressed in spleen and testis. The embryonic expression patterns of the NFI genes is complex, with NFI‐A transcripts appearing earliest—within 9 days postcoitum in the heart and developing brain. The four genes exhibit unique but overlapping patterns of expression during embryonic development, with high level expression of NFI‐A, NFI‐B, and NFI‐X transcripts in neocortex and extensive expression of the four genes in muscle, connective tissue, liver, and other organ systems. The four NFI gene products studied differ in their ability to activate expression of the NFI‐dependent MMTV promoter, with the NFI‐B protein being most active and the NFI‐A protein being least active. These data are discussed in the context of the developmental expression patterns of known NFI‐responsive genes. The differential activation of an NFI‐dependent promoter, together with the expression patterns observed for the four genes, indicate that the NFI proteins may play an important role in regulating tissue‐specific gene expression during mammalian embryogenesis. Dev. Dyn. 208:313–325, 1997.

Essential Role for NFI-C/CTF Transcription-Replication Factor in Tooth Root Development

ABSTRACT The mammalian tooth forms by a series of reciprocal epithelial-mesenchymal interactions. Although several signaling pathways and transcription factors have been implicated in regulating molar crown development, relatively little is known about the regulation of root development. Four genes encoding nuclear factor I (NFI) transcription-replication proteins are present in the mouse genome: Nfia, Nfib, Nfic, and Nfix. In order to elucidate its physiological role(s), we disrupted the Nfic gene in mice. Heterozygous animals appear normal, whereas Nfic−/− mice have unique tooth pathologies: molars lacking roots, thin and brittle mandibular incisors, and weakened abnormal maxillary incisors. Feeding in Nfic−/− mice is impaired, resulting in severe runting and premature death of mice reared on standard laboratory chow. However, a soft-dough diet mitigates the feeding impairment and maintains viability. Although Nfic is expressed in many organ systems, including the developing tooth, the tooth root development defects were the prominent phenotype. Indeed, molar crown development is normal, and well-nourished Nfic−/− animals are fertile and can live as long as their wild-type littermates. The Nfic mutation is the first mutation described that affects primarily tooth root formation and should greatly aid our understanding of postnatal tooth development.

Thioltransferase (glutaredoxin) reactivates the DNA-binding activity of oxidation-inactivated nuclear factor I.

The reversible oxidative inactivation of transcription factors has been proposed to be important in cellular responses to oxidant stress and in several signal transduction pathways. The nuclear factor I (NFI) family of transcription factors is sensitive to oxidative inactivation due to the presence of a conserved, oxidation-sensitive cysteine residue within the NFI DNA-binding domain. Here we show that restoration of the DNA-binding activity of oxidized NFI-C can be catalyzed in vitro by the cellular enzyme thioltransferase (glutaredoxin) coupled to GSH and GSSG reductase. To test whether GSH-dependent pathways play a role in the maintenance of NFI activity in vivo, we used buthionine sulfoximine, an agent that inhibits GSH synthesis, andN-acetylcysteine, an agent that can replenish intracellular GSH. Pretreatment of HeLa cells with buthionine sulfoximine greatly potentiated the inactivation of NFI by the oxidizing agent diamide. Inclusion of N-acetylcysteine in the culture medium during the recovery period following diamide treatment increased the extent of restoration of NFI activity. These results suggest that maintenance of the DNA-binding activity of NFI proteins during oxidant stress in vivo requires a GSH-dependent pathway, likely involving thioltransferase-catalyzed reduction of the oxidation-sensitive cysteine residue on NFI.

Sox9 and NFIA Coordinate a Transcriptional Regulatory Cascade during the Initiation of Gliogenesis

Transcriptional cascades that operate over the course of lineage development are fundamental mechanisms that control cellular differentiation. In the developing central nervous system (CNS), these mechanisms are well characterized during neurogenesis, but remain poorly defined during neural stem cell commitment to the glial lineage. NFIA is a transcription factor that plays a crucial role in the onset of gliogenesis; we found that its induction is regulated by the transcription factor Sox9 and that this relationship mediates the initiation of gliogenesis. Subsequently, Sox9 and NFIA form a complex and coregulate a set of genes induced after glial initiation. Functional studies revealed that a subset of these genes, Apcdd1 and Mmd2, perform key migratory and metabolic roles during astro-gliogenesis, respectively. In sum, these studies delineate a transcriptional regulatory cascade that operates during the initiation of gliogenesis and identifies a unique set of genes that regulate key aspects of astro-glial precursor physiology during development.

The transcription factor Nfix is essential for normal brain development

BackgroundThe Nuclear Factor I (NFI) multi-gene family encodes site-specific transcription factors essential for the development of a number of organ systems. We showed previously that Nfia-deficient mice exhibit agenesis of the corpus callosum and other forebrain defects; Nfib-deficient mice have defects in lung maturation and show callosal agenesis and forebrain defects resembling those seen in Nfia-deficient animals, while Nfic-deficient mice have defects in tooth root formation. Recently the Nfix gene has been disrupted and these studies indicated that there were largely uncharacterized defects in brain and skeletal development in Nfix-deficient mice.ResultsHere we show that disruption of Nfix by Cre-recombinase mediated excision of the 2nd exon results in defects in brain development that differ from those seen in Nfia and Nfib KO mice. In particular, complete callosal agenesis is not seen in Nfix-/- mice but rather there appears to be an overabundance of aberrant Pax6- and doublecortin-positive cells in the lateral ventricles of Nfix-/- mice, increased brain weight, expansion of the cingulate cortex and entire brain along the dorsal ventral axis, and aberrant formation of the hippocampus. On standard lab chow Nfix-/- animals show a decreased growth rate from ~P8 to P14, lose weight from ~P14 to P22 and die at ~P22. If their food is supplemented with a soft dough chow from P10, Nfix-/- animals show a lag in weight gain from P8 to P20 but then increase their growth rate. A fraction of the animals survive to adulthood and are fertile. The weight loss correlates with delayed eye and ear canal opening and suggests a delay in the development of several epithelial structures in Nfix-/- animals.ConclusionThese data show that Nfix is essential for normal brain development and may be required for neural stem cell homeostasis. The delays seen in eye and ear opening and the brain morphology defects appear independent of the nutritional deprivation, as rescue of perinatal lethality with soft dough does not eliminate these defects.

Nfix Regulates Fetal-Specific Transcription in Developing Skeletal Muscle

Skeletal myogenesis, like hematopoiesis, occurs in successive developmental stages that involve different cell populations and expression of different genes. We show here that the transcription factor nuclear factor one X (Nfix), whose expression is activated by Pax7 in fetal muscle, in turn activates the transcription of fetal specific genes such as MCK and beta-enolase while repressing embryonic genes such as slow myosin. In the case of the MCK promoter, Nfix forms a complex with PKC theta that binds, phosphorylates, and activates MEF2A. Premature expression of Nfix activates fetal and suppresses embryonic genes in embryonic muscle, whereas muscle-specific ablation of Nfix prevents fetal and maintains embryonic gene expression in the fetus. Therefore, Nfix acts as a transcriptional switch from embryonic to fetal myogenesis.