Nuclear Factor I X Research Articles

Distinct Effects of Allelic NFIX Mutations on Nonsense-Mediated mRNA Decay Engender Either a Sotos-like or a Marshall-Smith Syndrome

By using a combination of array comparative genomic hybridization and a candidate gene approach, we identified nuclear factor I/X (NFIX) deletions or nonsense mutation in three sporadic cases of a Sotos-like overgrowth syndrome with advanced bone age, macrocephaly, developmental delay, scoliosis, and unusual facies. Unlike the aforementioned human syndrome, Nfix-deficient mice are unable to gain weight and die in the first 3 postnatal weeks, while they also present with a spinal deformation and decreased bone mineralization. These features prompted us to consider NFIX as a candidate gene for Marshall-Smith syndrome (MSS), a severe malformation syndrome characterized by failure to thrive, respiratory insufficiency, accelerated osseous maturation, kyphoscoliosis, osteopenia, and unusual facies. Distinct frameshift and splice NFIX mutations that escaped nonsense-mediated mRNA decay (NMD) were identified in nine MSS subjects. NFIX belongs to the Nuclear factor one (NFI) family of transcription factors, but its specific function is presently unknown. We demonstrate that NFIX is normally expressed prenatally during human brain development and skeletogenesis. These findings demonstrate that allelic NFIX mutations trigger distinct phenotypes, depending specifically on their impact on NMD.

Abstract 1231: Nuclear Factor I family members interact with FoxA1 to regulate androgen responsive promoters

Abstract Forkhead (Fox) proteins are an important class of transcription factors (TFs) that act as molecular “pioneers” by increasing chromatin accessibility. The ability of FoxA1 to act in this manner allows for the subsequent nucleation of responsive promoters by additional, tissue specific TFs, allowing for gene regulation. Our laboratory has previously shown that FoxA1 interacts directly with the androgen receptor (AR), and that the expression of FoxA1 is essential for normal prostate maturation and differentiation. Thus, the ability of FoxA1 to enter a transcriptional complex with AR and other TFs contributes to tissue-specific gene expression in the prostate, and determining the components of FoxA1/AR TF complexes will identify additional targets for the treatment of castrate-resistant PCa. In order to identify additional proteins that interact with FoxA1, we ectopically expressed dual-affinity tagged FoxA1 in the human PCa cell line LNCaP. Following tandem purification and mass spectrometric analysis, we identified several FoxA1-interacting proteins, including Nuclear Factor I x (NFIx). NFIx is a member of the Nuclear Factor family of TFs, consisting of NFIa, NFIb, NFIc, and NFIx. Co-immunoprecipitation studies verify that NFI family members interact with FoxA1, and Chromatin immunoprecipitation (ChIP) experiments indicate NFI family members bind to androgen regulated, prostate-specific promoters. Finally, transient transfection experiments indicate that NFI family members modulate the androgenic stimulation of the Probasin and PSA promoters both in the presence and absence of FoxA1. In summary, we have identified NFIx as a novel FoxA1 interacting protein. NFI family members act to modulate the transcriptional activity of FoxA1 and AR on prostate-specific target promoters. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1231.

NFIX – one gene, two knockouts, multiple effects

A previous knockout of the transcription factor gene nuclear factor IX (NFIX) in mice produced impaired development of the corpus callosum and severe skeletal defects. A recent paper in BMC Developmental Biology reports an apparently similar NFIX knockout that produced marked differences in phenotype, raising intriguing general questions about the possible causes of such differences in mouse knockouts.

A Quick Method for Identifying the Molecular Targets of Potentially Diagnostic New Monoclonal Antibodies Using Protein Array Technology.

During the course of raising monoclonal antibodies, reagents are often produced that are not directed against the immunising antigen. These may pass unnoticed unless a screening step based on immunostaining of human tissue is included. Many of these reagents are auto-antibodies, often directed against intracellular targets (e.g. nuclear components), and the process of hybridoma production serves to “rescue” these self-reactive B cell clones. Such unexpected antibodies of this sort may prove of interest if their distribution is analysed on normal tissues and within the spectrum of leukaemia/lymphoma samples showing specific patterns of staining. However, before they can be used diagnostically or therapeutically, their target molecule needs to be identified, and this can be technically demanding. We describe a novel approach that can be used to define the targets of new monoclonal antibodies to intracellular molecules. The technique involves screening against protein arrays comprising thousands of recombinant human proteins expressed in bacteria. In our initial experiments we showed that a control monoclonal antibody against nucleophosmin identified 22 clones encoding this ubiquitous protein in a human foetal brain cDNA library. Monoclonal antibody JC1 (first published in 1992) was known to recognise an unidentified proliferation-associated nuclear molecule, and screening on the recombinant protein array defined nuclear factor I/X (NFIX) as its probable target. A second antibody (JJ166), reactive with cell nuclei, was shown by the same technique to recognise the mitotic spindle-associated molecule NUMA1 (the gene for which is involved in rare chromosomal translocations). To confirm these putative specificities, the antibodies were shown to react with Cos-1 cells that had been transiently transfected with the corresponding clones from the array (after transfer into a mammalian vector pcDNA4HisMaxC, incorporating an Xpress tag). This new method represents an effective and quick strategy for defining the protein targets of new monoclonal antibodies identified as having diagnostic or other potential value on the basis of their immunostaining patterns.

Localisation of the human nuclear factor I/X (NFI/X) gene to chromosome 19p13 and detection of five other related loci at 1p21-22, 1q42-43, 5q15, 11p13 and 20q13 by FISH

Nuclear factor I (NFI) is a member of a family of dimeric DNA-binding proteins that are involved both in the initiation of adenovirus DNA replication and in the stimulation of transcriptional activation. We have used fluorescence in situ hybridisation (FISH) to map one of four known genes encoding an NFI protein, the human NFI/X gene, to chromosome 19p1.3. Secondary sites of hybridisation observed at 5p1.5, 1q4.2-4.4, 1p2.1-2.2, and 20p1.3 most likely are attributable to partial sequence homologies with related NFI genes.