Human I-mfa Domain-containing Protein Research Articles

Abstract 2902: Epithelial-type CD133+ stem-like lung cancer cells emerge higher drug resistance through MDFIC-mediated Wnt/β-catenin signaling pathway

Abstract Background: The epithelial-to-mesenchymal transition (EMT) has been described to promote drug resistance and cancer stem cell (CSC) properties. Many studies demonstrated that a major proportion of circulating tumor cell (CTC) exhibits EMT and CSC characteristics. A recent finding revealed the presence of epithelial type (EpCAM+) CTC was associated with poor outcome, whereas the mesenchymal type (EpCAM-) CTC were not. We therefore hypothesize that the epithelial-type CSC may exhibit higher drug resistant ability. Methods: We isolated epithelial type (E+) and mesenchymal type (E-) CD133+ cells and CD133- cells from PC14 lung cancer cell line by fluorescence assisted cell sorting. We used western blot, QPCR, immunofluorescence, sphere formation assay and tumor xenograft assay to characterize the 4 subpopulations. The drug resistant ability was determined by cell viability assay and in vivo drug response assays. The drug resistant signature was identified by comparing the gene expression profile from four subpopulations and Gene Expression Omnibus (GEO) database. Knockdown and overexpression of MDFIC in PC14 subpopulations were established by using lentivirus vectors. Immunoprecipitation and subcellular fractionation were performed for drug resistant mechanism investigation. Results: The epithelial type PC14 CD133+ cells (E+/CD133+ subpopulation) exhibited higher sphere formation ability and was more resistant to the treatment of chemotherapy agents compared to the mesenchymal type CD133+ cells (E-CD133+ subpopulation) in vitro and in vivo. Gene expression profiling showed 86 genes were bioinformatically predicted as drug resistant signature and were correlated with the disease free survival of the patients with lung cancer. Among these genes, the mRNA level of 20 genes were significantly related to the patient’s prognosis in the GSE31210 dataset. Human I-mfa domain-containing protein (MDFIC) was highly expressed in E+/CD133+ subpopulation. Knockdown and overexpression of MDFIC modulates drug resistance ability in cancer cells. MDFIC increased the level of free β-catenin through binding and stabilizing the axin-GSK3-β-β-catenin destruction complex and increased the transcriptional activity of Wnt/β-catenin signaling. Conclusion: The epithelial-type CD133+ stem-like lung cancer cells are more resistant to the chemotherapy through MDFIC-mediated Wnt/β-catenin signaling activation. Citation Format: Chao-Ju Chen, Chih-Jen Yang, Ming-Shyang Huang, Yu-Peng Liu. Epithelial-type CD133+ stem-like lung cancer cells emerge higher drug resistance through MDFIC-mediated Wnt/β-catenin signaling pathway [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2902. doi:10.1158/1538-7445.AM2017-2902

RELT binds HIC and induces apoptosis by a mechanism distinct from TNFR1.

Abstract Receptor Expressed in Lymphoid Tissues (RELT) is a human Tumor Necrosis Factor Receptor (TNFR) that is expressed most prominently in cells and tissues of the hematopoietic system. RELT has two identified homologous binding partners RELL1 and RELL2. This study sought to further elucidate the function of RELT by identifying novel protein interactions with RELT family members. Human I-mfa domain-containing protein (HIC), also known as MDFIC, was identified in a yeast two-hybrid genetic screen using RELL1 as bait. HIC is a transcription factor that was initially identified to differentially regulate HTLV and HIV gene expression. HIC is now known to regulate many other cellular processes and the hic gene is located proximally to regions of chromosome 7 (7q31.1) frequently lost in Acute Myeloid Leukemia (AML) patients. HIC physically interacts with both RELT and RELL1, as determined by in vitro co-immunoprecipitations. HIC co-localizes with RELL1 at the plasma membrane and co-localizes with RELT in intracellular compartments. Deletion mutants of RELT were utilized to determine regions of RELT required for both the activation of p38 and the induction of apoptosis in HEK-293 cells, two previously described functions of RELT. Co-immunoprecipitation experiments utilizing the deletion mutants indicate that regions of RELT proximal to the plasma membrane are sufficient for physical interaction with HIC. Overexpression of RELT induces cleavage of PARP and Caspase-3 as determined by western blotting. Interestingly, induction of apoptotic morphology by RELT overexpression was not prevented if signaling by FADD or Caspase-8 was blocked, indicating RELT induces apoptosis by a pathway distinct from death-domain containing TNFRs such as TNFR1.

Abstract 2264: HIC (MDFIC) is a protein that interacts and colocalizes with the RELT family of TNFRs

Abstract Tumor necrosis factor receptors (TNFRs) have been recognized to be critically important for cellular processes such as apoptosis, cell proliferation and inflammatory responses. Aberrations in TNFR signaling have been implicated in a wide range of human diseases such as cancer, autoimmune diseases and developmental abnormalities. Receptor Expressed in Lymphoid Tissues (RELT) is a recently identified human TNFR that has two identified homologous binding partners RELL1 and RELL2. This study sought to further elucidate the function of RELT by identifying novel protein interactions with RELT family members. Human I-mfa domain-containing protein (HIC), also known as MDFIC, was identified in a genetic screen for novel RELL1 binding partners. HIC is a transcription factor that was initially identified to differentially regulate HTLV and HIV gene expression. HIC is now known to regulate many other cellular processes and the hic gene is located proximally to regions of chromosome 7 (7q31.1) frequently lost in Acute Myeloid Leukemia (AML) patients. Our current studies indicate that HIC physically interacts with both RELT and RELL1, as determined by in vitro co-immunoprecipitations. Immunohistochemistry was utilized to determine whether HIC co-localizes with RELT family members using Human Embryonic Kidney (HEK-293) cells. HIC was observed to co-localize with RELL1 at the plasma membrane of HEK-293 cells and appeared to co-localize with RELT in intracellular compartments. A series of deletion mutants of RELT were created that could be used to identify the regions of RELT required to bind to HIC. Site-directed mutagenesis was utilized to create eight (8) deletion mutants of RELT that lacked differing portions of the intracellular domain. Interestingly, mutants that were designed to lack more than 60 intracellular amino acids proximal to the plasma membrane were barely detectable by overexposing Western blots, and this may indicate that the regions of RELT immediately proximal to the plasma membrane are required for protein stability. Current ongoing studies involve using the created deletion mutants of RELT to identify which regions of RELT are required to bind HIC, and to identify the physiological significance of these protein interactions. It is interesting to note that the hematologically expressed RELT was previously identified to interact with PLSCR1, a protein that induces cell cycle arrest, differentiation and apoptosis of AML cells. Therefore the discovery that RELT also binds to HIC, a protein that is highly expressed in leukocytes and is located in a region of chromosome 7 frequently lost in AML patients, is intriguing and warrants further study. Citation Format: John K. Cusick, Cara Sumida, Matthew Checketts, Mary E. Reyland, Aaron T. Jacobs. HIC (MDFIC) is a protein that interacts and colocalizes with the RELT family of TNFRs. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2264. doi:10.1158/1538-7445.AM2014-2264

Intermolecular masking of the HIV-1 Rev NLS by the cellular protein HIC: Novel insights into the regulation of Rev nuclear import

BackgroundThe HIV-1 regulatory protein Rev, which is essential for viral replication, mediates the nuclear export of unspliced viral transcripts. Rev nuclear function requires active nucleocytoplasmic shuttling, and Rev nuclear import is mediated by the recognition of its Nuclear Localisation Signal (NLS) by multiple import factors, which include transportin and importin β. However, it remains unclear which nuclear import pathway(s) predominate in vivo, and the cellular environment that modulates Rev nucleocytoplasmic shuttling remains to be characterised.ResultsIn our study, we have identified the cellular protein HIC (Human I-mfa domain-Containing protein) as a novel interactor of HIV-1 Rev. We demonstrate that HIC selectively interferes with Rev NLS interaction with importin β and impedes its nuclear import and function, but does not affect Rev nuclear import mediated by transportin. Hence, the molecular determinants mediating Rev-NLS recognition by importin β and transportin appear to be distinct. Furthermore, we have employed HIC and M9 M, a peptide specifically designed to inhibit the transportin-mediated nuclear import pathway, to characterise Rev nuclear import pathways within different cellular environments. Remarkably, we could show that in 293T, HeLa, COS7, Jurkat, U937, THP-1 and CEM cells, Rev nuclear import is cell type specific and alternatively mediated by transportin or importin β, in a mutually exclusive fashion.ConclusionsRev cytoplasmic sequestration by HIC may represent a novel mechanism for the control of Rev function. These studies highlight that the multivalent nature of the Rev NLS for different import receptors enables Rev to adapt its nuclear trafficking strategy.

Human I-mfa domain proteins specifically interact with KSHV LANA and affect its regulation of Wnt signaling-dependent transcription

Kaposi’s sarcoma-associated herpes virus (KSHV)-encoded latency-associated nuclear antigen (LANA) protein has been reported to interact with glycogen synthase kinase 3β (GSK-3β) and to negatively regulate its activity, leading to stimulation of GSK-3β-dependent β-catenin degradation. We show here that the I-mfa domain proteins, HIC (human I-mfa domain-containing protein) and I-mfa (inhibitor of MyoD family a), interacted in vivo with LANA through their C-terminal I-mfa domains. This interaction affected the intracellular localization of HIC, inhibited the LANA-dependent transactivation of a β-catenin-regulated reporter construct, and decreased the level of the LANA·GSK-3β complex. These data reveal for the first time that I-mfa domain proteins interact with LANA and negatively regulate LANA-mediated activation of Wnt signaling-dependent transcription by inhibiting the formation of the LANA·GSK-3β complex.

The Complex Regulation of HIC (Human I-mfa Domain Containing Protein) Expression

Human I-mfa domain containing protein (HIC) differentially regulates transcription from viral promoters. HIC affects the Wnt pathway, the JNK/SAPK pathway and the activity of positive transcription elongation factor-b (P-TEFb). Studies exploring HIC function in mammalian cells used ectopically expressed HIC due to undetected endogenous HIC protein. HIC mRNA contains exceptionally long 5′ and 3′ untranslated regions (UTRs) compared to the average length of mRNA UTRs. Here we show that HIC protein is subject to strict repression at multiple levels. The HIC mRNA UTRs reduce the expression of HIC or of a reporter protein: The HIC 3′-UTR decreases both HIC and reporter mRNA levels, whereas upstream open reading frames located in the 5′-UTR repress the translation of HIC or of the reporter protein. In addition, ectopically expressed HIC protein is degraded by the proteasome, with a half-life of approximately 1 h, suggesting that upon activation, HIC expression in cells may be transient. The strict regulation of HIC expression at the levels of mRNA stability, translation efficiency and protein stability suggests that expression of the HIC protein and its involvement in the various pathways is required only under specific cellular conditions.

Expression profile and differential regulation of the Human I-mfa domain-Containing protein (HIC) gene in immune cells

The Human I-mfa domain-Containing protein, HIC, is a 246 amino acid protein that functions as a transcriptional regulator. Although the precise function of HIC remains to be clarified, the association of the HIC gene locus with myeloid neoplasms, its interactions with lymphotropic viruses such as EBV, HIV-1 and HTLV-1 and its expression in immune tissues suggest that HIC might have a modulatory role in immune cells. To further characterise the HIC functional relationship with the immune system, we sought to analyse the HIC gene expression profile in immune cells and to determine if immunomodulatory cytokines, such as interleukin (IL)-2, could regulate the expression of HIC mRNA. Relative quantitative real-time RT-PCR revealed that HIC mRNA is highly expressed in PBMCs and in various hematopoietic cell lines. The immunomodulatory cytokine IL-2 up-regulated HIC gene expression in PBMCs, CEM, MT-2 and U937 but markedly reduced HIC gene expression in Raji. Addition of cycloheximide indicated that the IL-2 effects were independent of de novo protein synthesis and that the HIC gene is a direct target of IL-2. Two cell lines (Jurkat and BJAB) displayed a distinct loss in HIC gene expression. However, when these cell lines were subjected to a combination of DNA methyltransferase and histone-deacetylase inhibitors, (5-aza-2-deoxycytidine and trichostatin A, respectively), HIC expression was de-repressed, indicating possible epigenetic control of HIC expression. Overall, our study describes that the immune expression of HIC is cell-specific, dynamic, and identifies the HIC gene as an IL-2 responsive gene. Furthermore, our de-repression studies support the hypothesis that HIC might represent a candidate tumor suppressor gene. Overall, this report provides new insights for a putative role of HIC in the modulation of immune and inflammatory responses and/or hematological malignancies.

Cellular mRNA Activates Transcription Elongation by Displacing 7SK RNA

The positive transcription elongation factor P-TEFb is a pivotal regulator of gene expression in higher cells. Originally identified in Drosophila, attention was drawn to human P-TEFb by the discovery of its role as an essential cofactor for HIV-1 transcription. It is recruited to HIV transcription complexes by the viral transactivator Tat, and to cellular transcription complexes by a plethora of transcription factors. P-TEFb activity is negatively regulated by sequestration in a complex with the HEXIM proteins and 7SK RNA. The mechanism of P-TEFb release from the inhibitory complex is not known. We report that P-TEFb-dependent transcription from the HIV promoter can be stimulated by the mRNA encoding HIC, the human I-mfa domain-containing protein. The 3′-untranslated region of HIC mRNA is necessary and sufficient for this action. It forms complexes with P-TEFb and displaces 7SK RNA from the inhibitory complex in cells and cell extracts. A 314-nucleotide sequence near the 3′ end of HIC mRNA has full activity and contains a predicted structure resembling the 3′-terminal hairpin of 7SK that is critical for P-TEFb binding. This represents the first example of a cellular mRNA that can regulate transcription via P-TEFb. Our findings offer a rationale for 7SK being an RNA transcriptional regulator and suggest a practical means for enhancing gene expression.

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Direct interaction of the human I-mfa domain-containing protein, HIC, with HIV-1 Tat results in cytoplasmic sequestration and control of Tat activity.

The primary function of the HIV-1 regulatory protein Tat, activation of transcription from the viral LTR, is highly regulated by complex interactions between Tat and a number of host cell proteins. Tat nuclear import, a process mediated by importin beta, is a prerequisite for its activity. Here, we report and characterize the interaction of the human inhibitor of MyoD family domain-containing protein (I-mfa), HIC, with Tat at a biochemical and a functional level. This interaction was shown to occur in vivo and in vitro and to involve the nuclear localization signal and the transactivation responsive element-binding domains of Tat and the I-mfa domain of HIC. Coexpression of HIC and Tat resulted in the down-regulation of transactivation of the HIV-1 LTR, and colocalization studies revealed the cytoplasmic sequestration of Tat by HIC. Functionally this sequestration appears to be the underlying mechanism of LTR transcriptional repression by HIC and represents a unique mechanism for the control of Tat activity and regulation of HIV-1 replication.

Identification of Epstein-Barr Virus RK-BARF0-Interacting Proteins and Characterization of Expression Pattern

The Epstein-Barr virus (EBV) BamHI A transcripts are a family of transcripts that are differentially spliced and can be detected in multiple EBV-associated malignancies. Several of the transcripts may encode proteins. One transcript of interest, RK-BARF0, is proposed to encode a 279-amino-acid protein with a possible endoplasmic reticulum-targeting sequence. In this study, the properties of RK-BARF0 were examined through identification of cellular-interacting proteins through yeast two-hybrid analysis and characterization of its expression in EBV-infected cells and tumors. In addition to the interaction previously identified with cellular Notch, it was determined that RK-BARF0 also bound cellular human I-mfa domain-containing protein (HIC), epithelin, and scramblase. An interaction between RK-BARF0 and Notch or epithelin induced proteasome-dependent degradation of Notch and epithelin but not of HIC or scramblase. Low levels of endogenous Notch expression in EBV-positive cell lines may correlate with RK-BARF0 expression. However, a screen of EBV-positive cell lines and tumors with an affinity-purified alpha-RK-BARF0 antiserum did not consistently detect RK-BARF0. These data suggest that while RK-BARF0 may have important cellular functions during EBV infection, and while the phenotype of EBV-positive cells suggest its expression, RK-BARF0 levels may be too low to detect.

The human I-mfa domain-containing protein, HIC, interacts with cyclin T1 and modulates P-TEFb-dependent transcription.

Positive transcription elongation factor b (P-TEFb) hyperphosphorylates the carboxy-terminal domain of RNA polymerase II, permitting productive transcriptional elongation. The cyclin T1 subunit of P-TEFb engages cellular transcription factors as well as the human immunodeficiency virus type 1 (HIV-1) transactivator Tat. To identify potential P-TEFb regulators, we conducted a yeast two-hybrid screen with cyclin T1 as bait. Among the proteins isolated was the human I-mfa domain-containing protein (HIC). HIC has been reported to modulate expression from both cellular and viral promoters via its C-terminal cysteine-rich domain, which is similar to the inhibitor of MyoD family a (I-mfa) protein. We show that HIC binds cyclin T1 in yeast and mammalian cells and that it interacts with intact P-TEFb in mammalian cell extracts. The interaction involves the I-mfa domain of HIC and the regulatory histidine-rich region of cyclin T1. HIC also binds Tat via its I-mfa domain, although the sequence requirements are different. HIC colocalizes with cyclin T1 in nuclear speckle regions and with Tat in the nucleolus. Expression of the HIC cDNA modulates Tat transactivation of the HIV-1 long terminal repeat (LTR) in a cell type-specific fashion. It is mildly inhibitory in CEM cells but stimulates gene expression in HeLa, COS, and NIH 3T3 cells. The isolated I-mfa domain acts as a dominant negative inhibitor. Activation of the HIV-1 LTR by HIC in NIH 3T3 cells occurs at the RNA level and is mediated by direct interactions with P-TEFb.

I-mfa domain proteins interact with Axin and affect its regulation of the Wnt and c-Jun N-terminal kinase signaling pathways.

I-mfa has been identified as an inhibitor of myogenic basic helix-loop-helix transcription factors, and a related human I-mfa domain-containing protein (HIC) also has been identified as a protein that regulates Tat- and Tax-mediated expression of viral promoters. HIC and I-mfa represent a family of proteins that share a highly conserved cysteine-rich domain, termed the I-mfa domain. We show here that both I-mfa domain proteins, HIC and I-mfa, interacted in vivo with the Axin complex through their C-terminal I-mfa domains. This interaction inhibited Axin-mediated downregulation of free levels of cytosolic beta-catenin. I-mfa and HIC also both directly interacted with lymphocyte enhancer factor (LEF); however, I-mfa but not HIC significantly inhibited reporter constructs regulated by beta-catenin. The overexpression of HIC but not I-mfa decreased the inhibitory effects of Axin on beta-catenin-regulated reporter constructs, while both HIC and I-mfa decreased Axin-mediated c-Jun N-terminal kinase (JNK) activation. These data reveal for the first time that I-mfa domain proteins interact with the Axin complex and affect Axin regulation of both the Wnt and the JNK activation pathways. Interestingly, HIC differs from I-mfa in that I-mfa affects both Axin function and T-cell factor- or LEF-regulated transcription in the Wnt signaling pathway while HIC affects primarily Axin function.

How the sequestration of a protein interferes with its mechanism of action: example of a new family of proteins characterized by a particular cysteine-rich carboxy-terminal domain involved in gene expression regulation.

We describe here a new family of proteins characterized by a particular cysteine-rich carboxy-terminal domain and involved in gene expression regulation. This family presently includes three members: I-mfa (inhibitor of MyoD family), HIC p40 and HIC p32 (human I-mfa domain-containing protein). I-mfa, by interacting with MyoD family members, represses both transcriptional activation and myogenesis mediated by these factors. HIC two isoforms, HIC p40 and HIC p32, are involved in the positive regulation of Tax-mediated HTLV-I (human T-cell leukemia virus type 1) promoter activation and in the negative regulation of Tat-mediated HIV-1 (human immunodeficiency virus type 1) promoter transcription. The common carboxy-terminal region of HIC p40 and HIC p32, which is clearly involved in these regulations, shares 77% homology with the carboxy-terminal domain of I-mfa. This suggests that I-mfa, HIC p40 and HIC p32 are part of a new family of proteins involved in gene expression regulation and characterized by a specific cysteine-rich carboxy-terminal domain. Moreover, the three proteins present different subcellular localizations: I-mfa and HIC p32 are mainly cytoplasmic while HIC p40 is mainly nucleolar. The specific localization of each member of this new family will be discussed, possibly explaining how they work. Effectively, a mechanism of protein sequestration in a particular compartment, cytoplasm or nucleolus, could be involved in their function, as it is the case for many other proteins. This relationship between sequestration and function regulation will be exemplified for several cellular factors.

Sequence requirement for the nucleolar localization of human I-mfa domain-containing protein (HIC p40)

The human I-mfa domain-containing protein (HIC) mRNA produces two protein isoforms, HIC p32 and p40, synthesized from alternative translational initiations. p32 translation is initiated from a standard AUG codon and p40 is an N-terminal extension of p32 generated from an upstream GUG codon. The two isoforms show different subcellular localization: p32 is distributed throughout the cytoplasm whereas p40 can be found both in the cytoplasm and the nucleolus. To investigate the possibility that p40 contains a nucleolus targeting sequence in its N-terminal region, COS cells were transfected with an eukaryotic expression vector coding for green fluorescent protein (GFP) fused to the p40 N terminus. The localization of this fusion protein in the nucleolus indicated that the N-terminal amino acids of p40 probably contain a nucleolar localization signal (NoLS). To find the structural motifs required for nucleolar localization of p40, deletion mutants were expressed in COS cells as fusion polypeptides with GFP. We defined a domain of 19 amino acids near the N terminus that contains an arginine-rich subdomain that conforms to other known NoLS. To demonstrate that this sequence is an authentic NoLS, the sequence was fused to GFP. This fusion protein was observed to migrate into the nucleolus. Taken together, our studies demonstrate that p40 contains a NoLS.

Molecular Cloning of a Novel Human I-mfa Domain-containing Protein That Differently Regulates Human T-cell Leukemia Virus Type I and HIV-1 Expression

Regulation of viral genome expression is the result of complex cooperation between viral proteins and host cell factors. We report here the characterization of a novel cellular factor sharing homology with the specific cysteine-rich C-terminal domain of the basic helix-loop-helix repressor protein I-mfa. The synthesis of this new factor, called HIC for Human I-mfa domain-Containing protein, is controlled at the translational level by two different codons, an ATG and an upstream non-ATG translational initiator, allowing the production of two protein isoforms, p32 and p40, respectively. We show that the HIC protein isoforms present different subcellular localizations, p32 being mainly distributed throughout the cytoplasm, whereas p40 is targeted to the nucleolus. Moreover, in trying to understand the function of HIC, we have found that both isoforms stimulate in T-cells the expression of a luciferase reporter gene driven by the human T-cell leukemia virus type I-long terminal repeat in the presence of the viral transactivator Tax. We demonstrate by mutagenesis that the I-mfa-like domain of HIC is involved in this regulation. Finally, we also show that HIC is able to down-regulate the luciferase expression from the human immunodeficiency virus type 1-long terminal repeat induced by the viral transactivator Tat. From these results, we propose that HIC and I-mfa represent two members of a new family of proteins regulating gene expression and characterized by a particular cysteine-rich C-terminal domain.