RESUMO
Catalytic activity of the imitation switch (ISWI) family of remodelers is critical for nucleosomal organization and DNA binding of certain transcription factors, including the insulator protein CTCF. Here we define the contribution of individual subcomplexes by deriving a panel of isogenic mouse stem cell lines, each lacking one of six ISWI accessory subunits. Individual deletions of subunits of either CERF, RSF, ACF, WICH or NoRC subcomplexes only moderately affect the chromatin landscape, while removal of the NURF-specific subunit BPTF leads to a strong reduction in chromatin accessibility and SNF2H ATPase localization around CTCF sites. This affects adjacent nucleosome occupancy and CTCF binding. At a group of sites with reduced chromatin accessibility, CTCF binding persists but cohesin occupancy is reduced, resulting in decreased insulation. These results suggest that CTCF binding can be separated from its function as an insulator in nuclear organization and identify a specific role for NURF in mediating SNF2H localization and chromatin opening at bound CTCF sites.
Assuntos
Adenosina Trifosfatases , Fator de Ligação a CCCTC , Cromatina , Proteínas Repressoras , Fatores de Transcrição , Fator de Ligação a CCCTC/metabolismo , Fator de Ligação a CCCTC/genética , Animais , Camundongos , Fatores de Transcrição/metabolismo , Fatores de Transcrição/genética , Proteínas Repressoras/metabolismo , Proteínas Repressoras/genética , Cromatina/metabolismo , Cromatina/genética , Adenosina Trifosfatases/metabolismo , Adenosina Trifosfatases/genética , Ligação Proteica , Linhagem Celular , Proteínas Cromossômicas não Histona/metabolismo , Proteínas Cromossômicas não Histona/genética , Nucleossomos/metabolismo , Nucleossomos/genética , Subunidades Proteicas/metabolismo , Subunidades Proteicas/genética , Proteínas de Ciclo Celular/metabolismo , Proteínas de Ciclo Celular/genética , Sítios de LigaçãoRESUMO
Chromatin accessibility is a hallmark of regulatory regions, entails transcription factor (TF) binding and requires nucleosomal reorganization. However, it remains unclear how dynamic this process is. In the present study, we use small-molecule inhibition of the catalytic subunit of the mouse SWI/SNF remodeler complex to show that accessibility and reduced nucleosome presence at TF-binding sites rely on persistent activity of nucleosome remodelers. Within minutes of remodeler inhibition, accessibility and TF binding decrease. Although this is irrespective of TF function, we show that the activating TF OCT4 (POU5F1) exhibits a faster response than the repressive TF REST. Accessibility, nucleosome depletion and gene expression are rapidly restored on inhibitor removal, suggesting that accessible chromatin is regenerated continuously and in a largely cell-autonomous fashion. We postulate that TF binding to chromatin and remodeler-mediated nucleosomal removal do not represent a stable situation, but instead accessible chromatin reflects an average of a dynamic process under continued renewal.
