scholarly journals Second-generation method for analysis of chromatin binding using formaldehyde crosslinking kinetics

2017 ◽  
Author(s):  
Hussain Zaidi ◽  
Elizabeth A. Hoffman ◽  
Savera J. Shetty ◽  
Stefan Bekiranov ◽  
David T. Auble

ABSTRACTFormaldehyde crosslinking underpins many of the most commonly used experimental approaches in the chromatin field, especially in capturing site-specific protein-DNA interactions. Extending such assays to assess the stability and binding kinetics of protein-DNA interactions is more challenging, requiring absolute measurements with a relatively high degree of physical precision. We previously described an experimental framework called CLK, which uses time-dependent formaldehyde crosslinking data to extract chromatin binding kinetic parameters. Many aspects of formaldehyde behavior in cells are unknown or undocumented, however, and could potentially impact analyses of CLK data. Here we report biochemical results that better define the properties of formaldehyde crosslinking in budding yeast cells. These results have the potential to inform interpretations of ‘standard’ chromatin assays including chromatin immunoprecipitation, and the chemical complexity we uncovered resulted in the development of an improved method for measuring binding kinetics using the CLK approach. Optimum conditions included an increased formaldehyde concentration and more robust glycine quench conditions. Notably, we find that formaldehyde crosslinking rates can vary dramatically for different protein-DNA interactions in vivo. Some interactions were crosslinked much faster than the time scale for macromolecular interaction, making them suitable for kinetic analysis. For other interactions, we find the crosslinking reaction occurred on the same time scale or slower than binding dynamics; for these it was in some cases possible to compute the in vivo equilibrium-binding constant but not on- and off-rates for binding. Selected TATA-binding protein-promoter interactions displayed dynamic behavior on the minute to several minutes time scale.

1991 ◽  
Vol 11 (1) ◽  
pp. 154-165 ◽  
Author(s):  
L Densmore ◽  
W E Payne ◽  
M Fitzgerald-Hayes

We have used in vivo genomic footprinting to investigate the protein-DNA interactions within the conserved DNA elements (CDEI, CDEII, and CDEIII) in the centromere from chromosome III of the yeast Saccharomyces cerevisiae. The in vivo footprint pattern obtained from wild-type cells shows that some guanines within the centromere DNA are protected from methylation by dimethyl sulfate. These results are consistent with studies demonstrating that yeast cells contain sequence-specific centromere DNA-binding proteins. Our in vivo experiments on chromosomes with mutant centromeres show that some mutations which affect chromosome segregation also alter the footprint pattern caused by proteins bound to the centromere DNA. The results of this study provide the first fine-structure map of proteins bound to centromere DNA in living yeast cells and suggest a direct correlation between these protein-DNA interactions and centromere function.


1991 ◽  
Vol 11 (1) ◽  
pp. 154-165
Author(s):  
L Densmore ◽  
W E Payne ◽  
M Fitzgerald-Hayes

We have used in vivo genomic footprinting to investigate the protein-DNA interactions within the conserved DNA elements (CDEI, CDEII, and CDEIII) in the centromere from chromosome III of the yeast Saccharomyces cerevisiae. The in vivo footprint pattern obtained from wild-type cells shows that some guanines within the centromere DNA are protected from methylation by dimethyl sulfate. These results are consistent with studies demonstrating that yeast cells contain sequence-specific centromere DNA-binding proteins. Our in vivo experiments on chromosomes with mutant centromeres show that some mutations which affect chromosome segregation also alter the footprint pattern caused by proteins bound to the centromere DNA. The results of this study provide the first fine-structure map of proteins bound to centromere DNA in living yeast cells and suggest a direct correlation between these protein-DNA interactions and centromere function.


1996 ◽  
Vol 93 (4) ◽  
pp. 1498-1503 ◽  
Author(s):  
D. S. Dimitrova ◽  
M. Giacca ◽  
F. Demarchi ◽  
G. Biamonti ◽  
S. Riva ◽  
...  

1998 ◽  
Vol 136 (2) ◽  
pp. 109-118 ◽  
Author(s):  
Masato Ikeda ◽  
Remco A Spanjaard ◽  
Elizabeth W Noordhoek ◽  
Akio Kawaguchi ◽  
Toshimasa Onaya ◽  
...  

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