Silencing of IFN-stimulated gene transcription is regulated by histone H1 and its chaperone TAF-I.

Chromatin structure and its alteration play critical roles in the regulation of transcription. However, the transcriptional silencing mechanism with regard to the chromatin structure at an unstimulated state of the interferon (IFN)-stimulated gene (ISG) remains unclear. Here we investigated the role of template activating factor-I (TAF-I, also known as SET) in ISG transcription. Knockdown (KD) of TAF-I increased ISG transcript and simultaneously reduced the histone H1 level on the ISG promoters during the early stages of transcription after IFN stimulation from the unstimulated state. The transcription factor levels on the ISG promoters were increased in TAF-I KD cells only during the early stages of transcription. Furthermore, histone H1 KD also increased ISG transcript. TAF-I and histone H1 double KD did not show the additive effect in ISG transcription, suggesting that TAF-I and histone H1 may act on the same regulatory pathway to control ISG transcription. In addition, TAF-I KD and histone H1 KD affected the chromatin structure near the ISG promoters. On the basis of these findings, we propose that TAF-I and its target histone H1 are key regulators of the chromatin structure at the ISG promoter to maintain the silent state of ISG transcription.

pp32, an INHAT component, is a transcription machinery recruiter for maximal induction of IFN-stimulated genes.

Type I interferon (IFN) plays a crucial role in establishing the cellular antiviral state by inducing transcription of IFN-stimulated genes (ISGs). Generally, histone acetyltransferases (HATs) are positive regulators of transcription, but histone deacetylase (HDAC) activity is essential for transcriptional induction of ISGs. pp32 is known to be a key component of the inhibitor of acetyltransferase (INHAT) complex that inhibits HAT-dependent transcriptional activation. Here, we show that pp32 is involved in the positive regulation of ISG transcription. pp32 interacted with signal transducer and activator of transcription 1 (STAT1) and STAT2 in an IFN-dependent manner. pp32 was not required for tyrosine phosphorylation and nuclear translocation of STATs, but was needed for binding of transcriptional complexes with ISG promoters and, thereby, for maximal transcription activation. pp32 was found to be associated with ISG promoters in IFN-untreated cells, and its binding amount fluctuated as a function of time after IFN treatment. short interfering RNA (siRNA)-mediated knockdown of pp32 expression reduced the histone acetylation level on ISG promoters, suggesting that pp32 plays a role in ISG transcription by a function other than that of INHAT. Taking these findings together, we propose that pp32 is involved in the formation of ISG transcription initiation complexes, possibly as their recruiter.

Nucleoporin 153 links nuclear pore complex to chromatin architecture by mediating CTCF and cohesin binding.

Nucleoporin proteins (Nups) have been proposed to mediate spatial and temporal chromatin organization during gene regulation. Nevertheless, the molecular mechanisms in mammalian cells are not well understood. Here, we report that Nucleoporin 153 (NUP153) interacts with the chromatin architectural proteins, CTCF and cohesin, and mediates their binding across cis-regulatory elements and TAD boundaries in mouse embryonic stem (ES) cells. NUP153 depletion results in altered CTCF and cohesin binding and differential gene expression - specifically at the bivalent developmental genes. To investigate the molecular mechanism, we utilize epidermal growth factor (EGF)-inducible immediate early genes (IEGs). We find that NUP153 controls CTCF and cohesin binding at the cis-regulatory elements and POL II pausing during the basal state. Furthermore, efficient IEG transcription relies on NUP153. We propose that NUP153 links the nuclear pore complex (NPC) to chromatin architecture allowing genes that are poised to respond rapidly to developmental cues to be properly modulated.

Enhancing of measles virus infection by magnetofection.

Magnetofection is a viral and non-viral gene delivery method using polyethyleneimine-conjugated super-paramagnetic nanoparticle under a magnetic field. Previous studies have indicated that magnetofection enhanced the infection of adenoviruses and retroviruses. It is shown that magnetofection enhances the infection of measles virus, a paramyxovirus. When cells expressing a measles virus receptor human SLAM were infected with a measles virus that encodes green fluorescent protein gene, magnetofection enhanced measles virus infection by 30- to 70-fold. The infection of SLAM-negative cells with measles virus was also enhanced by magnetofection, but to a lesser extent. These results indicate that magnetofection could be useful for isolation of measles virus from clinical specimens.

Expression of N-acetylglucosaminyltransferase V in the subserosal layer correlates with postsurgical survival of pathological tumor stage 2 carcinoma of the gallbladder.

BACKGROUND: N-Acetylglucosaminyltransferase V (GnT-V), an enzyme that catalyzes the ß1-6 branching of N-acetylglucosamine on asparagine-linked oligosaccharides of cellular proteins, enhances the malignant behaviors of carcinoma cells in experimental models. The aim of this study was to determine clinical significance of GnT-V expression in human pT2 gallbladder carcinoma with simple in vitro experiments. METHODS: Ninety patients with pT2 gallbladder carcinoma were included for this study. The in vitro and in vivo biological effects of GnT-V were investigated using gallbladder carcinoma cells with variable GnT-V expression levels induced by a small interfering RNA. RESULTS: Of the 90 cases, 57 showed positive staining and the remaining 33 demonstrated negative staining, the subcellular localization in the 57 cases was classified into the granular-type in 31 cases and the diffuse-type in 26 cases. In 76 cases with curative resection, postsurgical survival was significantly poorer in those showing positive staining than in those showing negative staining (P = 0.028). In all of the 76 cases, postsurgical recurrence was significantly more frequent in those showing diffuse-type localization than in those showing negative staining. Experimental analyses demonstrated that the down-regulation of GnT-V expression in gallbladder carcinoma cells induced suppression of cell growth in vitro. The expression levels of GnT-V in the cells were highly correlated with the rapid in vivo growth coupled with the enhanced angiogenesis, and the tendency to form liver metastasis. CONCLUSIONS: GnT-V expression in the subserosal layer of pT2 gallbladder carcinoma is correlated with the aggressiveness of the disease.

Stringent requirement for the C protein of wild-type measles virus for growth both in vitro and in macaques.

The P gene of measles virus (MV) encodes the P protein and three accessory proteins (C, V, and R). However, the role of these accessory proteins in the natural course of MV infection remains unclear. For this study, we generated a recombinant wild-type MV lacking the C protein, called wtMV(C-), by using a reverse genetics system (M. Takeda, K. Takeuchi, N. Miyajima, F. Kobune, Y. Ami, N. Nagata, Y. Suzaki, Y. Nagai, and M. Tashiro, J. Virol. 74:6643-6647). When 293 cells expressing the MV receptor SLAM (293/hSLAM) were infected with wtMV(C-) or parental wild-type MV (wtMV), the growth of wtMV(C-) was restricted, particularly during late stages. Enhanced green fluorescent protein-expressing wtMV(C-) consistently induced late-stage cell rounding and cell death in the presence of a fusion-inhibiting peptide, suggesting that the C protein can prevent cell death and is required for long-term MV infection. Neutralizing antibodies against alpha/beta interferon did not restore the growth restriction of wtMV(C-) in 293/hSLAM cells. When cynomolgus monkeys were infected with wtMV(C-) or wtMV, the number of MV-infected cells in the thymus was >1,000-fold smaller for wtMV(C-) than for wtMV. Immunohistochemical analyses showed strong expression of an MV antigen in the spleen, lymph nodes, tonsils, and larynx of a cynomolgus monkey infected with wtMV but dramatically reduced expression in the same tissues in a cynomolgus monkey infected with wtMV(C-). These data indicate that the MV C protein is necessary for efficient MV replication both in vitro and in cynomolgus monkeys.