Heterogeneous structure of the polyubiquitin gene UbC of HeLa S3 cells

doi:10.1016/0378-1119(96)00145-X

Gene

Volume 175, Issues 1–2, 10 October 1996, Pages 179-185

https://doi.org/10.1016/0378-1119(96)00145-X Get rights and content

Abstract

The nucleotide sequence of the polyubiquitin gene UbC of HeLa S3 cells and its upstream region was determined and characterized. Recognition sequences for the transcription factors HSF, NFκB, AP-1(c-jun), NF-IL6 and Spl were found in the upstream control region, a result consistent with the observation of a distinct regulatory response for the UbC gene compared with that of another polyubiquitin gene UbB. Employing a PCR procedure to amplify the entire coding region from genomic DNA, we found a heterogeneity in the repeat number (eight and nine repeats) of the ubiquitin coding units, which resulted from an apparent deletion of either the seventh or the eighth unit in the predominant nine-ubiquitin-unit coding gene. In addition, by comparison with the nucleotide sequence of the UbC gene of human leukocytes previously determined, we found a significant number of nucleotide discrepancies. However, these discrepancies could be substantially reduced by realigning the units so that the first and second ubiquitin units of the sequence determined here are translocated to the boundary between the eighth and the ninth units.

References (22)

R. Einspanier et al.
Cloning and sequence analysis of a cDNA encoding poly-ubiquitin in human ovarian granulosa cells
Biochem. Biophys. Res. Commun.
(1987)
D. Finley et al.
The yeast polyubiquitin gene is essential for resistance to high temperatures, starvation, and other stresses
Cell
(1987)
T. Hayashi et al.
Nucleotide sequence and expression of the rat polyubiquitin mRNA
Biochim. Biophys. Acta
(1994)
S. Jentsch et al.
Genetic analysis of the ubiquitin system
Biochim. Biophys. Acta
(1991)
M. Nenoi et al.
Evolutionarily conserved structure of the 3′ non-translated region of a Chinese hamster polyubiquitin gene
Biochim. Biophys. Acta
(1992)
M. Nenoi et al.
Novel structure of a Chinese hamster polyubiquitin gene
Biochim. Biophys. Acta
(1994)
P.M. Sharp et al.
Molecular evolution of ubiquitin genes
Trends Ecol. Evol.
(1987)
S. Akira et al.
IL-6 and NF-IL6 in acute-phase response and vital infection
Immunol. Rev.
(1992)
R.T. Baker et al.
The human ubiquitin gene family: structure of a gene and pseudogenes from the UbB subfamily
Nucleic Acids Res.
(1987)
R.T. Baker et al.
Unequal crossover generates variation in ubiquitin coding unit number at the human UbC polyubiquitin locus
Am. J. Hum. Genet.
(1989)

R.T. Baker et al.

The human ubiquitin 52 amino acid fusion protein gene shares several structural features with mammalian ribosomal protein genes

Nucleic Acids Res.

(1991)

Cited by (31)

A novel isoform of myosin 18A (Myo18Aγ) is an essential sarcomeric protein in mouse heart
2019, Journal of Biological Chemistry
Citation Excerpt :
Restriction and DNA-modifying enzymes (Fermentas, Thermo Fisher Scientific (Breda, The Netherlands) or New England Biolabs, Bioké (Leiden, The Netherlands)) and oligodeoxyribonucleotides (Sigma-Aldrich) were used, following the manufacturer's instructions. For live sarcomeric Z-line imaging, we constructed an expression plasmid named pLV.HsUBC.mScarlet-I∼MmCypher1c, in which the coding sequence of the murine Cypher 1c protein (37) was preceded by a human ubiquitin C gene promoter (38) and extended at the N terminus with the coding sequence of mScarlet-I (39) (AddGene plasmid number 85044). For live-cell imaging of the sarcomeric A-band, the expression plasmid pLV.HsUBC.RnMyl2∼mScarlet.WHVoPRE was made.
Whereas myosin 18B (Myo18B) is known to be a critical sarcomeric protein, the function of myosin 18A (Myo18A) is unclear, although it has been implicated in cell motility and Golgi shape. Here, we show that homozygous deletion (homozygous tm1a, tm1b, or tm1d alleles) of Myo18a in mouse is embryonic lethal. Reminiscent of Myo18b, Myo18a was highly expressed in the embryo heart, and cardiac-restricted Myo18a deletion in mice was embryonic lethal. Surprisingly, using Western blot analysis, we were unable to detect the known isoforms of Myo18A, Myo18Aα and Myo18Aβ, in mouse heart using a custom C-terminal antibody. However, alternative anti-Myo18A antibodies detected a larger than expected protein, and RNA-Seq analysis indicated that a novel Myo18A transcript is expressed in mouse ventricular myocytes (and human heart). Cloning and sequencing revealed that this cardiac isoform, denoted Myo18Aγ, lacks the PDZ-containing N terminus of Myo18Aα but includes an alternative N-terminal extension and a long serine-rich C terminus. EGFP-tagged Myo18Aγ expressed in ventricular myocytes localized to the level of A-bands in sarcomeres, and Myo18a knockout embryos at day 10.5 exhibited disorganized sarcomeres with wavy thick filaments. We additionally generated myeloid-restricted Myo18a knockout mice to investigate the role of Myo18A in nonmuscle cells, exemplified by macrophages, which express more Myo18Aβ than Myo18Aα, but no defects in cell shape, motility, or Golgi shape were detected. In summary, we have identified a previously unrecognized sarcomere component, a large novel isoform (denoted Myo18Aγ) of Myo18A. Thus, both members of class XVIII myosins are critical components of cardiac sarcomeres.
Dynamic transcription of ubiquitin genes under basal and stressful conditions and new insights into the multiple UBC transcript variants
2015, Gene
Citation Excerpt :
To date, the heat-triggered induction of the Ub-ribosomal fusion genes has not been documented. From a molecular point of view, this can be explained by the presence of heat shock elements (HSEs) in the promoters of polyubiquitin genes UBB and UBC, but not of monoubiquitin genes RPS27A and UBA52 (Vihervaara et al., 2013; Nenoi et al., 1996). Heat shock factors (HSFs) binding to HSEs are the master regulators of transcription under protein damaging stress conditions and HSF1/2-driven induction of polyubiquitin genes, which is a rapid means to increase Ub levels, has recently been addressed (Vihervaara et al., 2013; Crinelli, submitted paper).
Ubiquitin (Ub) is a small 76-amino acid protein that is engaged in many different pathways within the cell, including protein turnover. During proteotoxic stress, when the demand of clearing damaged/misfolded proteins strongly increases, cells activate Ub gene transcription to face the need of extra ubiquitin. This paper shows the contribution of the four ubiquitin coding genes (UBB, UBC, UBA52, RPS27A) to the ubiquitin RNA pool under basal and stressful conditions. Our results reveal that UBC and RPS27A represent the major fraction of the Ub transcriptome in different cell lines, but when converted to the coding potential, polyubiquitin genes UBC and UBB mainly contribute to determine the intracellular ubiquitin content under basal conditions. Both the polyubiquitin genes UBB and UBC are upregulated upon proteasome inhibition and oxidative stress, with markedly higher responses from the UBC promoter. A similar output, with lower fold-inductions, is detected in heat-stressed cells, with UBC acting as the main contributor to thermotolerance. By contrast, upon these stressors, the levels of UBA52 and RPS27A mRNAs remain unchanged. Remarkably, UV irradiation fails to induce Ub gene transcription, but rather seems to act at the post-transcriptional level, by stabilizing ubiquitin mRNAs at UV doses which induce rapid degradation of other RNA molecules. Moreover, the evidence that the UBC core promoter contains multiple transcription start sites and their responsiveness to stress, is here reported for the first time.
Preclinical safety and efficacy of an anti-HIV-1 lentiviral vector containing a short hairpin RNA to CCR5 and the C46 fusion inhibitor
2014, Molecular Therapy Methods and Clinical Development
Citation Excerpt :
The stability of the delivered LVsh5/C46 vector into target cells was confirmed by southern blot and sequencing analyses, with no unforeseen alterations to the genetic elements of LVsh5/C46. Splicing of the 812 bp intron within the 5′ UTR of the human UbC promoter was well documented previously.58,59 In the present study, the spliced UbC promoter was found to be sufficient to drive expression of C46 peptide to a detectable level and was effective in inhibiting HIV-1 infection.
Gene transfer has therapeutic potential for treating HIV-1 infection by generating cells that are resistant to the virus. We have engineered a novel self-inactivating lentiviral vector, LVsh5/C46, using two viral-entry inhibitors to block early steps of HIV-1 cycle. The LVsh5/C46 vector encodes a short hairpin RNA (shRNA) for downregulation of CCR5, in combination with the HIV-1 fusion inhibitor, C46. We demonstrate here the effective delivery of LVsh5/C46 to human T cell lines, peripheral blood mononuclear cells, primary CD4⁺ T lymphocytes, and CD34⁺ hematopoietic stem/progenitor cells (HSPC). CCR5-targeted shRNA (sh5) and C46 peptide were stably expressed in the target cells and were able to effectively protect gene-modified cells against infection with CCR5- and CXCR4-tropic strains of HIV-1. LVsh5/C46 treatment was nontoxic as assessed by cell growth and viability, was noninflammatory, and had no adverse effect on HSPC differentiation. LVsh5/C46 could be produced at a scale sufficient for clinical development and resulted in active viral particles with very low mutagenic potential and the absence of replication-competent lentivirus. Based on these in vitro results, plus additional in vivo safety and efficacy data, LVsh5/C46 is now being tested in a phase 1/2 clinical trial for the treatment of HIV-1 disease.
A potent enhancer element in the 5′-UTR intron is crucial for transcriptional regulation of the human ubiquitin C gene
2009, Gene
Ubiquitin (Ub) plays a crucial role in almost every aspect of cellular functions. It is encoded by four genes, of which UbC is known to meet cell demand for ubiquitin in both basal and stressful conditions. To understand the molecular mechanisms regulating UbC gene expression, we performed a functional characterization of the UbC promoter. Deletion analyses on the 5′ end of the − 916/+ 878 promoter region, excluded the functional importance of nt − 916/− 371 in the transcriptional regulation of the gene, while 3′ deletions revealed that intron removal (nt+ 65/+ 876) resulted in a marked reduction of promoter activity in all the reporter constructs, regardless of the cell types. Intron substitution with a heterologous chimeric intron failed to restore promoter activity, thus allowing to exclude that the splicing event, per se, can be responsible for the intron-mediated burst of transcription. Gel shift assays demonstrated nuclear factor binding with the + 137/+ 766 intron region. Reporter constructs carrying partial intron deletions confirmed that this sequence is, indeed, required for maximal transcriptional activity. Computer-based analysis found potential Sp1 binding motifs within the intron sequence and electrophoretic mobility shift and supershift assays demonstrated that both Sp1 and Sp3 transcription factors interact, in vitro, with the UbC intron, at multiple binding sites. Moreover, ectopic expression of Sp1 and Sp3 revealed that both transcription factors positively regulate UbC promoter activity. Collectively, our data highlight the very new evidence that the 5′-UTR intron is crucial in regulating UbC gene expression and provide insights into the pivotal role of Sp1/Sp3 binding to the intronic enhancer in the regulation of UbC transcription.
Activity of different vaccine-associated promoter elements in human dendritic cells
2008, Immunology Letters
Vaccine design approaches that target dendritic cells (DC) aim at achieving high levels of transgene expression. Careful selection of the promoter element driving the foreign gene is therefore important. We have constructed adenovirus vectors carrying the gene for enhanced green fluorescent protein (eGFP) driven by three different promoters, CMV, CMV5 and ubiquitin C (UbC) promoter, and analysed their activity in different populations of human DC, namely blood plasmacytoid (pDC) and myeloid DC (mDC), monocyte-derived DC (moDC), Langerhans (LC) and dermal type DC (dDC). Although the CMV5 promoter was more active than the other two promoters in the HeLa and 911HER cell lines, in human DC the highest level of transgene expression was seen with the CMV promoter. There was very low-level eGFP expression in all cell types transduced with the UbC promoter. Highest eGFP expression levels were observed in moDC, cultured mDC and LC and the lowest levels in pDC. Expression of eGFP was augmented in all DC populations upon stimulation with CD40 ligand (CD40L). These findings demonstrate that the CMV promoter is the most effective of the three promoters tested in a range of different human DC populations.
Transduction of myogenic cells by retargeted dual high-capacity hybrid viral vectors: robust dystrophin synthesis in duchenne muscular dystrophy muscle cells
2006, Molecular Therapy
Citation Excerpt :
The NruI site precedes the DMD ORF and lies immediately downstream of the simian virus 40 pA of the DsRedexpression unit. The UBC promoter fragment was amplified from pUbCR#1 [26] (a kind gift from M. Nenoi) by PCR using the Platinum TaqDNA polymerase High Fidelity enzyme mixture (Invitrogen) together with primers hUbiCR (5′ -CGTAAGCTTGTCTAACAAAAAAGCCAAAAACGG-3′) and M13F (5′-GACGTTGTAAAACGACGGCCAGT-3′). The amplified DNA was digested with HindIII (Fermentas) and blunt-ended with the Klenow fragment (Fermentas).
Duchenne muscular dystrophy (DMD) is caused by mutations in the dystrophin gene (DMD), making it amenable to gene- or cell-based therapies. Another possible treatment entails the combination of both principles by transplantation of autologous myogenic cells after their genetic complementation. This approach requires efficient and stable transduction of these cells with recombinant DMD. Recently, we generated a dual high-capacity (hc) adenovirus (Ad)–adeno-associated virus (AAV) hybrid vector (HV) that can deliver two full-length dystrophin-encoding modules into target cells. We showed that HV transduction of human cells containing AAV Rep proteins leads to the insertion of foreign DNA into the AAVS1 locus. Here, we improved HV entry into muscle cells from DMD patients. After having verified that these cells barely express the coxsackie B virus and Ad receptor (CAR), which constitutes the attachment molecule for Ad serotype 5 (Ad5) fibers, we equipped dual hcAd/AAV HV particles with Ad serotype 50 fiber domains to achieve CAR-independent uptake. These retargeted vectors complemented much more efficiently the genetic defect of dystrophin-defective myoblasts and myotubes than their isogenic counterparts with conventional Ad5 fibers. Importantly, the accumulation of β-dystroglycan along the membranes of vector-treated DMD myotubes indicated proper assembly of dystrophin-associated glycoprotein complexes.

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References (22)

Cloning and sequence analysis of a cDNA encoding poly-ubiquitin in human ovarian granulosa cells

Biochem. Biophys. Res. Commun.

The yeast polyubiquitin gene is essential for resistance to high temperatures, starvation, and other stresses

Cell

Nucleotide sequence and expression of the rat polyubiquitin mRNA

Biochim. Biophys. Acta

Genetic analysis of the ubiquitin system

Biochim. Biophys. Acta

Evolutionarily conserved structure of the 3′ non-translated region of a Chinese hamster polyubiquitin gene

Biochim. Biophys. Acta

Novel structure of a Chinese hamster polyubiquitin gene

Biochim. Biophys. Acta

Molecular evolution of ubiquitin genes

Trends Ecol. Evol.

IL-6 and NF-IL6 in acute-phase response and vital infection

Immunol. Rev.

The human ubiquitin gene family: structure of a gene and pseudogenes from the UbB subfamily

Nucleic Acids Res.

Unequal crossover generates variation in ubiquitin coding unit number at the human UbC polyubiquitin locus

Am. J. Hum. Genet.

The human ubiquitin 52 amino acid fusion protein gene shares several structural features with mammalian ribosomal protein genes

Nucleic Acids Res.

Cited by (31)

A novel isoform of myosin 18A (Myo18Aγ) is an essential sarcomeric protein in mouse heart

Dynamic transcription of ubiquitin genes under basal and stressful conditions and new insights into the multiple UBC transcript variants

Preclinical safety and efficacy of an anti-HIV-1 lentiviral vector containing a short hairpin RNA to CCR5 and the C46 fusion inhibitor

A potent enhancer element in the 5′-UTR intron is crucial for transcriptional regulation of the human ubiquitin C gene

Activity of different vaccine-associated promoter elements in human dendritic cells

Transduction of myogenic cells by retargeted dual high-capacity hybrid viral vectors: robust dystrophin synthesis in duchenne muscular dystrophy muscle cells