GSE160468
Human SMARCA5 Is Continuously Required to Maintain Nucleosome Spacing [CUT&RUN]
Genetic models suggested that SMARCA5 was required for DNA templated events including transcription, DNA replication and DNA repair.  We engineered a degron tag into the endogenous alleles of SMARCA5, a catalytic component of the imitation switch (ISWI) complexes, in three different human cell lines to define the effects of rapid degradation of this key regulator.  Degradation of SMARCA5 was associated with a rapid increase in global nucleosome repeat length, which may allow greater chromatin compaction. However, there were few changes in nascent transcription within the first 6hr of degradation. Nevertheless, we demonstrated a requirement for SMARCA5 to control nucleosome repeat length at G1/S and during the S phase. SMARCA5 co-localized with CTCF and H2A.Z, and we found a rapid loss of CTCF DNA binding and disruption of nucleosomal phasing around CTCF binding sites. This spatiotemporal analysis indicates that SMARCA5 is continuously required for maintaining nucleosomal spacing. Overall design: CUT&RUN was performed on biological replicates. For anti-Flag CUT&RUN, untreated cells expressing endogenous expressed SMARCA5-FKBP12F36V-1xFLAG on both alleles was used as postivie control versus SMARCA5- FKBP12F36V-1xFLAG cells  treated for 18hrs with 500nM dTAG-47. For anti-CTCF CUT&RUN, untreated cells expressing endogenous expressed SMARCA5-FKBP12F36V-1xFLAG on both alleles was used as postivie control versus  SMARCA5- FKBP12F36V-1xFLAG cells  treated for 2hr, 6hr, and 24hr with 500nM dTAG-47. For anti-H2AZ and anti-H3K27me3, in untreated Kasumi-1 cells expressing endogenous expressed SMARCA5-FKBP12F36V-1xFLAG. For anti-RUNX1 and anti-ETOZnf CUT&RUN, untreated Kasumi-1 cells expressing endogenous expressed SMARCA5-FKBP12F36V-1xFLAG on both alleles was used as postivie control versus Kasumi-1 SMARCA5- FKBP12F36V-1xFLAG cells  treated for 24hr with 500nM dTAG-47.