Project description:The pioneer transcription factor Pax7 contains two DNA binding domains (DBD), a paired and a homeo domain. Previous work on Pax7 and the related Pax3 showed that each DBD binds a cognate DNA sequence, thus defining two targets of binding and possibly modalities of action. Genomic targets of Pax7 pioneer action leading to chromatin opening are enriched for composite DNA target sites containing juxtaposed sites for both paired and homeo domains. The present work investigated the implication of the DBDs in pioneer action. We show that the composite sequence is a higher affinity binding site and that efficient binding to this site involves both DBDs of the same Pax7 molecule. This binding is not sensitive to cytosine methylation of the DNA sites consistent with pioneer action within nucleosomal heterochromatin. Introduction of single amino acid mutations in either paired or homeo domain that impair binding to cognate DNA sequences showed that both DBDs must be intact for pioneer action. In contrast, only the paired domain is required for low affinity binding of heterochromatin sites. Thus, Pax7 pioneer action on heterochromatin requires unique protein:DNA interactions that are more complex compared to its simpler DNA binding modalities at accessible enhancer target sites.
Project description:The pioneer transcription factor Pax7 contains two DNA binding domains (DBD), a paired and a homeo domain. Previous work on Pax7 and the related Pax3 showed that each DBD binds a cognate DNA sequence, thus defining two targets of binding and possibly modalities of action. Genomic targets of Pax7 pioneer action leading to chromatin opening are enriched for composite DNA target sites containing juxtaposed sites for both paired and homeo domains. The present work investigated the implication of the DBDs in pioneer action. We show that the composite sequence is a higher affinity binding site and that efficient binding to this site involves both DBDs of the same Pax7 molecule. This binding is not sensitive to cytosine methylation of the DNA sites consistent with pioneer action within nucleosomal heterochromatin. Introduction of single amino acid mutations in either paired or homeo domain that impair binding to cognate DNA sequences showed that both DBDs must be intact for pioneer action. In contrast, only the paired domain is required for low affinity binding of heterochromatin sites. Thus, Pax7 pioneer action on heterochromatin requires unique protein:DNA interactions that are more complex compared to its simpler DNA binding modalities at accessible enhancer target sites.
Project description:The pioneer transcription factor Pax7 contains two DNA binding domains (DBD), a paired and a homeo domain. Previous work on Pax7 and the related Pax3 showed that each DBD binds a cognate DNA sequence, thus defining two targets of binding and possibly modalities of action. Genomic targets of Pax7 pioneer action leading to chromatin opening are enriched for composite DNA target sites containing juxtaposed sites for both paired and homeo domains. The present work investigated the implication of the DBDs in pioneer action. We show that the composite sequence is a higher affinity binding site and that efficient binding to this site involves both DBDs of the same Pax7 molecule. This binding is not sensitive to cytosine methylation of the DNA sites consistent with pioneer action within nucleosomal heterochromatin. Introduction of single amino acid mutations in either paired or homeo domain that impair binding to cognate DNA sequences showed that both DBDs must be intact for pioneer action. In contrast, only the paired domain is required for low affinity binding of heterochromatin sites. Thus, Pax7 pioneer action on heterochromatin requires unique protein:DNA interactions that are more complex compared to its simpler DNA binding modalities at accessible enhancer target sites.
Project description:Pioneer transcription factors direct cell differentiation by deploying new enhancer repertoires through their unique ability to target and initiate remodelling of closed chromatin. The initial steps of their action remain undefined although pioneers were shown to interact with nucleosomal target DNA and with some chromatin remodelling complexes. We now define the sequence of events that enable the pioneer Pax7 with its unique abilities. Chromatin condensation exerted by linker histone H1 is the first constraint on Pax7 recruitment, and this establishes the initial speed of chromatin remodelling. The first step of pioneer action involves recruitment of the KDM1A (Lsd1) H3K9me2 demethylase for removal of this repressive mark, as well as recruitment of the MLL complex for deposition of the activating H3K4me1 mark. Further progression of pioneer action requires passage through cell division, and this involves dissociation of pioneer targets from perinuclear lamin B. Only then, the SWI/SNF remodeling complex and the coactivator p300 are recruited, leading to nucleosome displacement and enhancer activation. Thus, the unique features of pioneer actions are those occurring in the lamin-associated compartment of the nucleus. This model is consistent with prior work that showed a dependence on cell division for establishment of new cell fates.
Project description:Pioneer transcription factors direct cell differentiation by deploying new enhancer repertoires through their unique ability to target and initiate remodelling of closed chromatin. The initial steps of their action remain undefined although pioneers were shown to interact with nucleosomal target DNA and with some chromatin remodelling complexes. We now define the sequence of events that enable the pioneer Pax7 with its unique abilities. Chromatin condensation exerted by linker histone H1 is the first constraint on Pax7 recruitment, and this establishes the initial speed of chromatin remodelling. The first step of pioneer action involves recruitment of the KDM1A (Lsd1) H3K9me2 demethylase for removal of this repressive mark, as well as recruitment of the MLL complex for deposition of the activating H3K4me1 mark. Further progression of pioneer action requires passage through cell division, and this involves dissociation of pioneer targets from perinuclear lamin B. Only then, the SWI/SNF remodeling complex and the coactivator p300 are recruited, leading to nucleosome displacement and enhancer activation. Thus, the unique features of pioneer actions are those occurring in the lamin-associated compartment of the nucleus. This model is consistent with prior work that showed a dependence on cell division for establishment of new cell fates.