Nuclear S6K1 Enhances Oncogenic Wnt Signaling by Inducing Wnt/β-Catenin Transcriptional Complex Formation

Ribosomal protein S6 kinase 1 (S6K1), a key downstream effector of the mammalian target of rapamycin (mTOR), regulates diverse functions, such as cell proliferation, cell growth, and protein synthesis. Because S6K1 was previously known to be localized in the cytoplasm, its function has been mainly studied in the cytoplasm. However, the nuclear localization and function of S6K1 have recently been elucidated and other nuclear functions are expected to exist but remain elusive. Here, we show a novel nuclear role of S6K1 in regulating the expression of the Wnt target genes. Upon activation of the Wnt signaling, S6K1 translocated from the cytosol into the nucleus and subsequently bound to β-catenin and the cofactors of the Wnt/β-catenin transcriptional complex, leading to the upregulation of the Wnt target genes. The depletion or repression of S6K1 downregulated the Wnt target gene expression by inhibiting the formation of the Wnt/β-catenin transcriptional complex. The S6K1-depleted colon cancer cell lines showed lower transcription levels of the Wnt/β-catenin target genes and a decrease in the cell proliferation and invasion compared to the control cell lines. Taken together, these results indicate that nuclear S6K1 positively regulates the expression of the Wnt target genes by inducing the reciprocal interaction of the subunits of the transcriptional complex

Hexosamine Biosynthetic Pathway-Derived O-GlcNAcylation Is Critical for RANKL-Mediated Osteoclast Differentiation

O-linked-N-acetylglucosaminylation (O-GlcNAcylation) performed by O-GlcNAc transferase (OGT) is a nutrient-responsive post-translational modification (PTM) via the hexosamine biosynthetic pathway (HBP). Various transcription factors (TFs) are O-GlcNAcylated, affecting their activities and significantly contributing to cellular processes ranging from survival to cellular differentiation. Given the pleiotropic functions of O-GlcNAc modification, it has been studied in various fields; however, the role of O-GlcNAcylation during osteoclast differentiation remains to be explored. Kinetic transcriptome analysis during receptor activator of nuclear factor-kappaB (NF-κB) ligand (RANKL)-mediated osteoclast differentiation revealed that the nexus of major nutrient metabolism, HBP was critical for this process. We observed that the critical genes related to HBP activation, including Nagk, Gfpt1, and Ogt, were upregulated, while the global O-GlcNAcylation was increased concomitantly during osteoclast differentiation. The O-GlcNAcylation inhibition by the small-molecule inhibitor OSMI-1 reduced osteoclast differentiation in vitro and in vivo by disrupting the translocation of NF-κB p65 and nuclear factor of activated T cells c1 (NFATc1) into the nucleus by controlling their PTM O-GlcNAcylation. Furthermore, OSMI-1 had a synergistic effect with bone target therapy on osteoclastogenesis. Lastly, knocking down Ogt with shRNA (shOgt) mimicked OSMI-1’s effect on osteoclastogenesis. Targeting O-GlcNAcylation during osteoclast differentiation may be a valuable therapeutic approach for osteoclast-activated bone diseases

SNF5 Is an Essential Executor of Epigenetic Regulation during Differentiation

<div><p>Nucleosome occupancy controls the accessibility of the transcription machinery to DNA regulatory regions and serves an instructive role for gene expression. Chromatin remodelers, such as the BAF complexes, are responsible for establishing nucleosome occupancy patterns, which are key to epigenetic regulation along with DNA methylation and histone modifications. Some reports have assessed the roles of the BAF complex subunits and stemness in murine embryonic stem cells. However, the details of the relationships between remodelers and transcription factors in altering chromatin configuration, which ultimately affects gene expression during cell differentiation, remain unclear. Here for the first time we demonstrate that SNF5, a core subunit of the BAF complex, negatively regulates OCT4 levels in pluripotent cells and is essential for cell survival during differentiation. SNF5 is responsible for generating nucleosome-depleted regions (NDRs) at the regulatory sites of OCT4 repressed target genes such as PAX6 and NEUROG1, which are crucial for cell fate determination. Concurrently, SNF5 closes the NDRs at the regulatory regions of OCT4-activated target genes such as OCT4 itself and NANOG. Furthermore, using loss- and gain-of-function experiments followed by extensive genome-wide analyses including gene expression microarrays and ChIP-sequencing, we highlight that SNF5 plays dual roles during differentiation by antagonizing the expression of genes that were either activated or repressed by OCT4, respectively. Together, we demonstrate that SNF5 executes the switch between pluripotency and differentiation.</p> </div

Urinary Proteome Profile Predictive of Disease Activity in Rheumatoid Arthritis

Current serum biomarkers for rheumatoid arthritis (RA) are not highly sensitive or specific to changes of disease activities. Thus, other complementary biomarkers have been needed to improve assessment of RA activities. In many diseases, urine has been studied as a window to provide complementary information to serum measures. Here, we conducted quantitative urinary proteome profiling using liquid chromatographytandem mass spectrometry (LCMS/MS) and identified 134 differentially expressed proteins (DEPs) between RA and osteoarthritis (OA) urine samples. By integrating the DEPs with gene expression profiles in joints and mononuclear cells, we initially selected 12 biomarker candidates related to joint pathology and then tested their altered expression in independent RA and OA samples using enzyme-linked immunosorbent assay. Of the initial candidates, we selected four DEPs as final candidates that were abundant in RA patients and consistent with those observed in LCMS/MS analysis. Among them, we further focused on urinary soluble CD14 (sCD14) and examined its diagnostic value and association with disease activity. Urinary sCD14 had a diagnostic value comparable to conventional serum measures and an even higher predictive power for disease activity when combined with serum C-reactive protein. Thus, our urinary proteome provides a diagnostic window complementary to current serum parameters for the disease activity of RA.1

Knockdown of SNF5 enhances a stem cell like state and blocks differentiation.

<p>(A) SNF5, OCT4, EZH2, and loading control histone H3 were analyzed by western blot, 72 h post-transfection with SNF5 siRNA in NCCIT cells. (B and C) To get the nucleosome footprint, we have performed at least three biological replicates of NOMe-seq at 72 h post-transfection with SNF5 siRNA in NCCIT and selected ∼10 sequences in an unbiased manner to represented in the figures. (D) Stably infected SNF5 knockdown NCCIT cells were selected for 21 days with antibiotics and SNF5, OCT4, EZH2, and loading control histone H3 were subsequently analyzed by western blot. G401 cells were used for a SNF5 knockdown control. (E) At the same time point, cell morphology micrographs (200X) were taken. The data is representative of three biological experiments. (F) Apoptosis of SNF5 knockdown NCCIT cells after RA treatment was determined by flow cytometric analysis. The X axis indicates Annexin V and the Y axis indicates Propidium iodide (PI). The data are representative of three biological experiments (the mean +SEM).</p

SNF5 controls the balance between pluripotency and differentiation.

<p>(A and B) 2D matrix and heat plots depicting gene expression changes in SNF5 knockdown/SNF5 overexpression and RA 7 d treated NCCIT cells. Axes indicate degree of fold change, from the middle of axis. The numbers indicate the median fold change of genes in each column or row. The intensity of each square represents the number of genes that fall in that square. (C) Fold change of SNF5 target genes (over two fold changes in opposite direction) among previously defined ES signature genes <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003459#pgen.1003459-BenPorath1" target="_blank">[51]</a>.</p

OCT4 target genes show distinctive nucleosome occupancy patterns that underlie the potential for gene expression.

<p>(A) Genome-wide studies were performed in human embryonic stem cells (H1) using ENCODE and GEO data (wgEncodeHudsonalphaMethylSeqRegionsRep1H1hesc for DNA methylation, GSM518373 for OCT4 ChIP-Seq and wgEncodeUwDnaseSeqPeaksRep1H1es for DNaseI). The data comprised 100 bp windows of OCT4 binding regions (29740 sites), DNA methylated regions (43659 sites) and DNaseI hypersensitive regions (123778 sites). (B and E) H1 and NCCIT cells were exposed to 10 uM RA for the indicated days. The expression levels of OCT4, NANOG, PAX6 and NEUROG1 were determined by quantitative PCR (normalized to PCNA). Quantitative PCR data represent the average of three biological experiments (the mean +SEM) (C, D, F and G) Nucleosome occupancy at the <i>PAX6</i> and <i>NEUROG1</i> promoters was analyzed by NOMe-seq during differentiation of H1 and NCCIT cells. Blue circles represent GpC sites of the DNA (unfilled blue circles represent GpC sites which are inaccessible to GpC methyltransferase, teal-filled circles represent cytosines accessible to GpC methyltransferase). Pink bars represent regions of inaccessibility large enough to accommodate a nucleosome (around 150 bp). The data is representative of three biological experiments.</p

Overexpression of SNF5 disturbs epigenetic regulation and enhances differentiation.

<p>(A–D) Exogenous SNF5 was overexpressed in NCCIT cells and 72 h later, SNF5, OCT4, EZH2, and loading control histone H3 were analyzed by western blot (A). After exogenous SNF5 transfection, glycerol density centrifugation assay was performed. Fractions of 0.5 ml of the 10 ml 10∼30% glycerol gradient were collected and subjected to western bolt analysis for various BAF complex subunits (B). NOMe-seq was performed indicated OCT4 target regions (C and D). The data is representative of three biological experiments. (E) After overexpression, SNF5 binding at the DNA regulatory regions of OCT4 target genes were analyzed by quantitative PCR.</p