Periodic Reporting for period 1 - TF-bind Determinants (Genomic binding of transcription factors as a function of DNA affinity and chromatin.)
Periodo di rendicontazione: 2017-04-01 al 2019-03-31
The development of complex multicellular organisms requires varied expression patters (i.e. gene activation and silencing) in different cell types. Transcription factors (TFs) are proteins able to interpret DNA sequence by binding to specific motifs and ultimately enable the correct recruitment levels of transcriptional machinery at gene promoters. However, chromatin proteins such as nucleosomes represent a barrier for TF binding to genomic DNA in vivo. Here, we used sophisticated biochemical and genomic approaches to investigate to what extent TF and chromatin properties affect genomic binding in mouse embryonic stem (mES) cells.
As transcription factors are drivers of gene expression changes during differentiation and in disease settings, increased understanding of TF-sensitivities to chromatin will be of major importance to the development of targeted genomic therapeutics. These are potentially useful to optimise design parameters for synthetic transcriptional activators and repressors and are especially relevant to addressing the risk of off-target effects, which limit the applicability of these genomic therapeutics.
The overall objectives of the project were to gain a comprehensive understanding of the binding determinants of a model TF, as a function of the properties of the TF (i.e. affinity for DNA, expression level etc..) and chromatin marks enriched over potential binding motifs across the mammalian genome. By utilizing high throughput in vivo and in vitro approaches, we were able to assess binding of TFs as a function of chromatin, with the inclusive aim of establishing generalizable principle of TF-chromatin interactions.
As transcription factors are drivers of gene expression changes during differentiation and in disease settings, increased understanding of TF-sensitivities to chromatin will be of major importance to the development of targeted genomic therapeutics. These are potentially useful to optimise design parameters for synthetic transcriptional activators and repressors and are especially relevant to addressing the risk of off-target effects, which limit the applicability of these genomic therapeutics.
The overall objectives of the project were to gain a comprehensive understanding of the binding determinants of a model TF, as a function of the properties of the TF (i.e. affinity for DNA, expression level etc..) and chromatin marks enriched over potential binding motifs across the mammalian genome. By utilizing high throughput in vivo and in vitro approaches, we were able to assess binding of TFs as a function of chromatin, with the inclusive aim of establishing generalizable principle of TF-chromatin interactions.
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.
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.
In general, the presence or absence of a DNA motif is insufficient to predict binding of a TF and in fact, the vast majority of TFs only bind to a very small number (~1%-5%) of their cognate motifs genome-wide. Therefore, additional genomic and chromatin features encode TF binding features. Many such potential mechanisms have emerged, such as accessible chromatin or local sequence features (e.g. surrounding G+C nucleotide content), however, the majority of these finding are correlative in nature and have yet to be comprehensively tested.
To move beyond such correlations, we have manipulated both TF and chromatin properties and assessed binding of model TFs. These have yielded generalizable rules of genomic binding. In particular, binding can be assessed in genomic regions that were previously inaccessible as measured by chromatin accessibility assays (i.e. ATACseq) and is of great interest as it demonstrates the initial engagement of genomic DNA, i.e. TF pioneering activity. Such binding is thought to be at the top of local hierarchical relationships of regulatory regions by cooperating TFs.
These principles of cis-regulatory logic are expected to be of high impact to a broad audience familiar with TF biology.
To move beyond such correlations, we have manipulated both TF and chromatin properties and assessed binding of model TFs. These have yielded generalizable rules of genomic binding. In particular, binding can be assessed in genomic regions that were previously inaccessible as measured by chromatin accessibility assays (i.e. ATACseq) and is of great interest as it demonstrates the initial engagement of genomic DNA, i.e. TF pioneering activity. Such binding is thought to be at the top of local hierarchical relationships of regulatory regions by cooperating TFs.
These principles of cis-regulatory logic are expected to be of high impact to a broad audience familiar with TF biology.