Fig 1: Depletion of ZNF274 inhibits KAP1 recruitment and H3K9 trimethylation at target genes.A) Control siRNAs or specific siRNAs targeting ZNF274 were transfected into HelaS3 cells and western blot analysis of ZNF274 was performed; KAP1 and Actin antibodies were used as loading control. B) Shown are the binding patterns of KAP1, SETDB1, ZNF274, and H3K9me3 on a ZNF274 target gene (ZNF554) and on a zinc finger gene not bound by ZNF274 (ZNF556). C) Control siRNAs or specific siRNAs targeting ZNF274 were transfected into HelaS3 cells and ZNF274, KAP1, SETDB1 and H3K9me3 chromatin immunoprecipitation (ChIP) analyses were performed. The fold change in occupancy of ZNF274, KAP1, SETDB1, and H3K9me3 at the ZNF274 positive targets (ZNF180 and ZNF554) and negative sites (ZNF555 and ZNF556) for each factor was measured as the change in the mean enrichment for that factor from experiments with siRNA against ZNF274 over control siRNAs.
Fig 2: ZNF274 binds specifically to ZNF genes.A) The number of ZNF274 binding sites in K562 cells is shown for each chromosome (solid line). Also shown is the number of C2H2 ZNF genes encoded on each chromosome (dotted line). B) The ZNF274 target genes in K562 cells were analyzed using the DAVID gene ontology program. Shown are the enriched terms and P-value of enrichments of the target genes. C) Overlaps of the peaks sets of ZNF274, SETDB1, and KAP1 are shown; 85% of the 337 ZNF274 peaks, 52% of the 3237 SETDB1 peaks, and 54% of the 2027 KAP1 peaks overlap with H3K9me3. D) The ChIP-seq binding patterns of ZNF274, KAP1, SETDB1, and H3me3K9 are compared on chromosome 19. The number of tags reflecting the ChIP enrichments are plotted on the y axes and chromosomal coordinates are shown on the x axis.
Fig 3: Targeting dSaCas9-repressors to DUX4 increases chromatin repression at the locusChIP assays were performed using FSHD myocytes transduced with each dSaCas9-epigenetic regulator + individual optimal sgRNA targeting the DUX4 promoter or exon 1. (A–C) Chromatin was immunoprecipitated using antibodies specific for (A) HP1α or (B) KAP1 and analyzed by qPCR using primers to the promoter (Pro), transcription start site (TSS), or exon 3 of DUX4 or to MYOD1, or (C) antibodies specific for the elongating form of RNA Pol II (phospho-serine 2) and analyzed by qPCR using primers specific to DUX4 exon1/intron1 on chromosome 4 or to MYOD1. MYOD1 was used as a negative control for an active gene that should not be affected by CRISPRi targeted to DUX4. Locations of DUX4 primers are shown in Figure 1C. Data are presented as fold enrichment of the target region by each specific antibody normalized to α-histone H3, with enrichment for mock-infected cells set to 1. For all panels, each bar represents the average of at least three independent ChIP experiments. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001 are from comparing to enrichment at MYOD1.
Fig 4: Co-occupancy of ZNF274, KAP1 and SETDB1 at a ZNF 3′ end in vivo.A) ChIP-qPCR confirmation of a set of ZNF274 targets. Quantitative real-time PCR (qPCR) for 10 target regions identified by ChIP-seq and three negative regions (GAPDH, CDH1, and ZNF44) was performed. The fold enrichment of each site was calculated as 2 to the power of the cycle threshold (cT) difference between input chromatin and ChIP samples. The results in the graph are the mean of three independent replicates with standard deviation. Primers used in these experiments can be found in Supplementary Table S5. B) Sequential chromatin immunoprecipitation of ZNF274 and KAP1 in K562 cells. ZNF274 or KAP1 ChIP samples were sequentially immunoprecipitated using the indicated antibodies. The samples were analyzed by PCR and agarose gel electrophoresis with ethidium bromide staining using specific primer sets to ZNF180, a zinc finger gene bound by ZNF274; ZNF555, a zinc finger gene not bound by ZNF274, but bound by KAP1; STX16, and a non-zinc finger gene bound by neither ZNF274 nor KAP1.
Fig 5: A KRAB-domain containing protein ZNF274 interacts with the KAP1 corepressor both in vitro and in vivo.A) Schematics of ZNF263 and ZNF274 proteins are shown, indicating the position of the KRAB domains (potential KAP1-interacting domains), the SCAN domains, and the zinc fingers (DNA binding domains). B) The KRAB domains of ZNF274, ZNF263, and ZNF10 are compared at the amino acid level. C) Purified KRAB-GST fusion proteins are shown in the bottom panel. In the top panel, nuclear proteins from K562 cells that bound to the indicated KRAB-GST fusion proteins were analyzed using an antibody to KAP1. D) Co-immunoprecipitation of endogeneous ZNF274 with KAP1 and SETDB1, but not with G9a. Control IgG, KAP1, ZNF274, SETDB1, and G9a antibodies were used in immunoprecipitation reactions using K562 cell extracts and were analyzed by western blotting using the indicated antibodies.
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