Remodeling Ca2+ dynamics by targeting a promising E-box containing G-quadruplex at ORAI1 promoter in triple-negative breast cancer.

ORAI1 is an intrinsic component of store-operated calcium entry (SOCE) that strictly regulates Ca2+ influx in most non-excitable cells. ORAI1 is overexpressed in a wide variety of cancers, and its signal transduction has been associated with chemotherapy resistance. There is extensive proteomic interaction of ORAI1 with other channels and effectors, resulting in various altered phenotypes. However, the transcription regulation of ORAI1 is not well understood. We have found a putative G-quadruplex (G4) motif, ORAI1-Pu, in the upstream promoter region of the gene, having regulatory functions. High-resolution 3-D NMR structure elucidation suggests that ORAI1-Pu is a stable parallel-stranded G4, having a long 8-nt loop imparting dynamics without affecting the structural stability. The protruded loop further houses an E-box motif that provides a docking site for transcription factors like Zeb1. The G4 structure was also endogenously observed using Chromatin Immunoprecipitation (ChIP) with anti-G4 antibody (BG4) in the MDA-MB-231 cell line overexpressing ORAI1. Ligand-mediated stabilization suggested that the stabilized G4 represses transcription in cancer cell line MDA-MB-231. Downregulation of transcription further led to decreased Ca2+ entry by the SOCE pathway, as observed by live-cell Fura-2 Ca2+ imaging.

Polymorphic tandem DNA repeats activate the human telomerase reverse transcriptase gene.

Multiple independent sequence variants of the hTERT locus have been associated with telomere length and cancer risks in genome-wide association studies. Here, we identified an intronic variable number tandem repeat, VNTR2-1, as an enhancer-like element, which activated hTERT transcription in a cell in a chromatin-dependent manner. VNTR2-1, consisting of 42-bp repeats with an array of enhancer boxes, cooperated with the proximal promoter in the regulation of hTERT transcription by basic helix-loop-helix transcription factors and maintained hTERT expression during embryonic stem-cell differentiation. Genomic deletion of VNTR2-1 in MelJuSo melanoma cells markedly reduced hTERT transcription, leading to telomere shortening, cellular senescence, and impairment of xenograft tumor growth. Interestingly, VNTR2-1 lengths varied widely in human populations; hTERT alleles with shorter VNTR2-1 were underrepresented in African American centenarians, indicating its role in human aging. Therefore, this polymorphic element is likely a missing link in the telomerase regulatory network and a molecular basis for genetic diversities of telomere homeostasis and age-related disease susceptibilities.

Rewiring of Lactate-Interleukin-1ß Autoregulatory Loop with Clock-Bmal1: a Feed-Forward Circuit in Glioma.

A desynchronized circadian rhythm in tumors is coincident with aberrant inflammation and dysregulated metabolism. As their interrelationship in cancer etiology is largely unknown, we investigated the link among the three in glioma. The tumor metabolite lactate-mediated increase in the proinflammatory cytokine interleukin-1ß (IL-1ß) was concomitant with elevated levels of the core circadian regulators Clock and Bmal1. Small interfering RNA (siRNA)-mediated knockdown of Bmal1 and Clock decreased (i) lactate dehydrogenase A (LDHA) and IL-1ß levels and (ii) the release of lactate and proinflammatory cytokines. Lactate-mediated deacetylation of Bmal1 and its interaction with Clock regulate IL-1ß levels and vice versa. Site-directed mutagenesis and luciferase reporter assays indicated the functionality of E-box sites on LDHA and IL-1ß promoters. Sequential chromatin immunoprecipitation (ChIP-re-ChIP) revealed that lactate-IL-1ß cross talk positively affects the corecruitment of Clock-Bmal1 to these E-box sites. Clock-Bmal1 enrichment was accompanied by decreased H3K9me3 and increased H3K9ac and RNA polymerase II (Pol II) occupancy. The lactate-IL-1ß-Clock (LIC) loop positively regulated the expression of genes associated with the cell cycle, DNA damage, and cytoskeletal organization involved in glioma progression. TCGA (The Cancer Genome Atlas) data analysis suggested the presence of lactate-IL-1ß cross talk in other cancers. The responsiveness of stomach and cervical cancer cells to lactate inhibition followed the same trend as that exhibited by glioma cells. In addition, components of the LIC loop were found to be correlated with (i) patient survival, (ii) clinically actionable genes, and (iii) anticancer drug sensitivity. Our findings provide evidence for potential cancer-specific axis wiring of IL-1ß and LDHA through Clock-Bmal1, the outcome of which is to fuel an IL-1ß-lactate autocrine loop that drives proinflammatory and oncogenic signals.

Involvement of Scratch2 in GalR1-mediated depression-like behaviors in the rat ventral periaqueductal gray.

Galanin receptor1 (GalR1) transcript levels are elevated in the rat ventral periaqueductal gray (vPAG) after chronic mild stress (CMS) and are related to depression-like behavior. To explore the mechanisms underlying the elevated GalR1 expression, we carried out molecular biological experiments in vitro and in animal behavioral experiments in vivo. It was found that a restricted upstream region of the GalR1 gene, from -250 to -220, harbors an E-box and plays a negative role in the GalR1 promoter activity. The transcription factor Scratch2 bound to the E-box to down-regulate GalR1 promoter activity and lower expression levels of the GalR1 gene. The expression of Scratch2 was significantly decreased in the vPAG of CMS rats. Importantly, local knockdown of Scratch2 in the vPAG caused elevated expression of GalR1 in the same region, as well as depression-like behaviors. RNAscope analysis revealed that GalR1 mRNA is expressed together with Scratch2 in both GABA and glutamate neurons. Taking these data together, our study further supports the involvement of GalR1 in mood control and suggests a role for Scratch2 as a regulator of depression-like behavior by repressing the GalR1 gene in the vPAG.

Identification of the core promoter of ZNFO, an oocyte-specific maternal effect gene in cattle.

ZNFO is a Krüppel-associated box (KRAB) containing zinc finger transcription factor, which is exclusively expressed in bovine oocytes. Previous studies have demonstrated that ZNFO possesses an intrinsic transcriptional repressive activity and is essential for early embryonic development in cattle. However, the mechanisms regulating ZNFO transcription remain elusive. In the present study, the core promoter that controls the ZNFO basal transcription was identified. A 1.7 kb 5' regulatory region of the ZNFO gene was cloned and its promoter activity was confirmed by a luciferase reporter assay. A series of 5' deletion in the ZNFO promoter followed by luciferase reporter assays indicated that the core promoter region has to include the sequence located within 57 bp to 31 bp upstream of the transcription start site. Sequence analysis revealed that a putative USF1/USF2 binding site (GGTCACGTGACC) containing an E-box motif (CACGTG) is located within the essential region. Depletion of USF1/USF2 by RNAi and E-box mutation analysis demonstrated that the USF1/USF2 binding site is required for the ZNFO basal transcription. Furthermore, EMSA and super-shift assays indicated that the observed effects are dependent on the specific interactions between USF proteins and the ZNFO core promoter. From these results, it is concluded that USF1 and USF2 are essential for the basal transcription of the ZNFO gene.

The cooperation of cis-elements during M-cadherin promoter activation.

M-cadherin is a skeletal muscle-specific transmembrane protein mediating the cell-cell adhesion of myoblasts during myogenesis. It is expressed in the proliferating satellite cells and highly induced by myogenic regulatory factors (MRFs) during terminal myogenic differentiation. Several conserved cis-elements, including 5 E-boxes, 2 GC boxes, and 1 conserved downstream element (CDE) were identified in the M-cadherin proximal promoter. We found that E-box-3 and -4 close to the transcription initiation site (TIS) mediated most of its transactivation by MyoD, the strongest myogenic MRF. Including of any one of the other E-boxes restored the full activation by MyoD, suggesting an essential collaboration between E-boxes. Stronger activation of M-cadherin promoter than that of muscle creatine kinase (MCK) by MyoD was observed regardless of culture conditions and the presence of E47. Furthermore, MyoD/E47 heterodimer and MyoD ∼ E47 fusion protein achieved similar levels of activation in differentiation medium (DM), suggesting high affinity of MyoD/E47 to E-boxes 3/4 under DM. We also found that GC boxes and CDE positively affected MyoD mediated activation. The CDE element was predicted to be the target of the chromatin-modifying factor Meis1/Pbx1 heterodimer. Knockdown of Pbx1 significantly reduced the expression level of M-cadherin, but increased that of N-cadherin. Using ChIP assay, we further found significant reduction in MyoD recruitment to M-cadherin promoter when CDE was deleted. Taken together, these observations suggest that the chromatin-modifying function of Pbx1/Meis1 is critical to M-cadherin promoter activation before MyoD is recruited to E-boxes to trigger transcription.

Morphogenesis is transcriptionally coupled to neurogenesis during peripheral olfactory organ development.

Sense organs acquire their distinctive shapes concomitantly with the differentiation of sensory cells and neurons necessary for their function. Although our understanding of the mechanisms controlling morphogenesis and neurogenesis in these structures has grown, how these processes are coordinated remains largely unexplored. Neurogenesis in the zebrafish olfactory epithelium requires the bHLH proneural transcription factor Neurogenin 1 (Neurog1). To address whether Neurog1 also controls morphogenesis, we analysed the migratory behaviour of early olfactory neural progenitors in neurog1 mutant embryos. Our results indicate that the oriented movements of these progenitors are disrupted in this context. Morphogenesis is similarly affected by mutations in the chemokine receptor gene, cxcr4b, suggesting it is a potential Neurog1 target gene. We find that Neurog1 directly regulates cxcr4b through an E-box cluster located just upstream of the cxcr4b transcription start site. Our results suggest that proneural transcription factors, such as Neurog1, directly couple distinct aspects of nervous system development.

Circadian Clock Component Rev-erbα Regulates Diurnal Rhythm of UDP-Glucuronosyltransferase 1a9 and Drug Glucuronidation in Mice.

UDP-glucuronosyltransferases (UGTs) are a family of phase II enzymes that play an important role in metabolism and elimination of numerous endo- and xenobiotics. Here, we aimed to characterize diurnal rhythm of Ugt1a9 in mouse liver and to determine the molecular mechanisms underlying the rhythmicity. Hepatic Ugt1a9 mRNA and protein displayed robust diurnal rhythms in wild-type mice with peak levels at zeitgeber time (ZT) 6. Rhythmicity in Ugt1a9 expression was confirmed using synchronized Hepa-1c1c7 cells. We observed time-varying glucuronidation (ZT6 > ZT18) of propofol, a specific Ugt1a9 substrate, consistent with the diurnal pattern of Ugt1a9 protein. Loss of Rev-erbα (a circadian clock component) downregulated the Ugt1a9 expression and blunted its rhythm in mouse liver. Accordingly, propofol glucuronidation was reduced and its dosing time dependency was lost in Rev-erbα -/- mice. Dec2 (a transcription factor) was screened to be the potential intermediate that mediated Rev-erbα regulation of Ugt1a9. We confirmed Rev-erbα as a negative regulator of Dec2 in mice and in Hepa-1c1c7 cells. Based on promoter analysis and luciferase reporter assays, it was found that Dec2 trans-repressed Ugt1a9 via direct binding to an E-box-like motif in the gene promoter. Additionally, regulation of Ugt1a9 by Rev-erbα was Dec2-dependent. In conclusion, Rev-erbα generates and regulates rhythmic Ugt1a9 through periodical inhibition of Dec2, a transcriptional repressor of Ugt1a9. Our study may have implications for understanding of circadian clock-controlled drug metabolism and of metabolism-based chronotherapeutics. SIGNIFICANCE STATEMENT: Hepatic Ugt1a9 displays diurnal rhythmicities in expression and glucuronidation activity in mice. It is uncovered that Rev-erbα generates and regulates rhythmic Ugt1a9 through periodical inhibition of Dec2, a transcriptional repressor of Ugt1a9. The findings may have implications for understanding of circadian clock-controlled drug metabolism and of metabolism-based chronotherapeutics.

Alternatively Constructed Estrogen Receptor Alpha-Driven Super-Enhancers Result in Similar Gene Expression in Breast and Endometrial Cell Lines.

Super-enhancers (SEs) are clusters of highly active enhancers, regulating cell type-specific and disease-related genes, including oncogenes. The individual regulatory regions within SEs might be simultaneously bound by different transcription factors (TFs) and co-regulators, which together establish a chromatin environment conducting to effective transcription. While cells with distinct TF profiles can have different functions, how different cells control overlapping genetic programs remains a question. In this paper, we show that the construction of estrogen receptor alpha-driven SEs is tissue-specific, both collaborating TFs and the active SE components greatly differ between human breast cancer-derived MCF-7 and endometrial cancer-derived Ishikawa cells; nonetheless, SEs common to both cell lines have similar transcriptional outputs. These results delineate that despite the existence of a combinatorial code allowing alternative SE construction, a single master regulator might be able to determine the overall activity of SEs.

Long non-coding RNA ARAP1-AS1 promotes tumorigenesis and metastasis through facilitating proto-oncogene c-Myc translation via dissociating PSF/PTB dimer in cervical cancer.

Long non-coding RNA (lncRNA) is emerging as a pivotal regulator in tumorigenesis and aggressive progression. Here, we focused on an oncogenic lncRNA, ARAP1 antisense RNA 1 (ARAP1-AS1), which was notably upregulated in cervical cancer (CC) tissues, cell lines and serum. High ARAP1-AS1 expression was closely associated with larger tumor size, advanced FIGO stage as well as lymph node metastasis. Importantly, it was identified as an effective diagnostic and prognostic biomarker for CC. In vitro and in vivo assays showed that knockdown of ARAP1-AS1 inhibited, while overexpression of ARAP1-AS1 promoted CC cell growth and dissemination. Stepwise mechanistic dissection unveiled that ARAP1-AS1 could directly interact with PSF to release PTB, resulting in accelerating the internal ribosome entry site (IRES)-driven translation of proto-oncogene c-Myc, thereby facilitating CC development and progression. Moreover, c-Myc was able to transcriptionally activate ARAP1-AS1 by directly binding to the E-box motif located on ARAP1-AS1 promoter. Taken together, our findings clearly reveal the crucial role of ARAP1-AS1 in CC tumorigenesis and metastasis via regulation of c-Myc translation, targeting ARAP1-AS1 and its related regulatory loop implicates the therapeutic possibility for CC patients.

Acetylation of BMAL1 by TIP60 controls BRD4-P-TEFb recruitment to circadian promoters.

Many physiological processes exhibit circadian rhythms driven by cellular clocks composed of interlinked activating and repressing elements. To investigate temporal regulation in this molecular oscillator, we combined mouse genetic approaches and analyses of interactions of key circadian proteins with each other and with clock gene promoters. We show that transcriptional activators control BRD4-PTEFb recruitment to E-box-containing circadian promoters. During the activating phase of the circadian cycle, the lysine acetyltransferase TIP60 acetylates the transcriptional activator BMAL1 leading to recruitment of BRD4 and the pause release factor P-TEFb, followed by productive elongation of circadian transcripts. We propose that the control of BRD4-P-TEFb recruitment is a novel temporal checkpoint in the circadian clock cycle.

Non-coding cis-element of Period2 is essential for maintaining organismal circadian behaviour and body temperature rhythmicity.

Non-coding cis-regulatory elements are essential determinants of development, but their exact impacts on behavior and physiology in adults remain elusive. Cis-element-based transcriptional regulation is believed to be crucial for generating circadian rhythms in behavior and physiology. However, genetic evidence supporting this model is based on mutations in the protein-coding sequences of clock genes. Here, we report generation of mutant mice carrying a mutation only at the E'-box cis-element in the promoter region of the core clock gene Per2. The Per2 E'-box mutation abolishes sustainable molecular clock oscillations and renders circadian locomotor activity and body temperature rhythms unstable. Without the E'-box, Per2 messenger RNA and protein expression remain at mid-to-high levels. Our work delineates the Per2 E'-box as a critical nodal element for keeping sustainable cell-autonomous circadian oscillation and reveals the extent of the impact of the non-coding cis-element in daily maintenance of animal locomotor activity and body temperature rhythmicity.

PIWIL1 suppresses circadian rhythms through GSK3ß-induced phosphorylation and degradation of CLOCK and BMAL1 in cancer cells.

Circadian rhythms are maintained by series of circadian clock proteins, and post-translation modifications of clock proteins significantly contribute to regulating circadian clock. However, the underlying upstream mechanism of circadian genes that are responsible for circadian rhythms in cancer cells remains unknown. PIWIL1 participates in many physiological processes and current discoveries have shown that PIWIL1 is involved in tumorigenesis in various cancers. Here we report that PIWIL1 can suppress circadian rhythms in cancer cells. Mechanistically, by promoting SRC interacting with PI3K, PIWIL1 can activate PI3K-AKT signalling pathway to phosphorylate and inactivate GSK3ß, repressing GSK3ß-induced phosphorylation and ubiquitination of CLOCK and BMAL1. Simultaneously, together with CLOCK/BMAL1 complex, PIWIL1 can bind with E-BOX region to suppress transcriptional activities of clock-controlled genes promoters. Collectively, our findings first demonstrate that PIWIL1 negatively regulates circadian rhythms via two pathways, providing molecular connection between dysfunction of circadian rhythms and tumorigenesis.

A Critical E-box in Barhl1 3' Enhancer Is Essential for Auditory Hair Cell Differentiation.

Barhl1, a mouse homologous gene of Drosophila BarH class homeobox genes, is highly expressed within the inner ear and crucial for the long-term maintenance of auditory hair cells that mediate hearing and balance, yet little is known about the molecular events underlying Barhl1 regulation and function in hair cells. In this study, through data mining and in vitro report assay, we firstly identified Barhl1 as a direct target gene of Atoh1 and one E-box (E3) in Barhl1 3' enhancer is crucial for Atoh1-mediated Barhl1 activation. Then we generated a mouse embryonic stem cell (mESC) line carrying disruptions on this E3 site E-box (CAGCTG) using CRISPR/Cas9 technology and this E3 mutated mESC line is further subjected to an efficient stepwise hair cell differentiation strategy in vitro. Disruptions on this E3 site caused dramatic loss of Barhl1 expression and significantly reduced the number of induced hair cell-like cells, while no affections on the differentiation toward early primitive ectoderm-like cells and otic progenitors. Finally, through RNA-seq profiling and gene ontology (GO) enrichment analysis, we found that this E3 box was indispensable for Barhl1 expression to maintain hair cell development and normal functions. We also compared the transcriptional profiles of induced cells from CDS mutated and E3 mutated mESCs, respectively, and got very consistent results except the Barhl1 transcript itself. These observations indicated that Atoh1-mediated Barhl1 expression could have important roles during auditory hair cell development. In brief, our findings delineate the detail molecular mechanism of Barhl1 expression regulation in auditory hair cell differentiation.

Intrinsic cell-penetrating activity propels Omomyc from proof of concept to viable anti-MYC therapy.

Inhibiting MYC has long been considered unfeasible, although its key role in human cancers makes it a desirable target for therapeutic intervention. One reason for its perceived undruggability was the fear of catastrophic side effects in normal tissues. However, we previously designed a dominant-negative form of MYC called Omomyc and used its conditional transgenic expression to inhibit MYC function both in vitro and in vivo. MYC inhibition by Omomyc exerted a potent therapeutic impact in various mouse models of cancer, causing only mild, well-tolerated, and reversible side effects. Nevertheless, Omomyc has been so far considered only a proof of principle. In contrast with that preconceived notion, here, we show that the purified Omomyc mini-protein itself spontaneously penetrates into cancer cells and effectively interferes with MYC transcriptional activity therein. Efficacy of the Omomyc mini-protein in various experimental models of non-small cell lung cancer harboring different oncogenic mutation profiles establishes its therapeutic potential after both direct tissue delivery and systemic administration, providing evidence that the Omomyc mini-protein is an effective MYC inhibitor worthy of clinical development.

Transcriptional integration of the responses to iron availability in Arabidopsis by the bHLH factor ILR3.

Iron (Fe) homeostasis is crucial for all living organisms. In mammals, an integrated posttranscriptional mechanism couples the regulation of both Fe deficiency and Fe excess responses. Whether in plants an integrated control mechanism involving common players regulates responses both to deficiency and to excess is still to be determined. In this study, molecular, genetic and biochemical approaches were used to investigate transcriptional responses to both Fe deficiency and excess. A transcriptional activator of responses to Fe shortage in Arabidopsis, called bHLH105/ILR3, was found to also negatively regulate the expression of ferritin genes, which are markers of the plant's response to Fe excess. Further investigations revealed that ILR3 repressed the expression of several structural genes that function in the control of Fe homeostasis. ILR3 interacts directly with the promoter of its target genes, and repressive activity was conferred by its dimerisation with bHLH47/PYE. Last, this study highlighted that important facets of plant growth in response to Fe deficiency or excess rely on ILR3 activity. Altogether, the data presented herein support that ILR3 is at the centre of the transcriptional regulatory network that controls Fe homeostasis in Arabidopsis, in which it acts as both transcriptional activator and repressor.

HeLa E-Box Binding Protein, HEB, Inhibits Promoter Activity of the Lysophosphatidic Acid Receptor Gene Lpar1 in Neocortical Neuroblast Cells.

Lysophosphatidic acid (LPA) is an endogenous lysophospholipid with signaling properties outside of the cell and it signals through specific G protein-coupled receptors, known as LPA1-6. For one of its receptors, LPA1 (gene name Lpar1), details on the cis-acting elements for transcriptional control have not been defined. Using 5'RACE analysis, we report the identification of an alternative transcription start site of mouse Lpar1 and characterize approximately 3,500 bp of non-coding flanking sequence 5' of mouse Lpar1 gene for promoter activity. Transient transfection of cells derived from mouse neocortical neuroblasts with constructs from the 5' regions of mouse Lpar1 gene revealed the region between -248 to +225 serving as the basal promoter for Lpar1. This region also lacks a TATA box. For the region between -761 to -248, a negative regulatory element affected the basal expression of Lpar1. This region has three E-box sequences and mutagenesis of these E-boxes, followed by transient expression, demonstrated that two of the E-boxes act as negative modulators of Lpar1. One of these E-box sequences bound the HeLa E-box binding protein (HEB), and modulation of HEB levels in the transfected cells regulated the transcription of the reporter gene. Based on our data, we propose that HEB may be required for a proper regulation of Lpar1 expression in the embryonic neocortical neuroblast cells and to affect its function in both normal brain development and disease settings.

Structural basis for effects of CpA modifications on C/EBPß binding of DNA.

CCAAT/enhancer binding proteins (C/EBPs) regulate gene expression in a variety of cells/tissues/organs, during a range of developmental stages, under both physiological and pathological conditions. C/EBP-related transcription factors have a consensus binding specificity of 5'-TTG-CG-CAA-3', with a central CpG/CpG and two outer CpA/TpG dinucleotides. Methylation of the CpG and CpA sites generates a DNA element with every pyrimidine having a methyl group in the 5-carbon position (thymine or 5-methylcytosine (5mC)). To understand the effects of both CpG and CpA modification on a centrally-important transcription factor, we show that C/EBPß binds the methylated 8-bp element with modestly-increased (2.4-fold) binding affinity relative to the unmodified cognate sequence, while cytosine hydroxymethylation (particularly at the CpA sites) substantially decreased binding affinity (36-fold). The structure of C/EBPß DNA binding domain in complex with methylated DNA revealed that the methyl groups of the 5mCpA/TpG make van der Waals contacts with Val285 in C/EBPß. Arg289 recognizes the central 5mCpG by forming a methyl-Arg-G triad, and its conformation is constrained by Val285 and the 5mCpG methyl group. We substituted Val285 with Ala (V285A) in an Ala-Val dipeptide, to mimic the conserved Ala-Ala in many members of the basic leucine-zipper family of transcription factors, important in gene regulation, cell proliferation and oncogenesis. The V285A variant demonstrated a 90-fold binding preference for methylated DNA (particularly 5mCpA methylation) over the unmodified sequence. The smaller side chain of Ala285 permits Arg289 to adopt two alternative conformations, to interact in a similar fashion with either the central 5mCpG or the TpG of the opposite strand. Significantly, the best-studied cis-regulatory elements in RNA polymerase II promoters and enhancers have variable sequences corresponding to the central CpG or reduced to a single G:C base pair, but retain a conserved outer CpA sequence. Our analyses suggest an important modification-dependent CpA recognition by basic leucine-zipper transcription factors.

Lactate Promotes Myoblast Differentiation and Myotube Hypertrophy via a Pathway Involving MyoD In Vitro and Enhances Muscle Regeneration In Vivo.

Lactate is a metabolic substrate mainly produced in muscles, especially during exercise. Recently, it was reported that lactate affects myoblast differentiation; however, the obtained results are inconsistent and the in vivo effect of lactate remains unclear. Our study thus aimed to evaluate the effects of lactate on myogenic differentiation and its underlying mechanism. The differentiation of C2C12 murine myogenic cells was accelerated in the presence of lactate and, consequently, myotube hypertrophy was achieved. Gene expression analysis of myogenic regulatory factors showed significantly increased myogenic determination protein (MyoD) gene expression in lactate-treated cells compared with that in untreated ones. Moreover, lactate enhanced gene and protein expression of myosin heavy chain (MHC). In particular, lactate increased gene expression of specific MHC isotypes, MHCIIb and IId/x, in a dose-dependent manner. Using a reporter assay, we showed that lactate increased promoter activity of the MHCIIb gene and that a MyoD binding site in the promoter region was necessary for the lactate-induced increase in activity. Finally, peritoneal injection of lactate in mice resulted in enhanced regeneration and fiber hypertrophy in glycerol-induced regenerating muscles. In conclusion, physiologically high lactate concentrations modulated muscle differentiation by regulating MyoD-associated networks, thereby enhancing MHC expression and myotube hypertrophy in vitro and, potentially, in vivo.

Construction and rescue of Muscovy duck-origin goose parvovirus from an infectious clone containing an E-box deletion within the left terminal region.

To obtain a deletion mutant of Muscovy duck-origin goose parvovirus (MDGPV) and to analyze its biological characteristics, the pMDGPVPT plasmid, which contains a full-length DNA infectious clone of the MDGPV PT strain, was used in this study as the template. The E-box at nt 315 of the left inverted terminal repeat sequence (L-ITR) was deleted by overlap extension PCR to obtain the infectious recombinant plasmid p-PTΔE315. The p-PTΔE315 plasmid was transfected into 9-day-old non-immune Muscovy duck embryos via the yolk sac and the rescued deletion mutant virus r-PTΔE315 was generated. Experiments to demonstrate the novel deletion mutant virus' biological characteristics showed that r-PTΔE315 can cause typical lesions after infection of Muscovy duck embryos. Compared with its parent strain PT, the virulence of r-PTΔE315 and its proliferation ability in Muscovy duck embryos were attenuated, but its ability to replicate in MDEF cells was enhanced. This study laid the foundation for further understanding of the relationship between E-box deletion in the L-ITR and MDGPV virulence.