scholarly journals Single-molecule DNA unzipping reveals asymmetric modulation of a transcription factor by its binding site sequence and context

2017 ◽  
Author(s):  
Sergei Rudnizky ◽  
Hadeel Khamis ◽  
Omri Malik ◽  
Allison Squires ◽  
Amit Meller ◽  
...  
Keyword(s):  
Transcription Factor ◽  
Optical Tweezers ◽  
Single Molecule ◽  
Gene Promoter ◽  
Regulatory Elements ◽  
Base Pairs ◽  
Dna Unzipping ◽  
Functional Sites ◽  
Asymmetric Modulation ◽  
Flanking Sequences

ABSTRACTMost functional transcription factor (TF) binding sites deviate from their “consensus” recognition motif, although their sites and flanking sequences are often conserved across species. Here, we used single-molecule DNA unzipping with optical tweezers to study how Egr-1, a TF harbouring 3 zinc fingers (ZF1,ZF2 and ZF3), is modulated by the sequence and context of its functional sites in the Lhb gene promoter. We find that both the core 9 base pairs bound to Egr-1 in each of the sites, and the base pairs flanking them, modulate the affinity and structure of the protein-DNA complex. The effect of the flanking sequences is asymmetric, with a stronger effect for the sequence flanking ZF3. Characterization of the dissociation time of Egr-1 revealed that a local, mechanical perturbation of the interactions of ZF3 destabilizes the complex more effectively than a perturbation of the ZF1 interactions. Our results reveal a novel role for ZF3 in the interaction of Egr-1 with other proteins and the DNA, providing insight on the regulation of Lhb and other genes by Egr-1. Moreover, our findings reveal the potential of small changes in DNA sequence to alter transcriptional regulation, and may shed light on the organization of regulatory elements at promoters.

scholarly journals Single-Molecule DNA Unzipping Reveals Asymmetric Modulation of the Transcription Factor EGR-1 by its Binding Site Sequence and Context

2018 ◽  
Vol 114 (3) ◽  
pp. 683a
Author(s):  
Hadeel Khamis ◽  
Sergei Rudnizky ◽  
Omri Malik ◽  
Allison Squires ◽  
Amit Meller ◽  
...  
Keyword(s):  
Transcription Factor ◽  
Binding Site ◽  
Single Molecule ◽  
Dna Unzipping ◽  
Asymmetric Modulation

scholarly journals Single-molecule DNA unzipping reveals asymmetric modulation of a transcription factor by its binding site sequence and context

2017 ◽  
Vol 46 (3) ◽  
pp. 1513-1524 ◽  
Author(s):  
Sergei Rudnizky ◽  
Hadeel Khamis ◽  
Omri Malik ◽  
Allison H Squires ◽  
Amit Meller ◽  
...  
Keyword(s):  
Transcription Factor ◽  
Binding Site ◽  
Single Molecule ◽  
Dna Unzipping ◽  
Asymmetric Modulation

Regulatory elements controlling pituitary-specific expression of the human prolactin gene

1990 ◽  
Vol 10 (9) ◽  
pp. 4690-4700
Author(s):  
B Peers ◽  
M L Voz ◽  
P Monget ◽  
M Mathy-Hartert ◽  
M Berwaer ◽  
...  
Keyword(s):  
Regulatory Elements ◽  
Distal Region ◽  
Dnase I ◽  
Proximal Region ◽  
Base Pairs ◽  
Specific Expression ◽  
Positive Region ◽  
Dnase I Footprinting ◽  
Human Prolactin ◽  
Flanking Sequences

We have performed transfection and DNase I footprinting experiments to investigate pituitary-specific expression of the human prolactin (hPRL) gene. When fused to the chloramphenicol acetyltransferase (CAT) reporter gene, 5,000 base pairs of the 5'-flanking sequences of the hPRL gene were able to drive high cat gene expression in prolactin-expressing GH3B6 cells specifically. Deletion analysis indicated that this pituitary-specific expression was controlled by three main positive regulatory regions. The first was located just upstream from the TATA box between coordinates -40 and -250 (proximal region). We have previously shown that three motifs of this region bind the pituitary-specific Pit-1 factor. The second positive region was located in the vicinity of coordinates -1300 to -1750 (distal region). DNase I footprinting assays revealed that eight DNA motifs of this distal region bound protein Pit-1 and that two other motifs were recognized by ubiquitous factors, one of which seems to belong to the AP-1 (jun) family. The third positive region was located further upstream, between -3500 and -5000 (superdistal region). This region appears to enhance transcription only in the presence of the distal region.

scholarly journals Profound Nanoscale Structural and Biomechanical Changes in DNA Helix upon Treatment with Anthracycline Drugs

2020 ◽  
Vol 21 (11) ◽  
pp. 4142
Author(s):  
Aleksandra Kaczorowska ◽  
Weronika Lamperska ◽  
Kaja Frączkowska ◽  
Jan Masajada ◽  
Sławomir Drobczyński ◽  
...  
Keyword(s):  
Optical Tweezers ◽  
Single Molecule ◽  
Regulatory Elements ◽  
Dna Molecules ◽  
Anthracycline Antibiotics ◽  
Single Molecule Level ◽  
Force Microscopy ◽  
Atomic Force ◽  
Microscopy Analysis ◽  
Dna Regulatory Elements

In our study, we describe the outcomes of the intercalation of different anthracycline antibiotics in double-stranded DNA at the nanoscale and single molecule level. Atomic force microscopy analysis revealed that intercalation results in significant elongation and thinning of dsDNA molecules. Additionally, using optical tweezers, we have shown that intercalation decreases the stiffness of DNA molecules, that results in greater susceptibility of dsDNA to break. Using DNA molecules with different GC/AT ratios, we checked whether anthracycline antibiotics show preference for GC-rich or AT-rich DNA fragments. We found that elongation, decrease in height and decrease in stiffness of dsDNA molecules was highest in GC-rich dsDNA, suggesting the preference of anthracycline antibiotics for GC pairs and GC-rich regions of DNA. This is important because such regions of genomes are enriched in DNA regulatory elements. By using three different anthracycline antibiotics, namely doxorubicin (DOX), epirubicin (EPI) and daunorubicin (DAU), we could compare their detrimental effects on DNA. Despite their analogical structure, anthracyclines differ in their effects on DNA molecules and GC-rich region preference. DOX had the strongest overall effect on the DNA topology, causing the largest elongation and decrease in height. On the other hand, EPI has the lowest preference for GC-rich dsDNA. Moreover, we demonstrated that the nanoscale perturbations in dsDNA topology are reflected by changes in the microscale properties of the cell, as even short exposition to doxorubicin resulted in an increase in nuclei stiffness, which can be due to aberration of the chromatin organization, upon intercalation of doxorubicin molecules.

scholarly journals Interaction between two different regulatory elements activates the murine alpha A-crystallin gene promoter in explanted lens epithelia.

1987 ◽  
Vol 7 (5) ◽  
pp. 1807-1814 ◽  
Author(s):  
A B Chepelinsky ◽  
B Sommer ◽  
J Piatigorsky
Keyword(s):  
Dna Sequences ◽  
Gene Promoter ◽  
Regulatory Elements ◽  
Regulatory Sequences ◽  
Base Pairs ◽  
Hybrid Gene ◽  
Promoter Sequences ◽  
Hybrid Genes ◽  
Cat Gene ◽  
Cat Expression

Previous experiments have indicated that 5' flanking DNA sequences (nucleotides-366 to +46) are capable of regulating the lens-specific transcription of the murine alpha A-crystallin gene. Here we have analyzed these 5' regulatory sequences by transfecting explanted embryonic chicken lens epithelia with different alpha A-crystallin-CAT (chloramphenicol acetyltransferase) hybrid genes (alpha A-crystallin promoter sequences fused to the bacterial CAT gene in the pSVO-CAT expression vector). The results indicated the presence of a proximal (-88 to +46) and a distal (-111 to -88) domain which must interact for promoter function. Deletion experiments showed that the sequence between -88 and -60 was essential for function of the proximal domain in the explanted epithelia. A synthetic oligonucleotide containing the sequence between -111 and -84 activated the proximal domain when placed in either orientation 57 base pairs upstream from position -88 of the alpha A-crystallin-CAT hybrid gene.

Interaction between two different regulatory elements activates the murine alpha A-crystallin gene promoter in explanted lens epithelia

1987 ◽  
Vol 7 (5) ◽  
pp. 1807-1814
Author(s):  
A B Chepelinsky ◽  
B Sommer ◽  
J Piatigorsky
Keyword(s):  
Dna Sequences ◽  
Gene Promoter ◽  
Regulatory Elements ◽  
Regulatory Sequences ◽  
Base Pairs ◽  
Hybrid Gene ◽  
Promoter Sequences ◽  
Hybrid Genes ◽  
Cat Gene ◽  
Cat Expression

Previous experiments have indicated that 5' flanking DNA sequences (nucleotides-366 to +46) are capable of regulating the lens-specific transcription of the murine alpha A-crystallin gene. Here we have analyzed these 5' regulatory sequences by transfecting explanted embryonic chicken lens epithelia with different alpha A-crystallin-CAT (chloramphenicol acetyltransferase) hybrid genes (alpha A-crystallin promoter sequences fused to the bacterial CAT gene in the pSVO-CAT expression vector). The results indicated the presence of a proximal (-88 to +46) and a distal (-111 to -88) domain which must interact for promoter function. Deletion experiments showed that the sequence between -88 and -60 was essential for function of the proximal domain in the explanted epithelia. A synthetic oligonucleotide containing the sequence between -111 and -84 activated the proximal domain when placed in either orientation 57 base pairs upstream from position -88 of the alpha A-crystallin-CAT hybrid gene.

scholarly journals Interferon-responsive regulatory elements in the promoter of the human 2',5'-oligo(A) synthetase gene.

1987 ◽  
Vol 7 (12) ◽  
pp. 4498-4504 ◽  
Author(s):  
P Benech ◽  
M Vigneron ◽  
D Peretz ◽  
M Revel ◽  
J Chebath
Keyword(s):  
Dna Sequences ◽  
Regulatory Region ◽  
Gene Promoter ◽  
Regulatory Elements ◽  
Base Pairs ◽  
Independent Manner ◽  
E Gene ◽  
Constitutive Enhancer ◽  
Chloramphenicol Acetyltransferase Gene ◽  
Simplex Virus

The interferon (IFN)-activated human 2',5'-oligo(A) synthetase E gene contains 11 RNA starts and lacks TATA and CAAT signals. DNA sequences around the promoter make the expression of the chloramphenicol acetyltransferase gene (CAT) inducible over 20-fold by IFN. A 72-base-pair segment (E-IRS) immediately upstream of the RNA starts was defined as being required for IFN-activated expression of the E-gene promoter-CAT constructs and acts in a position-independent manner. It also confers IFN-activated enhancement to the herpes simplex virus thymidine kinase promoter. On this promoter, the 5' part of the E-IRS functions as a constitutive enhancer, while the last 16 base pairs of the E-IRS is sufficient to give IFN-induced expression. On the E-gene promoter, the constitutive enhancer and the IFN-activated sequence are both needed but can be separated. In addition, promoter competition experiments indicate a third regulatory region which helps to repress expression of the E gene in uninduced cells.

Interferon-responsive regulatory elements in the promoter of the human 2',5'-oligo(A) synthetase gene

1987 ◽  
Vol 7 (12) ◽  
pp. 4498-4504
Author(s):  
P Benech ◽  
M Vigneron ◽  
D Peretz ◽  
M Revel ◽  
J Chebath
Keyword(s):  
Dna Sequences ◽  
Regulatory Region ◽  
Gene Promoter ◽  
Regulatory Elements ◽  
Base Pairs ◽  
Independent Manner ◽  
E Gene ◽  
Constitutive Enhancer ◽  
Chloramphenicol Acetyltransferase Gene ◽  
Simplex Virus

The interferon (IFN)-activated human 2',5'-oligo(A) synthetase E gene contains 11 RNA starts and lacks TATA and CAAT signals. DNA sequences around the promoter make the expression of the chloramphenicol acetyltransferase gene (CAT) inducible over 20-fold by IFN. A 72-base-pair segment (E-IRS) immediately upstream of the RNA starts was defined as being required for IFN-activated expression of the E-gene promoter-CAT constructs and acts in a position-independent manner. It also confers IFN-activated enhancement to the herpes simplex virus thymidine kinase promoter. On this promoter, the 5' part of the E-IRS functions as a constitutive enhancer, while the last 16 base pairs of the E-IRS is sufficient to give IFN-induced expression. On the E-gene promoter, the constitutive enhancer and the IFN-activated sequence are both needed but can be separated. In addition, promoter competition experiments indicate a third regulatory region which helps to repress expression of the E gene in uninduced cells.

scholarly journals Regulatory elements controlling pituitary-specific expression of the human prolactin gene.

1990 ◽  
Vol 10 (9) ◽  
pp. 4690-4700 ◽  
Author(s):  
B Peers ◽  
M L Voz ◽  
P Monget ◽  
M Mathy-Hartert ◽  
M Berwaer ◽  
...  
Keyword(s):  
Regulatory Elements ◽  
Distal Region ◽  
Dnase I ◽  
Proximal Region ◽  
Base Pairs ◽  
Specific Expression ◽  
Positive Region ◽  
Dnase I Footprinting ◽  
Human Prolactin ◽  
Flanking Sequences

We have performed transfection and DNase I footprinting experiments to investigate pituitary-specific expression of the human prolactin (hPRL) gene. When fused to the chloramphenicol acetyltransferase (CAT) reporter gene, 5,000 base pairs of the 5'-flanking sequences of the hPRL gene were able to drive high cat gene expression in prolactin-expressing GH3B6 cells specifically. Deletion analysis indicated that this pituitary-specific expression was controlled by three main positive regulatory regions. The first was located just upstream from the TATA box between coordinates -40 and -250 (proximal region). We have previously shown that three motifs of this region bind the pituitary-specific Pit-1 factor. The second positive region was located in the vicinity of coordinates -1300 to -1750 (distal region). DNase I footprinting assays revealed that eight DNA motifs of this distal region bound protein Pit-1 and that two other motifs were recognized by ubiquitous factors, one of which seems to belong to the AP-1 (jun) family. The third positive region was located further upstream, between -3500 and -5000 (superdistal region). This region appears to enhance transcription only in the presence of the distal region.

Lack of excision of introns from primary transcripts of certain chicken vitellogenin II minigenes

1994 ◽  
Vol 72 (3-4) ◽  
pp. 84-94 ◽  
Author(s):  
Cynthia L. Brazolot Millan ◽  
Ann M. Verrinder Gibbins
Keyword(s):  
Cultured Cells ◽  
Primary Cultures ◽  
Regulatory Elements ◽  
Northern Blotting ◽  
Hormonal Control ◽  
Flanking Sequence ◽  
Base Pairs ◽  
Flanking Sequences ◽  
17Β Estradiol ◽  
Control Of Expression

Two truncated versions of the chicken vitellogenin II gene VTGII were designed and constructed to include all known essential regulatory elements of the complete gene. Both pCB123 and pCB123/4 contain 945 base pairs (bp) of the 5′-flanking sequence, introns and exons 1–3, and a subset of the remaining 32 exons of VTGII, inserted into a pBluescript SK (+/−)™ vector. pCB123/4 contains 752 bp of legitimate VTGII 3′-flanking sequences, while the 3′ end of pCB123 terminates at the VTGII cDNA end, followed by AT-tailing and vector sequences carried over during cloning. Expression of these plasmids was tested following their lipofection into primary cultures of chicken hepatocytes established from day 14 embryos. Poly(A)+ RNA derived from pCB123 was detected by Northern blotting and reverse transcription – polyacrylamide chain reaction. No evidence was observed for appropriate hormonal control of expression, despite the presence of 17β-estradiol or colipofection with the estrogen receptor clone pHEO. VTGII sequences at the 3′ end of pCB123/4 led to an apparent destabilization of the RNA transcript. Unexpectedly, unprocessed pCB123 transcripts of varying lengths accumulated in the cells. These experiments constitute the first reported attempts to express authentic VTGII coding sequences in cultured cells and highlight the dilemma of which introns to include in a minigene. Despite reports that some minigenes are expressed more efficiently if one or two introns are included, other minigenes may be expressed more effectively in the absence of introns. In the case of a complex gene with many introns, such as VTGII, there may be a preferential order in which introns are removed from the primary construct. The truncation of complex genes to give functional minigenes for transgenic studies may require considerable experimentation.Key words: chicken, vitellogenin, minigenes, introns, splicing.

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