Flanking Sequence Of Gene Research Articles

RB1 5́UTR contains an IRES related to cell cycle control and cancer progression

Retinoblastoma gene1 (RB1) is the first tumor suppressor gene that stands as the guardian of the gate of the G1 period and plays a central role in proliferation and differentiation. However, no reports focused on the possible internal ribosome entry site (IRES) function of the RB1 gene flanking sequence. In this study, we constructed a bicistronic reporter with the RB1 5′untranslated region (5́UTR) inserted between two reporter coding regions. We found RB1 5′UTR harbors an IRES and has higher activity in cancer cell lines than normal cells. Besides, RB1 IRES acquired the highest activity in the G0/G1 phase of the cell cycle, and the RB1 5′UTR mutation collected from retinoblastoma decreased IRES activity compared with RB1 5′UTR wild-type. These data indicated that RB1 IRES is a mechanism of stress regulation and is related to cell cycle control and cancer progression.

Meta-Analysis of Immune Induced Gene Expression Changes in Diverse Drosophila melanogaster Innate Immune Responses

Simple SummaryOrganisms can be infected by a wide range of pathogens, including bacteria, viruses, and parasites. Following infection, the host mounts an immune response to attempt to eliminate the pathogen. These responses are often specific to the type of pathogen and mediated by the expression of specialized genes. We have characterized the expression changes induced in host Drosophila fruit flies following infection by multiple types of pathogens, and identified a small number of genes that show expression changes in each infection. This includes genes that are known to be involved in pathogen resistance, and others that have not been previously studied as immune response genes. These findings provide new insight into transcriptional changes that accompany Drosophila immunity. They may suggest possible roles for the differentially expressed genes in innate immune responses to diverse classes of pathogens, and serve to identify candidate genes for further empirical study of these processes.Organisms are commonly infected by a diverse array of pathogens and mount functionally distinct responses to each of these varied immune challenges. Host immune responses are characterized by the induction of gene expression, however, the extent to which expression changes are shared among responses to distinct pathogens is largely unknown. To examine this, we performed meta-analysis of gene expression data collected from Drosophila melanogaster following infection with a wide array of pathogens. We identified 62 genes that are significantly induced by infection. While many of these infection-induced genes encode known immune response factors, we also identified 21 genes that have not been previously associated with host immunity. Examination of the upstream flanking sequences of the infection-induced genes lead to the identification of two conserved enhancer sites. These sites correspond to conserved binding sites for GATA and nuclear factor κB (NFκB) family transcription factors and are associated with higher levels of transcript induction. We further identified 31 genes with predicted functions in metabolism and organismal development that are significantly downregulated following infection by diverse pathogens. Our study identifies conserved gene expression changes in Drosophila melanogaster following infection with varied pathogens, and transcription factor families that may regulate this immune induction.

Molecular characterization, expression profile and transcriptional regulation of the CYP19 gene in goose ovarian follicles.

Cytochrome P450 Family 19 (CYP19) is a crucial enzyme to catalyze the conversion of androgens to estrogens. However, the regulatory mechanism of goose CYP19 gene remains poorly understood. The present study attempted to obtain the full-length coding sequence (CDS) and 5'-flanking sequence of CYP19 gene, to investigate its expression and distribution profiles in different sized follicles, and to analyze the transcriptional regulatory mechanism of CYP19 gene in goose. Results showed that its CDS consisted of 1512 nucleotides and the encoded amino acid sequence contained a classical P450 structural domain. Homology analysis showed that there were high homologies of nucleotide and amino acid sequences between goose and other avian species. Its promoter sequence spanned from -1925bp to the transcription start site (ATG) and several transcriptional factors were predicted in this region. Further analysis from luciferase assay showed that the luciferase activity was the highest spanning from -118 to -1 bp by constructing deletion promoter reporter vector. In addition, result from quantitative real-time polymerase chain reaction indicated that the mRNA level of CYP19 gene were highly expressed in theca layer of the fifth largest follicle, and the cellular location was in the theca externa cells by immunohistochemistry. Taken together, it could be concluded that the transcription activity of CYP19 gene was activated by transcriptional factors in its proximal region of promoter to promote the synthesis of estrogens, regulating the selection of pre-hierarchical into hierarchical follicle in goose.

Flanker: a tool for comparative genomics of gene flanking regions.

Analysing the flanking sequences surrounding genes of interest is often highly relevant to understanding the role of mobile genetic elements (MGEs) in horizontal gene transfer, particular for antimicrobial-resistance genes. Here, we present Flanker, a Python package that performs alignment-free clustering of gene flanking sequences in a consistent format, allowing investigation of MGEs without prior knowledge of their structure. These clusters, known as ‘flank patterns’ (FPs), are based on Mash distances, allowing for easy comparison of similarity across sequences. Additionally, Flanker can be flexibly parameterized to fine-tune outputs by characterizing upstream and downstream regions separately, and investigating variable lengths of flanking sequence. We apply Flanker to two recent datasets describing plasmid-associated carriage of important carbapenemase genes (bla OXA-48 and bla KPC-2/3) and show that it successfully identifies distinct clusters of FPs, including both known and previously uncharacterized structural variants. For example, Flanker identified four Tn4401 profiles that could not be sufficiently characterized using TETyper or MobileElementFinder, demonstrating the utility of Flanker for flanking-gene characterization. Similarly, using a large (n=226) European isolate dataset, we confirm findings from a previous smaller study demonstrating association between Tn1999.2 and bla OXA-48 upregulation and demonstrate 17 FPs (compared to the 5 previously identified). More generally, the demonstration in this study that FPs are associated with geographical regions and antibiotic-susceptibility phenotypes suggests that they may be useful as epidemiological markers. Flanker is freely available under an MIT license at https://github.com/wtmatlock/flanker.

A homozygous variant in a consanguineous pedigree with inherited protein S deficiency

Objective: To observe the clinical feature of familiar hereditary protein S deficiency, and to explore the related gene mutation. Methods: The blood samples were obtained from the proband and the family memebers(3 generations,6 persons). PROS1 gene of the proband and the family members was analyzed. The 15 exons and flanking sequence of PROS1 gene were analyzed by PCR and DNA sequencing. Results: Five out of 6 family members were diagnosed as having hereditary protein S deficiency. The proband suffered from pulmonary embolism. The others had no obvious thrombotic event. The gene sequencing revealed that the proband carried a c.-168C>T homozygous variant in the promoter of exon 1. His parents, brother and son all carried c.-168C>T heterozygosis variant at the same position. The gene of his wife was a wild type. Conclusion: A gene variant (c.-168C>T) of PROS1 was discovered in this Chinese family. Gene variant of PROS1 may result in protein S deficiency. Patients with protein S deficiency may suffer from vein thrombosis and(or) pulmonary embolism.

Identification of a novel LPL nonsense variant and further insights into the complex etiology and expression of hypertriglyceridemia-induced acute pancreatitis

BackgroundHypertriglyceridemia (HTG) is a leading cause of acute pancreatitis. HTG can be caused by either primary (genetic) or secondary etiological factors, and there is increasing appreciation of the interplay between the two kinds of factors in causing severe HTG.ObjectivesThe main aim of this study was to identify the genetic basis of hypertriglyceridemia-induced acute pancreatitis (HTG-AP) in a Chinese family with three affected members (the proband, his mother and older sister).MethodsThe entire coding and flanking sequences of LPL, APOC2, APOA5, GPIHBP1 and LMF1 genes were analyzed by Sanger sequencing. The newly identified LPL nonsense variant was subjected to functional analysis by means of transfection into HEK-293 T cells followed by Western blot and activity assays. Previously reported pathogenic LPL nonsense variants were collated and compared with respect to genotype and phenotype relationship.ResultsWe identified a novel nonsense variant, p.Gln118* (c.351C > T), in the LPL gene, which co-segregated with HTG-AP in the Chinese family. We provided in vitro evidence that this variant resulted in a complete functional loss of the affected LPL allele. We highlighted a role of alcohol abuse in modifying the clinical expression of the disease in the proband. Additionally, our survey of 12 previously reported pathogenic LPL nonsense variants (in 20 carriers) revealed that neither serum triglyceride levels nor occurrence of HTG-AP was distinguishable among the three carrier groups, namely, simple homozygotes, compound heterozygotes and simple heterozygotes.ConclusionsOur findings, taken together, generated new insights into the complex etiology and expression of HTG-AP.

A haplotype variant of Hu sheep follicle-stimulating hormone receptor promoter region decreases transcriptional activity.

In sheep, increased expression of the follicle-stimulating hormone receptor (FSHR) in the ovary is a common feature of ewes that carry the prolific allele. In this study, we demonstrated that polymorphisms in the core promoter of the FSHR gene are associated with the reproductive performance of Hu sheep and are involved in the transcriptional activity of FSHR. An approximately 1.5-kb region of the 5' flanking sequence of the Hu sheep FSHR gene was isolated and characterized, and its core promoter was located in the 5' regulatory region, from nucleotides -580 to -342. Four variants (c.-518T>C, c.-466C>T, c.-414A>G and c.-365C>T) were detected in this region, and six genotypes and three haplotypes were found in the Hu sheep population (n=245). An association analysis revealed that these polymorphisms are associated with the litter size of Hu ewes. Furthermore, a luciferase assay showed that the T-C-A-C- and C-T-G-T-type core promoters have higher transcriptional activity than does the T-C-G-C type. Notably, the putative binding site for the transcription factor Yin Yang 1 (YY1) was present at the A allele of nucleotide -414, but YY1 can significantly increase the transcriptional activity of the FSHR core promoter, which contains three different haplotypes. Taken together, our results establish that these variants might be involved in regulating the transcriptional activity of FSHR and litter size in Hu ewes and may provide a novel candidate marker for marker-assisted selection in sheep breeding.

Molecular biology of gonadotrophins

Regulation of gonadotrophin synthesis involves a complex interaction between hypothalamic and gonadal hormones. Chronic administration of oestrogens and androgens to gonadectomized animals blocked the postcastration rise in amounts of mRNA encoding gonadotrophin subunits. Removal of endogenous GnRH decreased amounts of mRNA encoding gonadotrophin subunits. Pulsatile administration of GnRH to GnRH-deficient animals increased amounts of mRNA encoding gonadotrophin subunits. Studies using transgenic mice and transient transfection assays identified at least eight cis-acting DNA sequences in the proximal 350 bp of 5' flanking sequence of the human alpha subunit gene that directed expression to gonadotrophs or conferred responsiveness to oestrogens, androgens or GnRH. Unique DNA-binding proteins were also identified which directed expression of the human alpha subunit gene specifically to the pituitary. Pituitary cell lines that express bovine gonadotrophin subunit genes are not currently available; thus, relatively little is known about the molecular mechanisms that regulate expression of bovine gonadotrophin subunit genes. Recent studies with transgenic mice harbouring bovine alpha, LH beta, or FSH beta subunit transgenes revealed that DNA sequences important for gonadotroph-specific expression and hormonal regulation resided within the proximal 5' flanking sequences.

The metastasis suppressor NME1 inhibits melanoma cell motility via direct transcriptional induction of the integrin beta-3 gene

Expression of the metastasis suppressor NME1 in melanoma is associated with reduced cellular motility, invasion, and metastasis, but mechanisms underlying these activities are not completely understood. Herein we report a novel mechanism through which NME1 drives formation of large, stable focal adhesions (FAs) in melanoma cells via induction of integrin β3 (ITGβ3), and in one cell line, concomitant suppression of integrin β1 (ITGβ1) transcripts. Forced expression of NME1 resulted in a strong activation of the promoter region (−301 to +13) of the ITGB3 gene. Chromatin immunoprecipitation (ChIP) analysis revealed the transcriptional induction was associated with direct recruitment of NME1 and an increase in the epigenetic activation mark, acetylation of histone 3 on lysine 27 (H3K27Ac) to a 1 kb stretch of 5'-flanking sequence of the ITGB3 gene. Unexpectedly, NME1 did not affect the amount either ITGβ1 or ITGβ3 proteins were internalized and recycled, processes commonly associated with regulating expression of integrins at the cell surface. The ability of NME1 to suppress motile and invasive phenotypes of melanoma cells was dependent on its induction of ITGβ3. Expression of ITGβ3 mRNA was associated with increased disease-free survival time in melanoma patients of the TCGA collection, consistent with its potential role as an effector of the metastasis suppressor function of NME1. Together, these data indicate metastasis suppressor activity of NME1 in melanoma is mediated by induction of ITGB3 gene transcription, with NME1-driven enrichment of ITGβ3 protein at the cell membrane resulting in attenuated cell motility through the stabilization of large focal adhesions.

Evolution and the flanking sequences of Β-lactamase resistance gene blaOKP

blaOKP genes are chromosomally encoded beta-lactamases that mediate several classes of antibiotics resistance. To investigate the evolution and flanking sequences of OKP beta-lactamase gene, the gene blaOKP and its flanking sequences from a newly isolated Klebsiella pneumoniae were studied using whole genome sequencing. The flanking sequences of different variant blaOKP genes and blaSHV, another plasmid-encoded beta-lactamase gene, were then compared. These studies show that the blaOKP and blaSHV genes evolve differently and belong to two different evolution branches. The blaOKP gene variants can be divided into subgroups: blaOKP-A and blaOKP-B. Although both blaOKP and blaSHV genes have no mobile genetic elements in their flanking sequences, their genetic environments are quite different. The blaOKP gene is adjacent to KdpC while blaSHV gene is flanked by RecF and ygbN-ygbM-ygbK. Furthermore, there are a variety of mobile genetic elements in the neighboring sequence plasmid-encoded blaSHV genes that are absent in blaOKP genes. These structural differences may slow the evolution of blaOKP gene. Collectively, we demonstrate that the evolution and flanking sequence of blaOKP gene are different from those of the blaSHV gene, which could be an important reason for its relatively slow evolution.

A case of tuberous sclerosis induced by a somatic mosaic mutation in the TSC2 gene

Objective To investigate clinical features and detect mutations in a case of tuberous sclerosis complex (TSC) caused by a somatic mosaic mutation in the TSC2 gene. Methods Peripheral blood samples were obtained from a patient with suspected TSC, his parents, and 200 unrelated healthy controls. Genomic DNA was extracted from these blood samples, polymerase chain reaction (PCR) and next-generation sequencing were performed to amplify all the exons and their flanking sequences of the TSC1 and TSC2 genes followed by DNA sequencing, so as to identify mutations in the TSC1 and TSC2 genes. DNA was also extracted from lesional skin tissues of the patient, and PCR was conducted to amplify the target fragment of the TSC2 gene followed by DNA sequencing. Results The patient clinically presented with facial angiofibroma, depigmented patches on the waist, periungual fibroma and angioleio-myolipoma of the kidney, which were consistent with the diagnosis of TSC. A mutation c.5130_5131insT (p.V1711Cfs*18) was identified in the TSC2 gene in the patient. A higher frequency of the mutation was found in the DNA of the tumor tissue than in that of the peripheral blood. No such a mutation was found in his parents′ DNA, unrelated healthy controls or any public database. Conclusion The somatic mosaic mutation c.5130_5131insT in the TSC2 gene is responsible for the phenotype of TSC in the patient. Key words: Tuberous sclerosis complex; Mosaicism; TSC2 gene

Analysis of keratin 1 gene mutation and phenotypes in a pair of twins with bullous congenital ichthyosiform erythroderma

Objective To identify mutations in keratin genes (KRT1 and KRT10) in a pair of twins with bullous congenital ichthyosiform erythroderma (BCIE) , and to explore the relationship between the causative genes and phenotypes. Methods Clinical data were collected from a pair of twins with BCIE and their family members. Peripheral blood samples were obtained from the twins, their old brother and parents, and DNA was extracted from these blood samples. Polymerase chain reaction (PCR) was performed to amplify all the coding exons and their flanking sequences of the KRT1 and KRT10 genes, and 100 unrelated healthy persons served as controls. Results The 11-year-old male proband presented with recurrent blisters, hypertrophy and desquamation all over the body for 11 years. His twin brother had similar skin lesions. Skin examination of the proband showed diffuse erythema covered with thick scaly crusts on the trunk and extremities. Blisters, bullae and erosions due to ruptured blisters were observed locally with tenderness on palpation. There were obvious hyperkeratotic and hard lesions on the big joints of the extremities. Diffuse hyperkeratosis could be seen on the palms and soles. A mutation c.591 + 1G > A was identified at position 1 in intron 1 of the KRT1 gene in the twins, but not in the 3 healthy family members or the 100 unrelated healthy controls. Conclusion The mutation c.591 + 1G > A at position 1 in intron 1 of the KRT1 gene may contribute to the clinical phenotype of the twins with BCIE. Key words: Ichthyosiform erythroderma, congenital; Keratin-1; Keratin-10; DNA mutational analysis

Placental transcriptome co-expression analysis reveals conserved regulatory programs across gestation

BackgroundMammalian development in utero is absolutely dependent on proper placental development, which is ultimately regulated by the placental genome. The regulation of the placental genome can be directly studied by exploring the underlying organisation of the placental transcriptome through a systematic analysis of gene-wise co-expression relationships.ResultsIn this study, we performed a comprehensive analysis of human placental co-expression using RNA sequencing and intergrated multiple transcriptome datasets spanning human gestation. We identified modules of co-expressed genes that are preserved across human gestation, and also identifed modules conserved in the mouse indicating conserved molecular networks involved in placental development and gene expression patterns more specific to late gestation. Analysis of co-expressed gene flanking sequences indicated that conserved co-expression modules in the placenta are regulated by a core set of transcription factors, including ZNF423 and EBF1. Additionally, we identified a gene co-expression module enriched for genes implicated in the pregnancy pathology preeclampsia. By using an independnet transcriptome dataset, we show that these co-expressed genes are differentially expressed in preeclampsia.ConclusionsThis study represents a comprehensive characterisation of placental co-expression and provides insight into potential transcriptional regulators that govern conserved molecular programs fundamental to placental development.

T-786C variation in the promoter sequence of human eNOS gene markedly influences its expression level.

This study investigated the role of the T-786C polymorphism (SNP) in the 5'-flanking sequence of the endothelial nitric oxide synthase gene (eNOS) on its expression level in vascular endothelium with the ultimate goal of shedding more light on the mechanisms by which genetic variations of eNOS might affect the vascular level of nitric oxide (NO). Sequences in the 5'-flanking region of eNOS gene were PCR-amplified using genomic DNA templates isolated from blood samples collected from cardiovascular disease (CVD) patients. Two sequence-versions carrying the same SNP site were used; a short (345 bp) and an extended one (1,594 bp), numbered relative to the translational start site. All sequences were cloned into a promoter-less vector (pGL3-basic), which carries the firefly luciferase gene as a reporter. Genotyping of the T-786C polymorphism was performed using Sanger sequencing of the insert region. Luminescence levels were then recorded 24-48 h after transfecting human endothelial cell line (EA.hy926). Three genotypes were identified in the subject samples; TT, TC, or CC. The highest expression levels associated with the TT genotype, followed by the TC genotype, then the CC genotype. The extended sequence version produced higher expression levels compared to the shorter version. Our results provide evidence that the T allele at the T-786C SNP site of the eNOS gene results in increased expression of the enzyme, and consequently might provide a protective mechanism from CVD. The extended promoter sequence of eNOS resulted in higher expression of the gene, suggesting the presence of some essential binding sites for transcription enhancing proteins.

A survey of FLS2 genes from multiple citrus species identifies candidates for enhancing disease resistance to Xanthomonas citri ssp. citri.

Pathogen-associated molecular patterns (PAMPs)-triggered immunity (PTI) is an important component of plant innate immunity. In a previous study, we showed that the PAMP flg22 from Xanthomonas citri ssp. citri (Xflg22), the causal agent of citrus canker, induced PTI in citrus, which correlated with the observed levels of canker resistance. Here, we identified and sequenced two bacterial flagellin/flg22 receptors (FLS2-1 and FLS2-2) from ‘Duncan’ grapefruit (Citrus paradisi, CpFLS2-1 and CpFLS2-2) and ‘Sun Chu Sha’ mandarin (C. reticulata, CrFLS2-1 and CrFLS2-2). We were able to isolate only one FLS2 from ‘Nagami’ kumquat (Fortunella margarita, FmFLS2-1) and gene flanking sequences suggest a rearrangement event that resulted in the deletion of FLS2-2 from the genome. Phylogenetic analysis, gene structure and presence of critical amino acid domains all indicate we identified the true FLS2 genes in citrus. FLS2-2 was more transcriptionally responsive to Xflg22 than FLS2-1, with induced expression levels higher in canker-resistant citrus than in susceptible ones. Interestingly, ‘Nagami’ kumquat showed the highest FLS2-1 steady-state expression levels, although it was not induced by Xflg22. We selected FmFLS2-1, CrFLS2-2 and CpFLS2-2 to further evaluate their capacity to enhance bacterial resistance using Agrobacterium-mediated transient expression assays. Both FmFLS2-1 and CrFLS2-2, the two proteins from canker-resistant species, conferred stronger Xflg22 responses and reduced canker symptoms in leaves of the susceptible grapefruit genotype. These two citrus genes will be useful resources to enhance PTI and achieve resistance against canker and possibly other bacterial pathogens in susceptible citrus types.

A novel heterozygous missense mutation in the SASH1 gene is associated with dyschromatosis universalis hereditaria in a pedigree

Objective To investigate clinical features and molecular genetic basis of dyschromatosis universalis hereditaria (DUH) in a pedigree. Methods A detailed genetic survey and a physical examination were performed for available members in a pedigree with DUH. In addition, reflectance confocal microscopy and histopathology were carried out to observe skin lesions of the proband. Blood samples were collected from 8 family members (including 5 patients with DUH and 3 unaffected members) and 200 unrelated healthy human controls. The FlexiGene DNA kit was used to extract genomic DNA from these blood samples, and PCR was performed to amplify all coding exons and their flanking sequences of SASH1 and ABCB6 genes followed by direct DNA sequencing. Results DUH was inherited in an autosomal dominant manner in this pedigree, and occurred within 1 year after birth in 9 patients. Lesions initially appeared on the face, then gradually spread to the whole body. Among 8 surviving patients, 7 presented with irregularly shaped light brown to puce macules complicated by uniform hypopigmentation of the remaining skin, and only the elder sister of the proband showed generalized hyperpigmented macules mingled with hypopigmented macules in a reticular pattern. No abnormality was observed in oral mucosa, nails or teeth, and general status was good in the 8 patients. DNA sequencing revealed a novel heterozygous missense mutation (c.1761C > G, p.Ser587Arg) in exon 15 of the SASH1 gene in the 5 available patients, but not in the 3 unaffected individuals or 200 healthy controls. No mutation was found in the ABCB6 gene in any of the 8 family members. Conclusions Heterogeneity exists in clinical features and molecular genetic basis of DUH. The heterozygous missense mutation p.S587R in the SASH1 gene might be responsible for DUH in this family. Key words: Autosomal-dominant; Heterozygote; Mutation; Genetics; Han nationality; Genes, SASH1; Dyschromatosis universalis hereditaria

Promoter regulatory domain identification of cassava starch synthase IIb gene in transgenic tobacco

Soluble starch synthase is a key enzyme in the starch biosynthesis pathway, and its enzyme activity significantly influences starch components in cassava storage root. However, studies on the regulation mechanism of soluble starch synthase gene are rare. In this study, we cloned the 5′ flanking sequence of the MeSSIIb gene and predicted the distribution of cis-elements. The region from −453 to −1 was considered the primary core promoter by the quantitative detection of GUS activity in transgenic tobacco plants containing 5′ truncated promoters fused with the GUS gene. Analysis results clarified that the region from −531 to −454 significantly repressed promoter activity. The region from −453 to −388 was a repressive domain of ethylene, and some unknown drought responsive cis-elements were located in the region from −387 to −1. These findings will provide useful information on the functional assay and transcriptional regulation mechanisms of the MeSSIIb gene.

Epigenetics Evolution and Replacement Histones: Evolutionary Changes at Drosophila H4r

Histone 4 replacement (H4r) can replace replication-dependent H4 in Drosophila. To study the evolution of epigenetic mechanisms, the H4 and H4r genes from 14 Drosophila species were compared with regard to gene arrangement, codon bias and flanking sequences. Although the amino acid sequences of H4 and H4r are identical or nearly identical, the gene structures are quite different. The H4r gene is a single copy gene located 3R88C9 in D. melanogaster between punt and CEP78K, as it is in 11 closely related Drosophila species, but not in the three distantly related species. The H4r gene, unlike the H4 gene, has two introns and generates polyadenylated transcripts. The codon usage bias at particular sites differed between H4r and H4. The H4r gene had more GC pairs at 3rd codon position. Strongly conserved signal sequence was not found in the 5’-region or 3’-region of the H4r gene. These results suggested that the post transcriptional process such as modifying histone at or after translation will be important for replacing histones and remodeling the chromatin. The evolutionary changes that affect gene structure and codon usage might be a key step to develop epigenetic systems by replacement histones.

HDAC inhibitors suppress c-Jun/Fra-1-mediated proliferation through transcriptionally downregulating MKK7 and Raf1 in neuroblastoma cells.

Activator protein 1 (AP-1) is a transcriptional factor composed of the dimeric members of bZIP proteins, which are frequently deregulated in human cancer cells. In this study, we aimed to identify an oncogenic AP-1 dimer critical for the proliferation of neuroblastoma cells and to investigate whether histone deacetylase inhibitors (HDACIs), a new generation of anticancer agents, could target the AP-1 dimer. We report here that HDACIs including trichostatin A, suberoylanilidehydroxamic acid, valproic acid and M344 can transcriptionally suppress both c-Jun and Fra-1, preceding their inhibition of cell growth. c-Jun preferentially interacting with Fra-1 as a heterodimer is responsible for AP-1 activity and critical for cell growth. Mechanistically, HDACIs suppress Fra-1 expression through transcriptionally downregulating Raf1 and subsequently decreasing MEK1/2-ERK1/2 activity. Unexpectedly, HDACI treatment caused MKK7 downregulation at both the protein and mRNA levels. Deletion analysis of the 5′-flanking sequence of the MKK7 gene revealed that a major element responsible for the downregulation by HDACI is located at −149 to −3 relative to the transcriptional start site. Knockdown of MKK7 but not MKK4 remarkably decreased JNK/c-Jun activity and proliferation, whereas ectopic MKK7-JNK1 reversed HDACI-induced c-Jun suppression. Furthermore, suppression of both MKK-7/c-Jun and Raf-1/Fra-1 activities was involved in the tumor growth inhibitory effects induced by SAHA in SH-SY5Y xenograft mice. Collectively, these findings demonstrated that c-Jun/Fra-1 dimer is critical for neuroblastoma cell growth and that HDACIs act as effective suppressors of the two oncogenes through transcriptionally downregulating MKK7 and Raf1.

P38 mitogen-activated protein kinase inhibits USP22 transcription in HeLa cells.

Elevated expression of ubiquitin-specific processing enzyme 22 (USP22) was identified in multiple types of human cancers, and was correlated with tumorigenesis and progression. Despite an increase in the numbers of studies in the physiological function of USP22, little is known regarding the regulation of its expression. The 5' flanking sequence of the USP22 gene was recently characterized. In the present study, USP22 transcription was regulated by p38 mitogen-activated protein kinase (MAPK). Treatment of human cervical carcinoma (HeLa) cells with SB203580, an inhibitor of p38 MAPK, enhanced basal USP22 promoter activity and mRNA abundance. Transfection of MAPK kinase 6 (MKK6), an upstream activator of p38 MAPK, resulted in a 40% decrease in USP22 mRNA, while the dominant negative MKK6 increased the transcription level of the USP22, similar to SB203580. Dual luciferase report assays showed that mutations of the Sp1 binding site ahead of the transcription start site abolished the promoting effect of the USP22 promoter by SB203580. Cisplatin, the activator of p38 MAPK, also suppressed USP22 expression. This suppression was blocked by SB203580. In conclusion, p38 MAPK acts as an upstream negative regulator of USP22 transcription in HeLa cells.