Histone H3K9 methyltransferase SETDB1 augments invadopodia formation to promote tumor metastasis.

Non-small cell lung cancer (NSCLC) is one of leading causes of cancer-related mortality worldwide, which harbors various accumulated genetic and epigenetic abnormalities. Histone methyltransferase SETDB1 is a pivotal epigenetic regulator whose focal amplification and upregulation are commonly detected in NSCLC. However, molecular mechanisms underlying the pro-oncogenic function of SETDB1 remain poorly characterized. Here, we demonstrate that SETDB1 augments the migration and invasion capabilities of NSCLC cells by reinforcing invadopodia formation and mediated ECM degradation. At the molecular level, SETDB1 suppresses the expression of FOXA2, a crucial tumor and metastasis suppressor via coordinated epigenetic mechanisms - SETDB1 not only catalyzes histone H3K9 methylation on FOXA2 genomic locus, but also recruits DNMT3A to regulate DNA methylation on CpG island. Consequently, depletion of Setdb1 in murine lung adenocarcinoma cells completely abolished their full and spontaneous metastatic capabilities in mouse xenograft models. These findings together establish the pro-metastasis activity of SETDB1 in NSCLC and elucidate the underlying cellular and molecular mechanisms.

Internal Associations of the Acidic Region of Upstream Binding Factor Control Its Nucleolar Localization.

Upstream binding factor (UBF) is a member of the high-mobility group (HMG) box protein family, characterized by multiple HMG boxes and a C-terminal acidic region (AR). UBF is an essential transcription factor for rRNA genes and mediates the formation of transcriptionally active chromatin in the nucleolus. However, it remains unknown how UBF is specifically localized to the nucleolus. Here, we examined the molecular mechanisms that localize UBF to the nucleolus. We found that the first HMG box (HMG box 1), the linker region (LR), and the AR cooperatively regulate the nucleolar localization of UBF1. We demonstrated that the AR intramolecularly associates with and attenuates the DNA binding activity of HMG boxes and confers the structured DNA preference to HMG box 1. In contrast, the LR was found to serve as a nuclear localization signal and compete with HMG boxes to bind the AR, permitting nucleolar localization of UBF1. The LR sequence binds DNA and assists the stable chromatin binding of UBF. We also showed that the phosphorylation status of the AR does not clearly affect the localization of UBF1. Our results strongly suggest that associations of the AR with HMG boxes and the LR regulate UBF nucleolar localization.

Tracking adenovirus genomes identifies morphologically distinct late DNA replication compartments.

In adenoviral virions, the genome is organized into a chromatin-like structure by viral basic core proteins. Consequently viral DNAs must be replicated, chromatinized and packed into progeny virions in infected cells. Although viral DNA replication centers can be visualized by virtue of viral and cellular factors, the spatiotemporal regulation of viral genomes during subsequent steps remains to be elucidated. In this study, we used imaging analyses to examine the fate of adenoviral genomes and to track newly replicated viral DNA as well as replication-related factors. We show de novo formation of a subnuclear domain, which we termed Virus-induced Post-Replication (ViPR) body, that emerges concomitantly with or immediately after disintegration of initial replication centers. Using a nucleoside analogue, we show that viral genomes continue being synthesized in morphologically distinct replication compartments at the periphery of ViPR bodies and are then transported inward. In addition, we identified a nucleolar protein Mybbp1a as a molecular marker for ViPR bodies, which specifically associated with viral core protein VII. In conclusion, our work demonstrates the formation of previously uncharacterized viral DNA replication compartments specific for late phases of infection that produce progeny viral genomes accumulating in ViPR bodies.

Reconstitution of human rRNA gene transcription in mouse cells by a complete SL1 complex.

An important characteristic of the transcription of a ribosomal RNA gene (rDNA) mediated by DNA-dependent RNA polymerase (Pol) I is its stringent species specificity. SL1/TIF-IB is a key complex for species specificity, but its functional complex has not been reconstituted. Here, we established a novel and highly sensitive monitoring system for Pol I transcription to reconstitute the SL1 activity in which a transcript harboring a reporter gene synthesized by Pol I is amplified and converted into translatable mRNA by the influenza virus RNA-dependent RNA polymerase. Using this monitoring system, we reconstituted Pol I transcription from the human rDNA promoter in mouse cells by expressing four human TATA-binding protein (TBP)-associated factors (TAFIs) in the SL1 complex. The reconstituted SL1 also re-activated human rDNA transcription in mouse A9 cells carrying an inactive human chromosome 21 that contains the rDNA cluster. Chimeric SL1 complexes containing human and mouse TAFIs could be formed, but these complexes were inactive for human rDNA transcription. We conclude that four human TAFIs are necessary and sufficient to overcome the barrier of species specificity for human rDNA transcription in mouse cells.

Upstream binding factor-dependent and pre-rRNA transcription-independent association of pre-rRNA processing factors with rRNA gene.

The nucleolus is the ribosome biogenesis center. The nucleolar structure is disrupted upon entry into mitosis and is formed in early G1 phase. To understand the molecular mechanisms of nucleolar assembly and disassembly, we have studied the mechanism of association between factors involved in pre-ribosome RNA (rRNA) processing and rRNA gene chromatin (r-chromatin). We found that the pre-rRNA transcription-processing linking factor Nopp140 and pre-rRNA processing factors such as DKC1 and fibrillarin (FBL) associate with r-chromatin during interphase, while Nopp140, DKC1, and FBL were released from r-chromatin in mitosis. The association of these factors with r-chromatin was found to be restored independent of pre-rRNA transcription in early G1 phase, but a mature nucleolar structure was not formed, suggesting that nucleolar assembly can be divided into at least two steps with respect to pre-rRNA transcription. Moreover, we found that the r-chromatin association of Nopp140, DKC1, and FBL was dependent on the transcription factor upstream binding factor (UBF). However, we demonstrated that UBF alone was not sufficient to recruit these pre-rRNA processing factors to r-chromatin. Thus, UBF is necessary but not sufficient for the associations between pre-rRNA processing factors and r-chromatin.

Regulation of nucleolar chromatin by B23/nucleophosmin jointly depends upon its RNA binding activity and transcription factor UBF.

Histone chaperones regulate the density of incorporated histone proteins around DNA transcription sites and therefore constitute an important site-specific regulatory mechanism for the control of gene expression. At present, the targeting mechanism conferring this site specificity is unknown. We previously reported that the histone chaperone B23/nucleophosmin associates with rRNA chromatin (r-chromatin) to stimulate rRNA transcription. Here, we report on the mechanism for site-specific targeting of B23 to the r-chromatin. We observed that, during mitosis, B23 was released from chromatin upon inactivation of its RNA binding activity by cdc2 kinase-mediated phosphorylation. The phosphorylation status of B23 was also shown to strongly affect its chromatin binding activity. We further found that r-chromatin binding of B23 was a necessary condition for B23 histone chaperone activity in vivo. In addition, we found that depletion of upstream binding factor (UBF; an rRNA transcription factor) decreased the chromatin binding affinity of B23, which in turn led to an increase in histone density at the r-chromatin. These two major strands of evidence suggest a novel cell cycle-dependent mechanism for the site-specific regulation of histone density via joint RNA- and transcription factor-mediated recruitment of histone chaperones to specific chromosome loci.