Dataset Information

Directing an artificial zinc finger protein to new targets by fusion to a non-DNA-binding domain.

ABSTRACT: Transcription factors are often regarded as having two separable components: a DNA-binding domain (DBD) and a functional domain (FD), with the DBD thought to determine target gene recognition. While this holds true for DNA bindingin vitro, it appears thatin vivoFDs can also influence genomic targeting. We fused the FD from the well-characterized transcription factor Krüppel-like Factor 3 (KLF3) to an artificial zinc finger (AZF) protein originally designed to target the Vascular Endothelial Growth Factor-A (VEGF-A) gene promoter. We compared genome-wide occupancy of the KLF3FD-AZF fusion to that observed with AZF. AZF bound to theVEGF-Apromoter as predicted, but was also found to occupy approximately 25,000 other sites, a large number of which contained the expected AZF recognition sequence, GCTGGGGGC. Interestingly, addition of the KLF3 FD re-distributes the fusion protein to new sites, with total DNA occupancy detected at around 50,000 sites. A portion of these sites correspond to known KLF3-bound regions, while others contained sequences similar but not identical to the expected AZF recognition sequence. These results show that FDs can influence and may be useful in directing AZF DNA-binding proteins to specific targets and provide insights into how natural transcription factors operate.

SUBMITTER: Lim WF

PROVIDER: S-EPMC4838343 | biostudies-literature | 2016 Apr

REPOSITORIES: biostudies-literature

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Directing an artificial zinc finger protein to new targets by fusion to a non-DNA-binding domain.

Lim Wooi F WF Burdach Jon J Funnell Alister P W AP Pearson Richard C M RC Quinlan Kate G R KG Crossley Merlin M

Nucleic acids research 20151215 7

Transcription factors are often regarded as having two separable components: a DNA-binding domain (DBD) and a functional domain (FD), with the DBD thought to determine target gene recognition. While this holds true for DNA bindingin vitro, it appears thatin vivoFDs can also influence genomic targeting. We fused the FD from the well-characterized transcription factor Krüppel-like Factor 3 (KLF3) to an artificial zinc finger (AZF) protein originally designed to target the Vascular Endothelial Grow ...[more]

PMID: 26673701

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Project description:Transcription factors are often regarded as having two distinct components: a DNA binding domain (DBD) to allow binding to the regulatory region of a target gene and a trans-acting functional domain (FD) to modulate gene expression. Recently, it is becoming clear that the DBDs of transcription factors alone are incapable of providing sufficient specificity to account for the highly complex genomic structures in eukaryotes. Other regions, outside of the DBD, may play a role in transcription factor DNA binding specificity in vivo. In order to test this hypothesis, we examined a model zinc finger transcription factor, KrÃ¼ppel-like factor 3 (KLF3) with a FD that recruits partner proteins for its repressive activity and a three zinc finger DBD. Previously, we showed that deletion of the entire KLF3 FD reduced DNA binding across the genome. This implied the importance of the FD for in vivo DNA binding specificity. In the current study, we fused the KLF3 FD onto an unrelated, but well characterised Artificial Zinc Finger (AZF) targeting a standard target gene, VEGF-A. We compared the genomic DNA binding profile of this chimeric KLF3 construct, termed KLF3FD-AZF, to that of the AZF alone. Chromatin Immunoprecipitation followed by next generation sequencing was used to assess genomic wide DNA-binding of these AZFs. Remarkably, in these gain-of-function experiments, we again observed that the KLF3 FD is critical for in vivo DNA binding specificity. The addition of KLF3 FD increased the number of binding sites by more than two fold compared to the AZF alone. KLF3 FD also directed more binding to the promoter regions. Further investigations of these acquired sites identified a substantial portion of these sites as the known endogenous KLF3 bound regions, whilst others contain sequences that are similar but not identical to the predicted AZF target sequence. These results clearly demonstrate that the specific localisation of this model transcription factor to target genes is not solely dependent on the DBD and that the regions outside of this domain may also play an important role. ChIP-Seq experiments were performed on four HEK293 cell lines, two each stably expressing AZF or KLF3FD.AZF or KLF3FD only (two biological replicates). Input samples were included as controls.

Project description:Transcription factors are often regarded as having two distinct components: a DNA binding domain (DBD) to allow binding to the regulatory region of a target gene and a trans-acting functional domain (FD) to modulate gene expression. Recently, it is becoming clear that the DBDs of transcription factors alone are incapable of providing sufficient specificity to account for the highly complex genomic structures in eukaryotes. Other regions, outside of the DBD, may play a role in transcription factor DNA binding specificity in vivo. In order to test this hypothesis, we examined a model zinc finger transcription factor, Krüppel-like factor 3 (KLF3) with a FD that recruits partner proteins for its repressive activity and a three zinc finger DBD. Previously, we showed that deletion of the entire KLF3 FD reduced DNA binding across the genome. This implied the importance of the FD for in vivo DNA binding specificity. In the current study, we fused the KLF3 FD onto an unrelated, but well characterised Artificial Zinc Finger (AZF) targeting a standard target gene, VEGF-A. We compared the genomic DNA binding profile of this chimeric KLF3 construct, termed KLF3FD-AZF, to that of the AZF alone. Chromatin Immunoprecipitation followed by next generation sequencing was used to assess genomic wide DNA-binding of these AZFs. Remarkably, in these gain-of-function experiments, we again observed that the KLF3 FD is critical for in vivo DNA binding specificity. The addition of KLF3 FD increased the number of binding sites by more than two fold compared to the AZF alone. KLF3 FD also directed more binding to the promoter regions. Further investigations of these acquired sites identified a substantial portion of these sites as the known endogenous KLF3 bound regions, whilst others contain sequences that are similar but not identical to the predicted AZF target sequence. These results clearly demonstrate that the specific localisation of this model transcription factor to target genes is not solely dependent on the DBD and that the regions outside of this domain may also play an important role.

Project description:Artificial transcription factors (ATFs) and genomic nucleases based on a DNA binding platform consist- ing of multiple zinc finger domains are currently be- ing developed for clinical applications. However, no genome-wide investigations into their binding speci- ficity have been performed. We have created six- finger ATFs to target two different 18 nt regions of the human SOX2 promoter; each ATF is constructed such that it contains or lacks a super KRAB do- main (SKD) that interacts with a complex contain- ing repressive histone methyltransferases. ChIP-seq analysis of the effector-free ATFs in MCF7 breast cancer cells identified thousands of binding sites, mostly in promoter regions; the addition of an SKD domain increased the number of binding sites M-bM-^HM-<5- fold, with a majority of the new sites located out- side of promoters. De novo motif analyses suggest that the lack of binding specificity is due to sub- sets of the finger domains being used for genomic interactions. Although the ATFs display widespread binding, few genes showed expression differences; genes repressed by the ATF-SKD have stronger bind- ing sites and are more enriched for a 12 nt motif. Interestingly, epigenetic analyses indicate that the transcriptional repression caused by the ATF-SKD is not due to changes in active histone modifications. ATF plasmids were designed and stably integrated into MCF7 cells as described in (Stolzenburg et al. 2012). Stable lines were grown at 30M-bM-^@M-^S80% confluency in DulbeccoM-bM-^@M-^Ys Modified EagleM-bM-^@M-^Ys Medium (Corning, Corning, NY) supplemented with 10% heat-inactivated fetal bovine serum (Invitrogen, Life Technologies, Grand Island, NY) and 1% penicillin/streptomycin; cells were selected using 5 M-BM-5g/ml puromycin (VWR, Radnor, PA) and 200 M-BM-5g/ml G418 (VWR, Radnor, PA). ATF expression was induced by treatment with media containing 1M-BM-5g/ml doxycycline (VWR, Radnor, PA) at 0 h, doxycycline media was refreshed at 48 h, and cells were harvested at 72 h. ATF expression was confirmed by hemagglutinin (HA) tag western blot prior to HA ChIP-seq, histone ChIP-seq and RNA-seq analysis. For HA-tag ChIP-seq, stable MCF7 cell lines were induced using 100 ng/ml doxycycline (Sigma) at 0 h, doxycycline media was refreshed at 48 h, and cells were harvested at 72 h by crosslinking in a final concentration of 1% formaldehyde. Crosslinking was stopped after 5 min by adding glycine to a final concentration of 125 mM. Crosslinked cells were washed in cold phosphate buffered saline, lysed using 1 mL low-salt IP buffer (150 mM NaCl, 50 mM Tris-HCl (pH7.5), 5 mM EDTA, NP-40 (0.5%), Triton X-100 (1%) containing protease inhibitors) and aliquoted at 1 M-CM-^W 10M-bM-^HM-'7 cells/mL. Cells are then sonicated to a fragment size range of 500M-bM-^@M-^S800 bp. Samples were then diluted in 1 mL low-salt buffer and incubated with 3 M-BM-5L of anti-HA antibody (Covance, Princeton, NJ). Three-hundred microliter Sepharose A beads (GE Healthcare Life Science) were used for pull-down. Samples were sequenced at the UNC-CH Genome Analysis Facil- ity (Chapel Hill, NC) on an HiSeq (Illumina, San Diego, CA) to read counts of 4.1M-bM-^@M-^S67.3 M total reads. For histone ChIP-seq, antibodies to H3K4me3 (Cell Signaling Tech- nologies CST9751), H3K9Ac (Cell Signaling Technologies CST9649) and H3K9me3 (Diagenode pAb-056-050) were used; samples were prepared as previously described (OM-bM-^@M-^YGeen et al. 2011).

Dataset Information

Directing an artificial zinc finger protein to new targets by fusion to a non-DNA-binding domain.

Publications

Directing an artificial zinc finger protein to new targets by fusion to a non-DNA-binding domain.

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