SP1 undergoes phase separation and activates RGS20 expression through super-enhancers to promote lung adenocarcinoma progression.

Lung adenocarcinoma (LUAD) is the leading cause of cancer-related death worldwide, but the underlying molecular mechanisms remain largely unclear. The transcription factor (TF) specificity protein 1 (SP1) plays a crucial role in the development of various cancers, including LUAD. Recent studies have indicated that master TFs may form phase-separated macromolecular condensates to promote super-enhancer (SE) assembly and oncogene expression. In this study, we demonstrated that SP1 undergoes phase separation and that its zinc finger 3 in the DNA-binding domain is essential for this process. Through Cleavage Under Targets & Release Using Nuclease (CUT&RUN) using antibodies against SP1 and H3K27ac, we found a significant correlation between SP1 enrichment and SE elements, identified the regulator of the G protein signaling 20 (RGS20) gene as the most likely target regulated by SP1 through SE mechanisms, and verified this finding using different approaches. The oncogenic activity of SP1 relies on its phase separation ability and RGS20 gene activation, which can be abolished by glycogen synthase kinase J4 (GSK-J4), a demethylase inhibitor. Together, our findings provide evidence that SP1 regulates its target oncogene expression through phase separation and SE mechanisms, thereby promoting LUAD cell progression. This study also revealed an innovative target for LUAD therapies through intervening in SP1-mediated SE formation.

Getah virus capsid protein undergoes co-condensation with viral genomic RNA to facilitate virion assembly.

Getah virus (GETV) belongs to the Alphavirus genus in the Togaviridae family and is a zoonotic arbovirus causing disease in both humans and animals. The capsid protein (CP) of GETV regulates the viral core assembly, but the mechanism underlying this process is poorly understood. In this study, we demonstrate that CP undergoes liquid-liquid phase separation (LLPS) with the GETV genome RNA (gRNA) in vitro and forms cytoplasmic puncta in cells. Two regions of GETV gRNA (nucleotides 1-4000 and 5000-8000) enhance CP droplet formation in vitro and the lysine-rich Link region of CP is essential for its phase separation. CP(K/R) mutant with all lysines in the Link region replaced by arginines exhibits improved LLPS versus wild type (WT) CP, but CP(K/E) mutant with lysines substituted by glutamic acids virtually loses condensation ability. Consistently, recombinant virus mutant with CP(K/R) possesses significantly higher gRNA binding affinity, virion assembly efficiency and infectivity than the virus with WT-CP. Overall, our findings provide new insights into the understanding of GETV assembly and development of new antiviral drugs against alphaviruses.

G-quadruplexes promote the motility in MAZ phase-separated condensates to activate CCND1 expression and contribute to hepatocarcinogenesis.

G-quadruplexes (G4s) can recruit transcription factors to activate gene expression, but detailed mechanisms remain enigmatic. Here, we demonstrate that G4s in the CCND1 promoter propel the motility in MAZ phase-separated condensates and subsequently activate CCND1 transcription. Zinc finger (ZF) 2 of MAZ is a responsible for G4 binding, while ZF3-5, but not a highly disordered region, is critical for MAZ condensation. MAZ nuclear puncta overlaps with signals of G4s and various coactivators including BRD4, MED1, CDK9 and active RNA polymerase II, as well as gene activation histone markers. MAZ mutants lacking either G4 binding or phase separation ability did not form nuclear puncta, and showed deficiencies in promoting hepatocellular carcinoma cell proliferation and xenograft tumor formation. Overall, we unveiled that G4s recruit MAZ to the CCND1 promoter and facilitate the motility in MAZ condensates that compartmentalize coactivators to activate CCND1 expression and subsequently exacerbate hepatocarcinogenesis.

Integrative genomic analyses of promoter G-quadruplexes reveal their selective constraint and association with gene activation.

G-quadruplexes (G4s) regulate DNA replication and gene transcription, and are enriched in promoters without fully appreciated functional relevance. Here we show high selection pressure on putative G4 (pG4) forming sequences in promoters through investigating genetic and genomic data. Analyses of 76,156 whole-genome sequences reveal that G-tracts and connecting loops in promoter pG4s display lower or higher allele frequencies, respectively, than pG4-flanking regions, and central guanines (Gs) in G-tracts show higher selection pressure than other Gs. Additionally, pG4-promoters produce over 72.4% of transcripts, and promoter G4-containing genes are expressed at relatively high levels. Most genes repressed by TMPyP4, a G4-ligand, regulate epigenetic processes, and promoter G4s are enriched with gene activation histone marks, chromatin remodeler and transcription factor binding sites. Consistently, cis-expression quantitative trait loci (cis-eQTLs) are enriched in promoter pG4s and their G-tracts. Overall, our study demonstrates selective constraint of promoter G4s and reinforces their stimulative role in gene expression.

Circular RNAs with protein-coding ability in oncogenesis.

As ubiquitously expressed transcripts in eukaryotes, circular RNAs (circRNAs) are covalently closed and lack a 5'-cap and 3'-polyadenylation (poly (A)) tail. Initially, circRNAs were considered non-coding RNA (ncRNA), and their roles as sponging molecules to adsorb microRNAs have been extensively reported. However, in recent years, accumulating evidence has demonstrated that circRNAs could encode functional polypeptides through the initiation of translation mediated by internal ribosomal entry sites (IRESs) or N6-methyladenosine (m6A). In this review, we collectively discuss the biogenesis, cognate mRNA products, regulatory mechanisms, aberrant expression and biological phenotypes or clinical relevance of all currently reported, cancer-relevant protein-coding circRNAs. Overall, we provide a comprehensive overview of circRNA-encoded proteins and their physiological and pathological functions.

Designing for downsizing: Home-based barriers and facilitators to reduce portion sizes for children.

Evidence confirms that parents know that they should limit non-core foods for their children since these tend to be high in energy density (HED), fat, salt and sugar. However, it is unclear how knowledge of portion size limits, such as the 100 kcal guide from Public Health England are applied in practice. To observe in real-time children's home food environment related to portion control and to explore with parents their reported portion size strategies, a mixed methods study was designed. Families with children aged 1-5 years were recruited (n = 21) to a three-part study: (1) to complete questionnaires and interviews on household food intake and portion control; (2) to report daily food intake for 4 days (n = 13) for one parent and their child(ren); (3) to observe home-based food provisioning via videorecording during dinner, breakfast and snack time (n = 6). Although the problem of large portion sizes of HED foods was recognised by mothers, strategies to downsize portions were not necessarily applied at home, as revealed in home observations and diaries. A mismatch between what was observed at home, what was reported in food diaries and what was said in interviews became apparent for some families. Mothers reported the need for greater support and guidance to downsize HED foods since they relied on pre-packaging as a guide to intake. Education and engagement were identified as important parameters for downsizing by mothers. One strategy which could be explored and applied by manufacturers is packaging design to faciliate the 100 kcal guidance using physical and engaging ways to assist parents in downsizing HED foods for their children. To facilitate effective government communication, innovative packaging design can be used to convey clear guidance and to tailor portion size messages for children. Packaging design, alongside government recommendations, can support parents' goals to achieve healthy eating and can reinforce guidance for portion norms through innovation involving learning, playful engagement, and interaction.

Phase-Separated Subcellular Compartmentation and Related Human Diseases.

In live cells, proteins and nucleic acids can associate together through multivalent interactions, and form relatively isolated phases that undertake designated biological functions and activities. In the past decade, liquid-liquid phase separation (LLPS) has gradually been recognized as a general mechanism for the intracellular organization of biomolecules. LLPS regulates the assembly and composition of dozens of membraneless organelles and condensates in cells. Due to the altered physiological conditions or genetic mutations, phase-separated condensates may undergo aberrant formation, maturation or gelation that contributes to the onset and progression of various diseases, including neurodegenerative disorders and cancers. In this review, we summarize the properties of different membraneless organelles and condensates, and discuss multiple phase separation-regulated biological processes. Based on the dysregulation and mutations of several key regulatory proteins and signaling pathways, we also exemplify how aberrantly regulated LLPS may contribute to human diseases.

YY1 Oligomerization Is Regulated by Its OPB Domain and Competes with Its Regulation of Oncoproteins.

Yin Yang 1 (YY1) plays an oncogenic role through regulating the expression of various cancer-related genes and activating key oncoproteins. Previous research reported that YY1 protein formed dimers or oligomers without definite biological implications. In this study, we first demonstrated the oncoprotein binding (OPB) and zinc finger (ZF) domains of YY1 as the regions involved in its intermolecular interactions. ZFs are well-known for protein dimerization, so we focused on the OPB domain. After mutating three hydrophobic residues in the OPB to alanines, we discovered that YY1(F219A) and YY1(3A), three residues simultaneously replaced by alanines, were defective of intermolecular interaction. Meanwhile, the OPB peptide could robustly facilitate YY1 protein oligomerization. When expressed in breast cancer cells with concurrent endogenous YY1 knockdown, YY1(F219A) and (3A) mutants showed better capacity than wt in promoting cell proliferation and migration, while their interactions with EZH2, AKT and MDM2 showed differential alterations, especially with improved EZH2 binding affinity. Our study revealed a crucial role of the OPB domain in facilitating YY1 oligomerization and suggested a mutually exclusive regulation between YY1-mediated enhancer formation and its activities in promoting oncoproteins.

A histidine cluster determines YY1-compartmentalized coactivators and chromatin elements in phase-separated enhancer clusters.

As an oncogenic transcription factor, Yin Yang 1 (YY1) regulates enhancer and promoter connection. However, gaps still exist in understanding how YY1 coordinates coactivators and chromatin enhancer elements to assemble enhancers and super-enhancers. Here, we demonstrate that a histidine cluster in YY1's transactivation domain is essential for its formation of phase separation condensates, which can be extended to additional proteins. The histidine cluster is also required for YY1-promoted cell proliferation, migration, clonogenicity and tumor growth. YY1-rich nuclear puncta contain coactivators EP300, BRD4, MED1 and active RNA polymerase II, and colocalize with histone markers of gene activation, but not that of repression. Furthermore, YY1 binds to the consensus motifs in the FOXM1 promoter to activate its expression. Wild-type YY1, but not its phase separation defective mutant, connects multiple enhancer elements and the FOXM1 promoter to form an enhancer cluster. Consistently, fluorescent in situ hybridization (FISH) assays reveal the colocalization of YY1 puncta with both the FOXM1 gene locus and its nascent RNA transcript. Overall, this study demonstrates that YY1 activates target gene expression through forming liquid-liquid phase separation condensates to compartmentalize both coactivators and enhancer elements, and the histidine cluster of YY1 plays a determinant role in this regulatory mechanism.

Disruption of YY1-EZH2 Interaction Using Synthetic Peptides Inhibits Breast Cancer Development.

Enhancer of zeste homolog 2 (EZH2) is a methyltransferase to mediate lysine 27 trimethylation in histone H3 (i.e., H3K27me3) and repress gene expression. In solid tumors, EZH2 promotes oncogenesis and is considered a therapeutic target. As a transcription factor, Yin Yang 1 (YY1) recruits EZH2 through its oncoprotein binding (OPB) domain to establish gene repression. In this study, we mapped the YY1 protein binding (YPB) domain on EZH2 to a region of 27 amino acids. Both YPB and OPB domain synthetic peptides could disrupt YY1EZH2 interaction, markedly reduce breast cancer cell viability, and efficiently inhibit tumor growth in a xenograft mouse model. We analyzed MDA-MB-231 cells treated with YPB, OPB, and control peptides by chromatin immunoprecipitation DNA sequencing (ChIP-seq) using an antibody against H3K27me3. YPB and OPB treatments altered H3K27me3 on 465 and 1137 genes, respectively, compared to the control. Of these genes, 145 overlapped between the two peptides. Among them, PTENP1, the PTEN pseudogene, showed reduced H3K27me3 signal when treated by either YPB or OPB peptide. Consistently, the two peptides enhanced both PTENP1 and PTEN expression with concomitantly reduced AKT activation. Further studies validated PTENP1's contribution to the anticancer activity of YPB and OPB peptides.

AKT1 is positively regulated by G-quadruplexes in its promoter and 3'-UTR.

AKT1 plays a key role in cell growth and survival, and its activation in cancers is mediated by different mechanisms. In this study, we investigated the potential of G-quadruplex (G4) formation by multiple consecutive G-tracts in the AKT1 promoter and its 3'-UTR. In circular dichroism analyses, synthetic oligonucleotides based on these G-tract regions showed molar ellipticity peaks at specific wavelengths of G4 structures. We verified G4 forming potential of these oligonucleotides using dimethyl sulfate footprinting, gel-shift and immunostaining assays. In reporter assays, mutations of the G-tracts in either the promoter or the 3'-UTR of AKT1 reduced expression mediated by these G-rich regions, suggesting positive regulation of AKT1 gene expression by these G4 structures. Furthermore, SP1 bound to its consensus sites regardless of the presence of G4 motifs in the AKT1 promoter, and both the G4 motifs and SP1 binding sites were needed to reach the strongest promoter strength.

Human MYC G-quadruplex: From discovery to a cancer therapeutic target.

Overexpression of the MYC oncogene is a molecular hallmark of both cancer initiation and progression. Targeting MYC is a logical and effective cancer therapeutic strategy. A special DNA secondary structure, the G-quadruplex (G4), is formed within the nuclease hypersensitivity element III1 (NHE III1) region, located upstream of the MYC gene's P1 promoter that drives the majority of its transcription. Targeting such G4 structures has been a focus of anticancer therapies in recent decades. Thus, a comprehensive review of the MYC G4 structure and its role as a potential therapeutic target is timely. In this review, we first outline the discovery of the MYC G4 structure and evidence of its formation in vitro and in cells. Then, we describe the functional role of G4 in regulating MYC gene expression. We also summarize three types of MYC G4-interacting proteins that can promote, stabilize and unwind G4 structures. Finally, we discuss G4-binding molecules and the anticancer activities of G4-stabilizing ligands, including small molecular compounds and peptides, and assess their potential as novel anticancer therapeutics.

The SNAIL1 promoter contains G-quadruplex structures regulating its gene expression and DNA replication.

SNAIL1 is a key regulator of epithelial-mesenchymal transition (EMT) and its expression is associated with tumor progression and poor clinical prognosis of cancer patients. Compared to the studies of SNAIL1 stability and its transcriptional regulation, very limited knowledge is available regarding effective approaches to directly target SNAIL1. In this study, we revealed the potential regulation of SNAIL1 gene expression by G-quadruplex structures in its promoter. We first revealed that the negative strand of the SNAIL1 promoter contained a multi-G-tract region with high potential of forming G-quadruplex structures. In circular dichroism studies, the oligonucleotide based on this region showed characteristic molar ellipticity at specific wavelengths of G-quadruplex structures. We also utilized native polyacrylamide gel electrophoresis, gel-shift assays, immunofluorescent staining, dimethyl sulfate footprinting and chromatin immunoprecipitation studies to verify the G-quadruplex structures formed by the oligonucleotide. In reporter assays, disruption of G-quadruplex potential increased SNAIL1 promoter-mediated transcription, suggesting that G-quadruplexes played a negative role in SNAIL1 expression. In a DNA synthesis study, we detected G-quadruplex-mediated retardation in the SNAIL1 promoter replication. Consistently, we discovered that the G-quadruplex region of the SNAIL1 promoter is highly enriched for mutations, implicating the clinical relevance of G-quadruplexes to the altered SNAIL1 expression in cancer cells.

Characterization of G-quadruplex formation in the ARID1A promoter.

As a member of the SWI/SNF family, ARID1A plays an essential role in modulating chromatin structure and gene expression. The tumor suppressive function of ARID1A has been well-defined and its downregulation in cancers is attributed to genomic deletion, DNA methylation and microRNA-mediated inhibition. In this study, we demonstrated that the negative strand of a C-rich region in the upstream vicinity of the human ARID1A transcription start site could form G-quadruplexes. Synthesized oligonucleotides based on the sequence of this region exhibited molar ellipticity at specific wavelengths characteristic of G-quadruplex structures in circular dichroism analyses. The formation of G-quadruplexes by these oligonucleotides were also proved by native polyacrylamide gel electrophoresis, DNA synthesis block assays, immunofluorescent staining and dimethyl sulfate footprinting studies. In reporter assays, mutations of the G-quadruplex forming sequence reduced ARID1A promoter-mediated transcription. Transfection of the oligonucleotide with the full length of G-quadruplex motif region, but not its partial sequences or the mutants, could both promote endogenous ARID1A expression and reduce cell proliferation.

Advances of Zinc Signaling Studies in Prostate Cancer.

Prostate cancer (PCa) is one of the most common cancers and the second leading cause of cancer-related death among men worldwide. Despite progresses in early diagnosis and therapeutic strategies, prognosis for patients with advanced PCa remains poor. Noteworthily, a unique feature of healthy prostate is its highest level of zinc content among all soft tissues in the human body, which dramatically decreases during prostate tumorigenesis. To date, several reviews have suggested antitumor activities of zinc and its potential as a therapeutic strategy of PCa. However, an overview about the role of zinc and its signaling in PCa is needed. Here, we review literature related to the content, biological function, compounds and clinical application of zinc in PCa. We first summarize zinc content in prostate tissue and sera of PCa patients with their clinical relevance. We then elaborate biological functions of zinc signaling in PCa on three main aspects, including cell proliferation, death and tumor metastasis. Finally, we discuss clinical applications of zinc-containing compounds and proteins involved in PCa signaling pathways. Based on currently available studies, we conclude that zinc plays a tumor suppressive role and can serve as a biomarker in PCa diagnosis and therapies.

"Wrap healthy snacks with cool packaging" - A qualitative study of mothers' portion size strategies for their children.

Offering large portions of high energy dense (HED) foods increases overall energy intake in children, a potentially important contributing factor to childhood overweight and obesity. Packaging offers a simple heuristic to encourage healthy eating for nutrient dense foods and to downsize portions of HED foods. However, it is not clear how parents use packaging for portion control, nor how packaging might be used as a solution to offset large portions. Therefore, the aim of the present study was to investigate mothers' portion strategies and how they use packaging to facilitate portion control for children. 21 mothers of 25 children aged 1-5 years participated in semi-structured interviews to identify strategies used by mothers for portioning snack and meal items. Mothers reported feeling confident in amounts offered to their children, and were unaware of, or did not apply, recommendations for age-appropriate portions of meal items and snacks. Mothers portioned according to child appetite, needs and characteristics, not necessarily age. They reported that their child was able to determine for themselves how much to consume. However, mothers also applied restrictions to some foods. No differences in considerations and strategies were found between different ethnic groups of British and Chinese mothers. Mothers reported that packaging was an important determinant of preferences and a useful, convenient means of portion control. To promote appropriate consumption norms in children, a packaging design concept is described to aid downsizing for a highly liked HED food. Future studies should examine how creative packaging solutions influence parents' feeding practices and how this might influence dietary quality through user testing.

YY1 Is an Inducer of Cancer Metastasis.

Yin Yang 1 (YY1) is a member of the GLI-Kruppel family of zinc finger proteins that plays vital roles in many biological processes, especially tumorigenesis. To date, ample evidence suggests a critical regulatory role of YY1 in tumor cell metastasis. The potential of YY1 as a valuable biomarker for cancer metastasis has been increasingly known. Here, we review the studies related to the expression, regulatory network, and clinical application of YY1 in cancer metastasis. We first summarize YY1 expression patterns in metastatic tumors. We then elaborate YY1-regulated mechanisms on five aspects, including epithelial-mesenchymal transition, cell migration and invasion, stemness, polyploidy, and genomic stability. Finally, we discuss the correlation between YY1 expression and clinical outcomes and therapeutic potential of YY1 in cancer treatment. Based on this review, we conclude that YY1 is a bona fide inducer of cancer metastasis and can serve as a clinical biomarker and therapeutic target for cancer treatment.

Nucleolin down-regulation is involved in ADP-induced cell cycle arrest in S phase and cell apoptosis in vascular endothelial cells.

High concentration of extracellular ADP has been reported to induce cell apoptosis, but the molecular mechanisms remain not fully elucidated. In this study, we found by serendipity that ADP treatment of human umbilical vein endothelial cells (HUVEC) and human aortic endothelial cells (HAEC) down-regulated the protein level of nucleolin in a dose- and time-dependent manner. ADP treatment did not decrease the transcript level of nucloelin, suggesting that ADP might induce nucleolin protein degradation. HUVEC and HAEC expressed ADP receptor P2Y13 receptor, but did not express P2Y1 or P2Y12 receptors. However, P2Y1, 12, 13 receptor antagonists MRS2179, PSB0739, MRS2211 did not inhibit ADP-induced down-regulation of nucleolin. Moreover, MRS2211 itself down-regulated nucleolin protein level. In addition, 2-MeSADP, an agonist for P2Y1, 12 and 13 receptors, did not down-regulate nucleolin protein. These results suggested that ADP-induced nucleolin down-regulation was not due to the activation of P2Y1, 12, or 13 receptors. We also found that ADP treatment induced cell cycle arrest in S phase, cell apoptosis and cell proliferation inhibition via nucleolin down-regulation. The over-expression of nucleolin by gene transfer partly reversed ADP-induced cell cycle arrest, cell apoptosis and cell proliferation inhibition. Furthermore, ADP sensitized HUVEC to cisplatin-induced cell death by the down-regulation of Bcl-2 expression. Taken together, we found, for the first time to our knowledge, a novel mechanism by which ADP regulates cell proliferation by induction of cell cycle arrest and cell apoptosis via targeting nucelolin.

P2Y11 impairs cell proliferation by induction of cell cycle arrest and sensitizes endothelial cells to cisplatin-induced cell death.

Extracellular ATP mediates a wide range of physiological effects, including cell proliferation, differentiation, maturation, and migration. However, the effect of ATP on cell proliferation has been contradictory, and the mechanism is not fully understood. In the current study, we found that extracellular ATP significantly inhibited the proliferation of human umbilical vein endothelial cells (HUVECs) and human aortic endothelial cells (HAECs). Treatment with ATP did not induce cell apoptosis but instead induced cell cycle arrest in S phase. ATP induced the phosphorylation of ERK1/2, but the ERK inhibitors, U0126 and PD9809, did not regulate the inhibition of cell proliferation induced by ATP. However, ATP-induced inhibition of cell proliferation was blocked by suramin, a nonspecific antagonist of the P2Y receptors, and endothelial cells expressed P2Y11, a P2Y receptor that specifically binds ATP. Moreover, the down-regulation of P2Y11 by RNA interference not only reversed the inhibition of cell proliferation but also ameliorated cell cycle arrest in S phase. In addition, P2Y11 sensitized endothelial cells to cisplatin-induced cell death by down-regulation of the expression of Bcl-2. Taken together, these results suggest that extracellular ATP impairs cell proliferation by triggering signaling to induce cell cycle arrest and sensitizes cell to death via P2Y11 in endothelial cells.

TLR4 activation induces nontolerant inflammatory response in endothelial cells.

In professional immune cells, Toll-like receptor 4 (TLR4) induces tightly regulated inflammatory response to avoid tissue damage via the induction of "endotoxin tolerance", which is a transient state of cell desensitization in response to lipopolysaccharide (LPS) restimulation after a prior LPS exposure. However, in endothelial cells, the regulation of TLR4-induced inflammation is not fully understood. In this study, we found that the gene transcripts for a lot of Toll-like receptors were expressed in various endothelial cells, including human umbilical vein endothelial cells (HUVEC), human aortic endothelial cell (HAEC), and mouse microvascular endothelial cells (bEND.3). Proteins of TLR4 and its coreceptor CD14 were also detected in HUVEC. LPS treatment significantly upregulated the expression of proinflammation cytokines such as IL-1ß, IL-6, and IL-8 only in HUVEC, but not in HAEC and bEND.3, suggesting that vein endothelial cells are important source of proinflammatory cytokines in response to LPS. Unexpectedly, "endotoxin tolerance" was not induced in endothelial cell, but was induced in control glial cells, as LPS pretreatment downregulated the cytokine expression in control glial cells, but did not in endothelial cells, when the cells were restimulated with LPS. The upregulation of cytokine gene expression was dependent on NF-κB signaling, and NF-κB inhibitor repressed the induction of cytokines. Two important signal molecules MyD88 and TRIF, which are TLR4 downstream and NF-κB upstream, were upregulated in vein endothelial cells but were downregulated in control glial cells. These results suggested that vein endothelial cells may play important roles in the pathophysiology of systemic inflammation-associated diseases such as sepsis and septic cardiomyopathy.