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.

Plakophilin3 loss leads to increased adenoma formation and rectal prolapse in APCmin mice.

Plakophilin3 (PKP3) loss leads to tumor progression and metastasis of colon cancer cells. The goal of this report was to determine if PKP3 loss led to increased disease progression in mice. We generated a colonocyte-specific knockout of PKP3 in APCmin mice, which led to increased adenoma formation, the formation of rectal prolapse, and a significant decrease in survival. The observed increase in rectal prolapse formation and decrease in survival correlated with an increase in the expression of Lipocalin2 (LCN2). Increased disease progression was observed even upon treatment with 5-fluorouracil (5FU). These results suggest that an increase in LCN2 expression might lead to therapy resistance and that LCN2 might serve as a potential therapeutic target in colorectal cancer.

LncRNA-mediated regulation of SOX9 expression in basal subtype breast cancer cells.

Triple-negative breast cancer (TNBC) is one of the most aggressive breast cancer (BC) subtypes with a poor prognosis and high recurrence rate. Recent studies have identified vital roles played by several lncRNAs (long noncoding RNAs) in BC pathobiology. Cell type-specific expression of lncRNAs and their potential role in regulating the expression of oncogenic and tumor suppressor genes have made them promising cancer drug targets. By performing a transcriptome screen in an isogenic TNBC/basal subtype BC progression cell line model, we recently reported ∼1800 lncRNAs that display aberrant expression during breast cancer progression. Mechanistic studies on one such nuclear-retained lncRNA, linc02095, reveal that it promotes breast cancer proliferation by facilitating the expression of oncogenic transcription factor, SOX9. Both linc02095 and SOX9 display coregulated expression in BC patients as well in basal subtype BC cell lines. Knockdown of linc02095 results in decreased BC cell proliferation, whereas its overexpression promotes cells proliferation. Linc02095-depleted cells display reduced expression of SOX9 concomitant with reduced RNA polymerase II occupancy at the SOX9 gene body as well as defective SOX9 mRNA export, implying that linc02095 positively regulates SOX9 transcription and mRNA export. Finally, we identify a positive feedback loop in BC cells that controls the expression of both linc02095 and SOX9 Thus, our results unearth tumor-promoting activities of a nuclear lncRNA linc02095 by facilitating the expression of key oncogenic transcription factor in BC.

Chromatin's Influence on Pre-Replication Complex Assembly and Function.

In all eukaryotes, the initiation of DNA replication requires a stepwise assembly of factors onto the origins of DNA replication. This is pioneered by the Origin Recognition Complex, which recruits Cdc6. Together, they bring Cdt1, which shepherds MCM2-7 to form the OCCM complex. Sequentially, a second Cdt1-bound hexamer of MCM2-7 is recruited by ORC-Cdc6 to form an MCM double hexamer, which forms a part of the pre-RC. Although the mechanism of ORC binding to DNA varies across eukaryotes, how ORC is recruited to replication origins in human cells remains an area of intense investigation. This review discusses how the chromatin environment influences pre-RC assembly, function, and, eventually, origin activity.

Upregulation of miR-22-3p contributes to plumbagin-mediated inhibition of Wnt signaling in human colorectal cancer cells.

The emergence of resistance and side effects for currently available drugs warrant the need for an alternative treatment regime for colorectal cancer (CRC). Several natural compounds, including plumbagin, have exhibited promising anti-cancer effects in different cancer models, although the molecular mechanisms underlying their effects remain at large. We previously reported plumbagin-mediated inhibition of Wnt signaling in CRC cells and to unravel the actual mechanism, we hypothesized the involvement of miRNAs as they have emerged as critical regulators of gene expression. We performed miRNA-microarray to check if plumbagin-mediated effects are through alteration of miRNA expression and found 32 DE-miRNAs (11 upregulated and 21 downregulated), and KEGG enrichment analysis indicated that their target mRNAs are associated with several pathways relevant to CRC and Wnt signaling. miRNA-mRNA network analysis by miRNET revealed a highly upregulated miRNA upon plumbagin treatment, miR-22-3p, having multiple Wnt-associated mRNAs interacting partners. 3'UTR luciferase assays revealed that miR-22-3p directly targeted the 3'UTR of BCL9L (a coactivator of CTNNB1). miR-22-3p inhibited Wnt signaling by downregulating the levels of BCL9L and that of CTNNB1 and several Wnt-associated proteins (TCF4, c-Myc, CCND1, Snail, Slug, and vimentin) in CRC cells. We also demonstrated that both miR-22-3p and plumbagin reduced colony formation and caused apoptotic cell death (detected by Annexin V/PI dual staining), possibly through increased ROS (detection by Dihydroethidium staining) and decreased MMP (detection by MitoTracker™ Orange staining) in CRC cells. These effects of plumbagin were partially rescued by antimiR-22-3p, suggesting the involvement of miR-22-3p in plumbagin-mediated effects. The present study revealed that alteration in miR-22-3p levels by plumbagin contributes to the induction of apoptosis and its inhibitory effects on Wnt signaling and colony formation, thus providing a mechanistic basis behind its anti-cancer potential.