DNA Sequences, Transcription Factors and Chromatin Structure

Denaturing High Performance Liquid Chromatography (DHPLC) is a relatively new method used for screening DNA sequences, characterized by high capacity to detect mutations/polymorphisms. This study is focused on the Transgenomic WAVETM DNA Fragment Analysis (based on DHPLC separation method) of a 485 bp fragment from human EC-SOD gene promoter in order to detect single nucleotide polymorphism (SNPs) associated with atherosclerosis and risk factors of cardiovascular disease. The fragment of interest was amplified by PCR reaction and analyzed by DHPLC in 100 healthy subjects and 70 patients characterized by atheroma. No different melting profiles were detected for the analyzed DNA samples. A combination of computational methods was used to predict putative transcription factors in the fragment of interest. Several putative transcription factors binding sites from the Ets-1 oncogene family: ETS member Elk-1, polyomavirus enhancer activator-3 (PEA3), protein C-Ets-1 (Ets-1), GABP: GA binding protein (GABP), Spi-1 and Spi-B/PU.1 related transcription factors, from the Krueppel-like family: Gut-enriched Krueppel-like factor (GKLF), Erythroid Krueppel-like factor (EKLF), Basic Krueppel-like factor (BKLF), GC box and myeloid zinc finger protein MZF-1 were identified in the evolutionary conserved regions. The bioinformatics results need to be investigated further in others studies by experimental approaches.

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MGA2 and SPT23 Are Modifiers of Transcriptional Silencing in Yeast

Genetics ◽

10.1093/genetics/156.3.933 ◽

2000 ◽

Vol 156 (3) ◽

pp. 933-941 ◽

Cited By ~ 1

Author(s):

Mary Lou Dula ◽

Scott G Holmes

Keyword(s):

Transcription Factors ◽

Chromatin Structure ◽

Transcriptional Control ◽

Transcriptional Silencing ◽

Sir Proteins

Abstract Transcriptional silencing at the HM loci and telomeres in yeast depends on several trans-acting factors, including Rap1p and the Sir proteins. The SUM1-1 mutation was identified by its ability to restore silencing to strains deficient in one or more of these trans-acting factors. The mechanism by which SUM1-1 bypasses the requirement for silencing proteins is not known. We identified four loci that when reduced in dosage in diploid strains increase the ability of SUM1-1 strains to suppress silencing defects. Two of the genes responsible for this effect were found to be MGA2 and SPT23. Mga2p and Spt23p were previously identified as functionally related transcription factors that influence chromatin structure. We find that deletion of MGA2 or SPT23 also increases the efficiency of silencing in haploid SUM1-1 strains. These results suggest that Mga2p and Spt23p are antagonists of silencing. Consistent with this proposal we find that deletion of MGA2 or SPT23 also suppresses the silencing defects caused by deletion of the SIR1 gene or by mutations in the HMR silencer sequences. However, we find that Mga2p and Spt23p can positively affect silencing in other contexts; deletion of either MGA2 or SPT23 decreases mating in strains bearing mutations in the HML-E silencer. Mga2p and Spt23p appear to be a novel class of factors that influence disparate pathways of transcriptional control by chromatin.

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TBP and SNAP50 transcription factors bind specifically to the Pr77 promoter sequence from trypanosomatid non-LTR retrotransposons

Parasites & Vectors ◽

10.1186/s13071-021-04803-5 ◽

2021 ◽

Vol 14 (1) ◽

Author(s):

Francisco Macías ◽

Raquel Afonso-Lehmann ◽

Patricia E. Carreira ◽

M. Carmen Thomas

Keyword(s):

Transcription Factors ◽

Protein Interactions ◽

Dna Sequences ◽

Direct Interaction ◽

Promoter Sequence ◽

Nuclear Proteins ◽

Hill Coefficient ◽

Small Nuclear Rna ◽

Mobile Dna ◽

Mobility Shift

Abstract Background Trypanosomatid genomes are colonized by active and inactive mobile DNA elements, such as LINE, SINE-like, SIDER and DIRE retrotransposons. These elements all share a 77-nucleotide-long sequence at their 5′ ends, known as Pr77, which activates transcription, thereby generating abundant unspliced and translatable transcripts. However, transcription factors that mediates this process have still not been reported. Methods TATA-binding protein (TBP) and small nuclear RNA-activating protein 50 kDa (SNAP50) recombinant proteins and specific antibodies raised against them were generated. Protein capture assay, electrophoretic mobility-shift assays (EMSA) and EMSA competition assays carried out using these proteins and nuclear proteins of the parasite together to specific DNA sequences used as probes allowed detecting direct interaction of these transcription factors to Pr77 sequence. Results This study identified TBP and SNAP50 as part of the DNA-protein complex formed by the Pr77 promoter sequence and nuclear proteins of Trypanosoma cruzi. TBP establishes direct and specific contact with the Pr77 sequence, where the DPE and DPE downstream regions are docking sites with preferential binding. TBP binds cooperatively (Hill coefficient = 1.67) to Pr77 and to both strands of the Pr77 sequence, while the conformation of this highly structured sequence is not involved in TBP binding. Direct binding of SNAP50 to the Pr77 sequence is weak and may be mediated by protein–protein interactions through other trypanosomatid nuclear proteins. Conclusions Identification of the transcription factors that mediate Pr77 transcription may help to elucidate how these retrotransposons are mobilized within the trypanosomatid genomes and their roles in gene regulation processes in this human parasite. Graphic abstract

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Chicken beta B1-crystallin gene expression: presence of conserved functional polyomavirus enhancer-like and octamer binding-like promoter elements found in non-lens genes.

Molecular and Cellular Biology ◽

10.1128/mcb.11.3.1488 ◽

1991 ◽

Vol 11 (3) ◽

pp. 1488-1499 ◽

Cited By ~ 34

Author(s):

H J Roth ◽

G C Das ◽

J Piatigorsky

Keyword(s):

Transcription Factors ◽

Hela Cell ◽

Dna Sequences ◽

Nuclear Extract ◽

Regulatory Elements ◽

Dnase I ◽

Flanking Sequence ◽

Mobility Shift ◽

Promoter Elements ◽

Dnase I Footprinting

Expression of the chicken beta B1-crystallin gene was examined. Northern (RNA) blot and primer extension analyses showed that while abundant in the lens, the beta B1 mRNA is absent from the liver, brain, heart, skeletal muscle, and fibroblasts of the chicken embryo, suggesting lens specificity. Promoter fragments ranging from 434 to 126 bp of 5'-flanking sequence (plus 30 bp of exon 1) of the beta B1 gene fused to the bacterial chloramphenicol acetyltransferase gene functioned much more efficiently in transfected embryonic chicken lens epithelial cells than in transfected primary muscle fibroblasts or HeLa cells. Transient expression of recombinant plasmids in cultured lens cells, DNase I footprinting, in vitro transcription in a HeLa cell extract, and gel mobility shift assays were used to identify putative functional promoter elements of the beta B1-crystallin gene. Sequence analysis revealed a number of potential regulatory elements between positions -126 and -53 of the beta B1 promoter, including two Sp1 sites, two octamer binding sequence-like sites (OL-1 and OL-2), and two polyomavirus enhancer-like sites (PL-1 and PL-2). Deletion and site-specific mutation experiments established the functional importance of PL-1 (-116 to -102), PL-2 (-90 to -76), and OL-2 (-75 to -68). DNase I footprinting using a lens or a HeLa cell nuclear extract and gel mobility shifts using a lens nuclear extract indicated the presence of putative lens transcription factors binding to these DNA sequences. Competition experiments provided evidence that PL-1 and PL-2 recognize the same or very similar factors, while OL-2 recognizes a different factor. Our data suggest that the same or closely related transcription factors found in many tissues are used for expression of the chicken beta B1-crystallin gene in the lens.

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Integrase-Specific Enhancement and Suppression of Retroviral DNA Integration by Compacted Chromatin Structure In Vitro

Journal of Virology ◽

10.1128/jvi.78.11.5848-5855.2004 ◽

2004 ◽

Vol 78 (11) ◽

pp. 5848-5855 ◽

Cited By ~ 39

Author(s):

Konstantin D. Taganov ◽

Isabel Cuesta ◽

René Daniel ◽

Lisa Ann Cirillo ◽

Richard A. Katz ◽

...

Keyword(s):

Transcription Factors ◽

Chromatin Structure ◽

Site Selection ◽

Integration Site ◽

Histone H1 ◽

Detectable Effect ◽

Host Chromosome ◽

Dna Integration ◽

Hiv 1

ABSTRACT Integration of viral DNA into the host chromosome is an obligatory step in retroviral replication and is dependent on the activity of the viral enzyme integrase. To examine the influence of chromatin structure on retroviral DNA integration in vitro, we used a model target comprising a 13-nucleosome extended array that includes binding sites for specific transcription factors and can be compacted into a higher-ordered structure. We found that the efficiency of in vitro integration catalyzed by human immunodeficiency virus type 1 (HIV-1) integrase was decreased after compaction of this target with histone H1. In contrast, integration by avian sarcoma virus (ASV) integrase was more efficient after compaction by either histone H1 or a high salt concentration, suggesting that the compacted structure enhances this reaction. Furthermore, although site-specific binding of transcription factors HNF3 and GATA4 blocked ASV DNA integration in extended nucleosome arrays, local opening of H1-compacted chromatin by HNF3 had no detectable effect on integration, underscoring the preference of ASV for compacted chromatin. Our results indicate that chromatin structure affects integration site selection of the HIV-1 and ASV integrases in opposite ways. These distinct properties of integrases may also affect target site selection in vivo, resulting in an important bias against or in favor of integration into actively transcribed host DNA.

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Regulation of Histone Deacetylase 4 Expression by the SP Family of Transcription Factors

Molecular Biology of the Cell ◽

10.1091/mbc.e05-08-0775 ◽

2006 ◽

Vol 17 (2) ◽

pp. 585-597 ◽

Cited By ~ 36

Author(s):

Fang Liu ◽

Nabendu Pore ◽

Mijin Kim ◽

K. Ranh Voong ◽

Melissa Dowling ◽

...

Keyword(s):

Transcription Factors ◽

Dna Sequences ◽

Chromatin Immunoprecipitation ◽

Histone Deacetylases ◽

Targeted Mutagenesis ◽

Cellular Functions ◽

Specific Expression ◽

Protein Levels ◽

Histone Deacetylase 4 ◽

First Time

Histone deacetylases mediate critical cellular functions but relatively little is known about mechanisms controlling their expression, including expression of HDAC4, a class II HDAC implicated in the modulation of cellular differentiation and viability. Endogenous HDAC4 mRNA, protein levels and promoter activity were all readily repressed by mithramycin, suggesting regulation by GC-rich DNA sequences. We validated consensus binding sites for Sp1/Sp3 transcription factors in the HDAC4 promoter through truncation studies and targeted mutagenesis. Specific and functional binding by Sp1/Sp3 at these sites was confirmed with chromatin immunoprecipitation (ChIP) and electromobility shift assays (EMSA). Cotransfection of either Sp1 or Sp3 with a reporter driven by the HDAC4 promoter led to high activities in SL2 insect cells (which lack endogenous Sp1/Sp3). In human cells, restored expression of Sp1 and Sp3 up-regulated HDAC4 protein levels, whereas levels were decreased by RNA-interference-mediated knockdown of either protein. Finally, variable levels of Sp1 were in concordance with that of HDAC4 in a number of human tissues and cancer cell lines. These studies together characterize for the first time the activity of the HDAC4 promoter, through which Sp1 and Sp3 modulates expression of HDAC4 and which may contribute to tissue or cell-line-specific expression of HDAC4.

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Comparative structure analysis of the ETSi domain of ERG3 and its complex with the E74 promoter DNA sequence

Acta Crystallographica Section F Structural Biology Communications ◽

10.1107/s2053230x1801110x ◽

2018 ◽

Vol 74 (10) ◽

pp. 656-663 ◽

Cited By ~ 2

Author(s):

Ruby Sharma ◽

Shanti P. Gangwar ◽

Ajay K. Saxena

Keyword(s):

Transcription Factors ◽

Dna Binding ◽

Dna Sequence ◽

Structure Analysis ◽

Dna Sequences ◽

Space Group ◽

Class 1 ◽

Comparative Structure ◽

Ets Domain ◽

Promoter Dna

ERG3 (ETS-related gene) is a member of the ETS (erythroblast transformation-specific) family of transcription factors, which contain a highly conserved DNA-binding domain. The ETS family of transcription factors differ in their binding to promoter DNA sequences, and the mechanism of their DNA-sequence discrimination is little known. In the current study, crystals of the ETSi domain (the ETS domain of ERG3 containing a CID motif) in space group P41212 and of its complex with the E74 DNA sequence (DNA9) in space group C2221 were obtained and their structures were determined. Comparative structure analysis of the ETSi domain and its complex with DNA9 with previously determined structures of the ERGi domain (the ETS domain of ERG containing inhibitory motifs) in space group P65212 and of the ERGi–DNA12 complex in space group P41212 were performed. The ETSi domain is observed as a homodimer in solution as well as in the crystallographic asymmetric unit. Superposition of the structure of the ETSi domain on that of the ERGi domain showed a major conformational change at the C-terminal DNA-binding autoinhibitory (CID) motif, while minor changes are observed in the loop regions of the ETSi-domain structure. The ETSi–DNA9 complex in space group C2221 forms a structure that is quite similar to that of the ERG–DNA12 complex in space group P41212. Upon superposition of the complexes, major conformational changes are observed at the 5′ and 3′ ends of DNA9, while the conformation of the core GGA nucleotides was quite conserved. Comparison of the ETSi–DNA9 structure with known structures of ETS class 1 protein–DNA complexes shows the similarities and differences in the promoter DNA binding and specificity of the class 1 ETS proteins.

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Serum stimulation of the c-fos enhancer induces reversible changes in c-fos chromatin structure

Molecular and Cellular Biology ◽

10.1128/mcb.10.3.1126-1133.1990 ◽

1990 ◽

Vol 10 (3) ◽

pp. 1126-1133

Author(s):

J L Feng ◽

B Villeponteau

Keyword(s):

Growth Factors ◽

Dna Sequences ◽

Chromatin Structure ◽

Transcriptional Activity ◽

Dnase I ◽

Enhancer Activity ◽

Serum Stimulation ◽

Fos Protein ◽

Dnase I Sensitivity ◽

Stimulation Of

Transcription of the proto-oncogene c-fos is known to be activated by growth factors in serum and subsequently repressed by the Fos protein. We show that generalized DNase I sensitivity of c-fos chromatin correlates closely with enhancer activity during induction, repression, and superinduction of the c-fos gene. Within 90 s of serum stimulation, proximal DNA sequences on both sides of the enhancer exhibit increased DNase I sensitivity. Within 5 min, elevated DNase I sensitivity spreads to chromatin at the distal 3' end of the c-fos gene. These results suggest that an open state of chromatin is propagated in both directions from the enhancer. The induced alterations in chromatin structure precede the increased transcriptional activity of the c-fos gene, suggesting that these changes in chromatin structure potentiate transcription.

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Centromeric Transcription: A Conserved Swiss-Army Knife

Genes ◽

10.3390/genes11080911 ◽

2020 ◽

Vol 11 (8) ◽

pp. 911

Author(s):

Ganesan Arunkumar ◽

Daniël P. Melters

Keyword(s):

Repetitive Dna ◽

Dna Sequences ◽

Chromatin Structure ◽

Repetitive Dna Sequences ◽

Mitotic Spindles ◽

Heterochromatin Formation ◽

Protein Loading ◽

Range Of Functions ◽

Centromeric Protein ◽

Essential Function

In most species, the centromere is comprised of repetitive DNA sequences, which rapidly evolve. Paradoxically, centromeres fulfill an essential function during mitosis, as they are the chromosomal sites wherein, through the kinetochore, the mitotic spindles bind. It is now generally accepted that centromeres are transcribed, and that such transcription is associated with a broad range of functions. More than a decade of work on this topic has shown that centromeric transcripts are found across the eukaryotic tree and associate with heterochromatin formation, chromatin structure, kinetochore structure, centromeric protein loading, and inner centromere signaling. In this review, we discuss the conservation of small and long non-coding centromeric RNAs, their associations with various centromeric functions, and their potential roles in disease.

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