Conserved Cis-Acting Range Extender Element Mediates Extreme Long-Range Enhancer Activity in Mammals.

While most mammalian enhancers regulate their cognate promoters over moderate distances of tens of kilobases (kb), some enhancers act over distances in the megabase range. The sequence features enabling such extreme-distance enhancer-promoter interactions remain elusive. Here, we used in vivo enhancer replacement experiments in mice to show that short- and medium-range enhancers cannot initiate gene expression at extreme-distance range. We uncover a novel conserved cis-acting element, Range EXtender (REX), that confers extreme-distance regulatory activity and is located next to a long-range enhancer of Sall1. The REX element itself has no endogenous enhancer activity. However, addition of the REX to other short- and mid-range enhancers substantially increases their genomic interaction range. In the most extreme example observed, addition of the REX increased the range of an enhancer by an order of magnitude, from its native 71kb to 840kb. The REX element contains highly conserved [C/T]AATTA homeodomain motifs. These motifs are enriched around long-range limb enhancers genome-wide, including the ZRS, a benchmark long-range limb enhancer of Shh. Mutating the [C/T]AATTA motifs within the ZRS does not affect its limb-specific enhancer activity at short range, but selectively abolishes its long-range activity, resulting in severe limb reduction in knock-in mice. In summary, we identify a sequence signature globally associated with long-range enhancer-promoter interactions and describe a prototypical REX element that is necessary and sufficient to confer extreme-distance gene activation by remote enhancers.

Evolution of the enhancer-rich regulatory region of the gene for the cell-type specific transcription factor POU1F1.

Precise spatio-temporal expression of genes in organogenesis is regulated by the coordinated interplay of DNA elements such as promoter and enhancers present in the regulatory region of a given locus. POU1F1 transcription factor plays a crucial role in the development of somatotrophs, lactotrophs and thyrotrophs in the anterior pituitary gland, and in maintaining high expression of growth hormone, prolactin and TSH. In mouse, expression of POU1F1 is controlled by a region fenced by two CTCF sites, containing 5 upstream enhancer elements, designated E-A (5' to 3'). Elements C, B and A correspond to elements shown previously to play a role in pituitary development and hormonal expression; functional roles for elements E and D have not been reported. We performed comparative sequence analysis of this regulatory region and discovered that three elements, B, C and E, are present in all vertebrate groups except Agnatha. One very long (>2 kb) element (A) is unique to mammals suggesting a specific change in regulation of the gene in this group. Using DNA accessibility assay (ATAC-seq) we showed that conserved elements in anterior pituitary of four non-mammals are open, suggesting functionality as regulatory elements. We showed that, in many non-mammalian vertebrates, an additional upstream exon closely follows element E, leading to alternatively spliced transcripts. Here, element E functions as an alternative promoter, but in mammals this feature is lost, suggesting conversion of alternative promoter to enhancer. Our work shows that regulation of POU1F1 changed markedly during the course of vertebrate evolution, use of a low number of enhancer elements combined with alternative promoters in non-mammalian vertebrates being replaced by use of a unique combination of regulatory units in mammals. Most importantly, our work suggests that evolutionary conversion of alternate promoter to enhancer could be one of the evolutionary mechanisms of enhancer birth.

Stress induced aging in mouse eye.

Aging, a universal process that affects all cells in an organism, is a major risk factor for a group of neuropathies called glaucoma, where elevated intraocular pressure is one of the known stresses affecting the tissue. Our understanding of molecular impact of aging on response to stress in retina is very limited; therefore, we developed a new mouse model to approach this question experimentally. Here we show that susceptibility to response to stress increases with age and is primed on chromatin level. We demonstrate that ocular hypertension activates a stress response that is similar to natural aging and involves activation of inflammation and senescence. We show that multiple instances of pressure elevation cause aging of young retina as measured on transcriptional and DNA methylation level and are accompanied by local histone modification changes. Our data show that repeated stress accelerates appearance of aging features in tissues and suggest chromatin modifications as the key molecular components of aging. Lastly, our work further emphasizes the importance of early diagnosis and prevention as well as age-specific management of age-related diseases, including glaucoma.

Chromatin Accessibility and Transcriptional Differences in Human Stem Cell-Derived Early-Stage Retinal Organoids.

Retinogenesis involves the specification of retinal cell types during early vertebrate development. While model organisms have been critical for determining the role of dynamic chromatin and cell-type specific transcriptional networks during this process, an enhanced understanding of the developing human retina has been more elusive due to the requirement for human fetal tissue. Pluripotent stem cell (PSC) derived retinal organoids offer an experimentally accessible solution for investigating the developing human retina. To investigate cellular and molecular changes in developing early retinal organoids, we developed SIX6-GFP and VSX2-tdTomato (or VSX2-h2b-mRuby3) dual fluorescent reporters. When differentiated as 3D organoids these expressed GFP at day 15 and tdTomato (or mRuby3) at day 25, respectively. This enabled us to explore transcriptional and chromatin related changes using RNA-seq and ATAC-seq from pluripotency through early retina specification. Pathway analysis of developing organoids revealed a stepwise loss of pluripotency, while optic vesicle and retina pathways became progressively more prevalent. Correlating gene transcription with chromatin accessibility in early eye field development showed that retinal cells underwent a clear change in chromatin landscape, as well as gene expression profiles. While each dataset alone provided valuable information, considering both in parallel provided an informative glimpse into the molecular nature eye development.

Dynamic lipid turnover in photoreceptors and retinal pigment epithelium throughout life.

The retinal pigment epithelium-photoreceptor interphase is renewed each day in a stunning display of cellular interdependence. While photoreceptors use photosensitive pigments to convert light into electrical signals, the RPE supports photoreceptors in their function by phagocytizing shed photoreceptor tips, regulating the blood retina barrier, and modulating inflammatory responses, as well as regenerating the 11-cis-retinal chromophore via the classical visual cycle. These processes involve multiple protein complexes, tightly regulated ligand-receptors interactions, and a plethora of lipids and protein-lipids interactions. The role of lipids in maintaining a healthy interplay between the RPE and photoreceptors has not been fully delineated. In recent years, novel technologies have resulted in major advancements in understanding several facets of this interplay, including the involvement of lipids in phagocytosis and phagolysosome function, nutrient recycling, and the metabolic dependence between the two cell types. In this review, we aim to integrate the complex role of lipids in photoreceptor and RPE function, emphasizing the dynamic exchange between the cells as well as discuss how these processes are affected in aging and retinal diseases.

Protein Kinase A in Human Retina: Differential Localization of Cß, Cα, RIIα, and RIIß in Photoreceptors Highlights Non-redundancy of Protein Kinase A Subunits.

Protein kinase A (PKA) signaling is essential for numerous processes but the subcellular localization of specific PKA regulatory (R) and catalytic (C) subunits has yet to be explored comprehensively. Additionally, the localization of the Cß subunit has never been spatially mapped in any tissue even though ∼50% of PKA signaling in neuronal tissues is thought to be mediated by Cß. Here we used human retina with its highly specialized neurons as a window into PKA signaling in the brain and characterized localization of PKA Cα, Cß, RIIα, and RIIß subunits. We found that each subunit presented a distinct localization pattern. Cα and Cß were localized in all cell layers (photoreceptors, interneurons, retinal ganglion cells), while RIIα and RIIß were selectively enriched in photoreceptor cells where both showed distinct patterns of co-localization with Cα but not Cß. Only Cα was observed in photoreceptor outer segments and at the base of the connecting cilium. Cß in turn, was highly enriched in mitochondria and was especially prominent in the ellipsoid of cone cells. Further investigation of Cß using RNA BaseScope technology showed that two Cß splice variants (Cß4 and Cß4ab) likely code for the mitochondrial Cß proteins. Overall, our data indicates that PKA Cα, Cß, RIIα, and RIIß subunits are differentially localized and are likely functionally non-redundant in the human retina. Furthermore, Cß is potentially important for mitochondrial-associated neurodegenerative diseases previously linked to PKA dysfunction.

Annulation-retro-Claisen cascade of bifunctional peroxides for the synthesis of lactone natural products.

A new and highly efficient annulation-retro-Claisen cascade, which involves the [4 + 1] or [5 + 1] annulation of α-benzoylacetates with bielectrophilic peroxides and a subsequent debenzoylation process under mild basic conditions, has been developed for the rapid construction of valuable tetrahydrofuran- and dihydropyran-2-carboxylates in good yields. By employing the new reaction, the unified total synthesis of γ- and δ-lactone natural products such as (±)-tanikolide, (±)-goniothalamins, (±)-7-epi-goniodiol, and (±)-plakolide A has been accomplished in 4-7 steps.

Methylation-associated silencing of miR-9-1 promotes nasopharyngeal carcinoma progression and glycolysis via HK2.

Characteristically, cancer cells metabolize glucose through aerobic glycolysis, known as the Warburg effect. Accumulating evidence suggest that during cancer formation, microRNAs (miRNAs) could regulate such metabolic reprogramming. In the present study, miR-9-1 was identified as significantly hypermethylated in nasopharyngeal carcinoma (NPC) cell lines and clinical tissues. Ectopic expression of miR-9-1 inhibited NPC cell growth and glycolytic metabolism, including reduced glycolysis, by reducing lactate production, glucose uptake, cellular glucose-6-phosphate levels, and ATP generation in vitro and tumor proliferation in vivo. HK2 (encoding hexokinase 2) was identified as a direct target of miR-9-1 using luciferase reporter assays and Western blotting. In NPC cells, hypermethylation regulates miR-9-1 expression and inhibits HK2 translation by directly targeting its 3' untranslated region. MiR-9-1 overexpression markedly reduced HK2 protein levels. Restoration of HK2 expression attenuated the inhibitory effect of miR-9-1 on NPC cell proliferation and glycolysis. Fluorescence in situ hybridization results indicated that miR-9-1 expression was an independent prognostic factor in NPC. Our findings revealed the role of the miR-9-1/HK2 axis in the metabolic reprogramming of NPC, providing a potential therapeutic strategy for NPC.

A Unified Catalytic Asymmetric (4+1) and (5+1) Annulation Strategy to Access Chiral Spirooxindole-Fused Oxacycles.

A unified catalytic asymmetric (N+1) (N=4, 5) annulation reaction of oxindoles with bifunctional peroxides has been achieved in the presence of a chiral phase-transfer catalyst (PTC). This general strategy utilizes peroxides as unique bielectrophilic four- or five-atom synthons to participate in the C-C and the subsequent umpolung C-O bond-forming reactions with one-carbon unit nucleophiles, thus providing a distinct method to access the valuable chiral spirooxindole-tetrahydrofurans and -tetrahydropyrans with good yields and high enantioselectivities under mild conditions. DFT calculations were performed to rationalize the origin of high enantioselectivity. The gram-scale syntheses and synthetic utility of the resultant products were also demonstrated.

Effects of Txk­mediated activation of NF­κB signaling pathway on neurological deficit and oxidative stress after ischemia­reperfusion in rats.

Ischemic stroke is an extremely mortal cerebrovascular disease, and neuroinflammation and oxidative stress emerge as important traits of ischemic stroke. However, as an inflammation­associated factor, Txk tyrosine kinases (Txk) has been poorly studied in neuroscience research. The aim of the present study was to investigate the role of Txk after ischemia­reperfusion (I/R) in vivo and in vitro, observe the association between Txk knockdown and neurological deficit and oxidative stress, and to explore whether the process was mediated by the activation of nuclear factor (NF)­κB signaling pathway. Middle cerebral artery occlusion (MCAO), oxygen and glucose deprivation/reperfusion (OGD/R) model and western blotting have been used to simulate the I/R injury to analyze the expression, and to approximate the localization of Txk, respectively. Brain infarct volume, neurological score, brain water content, apoptosis and oxidative stress assays in vivo and apoptosis, cellular viability, the LDH release and oxidative stress assays in vitro were observed using a Txk­knockdown lentivirus. Finally, NF­κB overexpression lentivirus was applied to discuss whether the role of Txk following I/R was regulated by the NF­κB signaling pathway. The results show that the Txk expression peaked at 24 h after MCAO and 6 h after OGD/R, respectively. Txk molecules gradually entered the nucleus after MCAO and OGD/R. The Txk­knockdown lentivirus resulted in decreased brain infarct volume, neurological score, brain water content, apoptosis and oxidative stress after MCAO in vivo. Besides, Txk knockdown decreased apoptosis, LDH release, oxidative stress, and increased cellular viability, after ODG in vitro. Finally, NF­κB overexpression reversed the process of neurological deficit and oxidative stress after Txk regulation in vivo and vitro. Overall, the present study suggests that Txk potentially regulates apoptosis, neurological deficit, and oxidative stress after I/R, by entering the nucleus. NF­κB maybe the downstream target factor of Txk.

Elovl2 Is Required for Robust Visual Function in Zebrafish.

Omega-3 and omega-6 polyunsaturated fatty acids (PUFAs) play critical roles in membrane stability and cell signaling within the retina. ELOVL2 (Elongation of Very Long Chain Fatty Acids-Like 2), an elongase involved in the synthesis of long chain polyunsaturated fatty acids (LC-PUFAs), has recently been implicated in regulating aging in the mammalian retina. In this work, we characterize the expression and function of elovl2 in the retina development in embryonic zebrafish. Whole mount in situ hybridization shows elovl2 is expressed in the Muller glia in embryonic and adult zebrafish. Lipidomics analysis of elovl2 crispants whole embryos at day 2 and eyes at day 7 demonstrated significant changes in lipids composition, especially on the level of lipids containing docosahexaenoic acid (DHA). Histological analysis of zebrafish lacking elovl2 revealed increased retinal thickness compared to controls at day 7 without gross disruptions of the retinal architecture. Finally, elovl2 crispants showed differences in the visual motor reflex light off (VMR-OFF) at day 7 compared to controls. In sum, inactivation of elovl2 in zebrafish embryos caused changes in lipid composition and in visual behavior, further confirming the important role of LC-PUFAs in healthy vision.

Reorganized 3D Genome Structures Support Transcriptional Regulation in Mouse Spermatogenesis.

Three-dimensional chromatin structures undergo dynamic reorganization during mammalian spermatogenesis; however, their impacts on gene regulation remain unclear. Here, we focused on understanding the structure-function regulation of meiotic chromosomes by Hi-C and other omics techniques in mouse spermatogenesis across five stages. Beyond confirming recent reports regarding changes in compartmentalization and reorganization of topologically associating domains (TADs), we further demonstrated that chromatin loops are present prior to and after, but not at, the pachytene stage. By integrating Hi-C and RNA-seq data, we showed that the switching of A/B compartments between spermatogenic stages is tightly associated with meiosis-specific mRNAs and piRNAs expression. Moreover, our ATAC-seq data indicated that chromatin accessibility per se is not responsible for the TAD and loop diminishment at pachytene. Additionally, our ChIP-seq data demonstrated that CTCF and cohesin remain bound at TAD boundary regions throughout meiosis, suggesting that dynamic reorganization of TADs does not require CTCF and cohesin clearance.

Rearrangement of macronucleus chromosomes correspond to TAD-like structures of micronucleus chromosomes in Tetrahymena thermophila.

The somatic macronucleus (MAC) and germline micronucleus (MIC) of Tetrahymena thermophila differ in chromosome numbers, sizes, functions, transcriptional activities, and cohesin complex location. However, the higher-order chromatin organization in T. thermophila is still largely unknown. Here, we explored the higher-order chromatin organization in the two distinct nuclei of T. thermophila using the Hi-C and HiChIP methods. We found that the meiotic crescent MIC has a specific chromosome interaction pattern, with all the telomeres or centromeres on the five MIC chromosomes clustering together, respectively, which is also helpful to identify the midpoints of centromeres in the MIC. We revealed that the MAC chromosomes lack A/B compartments, topologically associating domains (TADs), and chromatin loops. The MIC chromosomes have TAD-like structures but not A/B compartments and chromatin loops. The boundaries of the TAD-like structures in the MIC are highly consistent with the chromatin breakage sequence (CBS) sites, suggesting that each TAD-like structure of the MIC chromosomes develops into one MAC chromosome during MAC development, which provides a mechanism of the formation of MAC chromosomes during conjugation. Overall, we demonstrated the distinct higher-order chromatin organization in the two nuclei of the T. thermophila and suggest that the higher-order chromatin structures may play important roles during the development of the MAC chromosomes.

Early removal of senescent cells protects retinal ganglion cells loss in experimental ocular hypertension.

Experimental ocular hypertension induces senescence of retinal ganglion cells (RGCs) that mimics events occurring in human glaucoma. Senescence-related chromatin remodeling leads to profound transcriptional changes including the upregulation of a subset of genes that encode multiple proteins collectively referred to as the senescence-associated secretory phenotype (SASP). Emerging evidence suggests that the presence of these proinflammatory and matrix-degrading molecules has deleterious effects in a variety of tissues. In the current study, we demonstrated in a transgenic mouse model that early removal of senescent cells induced upon elevated intraocular pressure (IOP) protects unaffected RGCs from senescence and apoptosis. Visual evoked potential (VEP) analysis demonstrated that remaining RGCs are functional and that the treatment protected visual functions. Finally, removal of endogenous senescent retinal cells after IOP elevation by a treatment with senolytic drug dasatinib prevented loss of retinal functions and cellular structure. Senolytic drugs may have the potential to mitigate the deleterious impact of elevated IOP on RGC survival in glaucoma and other optic neuropathies.

Gemcitabine plus cisplatin versus fluorouracil plus cisplatin as a first-line concurrent chemotherapy regimen in nasopharyngeal carcinoma: a prospective, multi-institution, randomized controlled phase II study.

BACKGROUND: The objective of this study was to evaluate the efficacy and safety of gemcitabine plus cisplatin concurrent chemoradiotherapy (CCRT) in patients with nasopharyngeal carcinoma. METHOD: Patients with NPC were randomly assigned to the gemcitabine plus cisplatin (GP) group or fluorouracil plus cisplatin (PF) group. Primary end-point was disease-free survival (DFS); secondary endpoints: overall survival, distant metastasis-free survival (DMFS), locoregional relapse-free survival, and treatment-related adverse events. RESULTS: Seventy-six patients were prospectively enrolled and the median follow-up time was 41 months (9-61 months). Three-year DFS were similar between the GP and PF groups (73.7% vs. 60.5%, HR 0.66, 95% CI 0.30-1.44; P = 0.30). Distant metastasis was the most common failure form in PF compared with GP (P = 0.034). Three-year DMFS was significantly better in the GP group than PF group (89.5% vs. 71.1%, P = 0.045). Grade 3-4 gastrointestinal toxicities (vomiting and diarrhea) were significantly more common in the PF group; grade 3-4 neutropenia and thrombocytopenia were more common in the GP group. CONCLUSION: Gemcitabine plus cisplatin could be used as an alternative regimen in CCRT for nasopharyngeal carcinoma.

Correction to: The key role of CYC2 during meiosis in Tetrahymena Thermophila.

In the original publication, the funding information was incorrectly published. The correct funding information is provided in this correction. This work is supported by grants from the Projects of International Cooperation and Exchanges Ministry of Science and Technology of China (2013DFG32390) and the National Natural Science Foundation of China (31472059) to X.S. X.S is a recipient of the Young Thousand Talents program (KJ2070000026).

Candidate tumor suppressor ZNF154 suppresses invasion and metastasis in NPC by inhibiting the EMT via Wnt/ß-catenin signalling.

BACKGROUND: Nasopharyngeal carcinoma (NPC) is especially prevalent in southeast Asia and southern China, but its molecular mechanisms remain poorly characterized. DNA methylation is associated with initiation and progression of tumors, including NPC. Through a genome-wide DNA methylation screening approach, we discovered ZNF154, but its methylation status and roles in NPC have not been investigated. METHODS: The methylation status of ZNF154 in NPC was detected with Methylation specific-PCR (MSP) and Quantitative Sequenom MassARRAY. The invasion and migration capacities were examined by wound healing and transwell invasion assays. The role of ZNF154 in NPC metastasis was clarified with experimental metastasis assay in vivo. Western blotting analysis was used to investigate protein changes followed by ZNF154 over-expression. Kaplan-Meier analysis was performed to determine the association between ZNF154 methylation and prognosis in NPC. RESULTS: Compared to immortalized nasopharyngeal tissues and cells, ZNF154 expression was frequently downregulated in NPC tissues and cell lines due to promoter methylation. Demethylation treatment with 5-aza-2-deoxycytidine (5-Aza) restored ZNF154 expression in NPC cell lines. Ectopic overexpression of ZNF154 in NPC cells inhibited cell migration and invasion in vitro and lung nodule formation in an in vivo tumor metastasis assay. Mechanistic investigations suggested ZNF154 inhibits Wnt/ß-catenin signalling pathway activation and prevents the EMT in NPC. Furthermore, Kaplan-Meier analysis showed hypermethylation of the ZNF154 promoter was associated with significantly poorer disease-free survival (P = 0.032) and distant metastasis-free survival (P = 0.040) among patients with locoregionally advanced NPC. CONCLUSIONS: Taken together, these findings define a novel role for ZNF154 as a tumor suppressor in NPC.

Shining the Light on Senescence Associated LncRNAs.

Cellular senescence can be described as a complex stress response that leads to irreversible cell cycle arrest. This process was originally described as an event that primary cells go through after many passages of cells during cell culture. More recently, cellular senescence is viewed as a programmed process by which the cell displays a senescence phenotype when exposed to a variety of stresses. Cellular senescence has been implicated in tumor suppression and aging such that senescence may contribute to both tumor progression and normal tissue repair. Here, we review different forms of cellular senescence, as well as current biomarkers used to identify senescent cells in vitro and in vivo. Additionally, we highlight the role of senescence-associated long noncoding RNAs (lncRNAs).

Matched analysis of induction chemotherapy plus chemoradiotherapy versus induction chemotherapy plus radiotherapy alone in locoregionally advanced nasopharyngeal carcinoma: a multicenter study.

BACKGROUND: The relative efficacy of induction chemotherapy (IC) followed by concurrent chemoradiotherapy (CCRT) versus IC followed by radiotherapy (RT) alone in locoregionally advanced NPC remains unclear. METHODS: A total of 877 patients with locally advanced NPC who underwent IC/CCRT or IC/RT at four institutions in China between January 2004 and December 2010 were retrospectively assessed. IC was cisplatin-based combination chemotherapy; concurrent chemotherapy, single agent cisplatin. Two-dimensional conventional radiotherapy (2DCRT) was the radiotherapy technique. All patients were matched in an equal ratio using a pair-matched method. Overall survival (OS), disease-free survival (DFS), distant metastasis-free survival (DMFS), locoregional relapse-free survival (LRRFS) and toxicities were assessed. RESULTS: Eligible patients were matched (n = 642; 321 patients per arm) based on eight clinicopathological characteristics. Five-year OS, DFS, DMFS, and LRRFS were 76%, 70%, 86%, and 88% for IC/CCRT and 75%, 70%, 90%, and 91% for IC/RT, respectively. There were no statistically significant survival differences between arms (P>0.05), even in subgroup analysis. In multivariate analysis, treatment (IC/CCRT vs. IC/RT) was not an independent prognostic factor for any survival end-point. Grade 3/4 acute gastrointestinal toxicities (vomiting, nausea) and hematological toxicities (leucopenia/neutropenia, thrombocytopenia and anemia) were significantly more common in the IC/CCRT arm than IC/RT arm during RT. CONCLUSION: Overall, IC/CCRT failed to demonstrate any survival advantage but higher acute toxicities over IC/RT in locoregionally advanced NPC.

The key role of CYC2 during meiosis in Tetrahymena thermophila.

Meiotic recombination is carried out through a specialized pathway for the formation and repair of DNA double-strand breaks (DSBs) made by the Spo11 protein. The present study shed light on the functional role of cyclin, CYC2, in Tetrahymena thermophila which has transcriptionally high expression level during meiosis process. Knocking out the CYC2 gene results in arrest of meiotic conjugation process at 2.5-3.5 h after conjugation initiation, before the meiosis division starts, and in company with the absence of DSBs. To investigate the underlying mechanism of this phenomenon, a complete transcriptome profile was performed between wild-type strain and CYC2 knock-out strain. Functional analysis of RNA-Seq results identifies related differentially expressed genes (DEGs) including SPO11 and these DEGs are enriched in DNA repair/mismatch repair (MMR) terms in homologous recombination (HR), which indicates that CYC2 could play a crucial role in meiosis by regulating SPO11 and participating in HR.