Cell-based screen identifies porphyrins as FGFR3 activity inhibitors with therapeutic potential for achondroplasia and cancer.

Overactive fibroblast growth factor receptor 3 (FGFR3) signaling drives pathogenesis in a variety of cancers and a spectrum of short-limbed bone dysplasias, including the most common form of human dwarfism, achondroplasia (ACH). Targeting FGFR3 activity holds great promise as a therapeutic approach for treatment of these diseases. Here, we established a receptor/adaptor translocation assay system that can specifically monitor FGFR3 activation, and we applied it to identify FGFR3 modulators from complex natural mixtures. An FGFR3-suppressing plant extract of Amaranthus viridis was identified from the screen, and 2 bioactive porphyrins, pheophorbide a (Pa) and pyropheophorbide a, were sequentially isolated from the extract and functionally characterized. Further analysis showed that Pa reduced excessive FGFR3 signaling by decreasing its half-life in FGFR3-overactivated multiple myeloma cells and chondrocytes. In an ex vivo culture system, Pa alleviated defective long bone growth in humanized ACH mice (FGFR3ACH mice). Overall, our study presents an approach to discovery and validation of plant extracts or drug candidates that target FGFR3 activation. The compounds identified by this approach may have applications as therapeutics for FGFR3-associated cancers and skeletal dysplasias.

H3K4 Methylation in Aging and Metabolism.

During the process of aging, extensive epigenetic alterations are made in response to both exogenous and endogenous stimuli. Here, we summarize the current state of knowledge regarding one such alteration, H3K4 methylation (H3K4me), as it relates to aging in different species. We especially highlight emerging evidence that links this modification with metabolic pathways, which may provide a mechanistic link to explain its role in aging. H3K4me is a widely recognized marker of active transcription, and it appears to play an evolutionarily conserved role in determining organism longevity, though its influence is context specific and requires further clarification. Interestingly, the modulation of H3K4me dynamics may occur as a result of nutritional status, such as methionine restriction. Methionine status appears to influence H3K4me via changes in the level of S-adenosyl methionine (SAM, the universal methyl donor) or the regulation of H3K4-modifying enzyme activities. Since methionine restriction is widely known to extend lifespan, the mechanistic link between methionine metabolic flux, the sensing of methionine concentrations and H3K4me status may provide a cogent explanation for several seemingly disparate observations in aging organisms, including age-dependent H3K4me dynamics, gene expression changes, and physiological aberrations. These connections are not yet entirely understood, especially at a molecular level, and will require further elucidation. To conclude, we discuss some potential H3K4me-mediated molecular mechanisms that may link metabolic status to the aging process.

Local chromatin fiber folding represses transcription and loop extrusion in quiescent cells.

A longstanding hypothesis is that chromatin fiber folding mediated by interactions between nearby nucleosomes represses transcription. However, it has been difficult to determine the relationship between local chromatin fiber compaction and transcription in cells. Further, global changes in fiber diameters have not been observed, even between interphase and mitotic chromosomes. We show that an increase in the range of local inter-nucleosomal contacts in quiescent yeast drives the compaction of chromatin fibers genome-wide. Unlike actively dividing cells, inter-nucleosomal interactions in quiescent cells require a basic patch in the histone H4 tail. This quiescence-specific fiber folding globally represses transcription and inhibits chromatin loop extrusion by condensin. These results reveal that global changes in chromatin fiber compaction can occur during cell state transitions, and establish physiological roles for local chromatin fiber folding in regulating transcription and chromatin domain formation.

Histone dynamics during DNA replication stress.

Accurate and complete replication of the genome is essential not only for genome stability but also for cell viability. However, cells face constant threats to the replication process, such as spontaneous DNA modifications and DNA lesions from endogenous and external sources. Any obstacle that slows down replication forks or perturbs replication dynamics is generally considered to be a form of replication stress, and the past decade has seen numerous advances in our understanding of how cells respond to and resolve such challenges. Furthermore, recent studies have also uncovered links between defects in replication stress responses and genome instability or various diseases, such as cancer. Because replication stress takes place in the context of chromatin, histone dynamics play key roles in modulating fork progression and replication stress responses. Here, we summarize the current understanding of histone dynamics in replication stress, highlighting recent advances in the characterization of fork-protective mechanisms.

Expression of zebrafish anterior gradient 2 in the semicircular canals and supporting cells of otic vesicle sensory patches is regulated by Sox10.

AGR2 is a member of the protein disulfide isomerase (PDI) family, which is implicated in cancer cell growth and metastasis, asthma, and inflammatory bowel disease. Despite the contributions of this protein to several biological processes, the regulatory mechanisms controlling expression of the AGR2 gene in different organs remain unclear. Zebrafish anterior gradient 2 (agr2) is expressed in several organs, including the otic vesicles that contain mucus-secreting cells. To elucidate the regulatory mechanisms controlling agr2 expression in otic vesicles, we generated a Tg(-6.0 k agr2:EGFP) transgenic fish line that expressed EGFP in a pattern recapitulating that of agr2. Double immunofluorescence studies were used to demonstrate that Agr2 and GFP colocalize in the semicircular canals and supporting cells of all sensory patches in the otic vesicles of Tg(-6.0 k agr2:EGFP) embryos. Transient/stable transgenic analyses coupled with 5'-end deletion revealed that a 100 bp sequence within the -2.6 to -2.5 kbp region upstream of agr2 directs EGFP expression specifically in the otic vesicles. Two HMG-binding motifs were detected in this region. Mutation of these motifs prevented EGFP expression. Furthermore, EGFP expression in the otic vesicles was prevented by knockdown of the sox10 gene. This corresponded with decreased agr2 expression in the otic vesicles of sox10 morphants during different developmental stages. Electrophoretic mobility shift assays were used to show that Sox10 binds to HMG-binding motifs located within the -2.6 to -2.5 kbp region upstream of agr2. These results demonstrate that agr2 expression in the otic vesicles of zebrafish embryos is regulated by Sox10.

Integrative transcriptome sequencing identifies trans-splicing events with important roles in human embryonic stem cell pluripotency.

Trans-splicing is a post-transcriptional event that joins exons from separate pre-mRNAs. Detection of trans-splicing is usually severely hampered by experimental artifacts and genetic rearrangements. Here, we develop a new computational pipeline, TSscan, which integrates different types of high-throughput long-/short-read transcriptome sequencing of different human embryonic stem cell (hESC) lines to effectively minimize false positives while detecting trans-splicing. Combining TSscan screening with multiple experimental validation steps revealed that most chimeric RNA products were platform-dependent experimental artifacts of RNA sequencing. We successfully identified and confirmed four trans-spliced RNAs, including the first reported trans-spliced large intergenic noncoding RNA ("tsRMST"). We showed that these trans-spliced RNAs were all highly expressed in human pluripotent stem cells and differentially expressed during hESC differentiation. Our results further indicated that tsRMST can contribute to pluripotency maintenance of hESCs by suppressing lineage-specific gene expression through the recruitment of NANOG and the PRC2 complex factor, SUZ12. Taken together, our findings provide important insights into the role of trans-splicing in pluripotency maintenance of hESCs and help to facilitate future studies into trans-splicing, opening up this important but understudied class of post-transcriptional events for comprehensive characterization.

Heme oxygenase-1 induction by the ROS-JNK pathway plays a role in aluminum-induced anemia.

Aluminum (Al) overload is correlated with hypochromic anemia. It is possible that Al impedes heme biosynthesis and degradation by affecting the activity of biosynthetic enzymes. However, the molecular mechanisms by which Al affects these enzymes are unknown. Here, we show that long-term exposure of Sprague-Dawley rats to Al decreased hemoglobin concentration and the hematocrit level. In addition, the activity of aminolevulinic acid dehydratase (ALA-D) in rat liver was reduced, but heme oxygenase (HO) activity was enhanced, suggesting an impairment of heme homeostasis. The increase in HO activity was due to up-regulation of mRNA and protein of an inducible HO isozyme, HO-1. Furthermore, we found that reactive oxygen species (ROS)-mediated activation of c-Jun N-terminal kinase (JNK) was critical for HO-1 induction by Al, because ROS scavengers and JNK inhibitors abrogated enhancement of HO-1 by Al in rat hepatocytes. Thus, Al enhances HO-1 expression through the ROS-JNK pathway, which may enhance HO activity and accelerate degradation of heme, leading to hypochromic anemia.

Epithelial cell adhesion molecule regulation is associated with the maintenance of the undifferentiated phenotype of human embryonic stem cells.

Human embryonic stem cells (hESCs) are unique pluripotent cells capable of self-renewal and differentiation into all three germ layers. To date, more cell surface markers capable of reliably identifying hESCs are needed. The epithelial cell adhesion molecule (EpCAM) is a type I transmembrane glycoprotein expressed in several progenitor cell populations and cancers. It has been used to enrich cells with tumor-initiating activity in xenograft transplantation studies. Here, we comprehensively profile the expression of EpCAM by immunofluorescence microscopy, Western blotting, and flow cytometry using an anti-EpCAM monoclonal antibody (mAb) OC98-1. We found EpCAM to be highly and selectively expressed by undifferentiated rather than differentiated hESCs. The protein and transcript level of EpCAM rapidly diminished as soon as hESC had differentiated. This silencing was closely and exclusively associated with the radical transformation of histone modification at the EpCAM promoter. Moreover, we demonstrated that the dynamic pattern of lysine 27 trimethylation of histone 3 was conferred by the interplay of SUZ12 and JMJD3, both of which were involved in maintaining hESC pluripotency. In addition, we used chromatin immunoprecipitation analysis to elucidate the direct regulation by EpCAM of several reprogramming genes, including c-MYC, OCT-4, NANOG, SOX2, and KLF4, to help maintain the undifferentiation of hESCs. Collectively, our results suggest that EpCAM might be used as a surface marker for hESC. The expression of EpCAM may be regulated by epigenetic mechanisms, and it is strongly associated with the maintenance of the undifferentiated state of hESCs.

DNA methylation and histone modification regulate silencing of OPG during tumor progression.

The identification of molecules that are down-regulated in malignant phenotype is important for understanding tumor biology and their role in tumor suppression. We compared the expression profile of four normal nasal mucosal (NNM) epithelia and a series of nasopharyngeal cancinoma (NPC) cell lines using cDNA microarray and confirmed the actual expression of the selected genes, and found osteoprotegerin (OPG) to be ubiquitously deficient in NPC cells. We also found OPG to be down-regulated in various cancer cell lines, including oral, cervical, ovarian, lung, breast, pancreas, colon, renal, prostate cancer, and hepatoma. Administration of recombinant OPG (rOPG) brought about a reduction in cancer cell growth through apoptotic mechanism. We generated eleven monoclonal antibodies (MAbs) against OPG to study OPG's expression and biological functions in cancer cells. OPG was detected in the tumor stromal regions, but not in the cancer cell per se in surgical specimens of liver cancer. Quantitative reverse transcription-polymerase chain reaction (Q-RT-PCR) revealed that OPG was down-regulated in NPC tissues compared with normal nasal polyp (NNP) tissues. In addition, we showed OPG silencing to be associated with promoter methylation as well as histone modifications. In OPG-silenced cancer cell lines, the OPG gene promoter CpG dinucleotides were highly methylated. Compared to normal cells, silenced OPG gene in cancer cells were found to have reduced histone 3 lysine 4 tri-methylation (H3K4me3) and increased histone 3 lysine 27 tri-methylation (H3K27me3). Taken together, these results suggest that OPG silencing in carcinoma cancer cells occurs through epigenetic repression.

NOLC1, an enhancer of nasopharyngeal carcinoma progression, is essential for TP53 to regulate MDM2 expression.

Nasopharyngeal carcinoma (NPC) is one of the most common cancers among Chinese living in South China, Singapore, and Taiwan. At present, its etiological factors are not well defined. To identify which genetic alterations might be involved in NPC pathogenesis, we identified genes that were differentially expressed in NPC cell lines and normal nasomucosal cells using subtractive hybridization and microarray analysis. Most NPC cell lines and biopsy specimens were found to have higher expression levels of the gene encoding nucleolar and coiled-body phosphoprotein 1 (NOLC1) as compared with normal cells. Severe combined immunodeficiency mice bearing NPC xenografts derived from NOLC1-short hairpin-RNA-transfected animals were found to have 82% lower levels of tumor growth than control mice as well as marked tumor cell apoptosis. Measuring the expression levels of genes related to cell growth, apoptosis, and angiogenesis, we found that the MDM2 gene was down-regulated in the transfectants. Both co-transfection and chromatin immunoprecipitation experiments showed that tumor protein 53-regulated expression of the MDM2 gene requires co-activation of NOLC1. These findings suggest that NOLC1 plays a role in the regulation of tumorigenesis of NPC and demonstrate that both NOLC1 and tumor protein 53 work together synergistically to activate the MDM2 promoter in NPC cells.