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. 2010 Dec 8;5(12):e15082.
doi: 10.1371/journal.pone.0015082.

ZNF274 recruits the histone methyltransferase SETDB1 to the 3' ends of ZNF genes

Seth Frietze  1 , Henriette O'Geen, Kimberly R Blahnik, Victor X Jin, Peggy J Farnham
Affiliations

ZNF274 recruits the histone methyltransferase SETDB1 to the 3' ends of ZNF genes

Seth Frietze et al. PLoS One. .

Abstract

Only a small percentage of human transcription factors (e.g. those associated with a specific differentiation program) are expressed in a given cell type. Thus, cell fate is mainly determined by cell type-specific silencing of transcription factors that drive different cellular lineages. Several histone modifications have been associated with gene silencing, including H3K27me3 and H3K9me3. We have previously shown that genes for the two largest classes of mammalian transcription factors are marked by distinct histone modifications; homeobox genes are marked by H3K27me3 and zinc finger genes are marked by H3K9me3. Several histone methyltransferases (e.g. G9a and SETDB1) may be involved in mediating the H3K9me3 silencing mark. We have used ChIP-chip and ChIP-seq to demonstrate that SETDB1, but not G9a, is associated with regions of the genome enriched for H3K9me3. One current model is that SETDB1 is recruited to specific genomic locations via interaction with the corepressor TRIM28 (KAP1), which is in turn recruited to the genome via interaction with zinc finger transcription factors that contain a Kruppel-associated box (KRAB) domain. However, specific KRAB-ZNFs that recruit TRIM28 (KAP1) and SETDB1 to the genome have not been identified. We now show that ZNF274 (a KRAB-ZNF that contains 5 C2H2 zinc finger domains), can interact with KAP1 both in vivo and in vitro and, using ChIP-seq, we show that ZNF274 binding sites co-localize with SETDB1, KAP1, and H3K9me3 at the 3' ends of zinc finger genes. Knockdown of ZNF274 with siRNAs reduced the levels of KAP1 and SETDB1 recruitment to the binding sites. These studies provide the first identification of a KRAB domain-containing ZNF that is involved in recruitment of the KAP1 and SETDB1 to specific regions of the human genome.

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Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. SETDB1, but not G9a, colocalizes with H3K9me3 on chromosome 19.
(A) ChIP-chip binding patterns of KAP1, SETDB1, H3me3K9 and G9a are compared at a zinc finger gene cluster on chromosome 19. The log2 (ratio) values reflecting the ChIP enrichments are plotted on the y axes and chromosomal coordinates are shown on the x axis. (B) Peaks were called using the ChIP-chip array data for SETDB1, G9a, and KAP1 using the Mayacamas peak calling program. The total number of peaks for each data set are shown; the intersection indicates the number of SETDB1, KAP1, and G9a peaks that overlap with the H3K9me3 peaks.
Figure 2
Figure 2. 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.
Figure 3
Figure 3. ZNF274 binds to C2H2 zinc finger genes.
ChIP-seq binding patterns of ZNF274 from GM12878, HeLa, HepG2, and K562 cells are compared to the location of all C2H2 zinc finger genes 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.
Figure 4
Figure 4. 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.
Figure 5
Figure 5. 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.
Figure 6
Figure 6. ZNF274 binds to a gene containing multiple zinc finger domains.
The number of finger domains in the C2H2 genes bound by ZNF274 are shown (solid line), along with the number of finger domains found in all 712 C2H2 zinc finger genes encoded in the hg18 human genome build (dotted line).
Figure 7
Figure 7. A model for SETDB1 recruitment.
Shown is a schematic illustrating the current model for KAP1-mediated transcriptional repression. KAP1 is thought to be brought to the genome by interaction with a KRAB-ZNF, which binds site-specifically to DNA. KAP1 in turn is thought to recruit the histone methyltransferase SETDB1, which then specifically mediates trimethylation of lysine 9 of histone H3 near the KRAB-ZNF binding sites.
Figure 8
Figure 8. 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.
Figure 9
Figure 9. The H3K9me3 methylome.
A) Schematic showing that the histone methytransferase SETDB1 is responsible for a large proportion, but not all, of the H3K9me3 sites in K562 cells and that ZNF274 is responsible for recruiting SETDB1 to some, but not all, of the SETDB1 binding sites. B) The distance of all H3K9me3 peaks found in the top 10% of that dataset is shown relative to the center of the nearest SETDB1 peak. C) Shown is the binding pattern of SETDB1 and H3K9me3 for a region of chromosome 21, indicating that there are large regions of the genome covered by H3K9me3 but not by SETDB1.
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