BEND3 safeguards pluripotency by repressing differentiation-associated genes.

BEN domain-containing proteins are emerging rapidly as an important class of factors involved in modulating gene expression, yet the molecular basis of how they regulate chromatin function and transcription remains to be established. BEND3 is a quadruple BEN domain-containing protein that associates with heterochromatin and functions as a transcriptional repressor. We find that BEND3 is highly expressed in pluripotent cells, and the induction of differentiation results in the down-regulation of BEND3. The removal of BEND3 from pluripotent cells results in cells exhibiting upregulation of the differentiation-inducing gene expression signature. We find that BEND3 binds to the promoters of differentiation-associated factors and key cell cycle regulators, including CDKN1A, encoding the cell cycle inhibitor p21, and represses the expression of differentiation-associated genes by enhancing H3K27me3 decoration at these promoters. Our results support a model in which transcription repression mediated by BEND3 is essential for normal development and to prevent differentiation.

Orc6 is a component of the replication fork and enables efficient mismatch repair.

In eukaryotes, the origin recognition complex (ORC) is required for the initiation of DNA replication. The smallest subunit of ORC, Orc6, is essential for prereplication complex (pre-RC) assembly and cell viability in yeast and for cytokinesis in metazoans. However, unlike other ORC components, the role of human Orc6 in replication remains to be resolved. Here, we identify an unexpected role for hOrc6, which is to promote S-phase progression after pre-RC assembly and DNA damage response. Orc6 localizes at the replication fork and is an accessory factor of the mismatch repair (MMR) complex. In response to oxidative damage during S phase, often repaired by MMR, Orc6 facilitates MMR complex assembly and activity, without which the checkpoint signaling is abrogated. Mechanistically, Orc6 directly binds to MutSα and enhances the chromatin-association of MutLα, thus enabling efficient MMR. Based on this, we conclude that hOrc6 plays a fundamental role in genome surveillance during S phase.

Antagonism between splicing and microprocessor complex dictates the serum-induced processing of lnc-MIRHG for efficient cell cycle reentry.

Cellular quiescence and cell cycle reentry regulate vital biological processes such as cellular development and tissue homeostasis and are controlled by precise regulation of gene expression. The roles of long noncoding RNAs (lncRNAs) during these processes remain to be elucidated. By performing genome-wide transcriptome analyses, we identify differential expression of several hundreds of lncRNAs, including a significant number of the less-characterized class of microRNA-host-gene (MIRHG) lncRNAs or lnc-MIRHGs, during cellular quiescence and cell cycle reentry in human diploid fibroblasts. We observe that MIR222HG lncRNA displays serum-stimulated RNA processing due to enhanced splicing of the host nascent pri-MIR222HG transcript. The pre-mRNA splicing factor SRSF1 negatively regulates the microprocessor-catalyzed cleavage of pri-miR-222, thereby increasing the cellular pool of the mature MIR222HG Association of SRSF1 to pri-MIR222HG, including to a mini-exon, which partially overlaps with the primary miR-222 precursor, promotes serum-stimulated splicing over microRNA processing of MIR222HG Further, we observe that the increased levels of spliced MIR222HG in serum-stimulated cells promote the cell cycle reentry post quiescence in a microRNA-independent manner. MIR222HG interacts with DNM3OS, another lncRNA whose expression is elevated upon serum-stimulation, and promotes cell cycle reentry. The double-stranded RNA binding protein ILF3/2 complex facilitates MIR222HG:DNM3OS RNP complex assembly, thereby promoting DNM3OS RNA stability. Our study identifies a novel mechanism whereby competition between the splicing and microprocessor machinery modulates the serum-induced RNA processing of MIR222HG, which dictates cell cycle reentry.

PCNA-mediated stabilization of E3 ligase RFWD3 at the replication fork is essential for DNA replication.

RING finger and WD repeat domain-containing protein 3 (RFWD3) is an E3 ligase known to facilitate homologous recombination by removing replication protein A (RPA) and RAD51 from DNA damage sites. Further, RPA-mediated recruitment of RFWD3 to stalled replication forks is essential for interstrand cross-link repair. Here, we report that in unperturbed human cells, RFWD3 localizes at replication forks and associates with proliferating cell nuclear antigen (PCNA) via its PCNA-interacting protein (PIP) motif. PCNA association is critical for the stability of RFWD3 and for DNA replication. Cells lacking RFWD3 show slower fork progression, a prolonged S phase, and an increase in the loading of several replication-fork components on the chromatin. These findings all point to increased frequency of stalled forks in the absence of RFWD3. The S-phase defect is rescued by WT RFWD3, but not by the PIP mutant, suggesting that the interaction of RFWD3 with PCNA is critical for DNA replication. Finally, we observe reduced ubiquitination of RPA in cells lacking RFWD3. We conclude that the stabilization of RFWD3 by PCNA at the replication fork enables the polyubiquitination of RPA and its subsequent degradation for proper DNA replication.

Temporal association of ORCA/LRWD1 to late-firing origins during G1 dictates heterochromatin replication and organization.

DNA replication requires the recruitment of a pre-replication complex facilitated by Origin Recognition Complex (ORC) onto the chromatin during G1 phase of the cell cycle. The ORC-associated protein (ORCA/LRWD1) stabilizes ORC on chromatin. Here, we evaluated the genome-wide distribution of ORCA using ChIP-seq during specific time points of G1. ORCA binding sites on the G1 chromatin are dynamic and temporally regulated. ORCA association to specific genomic sites decreases as the cells progressed towards S-phase. The majority of the ORCA-bound sites represent replication origins that also associate with the repressive chromatin marks H3K9me3 and methylated-CpGs, consistent with ORCA-bound origins initiating DNA replication late in S-phase. Further, ORCA directly associates with the repressive marks and interacts with the enzymes that catalyze these marks. Regions that associate with both ORCA and H3K9me3, exhibit diminished H3K9 methylation in ORCA-depleted cells, suggesting a role for ORCA in recruiting the H3K9me3 mark at certain genomic loci. Similarly, DNA methylation is altered at ORCA-occupied sites in cells lacking ORCA. Furthermore, repressive chromatin marks influence ORCA's binding on chromatin. We propose that ORCA coordinates with the histone and DNA methylation machinery to establish a repressive chromatin environment at a subset of origins, which primes them for late replication.

PSIP1/p75 promotes tumorigenicity in breast cancer cells by promoting the transcription of cell cycle genes.

Breast cancer (BC) is a highly heterogeneous disease, both at the pathological and molecular level, and several chromatin-associated proteins play crucial roles in BC initiation and progression. Here, we demonstrate the role of PSIP1 (PC4 and SF2 interacting protein)/p75 (LEDGF) in BC progression. PSIP1/p75, previously identified as a chromatin-adaptor protein, is found to be upregulated in basal-like/triple negative breast cancer (TNBC) patient samples and cell lines. Immunohistochemistry in tissue arrays showed elevated levels of PSIP1 in metastatic invasive ductal carcinoma. Survival data analyses revealed that the levels of PSIP1 showed a negative association with TNBC patient survival. Depletion of PSIP1/p75 significantly reduced the tumorigenicity and metastatic properties of TNBC cell lines while its over-expression promoted tumorigenicity. Further, gene expression studies revealed that PSIP1 regulates the expression of genes controlling cell-cycle progression, cell migration and invasion. Finally, by interacting with RNA polymerase II, PSIP1/p75 facilitates the association of RNA pol II to the promoter of cell cycle genes and thereby regulates their transcription. Our findings demonstrate an important role of PSIP1/p75 in TNBC tumorigenicity by promoting the expression of genes that control the cell cycle and tumor metastasis.

Phosphorylation of Orc6 During Mitosis Regulates DNA Replication and Ribosome Biogenesis.

The human Origin Recognition Complex (ORC) is required not only for the initiation of DNA replication, but is also implicated in diverse cellular functions, including chromatin organization, centrosome biology, and cytokinesis. The smallest subunit of ORC, Orc6, is poorly conserved amongst eukaryotes. Recent studies from our laboratory have suggested that human Orc6 is not required for replication licensing, but is needed for S-phase progression. Further, ATR-dependent phosphorylation of Orc6 at T229 is implicated in DNA damage response during S-phase. In this study, we demonstrate that the CDK-dependent phosphorylation of Orc6 at T195 occurs during mitosis. While the phosphorylation at T195 does not seem to be required to exit mitosis, cells expressing the phosphomimetic T195E mutant of Orc6 impede S-phase progression. Moreover, the phosphorylated form of Orc6 associates with ORC more robustly, and Orc6 shows enhanced association with the ORC outside of G1, supporting the view that Orc6 may prevent the role of Orc1-5 in licensing outside of G1. Finally, Orc6 and the phosphorylated Orc6 localize to the nucleolar organizing centers and regulate ribosome biogenesis. Our results suggest that phosphorylated Orc6 at T195 prevents replication.

DNA Damage-Induced, S-Phase Specific Phosphorylation of Orc6 is Critical for the Maintenance of Genome Stability.

The smallest subunit of the human Origin Recognition Complex, hOrc6, is required for DNA replication progression and plays an important role in mismatch repair (MMR) during S-phase. However, the molecular details of how hOrc6 regulates DNA replication and DNA damage response remain to be elucidated. Orc6 levels are elevated upon specific types of genotoxic stress, and it is phosphorylated at Thr229, predominantly during S-phase, in response to oxidative stress. Many repair pathways, including MMR, mediate oxidative DNA damage repair. Defects in MMR are linked to Lynch syndrome, predisposing patients to many cancers, including colorectal cancer. Orc6 levels are known to be elevated in colorectal cancers. Interestingly, tumor cells show reduced hOrc6-Thr229 phosphorylation compared to adjacent normal mucosa. Further, elevated expression of wild-type and the phospho-dead forms of Orc6 results in increased tumorigenicity, implying that in the absence of this "checkpoint" signal, cells proliferate unabated. Based on these results, we propose that DNA-damage-induced hOrc6-pThr229 phosphorylation during S-phase facilitates ATR signaling in the S-phase, halts fork progression, and enables assembly of repair factors to mediate efficient repair to prevent tumorigenesis. Our study provides novel insights into how hOrc6 regulates genome stability.

Replication initiation: Implications in genome integrity.

In every cell cycle, billions of nucleotides need to be duplicated within hours, with extraordinary precision and accuracy. The molecular mechanism by which cells regulate the replication event is very complicated, and the entire process begins way before the onset of S phase. During the G1 phase of the cell cycle, cells prepare by assembling essential replication factors to establish the pre-replicative complex at origins, sites that dictate where replication would initiate during S phase. During S phase, the replication process is tightly coupled with the DNA repair system to ensure the fidelity of replication. Defects in replication and any error must be recognized by DNA damage response and checkpoint signaling pathways in order to halt the cell cycle before cells are allowed to divide. The coordination of these processes throughout the cell cycle is therefore critical to achieve genomic integrity and prevent diseases. In this review, we focus on the current understanding of how the replication initiation events are regulated to achieve genome stability.

The E3 ligase RFWD3 stabilizes ORC in a p53-dependent manner.

RFWD3 is an E3 ubiquitin ligase that plays important roles in DNA damage response and DNA replication. We have previously demonstrated that the stabilization of RFWD3 by PCNA at the replication fork enables ubiquitination of the single-stranded binding protein, RPA and its subsequent degradation for replication progression. Here, we report that RFWD3 associates with the Origin Recognition Complex (ORC) and ORC-Associated (ORCA/LRWD1), components of the pre-replicative complex required for the initiation of DNA replication. Overexpression of ORC/ORCA leads to the stabilization of RFWD3. Interestingly, RFWD3 seems to stabilize ORC/ORCA in cells expressing wild type p53, as the depletion of RFWD3 reduces the levels of ORC/ORCA. Further, the catalytic activity of RFWD3 is required for the stabilization of ORC. Our results indicate that the RFWD3 promotes the stability of ORC, enabling efficient pre-RC assembly.

ORCA/LRWD1 Regulates Homologous Recombination at ALT-Telomeres by Modulating Heterochromatin Organization.

Telomeres are maintained by telomerase or in a subset of cancer cells by a homologous recombination (HR)-based mechanism, Alternative Lengthening of Telomeres (ALT). The mechanisms regulating telomere-homeostasis in ALT cells remain unclear. We report that a replication initiator protein, Origin Recognition Complex-Associated (ORCA/LRWD1), by localizing at the ALT-telomeres, modulates HR activity. ORCA's localization to the ALT-telomeres is facilitated by its interaction to SUMOylated shelterin components. The loss of ORCA in ALT-positive cells elevates the levels of two mediators of HR, RPA and RAD51, and consistent with this, we observe increased ALT-associated promyelocytic leukemia body formation and telomere sister chromatid exchange. ORCA binds to RPA and modulates the association of RPA to telomeres. Finally, the loss of ORCA causes global chromatin decondensation, including at the telomeres. Our results demonstrate that ORCA acts as an inhibitor of HR by modulating RPA binding to ssDNA and inducing chromatin compaction.

The S-phase-induced lncRNA SUNO1 promotes cell proliferation by controlling YAP1/Hippo signaling pathway.

Cell cycle is a cellular process that is subject to stringent control. In contrast to the wealth of knowledge of proteins controlling the cell cycle, very little is known about the molecular role of lncRNAs (long noncoding RNAs) in cell-cycle progression. By performing genome-wide transcriptome analyses in cell-cycle-synchronized cells, we observed cell-cycle phase-specific induction of >2000 lncRNAs. Further, we demonstrate that an S-phase-upregulated lncRNA, SUNO1, facilitates cell-cycle progression by promoting YAP1-mediated gene expression. SUNO1 facilitates the cell-cycle-specific transcription of WTIP, a positive regulator of YAP1, by promoting the co-activator, DDX5-mediated stabilization of RNA polymerase II on chromatin. Finally, elevated SUNO1 levels are associated with poor cancer prognosis and tumorigenicity, implying its pro-survival role. Thus, we demonstrate the role of a S-phase up-regulated lncRNA in cell-cycle progression via modulating the expression of genes controlling cell proliferation.

Dietary Flavonoids Luteolin and Quercetin Suppressed Cancer Stem Cell Properties and Metastatic Potential of Isolated Prostate Cancer Cells.

A highly invasive Du145-III subline was isolated by three successive passages of the parental Du145 prostate tumor cell line (Du145-P) through a Boyden chamber with matrigel-coated membrane support. Du145-III cells showed great invasion potential based on their increased ability to spread/migrate and enhanced expression/secretion of the matrix metalloproteinase 9 (MMP9). Du145-III cells exerted vasculogenic mimicry (VM) properties, reminiscent of endothelial cell characteristics and expressed elevated levels of cancer stem cell (CSC) markers, including Nanog, Sox2, CD44 and ABCG2 and ability to self-renew. Of prominence, MMP9 was required for the induction of VM and for increased stemness in Du145-III cells. Using Du145-III as a model, the effects of dietary flavonoids, luteolin and quercetin, were evaluated on stemness and invasion capacity of Du145-III cells in relation to JNK signaling pathway activation. These flavonoids depressed the malignancy of highly invasive Du145-III cells, VM, anchorage-independent spheroid formation and expression of certain CSC markers. Since luteolin and quercetin were able to target CSC cells and prevent cancer cell invasiveness, may serve as potential anti-angiogenesis and anti-metastasis agents.

C1GALT1 overexpression promotes the invasive behavior of colon cancer cells through modifying O-glycosylation of FGFR2.

Core 1 ß1,3-galactosyltransferase (C1GALT1) transfers galactose (Gal) to N-acetylgalactosamine (GalNAc) to form Galß1,3GalNAc (T antigen). Aberrant O-glycans, such as T antigen, are commonly found in colorectal cancer. However, the role of C1GALT1 in colorectal cancer remains unclear. Here we showed that C1GALT1 was frequently overexpressed in colorectal tumors and is associated with poor survival. C1GALT1 overexpression promoted cell survival, migration, invasion, and sphere formation as well as tumor growth and metastasis of colon cancer cells. Conversely, knockdown of C1GALT1 with small interference (si) RNA was sufficient to suppress these malignant phenotypes in vitro and in vivo. Moreover, we are the first to show that fibroblast growth factor receptor (FGFR) 2 carried O-glycans in colon cancer cells. Mechanistic investigations showed that C1GALT1 modified the O-glycans on FGFR2 and enhanced bFGF-triggered activation of FGFR2 as well as increased bFGF-mediated malignant phenotypes. In addition, BGJ398, a selective inhibitor of FGFR, blocked the effects of C1GALT1. These findings suggest that C1GALT1 overexpression modifies O-glycans on FGFR2 and enhances its phosphorylation to promote the invasive behavior and cancer stem-like property in colon cancer cells, indicating a critical role of O-glycosylation in the pathogenesis of colorectal cancer.

ß1, 4-N-acetylgalactosaminyltransferase III modulates cancer stemness through EGFR signaling pathway in colon cancer cells.

Cancer stem cells are cancer cells characterized with tumor initiating capacity. ß1,4-N-acetylgalactosaminyltransferase III (B4GALNT3) synthesizes GalNAcß1-4GlcNAc (LacdiNAc) which contributes to self-renewal of mouse embryonic stem cells. We previously showed that B4GALNT3 overexpression enhances colon cancer cell malignant phenotypes in vitro and in vivo. However, the role of B4GALNT3 in cancer stemness remains unclear. We found that B4GALNT3 expression was positively correlated with advanced stages and poor survival in colorectal cancer patients. Knockdown of B4GALNT3 using small interfering (si) RNAs in colon cancer cell lines (HCT116, SW480, HCT15, and HT29 cells) decreased sphere formation and the expression of stem cell markers, OCT4 and NANOG. The expression of B4GALNT3 was upregulated in colonospheres. Interestingly, we found that B4GALNT3 primarily modified N-glycans of EGFR with LacdiNAc by Wisteria floribunda agglutinin (WFA) pull down assays. B4GALNT3 knockdown suppressed EGF-induced phosphorylation of EGFR and its downstream signaling molecules. Furthermore, EGF-induced degradation of EGFR was facilitated. In addition, EGF-induced migration and invasion were significantly suppressed by B4GALNT3 knockdown. Taken together, these data suggest B4GALNT3 regulates cancer stemness and the invasive properties of colon cancer cells through modifying EGFR glycosylation and signaling. Our results provide novel insights into the role of LacdiNAc in colorectal cancer development.

Impact of flavonoids on matrix metalloproteinase secretion and invadopodia formation in highly invasive A431-III cancer cells.

Metastasis is a major cause of mortality in cancer patients. Invadopodia are considered to be crucial structures that allow cancer cells to penetrate across the extracellular matrix (ECM) by using matrix metalloproteinases (MMPs). Previously, we isolated a highly invasive A431-III subline from parental A431 cells by Boyden chamber assay. The A431-III cells possess higher invasive and migratory abilities, elevated levels of MMP-9 and an enhanced epithelial-mesenchymal transition (EMT) phenotype. In this study, we discovered that A431-III cells had an increased potential to form invadopodia and an improved capacity to degrade ECM compared with the original A431 cells. We also observed enhanced phosphorylation levels of cortactin and Src in A431-III cells; these phosphorylated proteins have been reported to be the main regulators of invadopodia formation. Flavonoids, almost ubiquitously distributed in food plants and plant food products, have been documented to exhibit anti-tumor properties. Therefore, it was of much interest to explore the effects of flavonoid antioxidants on the metastatic activity of A431-III cells. Exposure of A431-III cells to two potent dietary flavonoids, namely luteolin (Lu) and quercetin (Qu), caused inhibition of invadopodia formation and decrement in ECM degradation. We conclude that Lu and Qu attenuate the phosphorylation of cortactin and Src in A431-III cells. As a consequence, there ensues a disruption of invadopodia generation and the suppression of MMP secretion. These changes, in concert, bring about a reduction in metastasis.