Binding of transcription factors creates hot spots for UV photoproducts in vivo

Cyclobutane dipyrimidines and less than mean value of 6-4 dipyrimidines are the two major classes of mutagenic DNA photoproducts produced by UV irradiation of cells. We developed a method to map cyclobutane dipyrimidines at the DNA sequence level in mammalian cells. The frequency of this class of photoproducts was determined at every dipyrimidine along the human phosphoglycerate kinase-1 (PGK1) promoter sequence and was compared to the UV-induced frequency distribution of mean value of 6-4 dipyrimidines. After irradiation of living cells containing active or inactive PGK1 genes, enzymatic or chemical cleavage at UV photoproducts, and amplification by ligation-mediated polymerase chain reaction, photofootprints were seen in all regions which bind transcription factors and appear as DNase I footprints. Photoproduct frequency within transcription factor binding sites was suppressed or enhanced relative to inactive genes or naked DNA with enhancements of up to 30-fold. Since photoproducts are mutagenic, this indicates that photoproduct (mutation?) hot spots may be tissue specific in mammals.

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Characterization of constitutive HSF2 DNA-binding activity in mouse embryonal carcinoma cells

Molecular and Cellular Biology ◽

10.1128/mcb.14.8.5309-5317.1994 ◽

1994 ◽

Vol 14 (8) ◽

pp. 5309-5317

Author(s):

S P Murphy ◽

J J Gorzowski ◽

K D Sarge ◽

B Phillips

Keyword(s):

Transcription Factors ◽

Heat Shock ◽

Dna Binding ◽

Mammalian Cells ◽

Embryonal Carcinoma ◽

Embryonic Stem ◽

Binding Activity ◽

Hsp70 Promoter ◽

Ec Cells

Two distinct murine heat shock transcription factors, HSF1 and HSF2, have been identified. HSF1 mediates the transcriptional activation of heat shock genes in response to environmental stress, while the function of HSF2 is not understood. Both factors can bind to heat shock elements (HSEs) but are maintained in a non-DNA-binding state under normal growth conditions. Mouse embryonal carcinoma (EC) cells are the only mammalian cells known to exhibit HSE-binding activity, as determined by gel shift assays, even when maintained at normal physiological temperatures. We demonstrate here that the constitutive HSE-binding activity present in F9 and PCC4.aza.R1 EC cells, as well as a similar activity found to be present in mouse embryonic stem cells, is composed predominantly of HSF2. HSF2 in F9 EC cells is trimerized and is present at higher levels than in a variety of nonembryonal cell lines, suggesting a correlation of these properties with constitutive HSE-binding activity. Surprisingly, transcription run-on assays suggest that HSF2 in unstressed EC cells does not stimulate transcription of two putative target genes, hsp70 and hsp86. Genomic footprinting analysis indicates that HSF2 is not bound in vivo to the HSE of the hsp70 promoter in unstressed F9 EC cells, although HSF2 is present in the nucleus and the promoter is accessible to other transcription factors and to HSF1 following heat shock. Thus trimerization and nuclear localization of HSF2 do not appear to be sufficient for in vivo binding of HSF2 to the HSE of the hsp70 promoter in unstressed F9 EC cells.

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Nucleotide Excision Repair in Human Cells

Journal of Biological Chemistry ◽

10.1074/jbc.m113.482257 ◽

2013 ◽

Vol 288 (29) ◽

pp. 20918-20926 ◽

Cited By ~ 52

Author(s):

Jinchuan Hu ◽

Jun-Hyuk Choi ◽

Shobhan Gaddameedhi ◽

Michael G. Kemp ◽

Joyce T. Reardon ◽

...

Keyword(s):

Nucleotide Excision Repair ◽

Genomic Dna ◽

Excision Repair ◽

Human Cells ◽

Naked Dna ◽

Nucleotide Excision ◽

Uv Photoproducts ◽

Mutant Cells

Nucleotide excision repair is the sole mechanism for removing the major UV photoproducts from genomic DNA in human cells. In vitro with human cell-free extract or purified excision repair factors, the damage is removed from naked DNA or nucleosomes in the form of 24- to 32-nucleotide-long oligomers (nominal 30-mer) by dual incisions. Whether the DNA damage is removed from chromatin in vivo in a similar manner and what the fate of the excised oligomer was has not been known previously. Here, we demonstrate that dual incisions occur in vivo identical to the in vitro reaction. Further, we show that transcription-coupled repair, which operates in the absence of the XPC protein, also generates the nominal 30-mer in UV-irradiated XP-C mutant cells. Finally, we report that the excised 30-mer is released from the chromatin in complex with the repair factors TFIIH and XPG. Taken together, our results show the congruence of in vivo and in vitro data on nucleotide excision repair in humans.

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Determination of the spectrum of mutations induced by defined-wavelength solar UVB (313-nm) radiation in mammalian cells by use of a shuttle vector

Molecular and Cellular Biology ◽

10.1128/mcb.8.12.5425-5431.1988 ◽

1988 ◽

Vol 8 (12) ◽

pp. 5425-5431

Author(s):

S M Keyse ◽

F Amaudruz ◽

R M Tyrrell

Keyword(s):

Hot Spots ◽

Mammalian Cells ◽

Important Determinant ◽

Shuttle Vector ◽

Common Mechanism ◽

Site Specificity ◽

Base Pairs ◽

Dna Photoproducts ◽

Supf Gene

Mutations induced by UVB (313-nm) radiation, a wavelength in the region of peak effectiveness for sunlight-induced skin cancer in humans, have been analyzed at the sequence level in simian cells by using a plasmid shuttle vector (pZ189). We find that significant differences exist between the types of mutations induced by this solar wavelength and those induced by nonsolar UVC (254-nm) radiation. Compared with 254-nm radiation, 313-nm radiation induces more deletions and insertions in the region sequenced. In addition, although the types of base substitutions induced by the two wavelengths are broadly similar (in both cases, the majority of changes occur at G-C base pairs and the G-C to A-T transition is predominant), an analysis of the distribution of these base changes within the supF gene following irradiation at 313 nm reveals additional hot spots for mutation not seen after irradiation at 254 nm. These hot spots are shown to arise predominantly at sites of mutations involving multiple base changes, a class of mutations which arises more frequently at the longer solar wavelength. Lastly, we observed that most of the sites at which mutational hot spots arise after both UVC and UVB irradiation of the shuttle vector are also sites at which mutations arise spontaneously. Thus, a common mechanism may be involved in determining the site specificity of mutations, in which the DNA structure may be a more important determinant than the positions of DNA photoproducts.

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Chromatin potentiates transcription

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1620312114 ◽

2017 ◽

Vol 114 (7) ◽

pp. 1536-1541 ◽

Cited By ~ 19

Author(s):

Shigeki Nagai ◽

Ralph E. Davis ◽

Pierre Jean Mattei ◽

Kyle Patrick Eagen ◽

Roger D. Kornberg

Keyword(s):

Transcription Factors ◽

Rna Polymerase Ii ◽

Tata Box ◽

Transcription Start ◽

Chromosomal Locus ◽

Positive Role ◽

Transcription Start Sites ◽

Naked Dna ◽

General Transcription Factors

Chromatin isolated from the chromosomal locus of the PHO5 gene of yeast in a transcriptionally repressed state was transcribed with 12 pure proteins (80 polypeptides): RNA polymerase II, six general transcription factors, TFIIS, the Pho4 gene activator protein, and the SAGA, SWI/SNF, and Mediator complexes. Contrary to expectation, a nucleosome occluding the TATA box and transcription start sites did not impede transcription but rather, enhanced it: the level of chromatin transcription was at least sevenfold greater than that of naked DNA, and chromatin gave patterns of transcription start sites closely similar to those occurring in vivo, whereas naked DNA gave many aberrant transcripts. Both histone acetylation and trimethylation of H3K4 (H3K4me3) were important for chromatin transcription. The nucleosome, long known to serve as a general gene repressor, thus also performs an important positive role in transcription.

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Characterization of constitutive HSF2 DNA-binding activity in mouse embryonal carcinoma cells.

Molecular and Cellular Biology ◽

10.1128/mcb.14.8.5309 ◽

1994 ◽

Vol 14 (8) ◽

pp. 5309-5317 ◽

Cited By ~ 53

Author(s):

S P Murphy ◽

J J Gorzowski ◽

K D Sarge ◽

B Phillips

Keyword(s):

Transcription Factors ◽

Heat Shock ◽

Dna Binding ◽

Mammalian Cells ◽

Embryonal Carcinoma ◽

Embryonic Stem ◽

Binding Activity ◽

Hsp70 Promoter ◽

Ec Cells

Two distinct murine heat shock transcription factors, HSF1 and HSF2, have been identified. HSF1 mediates the transcriptional activation of heat shock genes in response to environmental stress, while the function of HSF2 is not understood. Both factors can bind to heat shock elements (HSEs) but are maintained in a non-DNA-binding state under normal growth conditions. Mouse embryonal carcinoma (EC) cells are the only mammalian cells known to exhibit HSE-binding activity, as determined by gel shift assays, even when maintained at normal physiological temperatures. We demonstrate here that the constitutive HSE-binding activity present in F9 and PCC4.aza.R1 EC cells, as well as a similar activity found to be present in mouse embryonic stem cells, is composed predominantly of HSF2. HSF2 in F9 EC cells is trimerized and is present at higher levels than in a variety of nonembryonal cell lines, suggesting a correlation of these properties with constitutive HSE-binding activity. Surprisingly, transcription run-on assays suggest that HSF2 in unstressed EC cells does not stimulate transcription of two putative target genes, hsp70 and hsp86. Genomic footprinting analysis indicates that HSF2 is not bound in vivo to the HSE of the hsp70 promoter in unstressed F9 EC cells, although HSF2 is present in the nucleus and the promoter is accessible to other transcription factors and to HSF1 following heat shock. Thus trimerization and nuclear localization of HSF2 do not appear to be sufficient for in vivo binding of HSF2 to the HSE of the hsp70 promoter in unstressed F9 EC cells.

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Determination of the spectrum of mutations induced by defined-wavelength solar UVB (313-nm) radiation in mammalian cells by use of a shuttle vector.

Molecular and Cellular Biology ◽

10.1128/mcb.8.12.5425 ◽

1988 ◽

Vol 8 (12) ◽

pp. 5425-5431 ◽

Cited By ~ 41

Author(s):

S M Keyse ◽

F Amaudruz ◽

R M Tyrrell

Keyword(s):

Hot Spots ◽

Mammalian Cells ◽

Important Determinant ◽

Shuttle Vector ◽

Common Mechanism ◽

Site Specificity ◽

Base Pairs ◽

Dna Photoproducts ◽

Supf Gene

Mutations induced by UVB (313-nm) radiation, a wavelength in the region of peak effectiveness for sunlight-induced skin cancer in humans, have been analyzed at the sequence level in simian cells by using a plasmid shuttle vector (pZ189). We find that significant differences exist between the types of mutations induced by this solar wavelength and those induced by nonsolar UVC (254-nm) radiation. Compared with 254-nm radiation, 313-nm radiation induces more deletions and insertions in the region sequenced. In addition, although the types of base substitutions induced by the two wavelengths are broadly similar (in both cases, the majority of changes occur at G-C base pairs and the G-C to A-T transition is predominant), an analysis of the distribution of these base changes within the supF gene following irradiation at 313 nm reveals additional hot spots for mutation not seen after irradiation at 254 nm. These hot spots are shown to arise predominantly at sites of mutations involving multiple base changes, a class of mutations which arises more frequently at the longer solar wavelength. Lastly, we observed that most of the sites at which mutational hot spots arise after both UVC and UVB irradiation of the shuttle vector are also sites at which mutations arise spontaneously. Thus, a common mechanism may be involved in determining the site specificity of mutations, in which the DNA structure may be a more important determinant than the positions of DNA photoproducts.

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Global Profiling of 2-hydroxyisobutyrylome in Common Wheat

10.1101/2020.03.05.978155 ◽

2020 ◽

Author(s):

Ning Zhang ◽

Lingran Zhang ◽

Linjie Li ◽

Junyou Geng ◽

Lei Zhao ◽

...

Keyword(s):

Western Blot ◽

Common Wheat ◽

Protein Interactions ◽

Mammalian Cells ◽

Metabolic Regulation ◽

Trichostatin A ◽

Affinity Purification ◽

Phosphoglycerate Kinase ◽

Post Translational Modification

AbstractAs a novel post-translational modification (PTM), lysine 2-hydroxyisobutyrylation (Khib) has been found to play a role in active gene transcription in mammalian cells and yeast, but the function of Khib proteins in plants remains unknown. In this study, we used western blot to demonstrate that Khib is an evolutionarily-conserved PTM in wheat and its donators, with the highest Khib abundance occurring in hexaploidy wheat. Additionally, global profiling using affinity purification and mass spectroscopy of 2-hydroxyisobutyrylome revealed that there were 3348 lysine modification sites from 1074 proteins in common wheat (Triticum aestivum L.). Moreover, bioinformatic data indicated that Khib proteins participate in a wide variety of biological and metabolic pathways. Immunoprecipitation and western blot confirmed that Khib proteins had an in vivo origin. A comparison of Khib and other major PTMs revealed that Khib proteins were simultaneously modified by multiple PTMs. Using mutagenesis experiments and Co-IP, we demonstrated that Khib on K206 is a key regulatory modification of phosphoglycerate kinase enzymatic activity and found that de-Khib on K206 affects protein interactions. Furthermore, Khib production of low-molecular-weight proteins was a response to the deacetylase inhibitors nicotinamide and trichostatin A. This study provides evidence that enhances our current understanding of Khib in wheat plants, including the cooperation between this PTM and metabolic regulation.

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Gene regulation by Sp1 and Sp3

Biochemistry and Cell Biology ◽

10.1139/o04-045 ◽

2004 ◽

Vol 82 (4) ◽

pp. 460-471 ◽

Cited By ~ 290

Author(s):

Lin Li ◽

Shihua He ◽

Jian-Min Sun ◽

James R Davie

Keyword(s):

Gene Regulation ◽

Transcription Factors ◽

Dna Binding ◽

Mammalian Cells ◽

Cellular Localization ◽

Target Genes ◽

In Vivo Studies ◽

Binding Domains

The Sp family of transcription factors is united by a particular combination of three conserved Cys2His2 zinc fingers that form the sequence-specific DNA-binding domain. Within the Sp family of transcription factors, Sp1 and Sp3 are ubiquitously expressed in mammalian cells. They can bind and act through GC boxes to regulate gene expression of multiple target genes. Although Sp1 and Sp3 have similar structures and high homology in their DNA binding domains, in vitro and in vivo studies reveal that these transcription factors have strikingly different functions. Sp1 and Sp3 are able to enhance or repress promoter activity. Regulation of the transcriptional activity of Sp1 and Sp3 occurs largely at the post-translational level. In this review, we focus on the roles of Sp1 and Sp3 in the regulation of gene expression.Key words: Sp1, Sp3, gene regulation, sub-cellular localization.

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Vacuoles Induced in Mammalian Cells by Helicobacter Cytotoxin are Acidic Organelles

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100158388 ◽

1990 ◽

Vol 48 (3) ◽

pp. 168-169

Author(s):

M. H. Chestnut ◽

C. E. Catrenich

Keyword(s):

Acridine Orange ◽

Mammalian Cells ◽

Laser Scanning ◽

Laser Scanning Microscopy ◽

Confocal Laser ◽

Ulcer Disease ◽

Gastroduodenal Disease ◽

H Pylori

Helicobacter pylori is a non-invasive, Gram-negative spiral bacterium first identified in 1983, and subsequently implicated in the pathogenesis of gastroduodenal disease including gastritis and peptic ulcer disease. Cytotoxic activity, manifested by intracytoplasmic vacuolation of mammalian cells in vitro, was identified in 55% of H. pylori strains examined. The vacuoles increase in number and size during extended incubation, resulting in vacuolar and cellular degeneration after 24 h to 48 h. Vacuolation of gastric epithelial cells is also observed in vivo during infection by H. pylori. A high molecular weight, heat labile protein is believed to be responsible for vacuolation and to significantly contribute to the development of gastroduodenal disease in humans. The mechanism by which the cytotoxin exerts its effect is unknown, as is the intracellular origin of the vacuolar membrane and contents. Acridine orange is a membrane-permeant weak base that initially accumulates in low-pH compartments. We have used acridine orange accumulation in conjunction with confocal laser scanning microscopy of toxin-treated cells to begin probing the nature and origin of these vacuoles.

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