Initially, several candidate TFs were tested as potential models in mES cells, where variable expression and the expression of mutant versions of the TFs were tolerated without cellular lethality. These were subjected to antibody-free tagging chromatin immunoprecipitation sequencing (ChIPseq) pipelines for genomic binding analysis. In addition, recombinant versions of these TFs were expressed to carry out in vitro experiments. Comparative analysis of genomic binding in vivo and competitive in vitro binding measurements using purified recombinant proteins provided valuable insights to the extent to which the DNA sequence motifs alone can explain the binding of TFs on chromatin.
By utilizing these sophisticated binding profiles, analysis of chromatin marks that are enriched at binding sites where DNA sequence in insufficient to explain enrichment profiles revealed some correlations. To move beyond correlation alone, knockout models were developed to address TF binding in the absence of these chromatin marks. These included epigenetic marks that are generally regarded as active and repressive from the literature, respectively.
An additional approach was taken to examine the extent to which chromatin modification enables genomic binding of these model TFs. As many TFs associate with protein complexes that are able to enzymatically modify chromatin (e.g. histone methyltransferases/demethylases etc..) it is possible that these protein interactions enable and/or maintain genomic binding patterns. To directly assess this, expression libraries were generated to examine the extent to which direct fusion of catalytic protein domains would alter the binding profiles of model TFs.
Taken together, these experiments are likely to yield generalizable principle of TF-chromatin interactions, which will be submitted as soon as the project is completed along with any computational and/or technical resources generated that may be useful to those studying TF/chromatin biology.
