Pioneer factors for DNA replication initiation in metazoans.

Precise DNA replication is fundamental for genetic inheritance. In eukaryotes, replication initiates at multiple origins that are first "licensed" and subsequently "fired" to activate DNA synthesis. Despite the success in identifying origins with specific DNA motifs in Saccharomyces cerevisiae, no consensus sequence or sequences with a predictive value of replication origins have been recognized in metazoan genomes. Rather, epigenetic rules and chromatin structures are believed to play important roles in governing the selection and activation of replication origins. We propose that replication initiation is facilitated by a group of sequence-specific "replication pioneer factors," which function to increase chromatin accessibility and foster a chromatin environment that is conducive to the loading of the prereplication complex. Dysregulation of the function of these factors may lead to gene duplication, genomic instability, and ultimately the occurrence of pathological conditions such as cancer.

NFIB-MLL1 complex is required for the stemness and Dlx5-dependent osteogenic differentiation of C3H10T1/2 mesenchymal stem cells.

Despite significant progress in our understanding of the molecular mechanism of mesenchymal stem cell (MSC) differentiation, less is known about the factors maintaining the stemness and plasticity of MSCs. Here, we show that the NFIB-MLL1 complex plays key roles in osteogenic differentiation and stemness of C3H10T1/2 MSCs. We find that depletion of either NFIB or MLL1 results in a severely hampered osteogenic potential and failed activation of key osteogenic transcription factors, such as Dlx5, Runx2, and Osx, following osteogenic stimuli. In addition, the NFIB-MLL1 complex binds directly to the promoter of Dlx5, and exogenous expression of Myc-Dlx5, but not the activation of either the BMP- or the Wnt-signaling pathway, is sufficient to restore the osteogenic potential of cells depleted of NFIB or MLL1. Moreover, chromatin immunoprecipitation (ChIP) and ChIP-sequencing analysis showed that the NFIB-MLL1 complex mediates the deposition of trimethylated histone H3K4 at both Dlx5 and Cebpa, key regulator genes that function at the early stages of osteogenic and adipogenic differentiation, respectively, in uncommitted C3H10T1/2 MSCs. Surprisingly, the depletion of either NFIB or MLL1 leads to decreased trimethylated histone H3K4 and results in elevated trimethylated histone H3K9 at those developmental genes. Furthermore, gene expression profiling and ChIP-sequencing analysis revealed lineage-specific changes in chromatin landscape and gene expression in response to osteogenic stimuli. Taken together, these data provide evidence for the hitherto unknown role of the NFIB-MLL1 complex in the maintenance and lineage-specific differentiation of C3H10T1/2 MSCs and support the epigenetic regulatory mechanism underlying the stemness and plasticity of MSCs.

A phase 2 study of MK-2206 in patients with incurable adenoid cystic carcinoma (Alliance A091104).

BACKGROUND: Recurrent/metastatic adenoid cystic carcinoma (ACC) is a rare, incurable disease. MYB is a putative oncogenic driver in ACC that is often overexpressed through an MYB-NFIB rearrangement. The authors hypothesized that AKT inhibition with the allosteric inhibitor MK-2206 could decrease MYB expression and induce tumor regression in patients with incurable ACC (ClinicalTrials.gov identifier NCT01604772). METHODS: Patients with progressive, incurable ACC were enrolled and received MK-2206 150 mg weekly; escalation to 200 mg was allowed. The primary end point was confirmed response. Secondary end points were progression-free survival, overall survival, and safety. An exploratory analysis evaluating the effect of MK-2206 on MYB expression was conducted in a subset of patients. RESULTS: Sixteen patients were enrolled, and 14 were evaluable for efficacy. No confirmed responses were observed. Thirteen patients had stable disease, and one had disease progression as their best response. The median progression-free survival was 9.7 months (95% CI, 3.8-11.8 months), and the median overall survival was 18.0 months (95% CI, 11.8-29.9 months). Nine of 16 patients (56%) had at least one grade 3 treatment-related adverse event, and the most common were rash (38%), fatigue (19%), decreased lymphocyte count (13%), and hyperglycemia (13%). Twelve of 14 tumors (86%) had detectable MYB expression by immunohistochemistry, and seven of 14 tumors (50%) had an MYB-NFIB gene rearrangement. Serial biopsies revealed decreased MYB levels with MK-2206 in four of five patients. CONCLUSIONS: MK-2206 failed to induce clinical responses in patients with incurable ACC. AKT inhibition may diminish MYB protein levels, although the effect was highly variable among patients. Novel approaches to target MYB in ACC are needed.

Adenoid cystic carcinoma of the Bartholin's gland is underpinned by MYB- and MYBL1- rearrangements.

OBJECTIVE: Adenoid cystic carcinoma (AdCC) of the Bartholin's gland (AdCC-BG) is a very rare gynecologic vulvar malignancy. AdCC-BGs are slow-growing but locally aggressive and are associated with high recurrence rates. Here we sought to characterize the molecular underpinning of AdCC-BGs. METHODS: AdCC-BGs (n = 6) were subjected to a combination of RNA-sequencing, targeted DNA-sequencing, reverse-transcription PCR, fluorescence in situ hybridization (FISH) and MYB immunohistochemistry (IHC). Clinicopathologic variables, somatic mutations, copy number alterations and chimeric transcripts were assessed. RESULTS: All six AdCC-BGs were biphasic, composed of ductal and myoepithelial cells. Akin to salivary gland and breast AdCCs, three AdCC-BGs had the MYB::NFIB fusion gene with varying breakpoints, all of which were associated with MYB overexpression by IHC. Two AdCC-BGs were underpinned by MYBL1 fusion genes with different gene partners, including MYBL1::RAD51B and MYBL1::EWSR1 gene fusions, and showed MYB protein expression. Although the final AdCC-BG studied had MYB protein overexpression, no gene fusion was identified. AdCC-BGs harbored few additional somatic genetic alterations, and only few mutations in cancer-related genes were identified, including GNAQ, GNAS, KDM6A, AKT1 and BCL2, none of which were recurrent. Two AdCC-BGs, both with a MYB::NFIB fusion gene, developed metastatic disease. CONCLUSIONS: AdCC-BGs constitute a convergent phenotype, whereby activation of MYB or MYBL1 can be driven by the MYB::NFIB fusion gene or MYBL1 rearrangements. Our observations further support the notion that AdCCs, irrespective of organ site, constitute a genotypic-phenotypic correlation. Assessment of MYB or MYBL1 rearrangements may be used as an ancillary marker for the diagnosis of AdCC-BGs.

MiR-326-mediated overexpression of NFIB offsets TGF-ß induced epithelial to mesenchymal transition and reverses lung fibrosis.

Idiopathic Pulmonary Fibrosis (IPF) is a progressively fatal and incurable disease characterized by the loss of alveolar structures, increased epithelial-mesenchymal transition (EMT), and aberrant tissue repair. In this study, we investigated the role of Nuclear Factor I-B (NFIB), a transcription factor critical for lung development and maturation, in IPF. Using both human lung tissue samples from patients with IPF, and a mouse model of lung fibrosis induced by bleomycin, we showed that there was a significant reduction of NFIB both in the lungs of patients and mice with IPF. Furthermore, our in vitro experiments using cultured human lung cells demonstrated that the loss of NFIB was associated with the induction of EMT by transforming growth factor beta (TGF-ß). Knockdown of NFIB promoted EMT, while overexpression of NFIB suppressed EMT and attenuated the severity of bleomycin-induced lung fibrosis in mice. Mechanistically, we identified post-translational regulation of NFIB by miR-326, a miRNA with anti-fibrotic effects that is diminished in IPF. Specifically, we showed that miR-326 stabilized and increased the expression of NFIB through its 3'UTR target sites for Human antigen R (HuR). Moreover, treatment of mice with either NFIB plasmid or miR-326 reversed airway collagen deposition and fibrosis. In conclusion, our study emphasizes the critical role of NFIB in lung development and maturation, and its reduction in IPF leading to EMT and loss of alveolar structures. Our study highlights the potential of miR-326 as a therapeutic intervention for IPF. The schema shows the role of NFIB in maintaining the normal epithelial cell characteristics in the lungs and how its reduction leads to a shift towards mesenchymal cell-like features and pulmonary fibrosis. A In normal lungs, NFIB is expressed abundantly in the epithelial cells, which helps in maintaining their shape, cell polarity and adhesion molecules. However, when the lungs are exposed to factors that induce pulmonary fibrosis, such as bleomycin, or TGF-ß, the epithelial cells undergo epithelial to mesenchymal transition (EMT), which leads to a decrease in NFIB. B The mesenchymal cells that arise from EMT appear as spindle-shaped with loss of cell junctions, increased cell migration, loss of polarity and expression of markers associated with mesenchymal cells/fibroblasts. C We designed a therapeutic approach that involves exogenous administration of NFIB in the form of overexpression plasmid or microRNA-326. This therapeutic approach decreases the mesenchymal cell phenotype and restores the epithelial cell phenotype, thus preventing the development or progression of pulmonary fibrosis.

CircIQCH Contributes to the Progression of Breast Cancer by Elevating NFIB Through Decoying miR-139-5p.

Circular RNAs (circRNAs) are implicated in the progression of breast cancer (BC). However, the explorations on circRNA IQ motif containing H (circIQCH) in BC progression remain limited. Functional experiments were conducted using in vitro and murine xenograft model assays, respectively. Dual-luciferase reporter assay and RIP assay detected the associations among circIQCH, miR-139-5p, and nuclear factor IB (NFIB). CircIQCH was upregulated in BC, and the silencing of circIQCH repressed BC cell growth, metastasis, and autophagy, arrested cell cycle, promoted cell apoptosis in vitro, and blocked tumor growth in vivo. CircIQCH positively modulated NFIB expression by sponging miR-139-5p. Moreover, the deletion of miR-139-5p abated the action of circIQCH deficiency on BC cell malignant behaviors. Overexpression of miR-139-5p repressed the malignant characteristics of BC cells, while these impacts were abolished by elevating NFIB. Collectively, CircIQCH functioned as an oncogene in BC through upregulating NFIB expression by sponging miR-139-5p.

Transcriptional regulation of NDUFA4L2 by NFIB induces sorafenib resistance by decreasing reactive oxygen species in hepatocellular carcinoma.

Sorafenib is one a first-line therapeutic drugs for advanced hepatocellular carcinoma (HCC). However, only 30% of patients benefit from sorafenib due to drug resistance. We and other groups have revealed that nuclear factor I B (NFIB) regulates liver regeneration and carcinogenesis, but its role in drug resistance is poorly known. We found that NFIB was more upregulated in sorafenib-resistant SMMC-7721 cells compared to parental cells. NFIB knockdown not only sensitized drug-resistant cells to sorafenib but also inhibited the proliferation and invasion of these cells. Meanwhile, NFIB promoted the proliferation and invasion of HCC cells in vitro and facilitated tumor growth and metastasis in vivo. Knocking down NFIB synergetically inhibited tumor growth with sorafenib. Mechanically, gene expression profiling and subsequent verification experiments proved that NFIB could bind with the promoter region of a complex I inhibitor NDUFA4L2 and promote its transcription. Transcriptional upregulation of NDUFA4L2 by NFIB could thus inhibit the sorafenib-induced reactive oxygen species accumulation. Finally, we found that NFIB was highly expressed in HCC tissues, and high NFIB expression level was associated with macrovascular invasion, advanced tumor stage, and poor prognosis of HCC patients (n = 156). In summary, we demonstrated that NFIB could transcriptionally upregulate NDUFA4L2 to enhance both intrinsic and acquired sorafenib resistance of HCC cells by reducing reactive oxygen species induction.

The Landscape of MYB/MYBL1- and Peri-MYB/MYBL1-Associated Rearrangements in Adenoid Cystic Carcinoma.

Approximately 60% of adenoid cystic carcinoma (AdCC) cases are positive for MYB::NFIB or MYBL1::NFIB, whereas MYB/MYBL1 oncoprotein, a key driver of AdCC, is overexpressed in most cases. Juxtaposition of superenhancer regions in NFIB and other genes into the MYB/MYBL1 locus is an attractive oncogenic hypothesis for AdCC cases, either negative or positive for MYB/MYBL1::NFIB. However, evidence supporting this hypothesis is insufficient. We examined 160 salivary AdCC cases for rearrangements in MYB/MYBL1 loci and peri-MYB/MYBL1 areas (centromeric and telomeric areas of 10 Mb each) using formalin-fixed, paraffin-embedded tumor sections. For the detection of the rearrangements, we employed conventional fluorescence in situ hybridization split and fusion assays and a 5 Mb fluorescence in situ hybridization split assay. The latter is a novel assay that enabled us to detect any possible splits within a 5 Mb distance of a chromosome. We found MYB/MYBL1- and peri-MYB/MYBL1-associated rearrangements in 149/160 patients (93%). AdCC cases positive for rearrangements in MYB, MYBL1, the peri-MYB area, and the peri-MYBL1 area numbered 105 (66%), 20 (13%), 19 (12%), and 5 (3%), respectively. In 24 peri-MYB/MYBL1 rearrangement-positive cases, 14 (58%) were found to have a juxtaposition of the NFIB or RAD51B locus into the MYB/MYBL1 loci. On comparing with a tumor group positive for MYB::NFIB, a hallmark of AdCC, other genetically classified tumor groups had similar features of overexpression of the MYB transcript and MYB oncoprotein as detected by semiquantitative RT-qPCR and immunohistochemistry, respectively. In addition, clinicopathological and prognostic features were similar among these groups. Our study suggests that peri-MYB/MYBL1 rearrangements may be a frequent event in AdCC and may result in biological and clinicopathological consequences comparable to MYB/MYBL1 rearrangements. The landscape of MYB/MYBL1 and peri-MYB/MYBL1 rearrangements shown here strongly suggests that juxtaposition of superenhancers into MYB/MYBL1 or peri-MYB/MYBL1 loci is an alteration that acts as a key driver for AdCC oncogenesis and may unify MYB/MYBL1 rearrangement-positive and negative cases.

Molecular characterisation of tumours of the lacrimal apparatus.

AIMS: Malignant tumours of the lacrimal apparatus are rare and frequently show a poor prognosis, with no clear therapeutic standards. Characterisation of the genetic landscape of these rare tumours is sparse, and therefore therapeutics generally follow those of their common salivary gland counterparts. To further clarify the pathophysiology and discover potential therapeutic targets, we investigated the genetic landscape of eight tumours of the lacrimal apparatus. METHODS AND RESULTS: DNA and RNA sequencing were performed to identify genetic mutations and gene fusions. Immunohistochemistry, fluorescence in-situ hybridisation and reverse transcription-polymerase chain reaction followed by Sanger sequencing were performed to confirm the identified molecular alterations. Genetic alterations were detected in six tumours. Among five adenoid cystic carcinomas (ACC), four had confirmed alterations of MYB or MYBL1 genes, including a MYB::NFIB fusion, a MYBL1::NFIB fusion, a MYB amplification and a novel NFIB::THSD7B fusion. Mutations in genes encoding epigenetic modifiers, as well as NOTCH1, FGFR2 and ATM mutations, were also identified in ACCs. A carcinoma ex pleomorphic adenoma showed TP53 and CIC mutations and an amplification of ERBB2. A transitional cell carcinoma was associated with HPV16 infection. No genetic alteration was found for one adenocarcinoma, not otherwise specified. CONCLUSIONS: Our study highlights the variety of molecular alterations associated with lacrimal system tumours and emphasises the importance of molecular testing in these tumours, which can reveal potentially targetable mutations. Our results also reinforce the hypothesis of a common physiopathology of all ACCs, regardless of their primary location.

Marked intrafamilial variability of clinical and neuroimaging manifestations in NFIB-related developmental disorder.

NFIB belongs to the nuclear factor I (NFI) family of transcription factors that, by activating or repressing gene expression during embryogenesis, has a relevant role in the development of several organs including the brain. Heterozygous pathogenic variants of NFIB have recently been associated with developmental delay and mild-to-moderate intellectual disability, macrocephaly, nonspecific facial dysmorphisms, and corpus callosum dysgenesis. We identified a heterozygous missense variant in the NFIB gene in a 15-year-old boy with neurodevelopmental disorder and brain malformations, who inherited the variant from his substantially healthy mother presenting only minor physical and neuroanatomical defects.

Further characterization of NFIB-associated phenotypes: Report of two new individuals.

Nuclear Factor I B (NFIB) haploinsufficiency has recently been identified as a cause of intellectual disability (ID) and macrocephaly. Here we report on two new individuals carrying a microdeletion in the chromosomal region 9p23-p22.3 containing NFIB. The first is a 7-year 9-month old boy with developmental delays, ID, definite facial anomalies, and brain and spinal cord magnetic resonance imaging findings including periventricular nodular heterotopia, hypoplasia of the corpus callosum, arachnoid cyst in the left middle cranial fossa, syringomyelia in the thoracic spinal cord and distal tract of the conus medullaris, and a stretched appearance of the filum terminale. The second is a 32-year-old lady (the proband' mother) with dysmorphic features, and a history of learning disability, hypothyroidism, poor growth, left inguinal hernia, and panic attacks. Her brain magnetic resonance imaging findings include a dysmorphic corpus callosum, and a small cyst in the left choroidal fissure that marks the hippocampal head. Array-based comparative genomic hybridization identified, in both, a 232 Kb interstitial deletion at 9p23p22.3 including several exons of NFIB and no other known genes. Our two individuals add to the knowledge of this rare disorder through the addition of new brain and spinal cord MRI findings and dysmorphic features. We propose that NFIB haploinsufficiency causes a clinically recognizable malformation-ID syndrome.

Circ_0082182 upregulates the NFIB level via sponging miR-326 to promote oxaliplatin resistance and malignant progression of colorectal cancer cells.

Circular RNAs (circRNAs) are key regulators in tumor metastasis and drug resistance. This study was designed to investigate circ_0082182 function and mechanism in oxaliplatin (OXA) resistance and cancer progression of colorectal cancer (CRC). The circ_0082182, microRNA-326 (miR-326), and nuclear factor I B (NFIB) levels were quantified by reverse transcription-quantitative polymerase chain reaction (RT-qPCR). Cell sensitization was analyzed by Cell Counting Kit-8 assay. The proliferation ability was determined via EdU assay, and apoptosis was measured by flow cytometry. Transwell assay and wound healing assay were performed to assess cell invasion and migration. The protein level was examined through Western blot. The binding interaction was conducted via dual-luciferase reporter assay. Xenograft tumor assay was used to explore the circ_0082182 function in vivo. The circ_0082182 level was upregulated in OXA-resistant CRC samples and cells. Downregulation of circ_0082182 suppressed OXA resistance, proliferation, invasion, and migration but promoted apoptosis of OXA-resistant CRC cells. Circ_0082182 acted as a sponge for miR-326. The regulatory role of circ_0082182 was ascribed to the miR-326 sponging function. MiR-326 directly targeted NFIB to impede OXA resistance and cancer progression in CRC cells. NFIB level was regulated by circ_0082182 via sponging miR-326. Circ_0082182 promoted tumor growth in OXA-resistant xenograft tumor model through mediating the miR-326/NFIB axis. These data suggested that circ_0082182 elevated the NFIB expression to regulate OXA resistance and CRC progression by absorbing miR-326.

A Review of the Molecular Landscape of Adenoid Cystic Carcinoma of the Lacrimal Gland.

Adenoid cystic carcinoma (ACC) has a worldwide incidence of three to four cases per million population. Although more cases occur in the minor and major salivary glands, it is the most common lacrimal gland malignancy. ACC has a low-grade, indolent histological appearance, but is relentlessly progressive over time and has a strong proclivity to recur and/or metastasise. Current treatment options are limited to complete surgical excision and adjuvant radiotherapy. Intra-arterial systemic therapy is a recent innovation. Recurrent/metastatic disease is common due to perineural invasion, and it is largely untreatable as it is refractory to conventional chemotherapeutic agents. Given the rarity of this tumour, the molecular mechanisms that govern disease pathogenesis are poorly understood. There is an unmet, critical need to develop effective, personalised targeted therapies for the treatment of ACC in order to reduce morbidity and mortality associated with the disease. This review details the evidence relating to the molecular underpinnings of ACC of the lacrimal gland, including the MYB-NFIB chromosomal translocations, Notch-signalling pathway aberrations, DNA damage repair gene mutations and epigenetic modifications.

Exosomal miR-626 promotes the malignant behavior of oral cancer cells by targeting NFIB.

BACKGROUND: Tumor-derived exosomes, as emerging regulators of intercellular communication, are important for tumorigenesis and development in multiple tumors. The purpose of this study was to investigate whether exosomal miR-626 exists. More importantly, if exosomal miR-626 exists, the mechanism by which it is transferred into neighboring cancer cells and contributes to tumor progression needs to be clarified. METHODS AND RESULTS: The expression of miRNA and mRNA are analyzed by RT-qPCR. Proliferation, colony formation, wound healing, cell cycle are carried out to assess the function of exosomal miR-626. Furthermore, a xenograft experiment is utilized to conform the cancer-promoting role of exosomal miR-626 in oral cancer. Here, we showed that miR-626 is upregulated in oral cancer-derived exosomes and can be transferred between oral cancer cells. Exosomal miR-626 promotes cancer cell proliferation, colony formation, migration and G0/G1-to-S phase transition. Nuclear factor I/B (NFIB), a tumor suppressor gene in various cancers, was predicted to be a potential target of miR-626 by using three algorithms. Luciferase reporter assay data revealed that miR-626 can directly bind to the 3'-UTR of NFIB and subsequently suppress its expression and downstream signaling. Restoration of NFIB expression rescued the malignant phenotype induced by exosomal miR-626. In addition, exosomal miR-626 administration facilitated cancer growth in a xenograft tumor model, accompanied by downregulation of NFIB expression. CONCLUSIONS: Our data demonstrate that exosomal miR-626 can facilitate the development of oral cancer by inhibiting the expression of its target NFIB. Exosomal miR-626 might be a therapeutic target for oral cancer.

Sevoflurane inhibits the malignant phenotypes of glioma through regulating miR-146b-5p/NFIB axis.

PURPOSE: Sevoflurane is a common used inhaled anesthetic that was reported to regulate the progression of multiple cancers. Here, we aimed to investigate the function and regulatory mechanism underlying sevoflurane in glioma cells. METHODS: A172 and U251 cells were treated with different concentrations of sevoflurane. Colony formation, EdU satining and 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide (MTT), flow cytometry, and transwell assays were performed to evaluate cell proliferation, apoptosis, migration and invasion, respectively. Circ_VCAN, microRNA-146b-5p (miR-146b-5p) and nuclear factor I B (NFIB) expression levels were assessed by real-time quantitative PCR (RT-qPCR) or western blot. Bioinformatics analysis and dual-luciferase reporter assay were applied to evaluate the correlation between miR-146b-5p and circ_VCAN or NFIB. A xenograft glioma mice model was established to verify the effect of sevoflurane on tumor growth in vivo. RESULTS: Sevoflurane (Sev) inhibited proliferation, migration, invasion, and elevated apoptosis of A172 and U251 cells. Sevoflurane treatment inhibited the expression of circ_VCAN and NFIB, but elevated the expression of miR-146b-5p in glioma cells. Overexpression of circ_VCAN alleviated the inhibition effects of sevoflurane on the malignant phenotypes of glioma in vitro and in vivo. Besides, miR-146b-5p is a target of circ_VCAN and negatively regulated NFIB expression. Overexpression of miR-146b-5p partly reversed the effects of circ_VCAN in Sev-treated glioma cells. Furthermore, miR-146b-5p deletion enhanced glioma progression in sevoflurane treated glioma cells by targeting NFIB. Moreover, circ_VCAN could upregulate NFIB expression by sponging miR-146b-5p in Sev-treated glioma cells. CONCLUSION: Sevoflurane alleviated proliferation, migration and invasion, but enhanced apoptosis of glioma cells through regulating circ_VCAN/miR-146b-5p/NFIB axis.

CircATL2 enhances paclitaxel resistance of ovarian cancer via impacting miR-506-3p/NFIB axis.

Circular RNAs (circRNAs) play vital regulatory roles in the development of ovarian cancer (OC). However, the functions of circRNA Atlastin GTPase 2 (circATL2) in paclitaxel (PTX) resistance of OC are still unclear. As a result, circATL2 was upregulated in PTX-resistant OC tissues and cells. CircATL2 knockdown reduced IC50 of PTX, inhibited colony formation ability and promoted cell cycle arrest and apoptosis in PTX-resistant OC cells. Silencing of circATL2 restrained PTX resistance in vivo. Furthermore, miR-506-3p could be targeted by circATL2 and miR-506-3p inhibition reversed the impacts of circATL2 knockdown on PTX resistance and cell progression in PTX-resistant OC cells. NFIB was identified as the target of miR-506-3p. MiR-506-3p overexpression suppressed PTX resistance and malignant behaviors of PTX-resistant OC cells, with NFIB elevation rescued the impacts. To summarize, circATL2 promoted the resistance of OC to PTX by sponging miR-506-3p to upregulate NFIB expression, providing a new sight in chemoresistance of OC.

Transcriptional Regulation of RIP2 Gene by NFIB Is Associated with Cellular Immune and Inflammatory Response to APEC Infection.

Avian pathogenic E. coli (APEC) can cause localized or systemic infection, resulting in large economic losses per year, and impact health of humans. Previous studies showed that RIP2 (receptor interacting serine/threonine kinase 2) and its signaling pathway played an important role in immune response against APEC infection. In this study, chicken HD11 cells were used as an in vitro model to investigate the function of chicken RIP2 and the transcription factor binding to the RIP2 core promoter region via gene overexpression, RNA interference, RT-qPCR, Western blotting, dual luciferase reporter assay, CHIP-PCR, CCK-8, and flow cytometry assay following APEC stimulation. Results showed that APEC stimulation promoted RIP2 expression and cells apoptosis, and inhibited cells viability. Knockdown of RIP2 significantly improved cell viability and suppressed the apoptosis of APEC-stimulated cells. Transcription factor NFIB (Nuclear factor I B) and GATA1 (globin transcription factor 1) binding site was identified in the core promoter region of RIP2 from -2300 bp to -1839 bp. However, only NFIB was confirmed to be bound to the core promoter of RIP2. Overexpression of NFIB exacerbated cell injuries with significant reduction in cell viability and increased cell apoptosis and inflammatory cytokines levels, whereas opposite results were observed in NFIB inhibition treatment group. Moreover, RIP2 was up-regulated by NFIB overexpression, and RIP2 silence mitigated the effect of NFIB overexpression in cell apoptosis, inflammation, and activation of NFκB signaling pathways. This study demonstrated that NFIB overexpression accelerated APEC-induced apoptosis and inflammation via up-regulation of RIP2 mediated downstream pathways in chicken HD11 cells.

NFIB induces functional astrocytes from human pluripotent stem cell-derived neural precursor cells mimicking in vivo astrogliogenesis.

The ability to generate astrocytes from human pluripotent stem cells (hPSCs) offers a promising cellular model to study the development and physiology of human astrocytes. The extant methods for generating functional astrocytes required long culture periods and there remained much ambiguity on whether such paradigms follow the innate developmental program. In this report, we provided an efficient and rapid method for generating physiologically functional astrocytes from hPSCs. Overexpressing the nuclear factor IB in hPSC-derived neural precursor cells induced a highly enriched astrocyte population in 2 weeks. RNA sequencing and functional analyses demonstrated progressive transcriptomic and physiological changes in the cells, resembling in vivo astrocyte development. Further analyses substantiated previous results and established the MAPK pathway necessary for astrocyte differentiation. Hence, this differentiation paradigm provides a prospective in vitro model for human astrogliogenesis studies and the pathophysiology of neurological diseases concerning astrocytes.

NFIB Haploinsufficiency Is Associated with Intellectual Disability and Macrocephaly.

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.

Nfib promotes chondrocyte proliferation and inhibits differentiation by mildly regulating Sox9 and its downstream genes.

BACKGROUND: Chondrocyte proliferation and differentiation play pivotal roles in regulating cartilage formation, endochondral bone formation, and repair. Cartilage damage and underdevelopment may cause severe joint diseases. Various transcription factors regulate cartilage development. Nuclear factor 1 B (Nfib) is a transcription factor that plays a regulatory role in various organs. However, the effect and mechanism of Nfib on the proliferation and differentiation of chondrocytes in cartilage are still largely unknown. METHODS AND RESULTS: In the present study, we investigated the gene expression patterns in primary chondrocytes with Nfib overexpression or silencing by RNA sequencing (RNA-seq) technology. The results showed that Nfib overexpression significantly up-regulated genes that are related to chondrocyte proliferation and extracellular matrix (ECM) synthesis and significantly down-regulated genes related to chondrocyte differentiation and ECM degradation. However, with Nfib silencing, the genes involved in promoting chondrocyte differentiation were significantly up-regulated, whereas those involved in promoting chondrocyte proliferation were significantly down-regulated. Furthermore, quantitative real-time PCR (qRT-PCR), western blot, alcian blue staining and immunofluorescence staining assays further confirmed that Nfib potentially promotes chondrocyte proliferation and extracellular synthesis but inhibits differentiation. CONCLUSIONS: The molecular mechanism of Nfib in promoting chondrocyte proliferation and inhibiting differentiation was probably achieved by stimulating Sox9 and its downstream genes. Thus, this study adds new insights regarding the underlying molecular mechanism of transcriptional regulation in cartilage.