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AXL which is a chemosensitizer protein for breast cancer cells in response to epidermal growth factor receptor-tyrosine kinase inhibitor and suppresses tumor growth. The clinical information show nuclear factor I (NFI)-C and NFI-X expression correlate with AXL expression in breast cancer patients. Following, we establish serial deletions of AXL promoter to identify regions required for Adenovirus-5 early region 1A (E1A)-mediated AXL suppression. All of the NFI family members were extensively studied for their expression and functions in regulating AXL. Moreover, E1A post-transcriptionally downregulates AXL expression through NFI. NFI-C and NFI-X, not NFI-A and NFI-B, resulting in cell death in response to EGFR-TKI. Our finding suggests that NFI-C and NFI-X are crucial regulators for AXL and significantly correlated with poor survival of breast cancer patients.

Noxious mechanical information is transmitted through molecularly distinct nociceptors, with pinprick-evoked sharp sensitivityviaA-fiber nociceptors marked by developmental expression of the neuropeptide Y receptor 2 (Npy2r) and von Frey filament-evoked punctate pressure informationviaunmyelinated C fiber nociceptors marked by MrgprD. However, the molecular programs controlling their development are only beginning to be understood. Here we demonstrate that Npy2r-expressing sensory neurons are in fact divided into two groups, based on transient or persistent Npy2r expression. Npy2r-transient neurons are myelinated, likely including A-fiber nociceptors, whereas Npy2r-persistent ones belong to unmyelinated pruriceptors that co-express Nppb. We then showed that the transcription factors NFIA and Runx1 are necessary for the development of Npy2r-transient A-fiber nociceptors and MrgprD+C-fiber nociceptors, respectively. Behaviorally, mice with conditional knockout ofNfia, but notRunx1showed a marked attenuation of pinprick-evoked nocifensive responses. Our studies therefore identify a transcription factor controlling the development of myelinated nociceptors.

Malignant astrocytomas are composed of heterogeneous cell populations. Compared to grade IV glioblastoma, low-grade astrocytomas have more differentiated cells and are associated with a better prognosis. Therefore, inducing cellular differentiation to alter the behaviour of high-grade astrocytomas may serve as a therapeutic strategy. The nuclear factor one (NFI) transcription factors are essential for normal astrocytic differentiation. Here, we investigate whether family members NFIA and NFIB act as effectors of cellular differentiation in glioblastoma. We analysed expression of NFIA and NFIB in mRNA expression data of high-grade astrocytoma and with immunofluorescence co-staining. Furthermore, we induced NFI expression in patient-derived subcutaneous glioblastoma xenografts via in vivo electroporation. The expression of NFIA and NFIB is reduced in glioblastoma as compared to lower grade astrocytomas. At a cellular level, their expression is associated with differentiated and mature astrocyte-like tumour cells. In vivo analyses consistently demonstrate that expression of either NFIA or NFIB is sufficient to promote tumour cell differentiation in glioblastoma xenografts. Our findings indicate that both NFIA and NFIB may have an endogenous pro-differentiative function in astrocytomas, similar to their role in normal astrocyte differentiation. Overall, our study establishes a basis for further investigation of targeting NFI-mediated differentiation as a potential differentiation therapy.

YAP1 fusion-positive supratentorial ependymomas predominantly occur in infants, but the molecular mechanisms of oncogenesis are unknown. Here we show YAP1-MAMLD1 fusions are sufficient to drive malignant transformation in mice, and the resulting tumors share histo-molecular characteristics of human ependymomas. Nuclear localization of YAP1-MAMLD1 protein is mediated by MAMLD1 and independent of YAP1-Ser127 phosphorylation. Chromatin immunoprecipitation-sequencing analyses of human YAP1-MAMLD1-positive ependymoma reveal enrichment of NFI and TEAD transcription factor binding site motifs in YAP1-bound regulatory elements, suggesting a role for these transcription factors in YAP1-MAMLD1-driven tumorigenesis. Mutation of the TEAD binding site in the YAP1 fusion or repression of NFI targets prevents tumor induction in mice. Together, these results demonstrate that the YAP1-MAMLD1 fusion functions as an oncogenic driver of ependymoma through recruitment of TEADs and NFIs, indicating a rationale for preclinical studies to block the interaction between YAP1 fusions and NFI and TEAD transcription factors., The molecular mechanisms driving proliferation in the pediatric brain cancer epdendymoma are poorly understood. Here the authors show that a YAP1- MAMLD1 fusion drives tumor formation in mice and show that the fusion protein can collaborate with the TEAD and NFI transcription factors.

Glioblastoma (GBM) is a brain tumor that remains largely incurable because of its highly-infiltrative properties. Nuclear factor I (NFI)-type transcription factors regulate genes associated with GBM cell migration and infiltration. We have previously shown that NFI activity depends on the NFI phosphorylation state and that calcineurin phosphatase dephosphorylates and activates NFI. Calcineurin is cleaved and activated by calpain proteases whose activity is, in turn, regulated by an endogenous inhibitor, calpastatin (CAST). The CAST gene is a target of NFI in GBM cells, with differentially phosphorylated NFIs regulating the levels of CAST transcript variants. Here, we uncovered an NFIB–calpain 1-positive feedback loop mediated through CAST and calcineurin. In NFI-hyperphosphorylated GBM cells, NFIB expression decreased the CAST–to–calpain 1 ratio in the cytoplasm. This reduced ratio increased autolysis and activity of cytoplasmic calpain 1. Conversely, in NFI-hypophosphorylated cells, NFIB expression induced differential subcellular compartmentalization of CAST and calpain 1, with CAST localizing primarily to the cytoplasm and calpain 1 to the nucleus. Overall, this altered compartmentalization increased nuclear calpain 1 activity. We also show that nuclear calpain 1, by cleaving and activating calcineurin, induces NFIB dephosphorylation. Of note, knockdown of calpain 1, NFIB, or both increased GBM cell migration and up-regulated the pro-migratory factors fatty acid–binding protein 7 (FABP7) and Ras homolog family member A (RHOA). In summary, our findings reveal bidirectional cross-talk between NFIB and calpain 1 in GBM cells. A physiological consequence of this positive feedback loop appears to be decreased GBM cell migration.

It has been reported that high mobility group nucleosomal binding domain 2 (HMGN2) is a nucleus-related protein that regulates gene transcription and plays a critical role in bacterial clearance. An elevated level of HMGN2 reduced integrin α5/β1 expression of human pulmonary epithelial A549 cells was demonstrated during Klebsiella pneumoniae infection, thus weakening bacterial adhesion and invasion. However, the mechanism by which HMGN2 regulates integrin expression remains unclear. This study found that a transcription factor-nuclear factor I (NFI), which serves as the potential target of HMGN2 regulated integrin expression. The results showed that HMGN2 was able to promote NFIA and NFIB expression by increasing H3K27 acetylation of NFIA/B promoter regions. The integrin α5/β1 expression was significantly enhanced by knockdown of NFIA/B via a siRNA approach. Meanwhile, NFIA/B silence could also compromise the inhibition effect of HMGN2 on the integrin α5/β1 expression. Mechanistically, it was demonstrated that HMGN2 facilitated the recruitment of NFI on the promoter regions of integrin α5/β1 according to the chromatin immunoprecipitation assay. In addition, it was further demonstrated that the knockdown of NFIA/B induced more adhesion of Klebsiella pneumoniae on pulmonary epithelial A549 cells, which could be reversed by the application of an integrin inhibitor RGD. The results revealed a regulatory role of HMGN2 on the transcription level of integrin α5/β1, indicating a potential treatment strategy against Klebsiella pneumoniae-induced infectious lung diseases.

The nuclear factor I (NFI) family of transcription factors play an important role in normal development of multiple organs. Three NFI family members are highly expressed in the brain, and deletions or sequence variants in two of these, NFIA and NFIX, have been associated with intellectual disability (ID) and brain malformations. NFIB, however, has not previously been implicated in human disease. Here, we present a cohort of 18 individuals with mild ID and behavioral issues who are haploinsufficient for NFIB. Ten individuals harbored overlapping microdeletions of the chromosomal 9p23-p22.2 region, ranging in size from 225 kb to 4.3 Mb. Five additional subjects had point sequence variations creating a premature termination codon, and three subjects harbored single-nucleotide variations resulting in an inactive protein as determined using an in vitro reporter assay. All individuals presented with additional variable neurodevelopmental phenotypes, including muscular hypotonia, motor and speech delay, attention deficit disorder, autism spectrum disorder, and behavioral abnormalities. While structural brain anomalies, including dysgenesis of corpus callosum, were variable, individuals most frequently presented with macrocephaly. To determine whether macrocephaly could be a functional consequence of NFIB disruption, we analyzed a cortex-specific Nfib conditional knockout mouse model, which is postnatally viable. Utilizing magnetic resonance imaging and histology, we demonstrate that Nfib conditional knockout mice have enlargement of the cerebral cortex but preservation of overall brain structure and interhemispheric connectivity. Based on our findings, we propose that haploinsufficiency of NFIB causes ID with macrocephaly. ispartof: AMERICAN JOURNAL OF HUMAN GENETICS vol:103 issue:5 pages:752-768 ispartof: location:United States status: published

Emerging evidence indicates that nuclear factor I/B (NFIB), a transcription factor required for proper development and regulation of cellular differentiation in several tissues, also plays critical roles in cancer. Despite being a metastatic driver in small cell lung cancer and melanoma, it has become apparent that NFIB also exhibits tumour suppressive functions in many malignancies. The contradictory contributions of NFIB to both the inhibition and promotion of tumour development and progression, corroborates its diverse and context-dependent roles in many tissues and cell types. Considering the frequent involvement of NFIB in cancer, a better understanding of its multifaceted nature may ultimately benefit the development of novel strategies for the management of a broad spectrum of malignancies. Here we discuss recent findings which bring to light NFIB as a crucial and paradoxical player in cancer., Highlights • NFIB, a versatile regulator of cell differentiation, is emerging as a crucial driver of cancer metastasis. • Paradoxically, NFIB also exhibits tumour suppressive functions in several cancer types. • A deeper understanding of the multifaceted and context-dependent nature of NFIB has the potential to improve the clinical management of a variety of cancers.

Reactive astrocytes are associated with every form of neurological injury. Despite their ubiquity, the molecular mechanisms controlling their production and diverse functions remain poorly defined. Because many features of astrocyte development are recapitulated in reactive astrocytes, we investigated the role of nuclear factor I-A (NFIA), a key transcriptional regulator of astrocyte development whose contributions to reactive astrocytes remain undefined. Here, we show that NFIA is highly expressed in reactive astrocytes in human neurological injury and identify unique roles across distinct injury states and regions of the CNS. In the spinal cord, after white matter injury (WMI), NFIA-deficient astrocytes exhibit defects in blood-brain barrier remodeling, which are correlated with the suppression of timely remyelination. In the cortex, after ischemic stroke, NFIA is required for the production of reactive astrocytes from the subventricular zone (SVZ). Mechanistically, NFIA directly regulates the expression of thrombospondin 4 (Thbs4) in the SVZ, revealing a key transcriptional node regulating reactive astrogenesis. Together, these studies uncover critical roles for NFIA in reactive astrocytes and illustrate how region- and injury-specific factors dictate the spectrum of reactive astrocyte responses.

International audience; Anal carcinomas (AC) are associated with human papillomavirus (HPV) DNA sequences, but little is known about the physical state of the viral genome in carcinoma cells. To define the integration status and gene(s) targeted by viral insertions in AC, tumor DNAs extracted from 35 tumor specimen samples in patients with HPV16‐associated invasive carcinoma were analyzed using the detection of integrated papillomavirus sequences‐PCR approach. The genomic status at integration sites was assessed using comparative genomic hybridization‐array assay and gene expression using reverse transcription quantitative PCR (RT‐qPCR). HPV16 DNA was found integrated in 25/35 (71%) cases and the integration locus could be determined at the molecular level in 19 cases (29 total integration loci). HPV DNA was inserted on different chromosomes, but 5 cases harbored viral sequences at 19p13.2, within the nuclear factor I X (NFIX) locus. Viral DNA mapped between the most distal and the two proximal alternatively expressed exons of this gene in three cases (CA21, CA04, and CA35) and upstream of this gene (663 kb and 2.3 Mb) in the others. CGH arrays showed genomic gains/amplifications at the NFIX region, associated with HPV within the gene and RT‐qPCR, revealed NFIX mRNA overexpression. Other genes targeted by integration were IL20RB, RPS6KA2, MSRA1, PIP5K1B, SLX4IP, CECR1, BCAR3, ATF6, CSNK1G1, APBA2, AGK, ILF3, PVT1, TRMT1, RAD51B, FASN, CCDC57, DSG3, and ZNF563. We identified recurrent targeting of NFIX by HPV16 insertion in anal carcinomas, supporting a role for this gene in oncogenesis, as reported for non‐HPV tumors.

Malignant glioma (MG) is the most lethal primary brain tumor. In addition to having inherent resistance to radiation treatment and chemotherapy, MG cells are highly infiltrative, rendering focal therapies ineffective. Genes involved in MG cell migration and glial cell differentiation are up-regulated by hypophosphorylated nuclear factor I (NFI), which is dephosphorylated by the phosphatase calcineurin in MG cells. Calcineurin is cleaved and thereby activated by calpain proteases, which are, in turn, inhibited by calpastatin (CAST). Here, we show that the CAST gene is a target of NFI and has NFI-binding sites in its intron 3 region. We also found that NFI-mediated regulation of CAST depends on NFI's phosphorylation state. We noted that occupation of CAST intron 3 by hypophosphorylated NFI results in increased activation of an alternative promoter. This activation resulted in higher levels of CAST transcript variants, leading to increased levels of CAST protein that lacks the N-terminal XL domain. CAST was primarily present in the cytoplasm of NFI-hypophosphorylated MG cells, with a predominantly perinuclear immunostaining pattern. NFI knockdown in NFI-hypophosphorylated MG cells increased CAST levels at the plasma membrane. These results suggest that NFI plays an integral role in the regulation of CAST variants and CAST subcellular distribution. Along with the previous findings indicating that NFI activity is regulated by calcineurin, these results provide a foundation for further investigations into the possibility of regulatory cross-talk between NFI and the CAST/calpain/calcineurin signaling pathway in MG cells.

Because it accounts for 70% of all eye cancers, uveal melanoma (UM) is therefore the most common primary ocular malignancy. In this study, we investigated the molecular mechanisms leading to the aberrant expression of the gene encoding the serotonin receptor 2B (HTR2B), one of the most discriminating among the candidates from the class II gene signature, in metastatic and non-metastatic UM cell lines. Transfection analyses revealed that the upstream regulatory region of the HTR2B gene contains a combination of alternative positive and negative regulatory elements functional in HTR2B&minus, but not in HTR23B+ UM cells. We demonstrated that both the transcription factors nuclear factor I (NFI) and Runt-related transcription factor I (RUNX1) interact with regulatory elements from the HTR2B gene to either activate (NFI) or repress (RUNX1) HTR2B expression in UM cells. The results of this study will help understand better the molecular mechanisms accounting for the abnormal expression of the HTR2B gene in uveal melanoma.

Introduction Stem cells isolated from the root apical papilla of human teeth (stem cells from the apical papilla [SCAPs]) are capable of forming tooth root dentin and are a feasible source for bioengineered tooth root regeneration. In this study, we examined the effect of acetylsalicylic acid (ASA) on odontogenic differentiation of SCAPs in vitro and in vivo. Methods SCAPs were cultured under odontogenic conditions supplemented with or without ASA. ASA-treated SCAPs were also subcutaneously transplanted into immunocompromised mice. Results ASA accelerates in vitro and in vivo odontogenic differentiation of SCAPs associated with down-regulation of runt-related nuclear factor 2 and up-regulation of specificity protein 7, nuclear factor I C, and dentin phosphoprotein. ASA up-regulated the phosphorylation of AKT in the odontogenic SCAPs. Of interest, pretreatments with phosphoinositide 3-kinase inhibitor LY294402 and small interfering RNA for AKT promoted ASA-induced in vitro and in vivo odontogenic differentiation of SCAPs. LY294402 and small interfering RNA for AKT also suppressed the ASA-induced expression of runt-related nuclear factor 2 and enhanced ASA-induced expression of specificity protein 7, nuclear factor I C, and dentin phosphoprotein in SCAPs. Conclusions These findings suggest that a combination of ASA treatment and suppressive regulation of the phosphoinositide 3-kinase–AKT signaling pathway is a novel approach for SCAP-based tooth root regeneration.

The nuclear factor I/X (NFIX) plays important roles in cell differentiation, but its function in cancer is still unclear. Besides, accumulating studies reported the important role of microRNAs (miRNAs) in the regulation of gene expression, among of which, the miR-1290 has been widely reported in various cancers. In this study, we investigated the mechanism through which NFIX was regulated by miRNAs. Firstly, we found that the NFIX protein and mRNA levels were consistently down-regulated in ESCC tissues suggesting that a post-transcriptional mechanism maybe involved in the regulation of NFIX. Since microRNAs (miRNAs) are powerful post-transcriptional regulators of gene expression, we performed bioinformatic analyses to search for miRNAs that potentially target NFIX. We identified the specific targeting site of miR-1290 in the 3'-UTR of NFIX and the inverse correlation between the levels of miR-1290 and NFIX protein and mRNA in ESCC tissue samples was then confirmed. By overexpressing or silencing miR-1290 in ESCC cells, we experimentally validated that miR-1290 directly binds to the 3'-UTR of the NFIX transcript and degrade the NFIX mRNA to regulate NFIX expression. Furthermore, the biological consequences that miR-1290 mediated by targeting NFIX were examined in vitro. We demonstrated that miR-1290 could promote proliferation, migration and invasion via the negative regulation of NFIX expression. Taken together, our findings suggested that miR-1290 functions as a tumor oncogene in the progression of ESCC by targeting NFIX.

The Nuclear Factor I-X (NFIX) is a member of the nuclear factor I (NFI) protein family and is deleted or mutated in a subset of patients with a peculiar overgrowth condition resembling Sotos Syndrome as well as in patients with Marshall-Smith syndrome. We identified three additional patients with this phenotype each carrying a different new mutation affecting the DNA-binding/dimerization domain of the NFIX protein. The present report further adds weight to the hypothesis that mutations in DNA-binding/dimerization domain are likely to cause haploinsufficiency of the NFIX protein and confirms that NFIX is the second gene that should be tested in individuals with overgrowth conditions resembling Sotos syndrome, previously tested negative for NSD1 mutations. We then propose to consider this overgrowth syndrome (namely Malan syndrome) and Marshall-Smith syndrome NFIX-related diseases.

Previous studies have reported that vitamin C (VC) promotes neural stem/precursor cell (NSC) differentiation toward dopamine (DA) neurons via DNA hydroxymethylation-induced transcriptional activation of DA neuron-specific genes. To further understand the VC effects on NSC differentiation, we profiled the transcriptome and DNA methylome/hydroxymethylome using high-throughput sequencing. Interestingly, RNA sequencing analyses have shown that, in addition to DA neuronal genes, astrocytic genes Gfap, Slc1a3, and S100a16 were also upregulated in NSC cultures differentiated with VC treatment. Consistently, enhanced GFAP+ astrocytic yields were manifested in the differentiated cultures with VC treatment, collectively indicating that VC promotes astrocytic differentiation. In genome-wide hydroxymethylome analyses, VC treatment induces enrichment of DNA hydroxymethylation (5-hydroxymethyl cytosine; 5hmC) near the consensus binding motifs of nuclear factor I (NFI). Furthermore, we showed that VC significantly enhanced recruitment of NFI and STAT3, key transcription factors for astrogenesis, in the 5hmC-enriched regions of the astrocyte-specific genes. These findings suggest that VC play important roles in astrocytogenesis during brain development.

To evaluate the role and the molecular mechanism of miR-30d in non-small cell lung cancer (NSCLC). qRT-PCR was used to detect miR-30d expression in NSCLC tissues and cell lines. miR-30d was frequently down-regulated in NSCLC and its expression was associated with clinicopathological features of NSCLCC patients. Over-expression of miR-30d notably inhibited cell migration and invasion in NSCLC cell lines. miR-30d could negatively regulate Nuclear factor I B (NFIB) by directly targeting its 3′-UTR, which was confirmed by luciferase assay. NFIB also reversed miR-30d-mediated suppression on the migration and invasion in NSCLC cell lines. miR-30d suppressed cell migration and invasion by directly targeting NFIB in NSCLC, and NFIB could partially abrogated the inhibition of biological functions by miR-30d.

The nuclear factor I (NFI) transcription factors play important roles during normal development and have been associated with developmental abnormalities in humans. All four family members, NFIA, NFIB, NFIC and NFIX, have a homologous DNA binding domain and function by regulating cell proliferation and differentiation via the transcriptional control of their target genes. More recently, NFI genes have also been implicated in cancer based on genomic analyses and studies of animal models in a variety of tumours across multiple organ systems. However, the association between their functions in development and in cancer is not well described. In this review, we summarise the evidence suggesting a converging role for the NFI genes in development and cancer. Our review includes all cancer types in which the NFI genes are implicated, focusing predominantly on studies demonstrating their oncogenic or tumour suppressive potential. We conclude by presenting the challenges impeding our understanding of NFI function in cancer biology, and demonstrate how a developmental perspective may contribute towards overcoming such hurdles. (C) 2017 Elsevier B.V. All rights reserved.

Retroviral vector-mediated gene therapy is promising, but genotoxicity has limited its use in the clinic. Genotoxicity is highly dependent on the retroviral vector used, and foamy viral (FV) vectors appear relatively safe. However, internal promoters may still potentially activate nearby genes. We developed insulated FV vectors, using four previously described insulators: a version of the well-studied chicken hypersensitivity site 4 insulator (650cHS4), two synthetic CCCTC-binding factor (CTCF)-based insulators, and an insulator based on the CCAAT box-binding transcription factor/nuclear factor I (7xCTF/NF1). We directly compared these insulators for enhancer-blocking activity, effect on FV vector titer, and fidelity of transfer to both proviral long terminal repeats. The synthetic CTCF-based insulators had the strongest insulating activity, but reduced titers significantly. The 7xCTF/NF1 insulator did not reduce titers but had weak insulating activity. The 650cHS4-insulated FV vector was identified as the overall most promising vector. Uninsulated and 650cHS4-insulated FV vectors were both significantly less genotoxic than gammaretroviral vectors. Integration sites were evaluated in cord blood CD34(+) cells and the 650cHS4-insulated FV vector had fewer hotspots compared with an uninsulated FV vector. These data suggest that insulated FV vectors are promising for hematopoietic stem cell gene therapy.

Androgen receptor (AR) action throughout prostate development and in maintenance of the prostatic epithelium is partly controlled by interactions between AR and forkhead box (FOX) transcription factors, particularly FOXA1. We sought to identity additional FOXA1 binding partners that may mediate prostate-specific gene expression. Here we identify the nuclear factor I (NFI) family of transcription factors as novel FOXA1 binding proteins. All four family members (NFIA, NFIB, NFIC, and NFIX) can interact with FOXA1, and knockdown studies in androgen-dependent LNCaP cells determined that modulating expression of NFI family members results in changes in AR target gene expression. This effect is probably mediated by binding of NFI family members to AR target gene promoters, because chromatin immunoprecipitation (ChIP) studies found that NFIB bound to the prostate-specific antigen enhancer. Förster resonance energy transfer studies revealed that FOXA1 is capable of bringing AR and NFIX into proximity, indicating that FOXA1 facilitates the AR and NFI interaction by bridging the complex. To determine the extent to which NFI family members regulate AR/FOXA1 target genes, motif analysis of publicly available data for ChIP followed by sequencing was undertaken. This analysis revealed that 34.4% of peaks bound by AR and FOXA1 contain NFI binding sites. Validation of 8 of these peaks by ChIP revealed that NFI family members can bind 6 of these predicted genomic elements, and 4 of the 8 associated genes undergo gene expression changes as a result of individual NFI knockdown. These observations suggest that NFI regulation of FOXA1/AR action is a frequent event, with individual family members playing distinct roles in AR target gene expression.

Aims To investigate the underlying mechanism by which glioblastoma (GBM) cells gain temozolomide (TMZ) resistance and to clarify novel therapeutic targets and new prognostic biomarkers for GBM. Main methods A genome-wide hierarchical bi-clustering based on previously published microarray databases identified Nuclear Factor I A (NFIA) as one of the most significantly upregulated genes correlated to TMZ resistance in GBM. Then, the potential biological functions of NFIA in oncogenesis and chemoresistance were clarified by qRT-PCR, Western blotting and in vivo xenograft models with artificially induced TMZ-resistant U87 cells. Additionally, immunohistochemistry (IHC) assays were performed to explore the clinical significance of NFIA in glioma patients. Last, luciferase reporter assay was performed to study the transcriptional regulation of NFIA on the nuclear factor κb (NF-kB) pathway. Key findings NFIA was correlated with TMZ resistance in GBM. Clinically, elevated NFIA expression was significantly correlated with adverse outcomes of glioma patients, especially in GBM patients. Moreover, NFIA contributed to the acquired TMZ resistance of GBM cells, while suppression of NFIA via lentivirus reduced cell proliferation, tumorigenesis and resistance to TMZ of GBM. Additionally, NFIA promoted transcription activity that regulated the expression of NF-kB. Last, NFIA induced phosphorylation of NF-kB p65 at serine 536, thus inducing TMZ resistance in GBM cells. Altogether, our study suggests that NFIA-dependent transcriptional regulation of NF-kB contributes to acquired TMZ resistance in GBM. Significance Abnormally activated NFIA-NF-kB signaling was strongly correlated with acquired TMZ resistance and poor prognosis in GBM, and it could be a new therapeutic target for TMZ-resistant GBM.

// Nan Wu 1 , Deshui Jia 1 , Ali H. Ibrahim 1 , Cindy J. Bachurski 2 , Richard M. Gronostajski 3 and David MacPherson 1,4 1 Divisions of Human Biology and Public Health Sciences, Fred Hutchinson Cancer Research Center, Seattle, WA, USA 2 Division of Pulmonary Biology, Cincinnati Children’s Hospital Research Foundation, Cincinnati, OH, USA 3 Department of Biochemistry, Program in Genetics, Genomics & Bioinformatics, University at Buffalo, Buffalo, NY, USA 4 Department of Genome Sciences, University of Washington, Seattle, WA, USA Correspondence to: David MacPherson, email: // Keywords : oncogene, metastasis, mouse model, nuclear factor I, NFI Received : June 28, 2016 Accepted : August 20, 2016 Published : August 24, 2016 Abstract Small cell lung cancer (SCLC) is a highly aggressive neuroendocrine tumor type that is typically metastatic upon diagnosis. We have a poor understanding of the factors that control SCLC progression and metastasis. TheNFIB transcription factor is frequently amplified in mouse models of SCLC, but clear evidence that NFIB promotes SCLC in vivo is lacking. We report that in mouse models, Nfib amplifications are far more frequent in liver metastases over primary SCLC, suggesting roles in tumor progression/metastasis. Overexpression of Nfib in a sensitized mouse model led to acceleration of SCLC, indicating that Nfib functions as a bona fide oncogene. Suppression of Nfib expression in cell lines derived from the doxycycline-inducible Rb/p53/TET-Nfib model led to increased apoptosis and suppression of proliferation. Transcriptional analysis revealed that Nfib regulates the expression of genes related to axon guidance, focal adhesion and extracellular matrix-receptor interactions. These data indicate that Nfib is a potent oncogene in SCLC, and the enrichment of Nfib amplifications in liver metastases over primary SCLC points to Nfib as a candidate driver of SCLC metastasis.

Small cell lung cancer (SCLC) is an aggressive cancer often diagnosed after it has metastasized. Despite the need to better understand this disease, SCLC remains poorly characterized at the molecular and genomic levels. Using a genetically engineered mouse model of SCLC driven by conditional deletion of Trp53 and Rb1 in the lung, we identified several frequent, high-magnitude focal DNA copy number alterations in SCLC. We uncovered amplification of a novel, oncogenic transcription factor, Nuclear factor I/B (Nfib), in the mouse SCLC model and in human SCLC. Functional studies indicate that NFIB regulates cell viability and proliferation during transformation., National Cancer Institute (U.S.) (grant P30-CA14051), David H. Koch Institute for Integrative Cancer Research at MIT (Ludwig Center for Molecular Oncology), Howard Hughes Medical Institute, Alfred P. Sloan Foundation (Research Fellowship), International Association for the Study of Lung Cancer

Transcription factors of the nuclear factor 1 (NFI) family regulate normal brain development in vertebrates. However, multiple splice variants of four NFI isoforms exist, and their biological functions have yet to be elucidated. Here, we cloned and analyzed human NFI-X3, a novel splice variant of the nfix gene, which contains a unique transcriptional activation (TA) domain completely conserved in primates. In contrast to previously cloned NFI-X1, overexpression of NFI-X3 potently activates NFI reporters, including glial fibrillary acidic protein (GFAP) reporter, in astrocytes and glioma cells. The GAL4 fusion protein containing the TA domain of NFI-X3 strongly activates the GAL4 reporter, whereas the TA domain of NFI-X1 is ineffective. The expression of NFI-X3 is dramatically up-regulated during the differentiation of neural progenitors to astrocytes and precedes the expression of astrocyte markers, such as GFAP and SPARCL1 (Secreted Protein, Acidic and Rich in Cysteines-like 1). Overexpression of NFI-X3 dramatically up-regulates GFAP and SPARCL1 expression in glioma cells, whereas the knockdown of NFI-X3 diminishes the expression of both GFAP and SPARCL1 in astrocytes. Although activation of astrocyte-specific genes involves DNA demethylation and subsequent increase of histone acetylation, NFI-X3 activates GFAP expression, in part, by inducing alterations in the nucleosome architecture that lead to the increased recruitment of RNA polymerase II.

Summary: Notch is activated globally in pancreatic progenitors; however, for progenitors to differentiate into endocrine cells, they must escape Notch activation to express Neurogenin-3. Here, we find that the transcription factor nuclear factor I/A (NFIA) promotes endocrine development by regulating Notch ligand Dll1 trafficking. Pancreatic deletion of NFIA leads to cell fate defects, with increased duct and decreased endocrine formation, while ectopic expression promotes endocrine formation in mice and human pancreatic progenitors. NFIA-deficient mice exhibit dysregulation of trafficking-related genes including increased expression of Mib1, which acts to target Dll1 for endocytosis. We find that NFIA binds to the Mib1 promoter, with loss of NFIA leading to an increase in Dll1 internalization and enhanced Notch activation with rescue of the cell fate defects after Mib1 knockdown. This study reveals NFIA as a pro-endocrine factor in the pancreas, acting to repress Mib1, inhibit Dll1 endocytosis and thus promote escape from Notch activation. : Scavuzzo et al. show NFIA is expressed at the onset of the secondary transition in pancreatic progenitors to regulate endocrine fate induction. NFIA binds the Mib1 promoter, with increased Mib1 in NFIA-deficient cells, more Dll1 endocytosis, and higher Notch activation, leading to formation of more ducts and fewer endocrine cells. Keywords: development, pancreas, cell fate, endocrine cell, Notch signaling, NFIA, Mib1, endocytosis

Although the regulation of thyroid stimulating hormone β-subunit gene (TSHβ) has been intensively studied, the functions of transcription factors involved are not fully understood. The authors found that the −615/−516 promoter region of the TSHβ interacts specifically with nuclear proteins derived from pituitary tissue or from cultured thyrotroph cells. The actual binding site at the nucleotide level, as revealed by DNase I protection assay, includes the consensus sequence for nuclear factor I (NFI). RT-PCR analysis indicated that NFI-B expression is restricted to thyrotroph cells in the anterior pituitary. EMSA and ChIP analysis showed that NFI-B binds most efficiently to the −588/−560 region of TSHβ promoter. The forced expressions of NFI-B markedly reduced TSHβ promoter activity and its mRNA expression. Furthermore, it was also shown that the −588/−560 region is involved in the insulin-mediated repression of the TSHβ. It was of particular interest to observe that NFI-B was recruited to the −588/−560 region of the TSHβ promoter in an insulin-dependent manner. Taken together, this study provides new insights of the delicate regulations of energy metabolism and hormonal homeostasis.

Insulin-like growth factor binding protein 5 (IGFBP5) is expressed in many cell types including osteoblasts and modulates IGF activities. IGFBP5 may affect osteoblasts and bone formation, in part by mechanisms independent of binding IGFs. The highly conserved IGFBP5 proximal promoter within 100 nucleotides of the start of transcription contains functional cis regulatory elements for C/EBP, Myb and AP-2. We report evidence for a functional Nuclear Factor I (NFI) cis element that mediates activation or repression of IGFBP5 transcription by the NFI gene family. All four NFI genes were expressed in human osteoblast cultures and osteosarcoma cell lines. Co-transfection with human IGFBP5 promoter luciferase reporter and murine Nfi expression vectors showed that Nfib was the most active in stimulating transcription. Nfix was less active and Nfia and Nfic were inhibitory. Knockdown of NFIB and NFIC expression using siRNA decreased and increased IGFBP5 expression, respectively. Analysis of IGFBP5 promoter deletion and mutation reporter constructs identified a functional NFI cis element. All four NFI proteins bound the NFI site in electrophoretic mobility shift experiments and NFI-B bound in Chromatin Immunoprecipitation assays. Results suggest that NFI proteins are important regulators of IGFBP5 expression in human osteoblasts and thus in modulating IGFBP5 functions in bone.

We have previously shown that the basic helix-loop-helix (bHLH) transcription factors coordinate Na(V) 1.4 Na(+) channel gene expression in skeletal muscle, but the identity of the co-factors they direct is unknown. Using C2C12 muscle cells as a model system, we test the hypothesis that the bHLH factors counteract negative regulation exerted through a repressor E box (-90/-85) by recruiting positive-acting transcription factors to the nucleotides (-135/-57) surrounding the repressor E box. We used electrophoretic mobility shift assays to identify candidate factors that bound the repressor E box or these adjacent regions. Repressor E box-binding factors included the known transcription factor, ZEB/AREB6, and a novel repressor E box-binding factor designated REB. Mutations of the repressor E box that interfere with the binding of these factors prevented repression. The transcription factor, nuclear factor I (NFI), bound immediately upstream and downstream of the repressor E box. Mutation of the NFI-binding sites diminished the ability of myogenin and MRF4 to counteract repression. Based on these observations we suggest that bHLH factors recruit NFI to enhance skeletal muscle Na(+) channel expression.

The transcription factor nuclear factor I (NFI) has been shown previously both in vivo and in vitro to be involved in the cooperative regulation of whey acidic protein (WAP) gene transcription along with the glucocorticoid receptor and STAT5. In addition, one of the specific NFI isoforms, NFI-B2, was demonstrated in transient co-transfection experiments in JEG cells, which lack endogenous NF-I, to be preferentially involved in the cooperative regulation of WAP gene expression. A comparison of the DNA-binding specificities of the different NFI isoforms only partially explained their differential ability to activate the WAP gene transcription. Here, we analyzed the transactivation regions of two NFI isoforms by making chimeric proteins between the NFI-A and B isoforms. Though, their DNA- binding specificities were not altered as compared to the corresponding wild type transcription factors, the C-terminal region of the NFI-B isoform was shown to preferentially activate WAP gene transcription in cooperation with GR and STAT5 in transient co-transfection assays in JEG-3 cells. Furthermore, determination of serine and threonine–specific glycosylation (O-linked N- acetylglucosamine) of the C-terminus of the NFI-B isoform suggested that the secondary modification by O-GlcNAc might play a role in the cooperative regulation of WAP gene transcription by NFI-B2 and STAT5.

Recent emerging evidence indicates that changes in gene expression levels are linked to human evolution. We have previously reported a human-specific nucleotide in the promoter sequence of the calreticulin (CALR) gene at position -220C, which is the site of action of valproic acid. Reversion of this nucleotide to the ancestral A-allele has been detected in patients with degrees of deficit in higher brain cognitive functions. This mutation has since been reported in the 1000 genomes database at an approximate frequency of

The Nuclear factor I (NFI) family of transcription factors regulates proliferation and differentiation throughout the developing central nervous system. In the developing telencephalon of humans and mice, reduced Nfi expression is associated with agenesis of the corpus callosum and other neurodevelopmental defects. Currently, little is known about how Nfi expression is regulated during early telencephalic development. PAX6, a transcription factor important for telencephalic development, has been proposed as an upstream regulator of Nfi expression in the neocortex. Here we demonstrate that, in the developing neocortex of mice, NFIA and NFIB are endogenously expressed in gradients with high caudo-medial to low rostro-lateral expression and are most highly expressed in the cortical plate. We found that this expression pattern deviates from that of PAX6, suggesting that PAX6 does not drive Nfi expression. This is supported by in vitro reporter assays showing that PAX6 over-expression does not regulate Nfi promoter activity. Similarly, we also found that in the Pax6 Small Eye mutant, no changes in Nfi mRNA or protein expression are observed in the neocortical ventricular zone where PAX6 and the NFIs are expressed. Together these data demonstrate that in mice, PAX6 is not a transcriptional activator of Nfi expression during neocortical development.

The LHX3 transcription factor is required for pituitary and nervous system development in mammals. Mutations in the human gene are associated with hormone-deficiency diseases. The gene generates two mRNAs, hLHX3a and hLHX3b, which encode three proteins with different properties. Here, the cis elements and trans-acting factors that regulate the basal transcription of the two mRNAs are characterized. A comparative approach was taken featuring analysis of seven mammalian Lhx3 genes, with a focus on the human gene. Two conserved, TATA-less, GC-rich promoters that are used to transcribe the mRNAs precede exons 1a and 1b of hLHX3. Transcription start sites were mapped for both promoters. Deletion experiments showed most activity for reporter genes containing the basal promoters in the context of −2.0 kb of hLHX3a and 1.8 kb of intron 1a (hLHX3b). Transfection, site-directed mutation, electrophoretic mobility shift, Southwestern blot, and chromatin immunoprecipitation approaches were used to characterize the interaction of transcription factors with conserved elements in the promoters. Specificity protein 1 is a regulator of both promoters through interaction with GC boxes. In addition, a distal element within intron 1a that is recognized by nuclear factor I is critical for hLHX3b promoter function. We conclude that dual promoters allow regulated production of two hLHX3 mRNAs.

Nervous system formation requires the elaboration of a complex series of differentiation events in both a spatially and maturation-regulated manner. A fundamental question is how neuronal subtype specification and developmental gene expression are controlled within maturing neurons. The alpha6 subunit of the gamma-aminobutyric acid type A (GABA(A)) receptor (GABRA6) is preferentially expressed in cerebellar granule neurons and is part of an intrinsic program directing their differentiation. We have employed a lentiviral approach to examine the transcriptional mechanisms controlling neuronal subtype-selective expression of this gene. These studies demonstrated that nuclear factor I (NFI) proteins are required for both transgenic GABRA6 promoter activity as well as endogenous expression of this gene in cerebellar granule neurons. Chromatin immunoprecipitation also showed that NFI proteins are bound to the GABRA6 promoter in these cells in vivo. Furthermore, analyses of gene knockout mice revealed that Nfia is specifically required for normal expression of the GABRA6 gene in cerebellar granule neurons. NFI expression and DNA binding activity are highly enriched in granule neurons, implicating this transcription factor family in the neuronal subtype-selective expression of the GABRA6 gene. These studies define a new role for NFI proteins as neuronal subtype-enriched transcriptional regulators that participate in an intrinsic transcriptional program directing the differentiation of cerebellar granule neurons.

Human papillomaviruses (HPVs) are present in virtually all cervical cancers. An important step in the development of malignant disease, including cervical cancer, involves a loss of sensitivity to transforming growth factor β (TGF-β). HPV type 16 (HPV16) early gene expression, including that of the E6 and E7 oncoprotein genes, is under the control of the upstream regulatory region (URR), and E6 and E7 expression in HPV16-immortalized human epithelial cells is inhibited at the transcriptional level by TGF-β. While the URR contains a myriad of transcription factor binding sites, including seven binding sites for nuclear factor I (NFI), the specific sequences within the URR or the transcription factors responsible for TGF-β modulation of the URR remain unknown. To identify potential transcription factors and binding sites involved in TGF-β modulation of the URR, we performed DNase I footprint analysis on the HPV16 URR using nuclear extracts from TGF-β-sensitive HPV16-immortalized human keratinocytes (HKc/HPV16) treated with and without TGF-β. Differentially protected regions were found to be located around NFI binding sites. Electrophoretic mobility shift assays, using the NFI binding sites as probes, showed decreased binding upon TGF-β treatment. This decrease in binding was not due to reduced NFI protein or NFI mRNA levels. Mutational analysis of individual and multiple NFI binding sites in the URR defined their role in TGF-β sensitivity of the promoter. Overexpression of the NFI family members in HKc/HPV16 decreased the ability of TGF-β to inhibit the URR. Since the oncoprotein Ski has been shown to interact with and increase the transcriptional activity of NFI and since cellular Ski levels are decreased by TGF-β treatment, we explored the possibility that Ski may provide a link between TGF-β signaling and NFI activity. Anti-NFI antibodies coimmunoprecipitated endogenous Ski in nuclear extracts from HKc/HPV16, confirming that NFI and Ski interact in these cells. Ski levels dramatically decreased upon TGF-β treatment of HKc/HPV16, and overexpression of Ski eliminated the ability of TGF-β to inhibit the URR. Based on these studies, we propose that TGF-β inhibition of HPV16 early gene expression is mediated by a decrease in Ski levels, which in turn dramatically reduces NFI activity.

An analysis of the human chromosome 22 genomic sequence shows that both Z-DNA forming regions (ZDRs) and promoter sites for nuclear factor-I (NFI) are correlated with the locations of known and predicted genes across the chromosome and accumulate around the transcriptional start sites of the known genes. Thus, the occurrence of Z-DNA across human genomic sequences mirrors that of a known eukaryotic transcription factor. In addition, 43 of the 383 fully annotated chromosomal genes have ZDRs within 2 nucleosomes upstream of strong NFIs. This suggests a distinct class of human genes that may potentially be transcriptionally regulated by a mechanism that couples Z-DNA with NFI activation, similar to the mechanism previously elucidated for the human colony stimulation factor-I promoter [Liu et al. (2001) Cell, 106, 309–318]. The results from this study will facilitate the design of experimental studies to test the generality of this mechanism for other genes in the cell.

Surfactant protein C (SP-C; Sftpc) gene expression is restricted to pulmonary type II epithelial cells. The proximal SP-C promoter region contains critical binding sites for nuclear factor I (NFI) and thyroid transcription factor 1 (TTF-1; also called Nkx2.1). To test the hypothesis that NFI isoforms interact with TTF-1 to differentially regulate SP-C transcription, we performed transient transfection assays in JEG-3 cells, a choriocarcinoma cell line with negligible endogenous NFI or TTF-1 activity. Cotransfection of NFI family members with TTF-1 induced synergistic activation of the SP-C promoter that was further enhanced by p300. TTF-1 directly interacts with the conserved DNA binding and dimerization domain of all NFI family members in coimmunoprecipitation and mammalian two-hybrid experiments. To determine whether SP-C expression is regulated by NFI in vivo, a chimeric fusion protein containing the DNA binding and dimerization domain of NFI-A and the Drosophila engrailed transcriptional repression domain (NFIen) was conditionally expressed in mice under control of a doxycycline-inducible transgene. Induction of NFIen in a subset of type II cells inhibited SP-C gene expression without affecting expression of TTF-1 in doxycycline-treated double-transgenic mice. Taken together, these findings support the hypothesis that NFI family members interact with TTF-1 to regulate type II cell function.

Transcription factors of the SMAD family relay signals from cell surface receptors to the nucleus in response to TGF-beta related soluble factors. Members of the nuclear factor I/CAAT box binding family (NFI/CTF) have been implicated as regulators of diverse biological processes such as adenovirus replication and transcription of TGF-responsive genes. There are highly conserved DNA binding domains in SMAD and NFI/CTF transcription factors that allow sequence specific DNA binding for members of each family. However, no homology relationship has been established for the DNA binding domains present in these families. For a better understanding of the structure and evolution of SMAD genes, we carried out a sensitive PSI-BLAST database search. This revealed significant similarities between the DNA binding domains of SMADs and NFI/CTF transcription factors. Enhanced graphic matrix analysis and multiple sequence alignment of the amino acid sequences of the SMAD and NFI/CTF DNA binding domains also show that these two classes of domains share considerable structural similarity. These results strongly suggest that these two classes of factors share a homologous DNA binding domain presumably resulting from a common ancestry. In contrast, the C-terminal transcription modulation domains of both SMAD and NFI/CTF families do not show any sequence similarity. Based on the structural relationship of their DNA binding domains, we propose that the SMAD and NFI/CTF transcription factors belong to new superfamily of genes.

The CCAAT-binding transcription factor (CTF)/nuclear factor I (NF-I) group of cellular DNA-binding proteins recognizes the sequence GCCAAT and is implicated in eukaryotic transcription, as well as DNA replication. Molecular analysis of human CTF/NF-I cDNA clones revealed multiple mRNA species that contain alternative coding regions, apparently as a result of differential splicing. Expression and functional analysis established that individual gene products can bind to GCCAAT recognition sites and serve as both promoter-selective transcriptional activators and initiation factors for DNA replication. The interaction between CTF2 and p53/p73 was shown to modulate their ability to regulate transcription of their respective target genes. In the present paper, we report that p53 down-regulates the activity of the high mobility group 1 (HMG1) gene promoter, whereas p73α up-regulates the activity of this promoter. Furthermore, CTF2 transactivates p53-induced p21 promoter activity, but inhibits p73α-induced p21 promoter activity. Using deletion mutants, we found that the DNA-binding domains of both p53 and p73α are required for physical interaction with CTF2 via the regions between amino acid residues 161 and 223, and 228 and 312 respectively. CTF2 enhances the DNA-binding activity of p53 and inhibits the DNA-binding activity of p73α. These results provide novel information on the functional interplay between CTF2 and p53/p73 as important determinants of their function in cell proliferation, apoptosis, DNA repair and cisplatin resistance.

Gene expression of the internal and long terminal repeat promoters of the spuma retrovirus is specifically activated by the transactivator Bel1, the key regulator of viral gene expression. Bel1 directly binds to and activates DNA target sites of viral promoters and those of distinct cellular genes. To determine the contribution of cellular transcription factors to viral transactivation, the viral internal promoter (IP) was analyzed by transient expression, electrophoretic mobility shift assays), and supershifts. Here we report that Bel1-mediated transactivation of the full-length and shortened versions of the Bel1 response element (BRE) were repressed by nuclear factor I (NFI). Electrophoretic mobility shift assays using nuclear extracts from transfected 293T cells revealed that different DNA-protein complexes consisting of DNA target sites of NFI and Bel1 proteins were formed. The specificity of the repressor and transactivator DNA binding was shown by NFI- and Bel1-specific antibodies that led to supershifts of the different nuclear protein-oligodeoxynucleotide complexes. The specificity of the complexes was confirmed by using unlabeled, shortened, and mutated IP.BRE oligodeoxynucleotides in competition experiments with the authentic IP.BRE. Cotransfection of the infectious spumavirus DNA genome with a human NFI-X1 expression plasmid into cell cultures greatly reduced the expression of viral structural and Bel1 proteins. These data demonstrate the relevance of NFI-mediated repression of Bel1-driven transactivation in vivo.

The Nuclear Factor I-X (NFIX) is a member of the nuclear factor I (NFI) family proteins, which are implicated as site-specific DNA-binding proteins and is deleted or mutated in a subset of patients with Sotos-like overgrowth syndrome and in patients with Marshall–Smith syndrome. We evaluated an additional patient with clinical features of Sotos-like syndrome by sequencing analysis of the NFIX gene and identified a 21 nucleotide in frame deletion predicting loss of 7 amino acids in the DNA-binding/dimerization domain of the NFIX protein. The deleted residues are all evolutionally conserved amino acids. The present report further confirms that mutations in DNA-binding/dimerization domain cause haploinsufficiency of the NFIX protein and strongly suggests that in individuals with Sotos-like features unrelated to NSD1 changes genetic testing of NFIX should be considered.

Expression of a 74-kDa nuclear factor I (NFI) protein is triggered in early involution in the mouse mammary gland, and its expression correlates with enhanced occupation of a twin (NFI) binding element in the clusterin promoter, a gene whose transcription is induced at this time (Furlong, E. E., Keon, N. K., Thornton, F. D., Rein, T., and Martin, F. (1996) J. Biol. Chem. 271, 29688-29697). We now identify this 74-kDa NFI as an NFIC isoform based on its interaction in Western analysis with two NFIC-specific antibodies. A transition from the expression of a 49-kDa NFIC in lactation to the expression of the 74-kDa NFIC in early involution is demonstrated. We show that the 74-kDa NFIC binds specifically to concanavalin A (ConA) and that this binding can be reversed by the specific ConA ligands, methyl alpha-D-mannopyranoside and methyl alpha-D-glucopyranoside. In addition, its apparent molecular size was reduced to approximately 63 kDa by treatment with the peptide N-glycosidase. The 49-kDa lactation-associated NFIC did not bind ConA nor was it affected by peptide N-glycosidase. Tunicamycin, a specific inhibitor of N-glycosylation, blocked formation of the 74-kDa NFI in involuting mouse mammary gland in vivo when delivered from implanted Elvax depot pellets. Finally, the production of the ConA binding activity could be reiterated in "mammospheres" formed from primary mouse mammary epithelial cells associated with a laminin-rich extracellular matrix. Synthesis of the 74-kDa NFIC was also inhibited in this setting by tunicamycin. Thus, involution triggers the production of an NFIC isoform that is post-translationally modified by N-glycosylation. We further show, by using quantitative competitive reverse transcriptase-PCR, that there is increased expression of the major mouse mammary NFIC mRNA transcript, mNFIC2, in early involution, suggesting that the involution-associated change in NFIC expression also has a transcriptional contribution.

Cyclin-dependent kinases are either post-transcriptionally regulated by interacting with cyclins and cyclin-dependent kinase inhibitors or are transcriptionally regulated by transcription factors, but the latter mechanism has not been extensively investigated. Dysregulated transcription factors resulting from aberrantly expressed microRNAs play critical roles in tumor development and progression. Our previous work identified miR-365 as an oncogenic microRNA that promotes the development of cutaneous squamous cell carcinoma via repression of cyclin-dependent kinase 6, while miR-365 also targets nuclear factor I/B. However, the underlying mechanism(s) of the interaction between nuclear factor I/B and cyclin-dependent kinase 6 are unclear. In this work, we demonstrate that miR-365-regulated nuclear factor I/B transcriptionally inhibits cyclin-dependent kinases 6 and 4 by binding to their promoter regions. In vivo and in vitro experiments demonstrate that the loss of nuclear factor I/B after miR-365 expression or treatment with small interfering RNAs results in the upregulation of cyclin-dependent kinases 6 and 4. This upregulation, in turn, enhances the phosphorylation of retinoblastoma protein and tumor progression. Characterizing this transcriptional repression of cyclin-dependent kinases 6 and 4 by nuclear factor I/B contributes to the understanding of the transcriptional regulation of cyclin-dependent kinases by transcription factors and also facilitates the development of new therapeutic regimens to improve the clinical treatment of cutaneous squamous cell carcinoma.

Aberrant expression of microRNAs plays vital roles in tumor development and progression. As transcription factors (TFs) are the critical components of signaling cascades, specific targeting effects of microRNAs to specific TFs may determine the role of microRNAs in different cancers. In this study, we identified Nuclear Factor I/B (NFIB) as one of the targets of miR-365 which was previously verified as an onco-miR in cutaneous squamous cell carcinoma (CSCC). Down-regulation of NFIB was a general feature in both CSCC cell lines and tumors from patients which show drastically up-regulated miR-365 expression levels. The siRNA-based knockdown of NFIB mimic the carcinogenic transformation of normal cells by ectopically expression of miR-365 which indicates depletion of NFIB is necessary for miR-365 to exert its pro-carcinogenic function. NFIB may represent a functional barrier targeted by miR-365 to the development of CSCC. Further studies also discovered a conserved feedback regulatory circuitry formed by NFIB and miR-365 in CSCC development which may be potentially utilized as therapeutic target to improve the clinical CSCC treatment.

The activity of the nuclear factor I/CCAAT transcription factor (NFI/CTF) is negatively regulated by oxidative stress. The addition of relatively high (millimolar) H2O2 concentrations inactivates cellular NFI DNA-binding activity whereas lower concentrations can repress NFI/CTF transactivating function. We have investigated the mechanism of this regulation using Gal4 fusion proteins and transfection assays. We show that micromolar H2O2 concentrations repress the transactivating domain of NFI/CTF in a dose-dependent manner and are less or not active on other transcription factors' transactivating domains. Studies using deletions and point mutations pointed to the critical role of Cys-427. Indeed, when this cysteine is mutated into a serine, the repression by H2O2 is totally blunted. Mutation of other cysteine, serine and tyrosine residues within the transactivating domain had no clear effect on the repression by H2O2. Finally, treatment of cells with the thiol-alkylating reagent N-ethylmaleimide leads to a decrease in the transactivating function, which is dependent on Cys-427. This study shows that transactivating domains of transcription factors can constitute very sensitive targets of oxidative stress and highlights the critical role of these domains.

DNase I footprint analysis of the human placental lactogen-A (hPL-A) promoter using nuclear extracts from purified human trophoblast cells and BeWo choriocarcinoma cells revealed five protected regions within the proximal 325 bp. Two of the protected regions, FP4 (−289–−267) and FP5 (−167–−154), are homologous to regions on the human growth hormone (hGH) promoter that bind transcription factors AP-2 and/or NFI. Competitive gel shift assays and supershift assays indicated that FP4 forms complexes with activator protein-2 (AP-2) and nuclear factor I (NFI), while FP5 forms a complex with AP-2 alone. In transient transfection studies in human trophoblast cells, hPL promoter constructs containing point mutations in the AP-2 binding sites of FP4 and/or FP5 were 60–80% less active than plasmids containing the wild-type promoter. A mutation in the NFI binding site of FP4, however, had little effect on promoter activity in these cells. Overexpression of AP-2 in HepG2 cells co-transfected with the wild-type hPL promoter resulted in a significant increase in promoter activity. Taken together, these findings suggest a critical role for AP-2 in the regulation of hPL gene expression.

Cytochrome P450 1A1 (CYP1A1), like many monooxygenases, can produce reactive oxygen species during its catalytic cycle. Apart from the well-characterized xenobiotic-elicited induction, the regulatory mechanisms involved in the control of the steady-state activity of CYP1A1 have not been elucidated. We show here that reactive oxygen species generated from the activity of CYP1A1 limit the levels of induced CYP1A1 mRNAs. The mechanism involves the repression of the CYP1A1 gene promoter activity in a negative-feedback autoregulatory loop. Indeed, increasing the CYP1A1 activity by transfecting CYP1A1 expression vectors into hepatoma cells elicited an oxidative stress and led to the repression of a reporter gene driven by the CYP1A1 gene promoter. This negative autoregulation is abolished by ellipticine (an inhibitor of CYP1A1) and by catalase (which catalyzes H2O2 catabolism), thus implying that H2O2 is an intermediate. Down-regulation is also abolished by the mutation of the proximal nuclear factor I (NFI) site in the promoter. The transactivating domain of NFI/CTF was found to act in synergy with the arylhydrocarbon receptor pathway during the induction of CYP1A1 by 2,3,7,8-tetrachloro-p-dibenzodioxin. Using an NFI/CTF-Gal4 fusion, we show that NFI/CTF transactivating function is decreased by a high activity of CYP1A1. This regulation is also abolished by catalase or ellipticine. Consistently, the transactivating function of NFI/CTF is repressed in cells treated with H2O2 ,a novel finding indicating that the transactivating domain of a transcription factor can be targeted by oxidative stress. In conclusion, an autoregulatory loop leads to the fine tuning of the CYP1A1 gene expression through the down-regulation of NFI activity by CYP1A1-based H2O2 production. This mechanism allows a limitation of the potentially toxic CYP1A1 activity within the cell. Cytochrome P450 monooxygenases are drug-metabolizing enzymes that play a major role in the detoxification and elimination of hydrophobic xenobiotics. Paradoxically, these enzymes also generate reactive metabolites which can form DNA adducts and lead to mutations (26). CYP1A1 is a ubiquitous member of the P450 superfamily which is among the products

The proto-oncoprotein Bcl-3 is a member of the IkappaB family and is present predominantly in the nucleus. To gain insight into specific nuclear functions of Bcl-3 we have isolated proteins that interact with its ankyrin repeat domain. Using the yeast two-hybrid-system we identified four novel binding partners of Bcl-3 in addition to NF-kappaB p50 and p52, previously known to associate with Bcl-3. The novel Bcl-3 interactors Jab1, Pirin, Tip60 and Bard1 are nuclear proteins which also bind to other transcription factors including c-Jun, nuclear factor I (NFI), HIV-1 Tat or the tumor suppressor and PolII holoenzyme component Brca1, respectively. Bcl-3, p50, and either Bard1, Tip60 or Pirin are sequestered into quarternary complexes on NF-kappaB DNA binding sites, whereas Jab1 enhances p50-Bcl-3-DNA complex formation. Furthermore, the histone acetylase Tip60 enhances Bcl-3-p50 activated transcription through an NF-kappaB binding site, indicating that quarternary complexes containing Bcl-3 interactors modulate NF-kappaB driven gene expression. These data implicate Bcl-3 as an adaptor between NF-kappaB p50/p52 and other transcription regulators and suggest that its gene activation function may at least in part be due to recruitment of the Tip60 histone actetylase.

The mineralocorticoid receptor (MR) and glucocorticoid receptor (GR) belong to the steroid/thyroid hormone superfamily of ligand-induced transcription factors. Both activate the human Na/K ATPase alpha1 and beta1 genes transcriptionally. To assess the role of the transcription factor Sp1 and the nuclear factor I (NF-I), in MR- and GR-mediated gene expression using the human Na/K ATPase beta1 full-length promoter, we have examined the functions of Sp-I and NF-I functions in two different cell lines, COS-1 and T-84. By transient transfections we have shown that Sp-I significantly enhances MR and GR expression, whereas NF-I had negligible effect. We propose that the transcriptional enhancement could be through a direct interaction physically between MR or GR with Sp1 that allows other factors to bind the responsive element resulting in synergistic upregulation of transcription.

Transcription of the surfactant protein-C (SP-C) gene is restricted to Type II epithelial cells in the adult lung. We have shown previously that the 0.32-kilobase pair (kb) mouse SP-C promoter is functional in transient transfection assays of the lung epithelial cell-derived cell line, MLE-15, and that thyroid transcription factor 1 (TTF-1) transactivates promoter activity. The 0.32-kb SP-C promoter can be separated into a proximal promoter region (-230 to +18) and an enhancer region (-318 to -230). Three DNase I footprints were mapped in the promoter region (C1 through C3) and two in the enhancer region (C4 and C5). We now show that nuclear factor I (NFI) family members bind to both individual NFI half-sites in footprints C1, C3, and C5, and to a composite site in footprint C4 by competition gel retardation and antibody supershift analyses. Mutational analysis of the 0.32-kb mouse SP-C promoter and transient transfection of MLE-15 cells demonstrated that the NFI binding sites are required for promoter activity in this cell type. Site-specific mutation of the proximal or distal NFI sites drastically reduced transactivation by a co-transfected NFI-A expression vector in HeLa cells. These data indicate that NFI family member(s), binding to sites in both the promoter and enhancer regions, regulate SP-C gene expression in a process independent of TTF-1.