Unknown,Transcriptomics,Genomics,Proteomics

Dataset Information

Genome-wide binding profiles of KLF3 and KLF3 mutants in MEF cells

ABSTRACT: Transcription factors are often regarded as being comprised of a DNA-binding domain and a functional domain. The two domains are considered separable and autonomous, with the DNA-binding domain directing the factor to its target genes and the functional domain imparting transcriptional regulation. We have examined a typical Zinc Finger (ZF) transcription factor from the Krüppel-like factor (KLF) family, KLF3. This factor has an N-terminal repression domain that binds the co-repressor C-terminal binding protein (CtBP), and a DNA-binding domain composed of three classical (ZFs) at its C-terminus. We established a system to compare the genomic occupancy profile of wildtype KLF3 with two mutants affecting the N-terminal functional domain: a mutant unable to contact its cofactor CtBP and a mutant lacking the entire N-terminal domain, but retaining the ZFs intact. We used chromatin immunoprecipitation followed by sequencing (ChIP-seq) to assess binding across the genome in murine embryonic fibroblasts. Our results define the in vivo recognition site for KLF3 and the two mutants as a typical CACCC-like element. Unexpectedly, we observe that mutations in the N-terminal functional domain severely affect DNA binding. In general, both mutations reduce binding but there are also instances where binding is retained or even increased. These results provide a clear demonstration that the correct localization of transcription factors to their target genes is not solely dependent on their DNA-contact domains. This informs our understanding of how transcription factors operate and is of relevance to the design of artificial ZF proteins. ChIP-seq was performed on the three samples, KLF3, ΔDL and DBD in duplicate (biological replicates). Input samples were used as controls.

ORGANISM(S): Mus musculus

SUBMITTER: Jon Burdach

PROVIDER: E-GEOD-44748 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Publications

Regions outside the DNA-binding domain are critical for proper in vivo specificity of an archetypal zinc finger transcription factor.

Burdach Jon J Funnell Alister P W AP Mak Ka Sin KS Artuz Crisbel M CM Wienert Beeke B Lim Wooi F WF Tan Lit Yeen LY Pearson Richard C M RC Crossley Merlin M

Nucleic acids research 20131007 1

Transcription factors (TFs) are often regarded as being composed of a DNA-binding domain (DBD) and a functional domain. The two domains are considered separable and autonomous, with the DBD directing the factor to its target genes and the functional domain imparting transcriptional regulation. We examined an archetypal zinc finger (ZF) TF, Krüppel-like factor 3 with an N-terminal domain that binds the corepressor CtBP and a DBD composed of three ZFs at its C-terminus. We established a system to ...[more]

PMID: 24106088

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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:Precise control of the innate immune response is required for resistance to microbial infections and maintenance of normal tissue homeostasis. Because this response involves coordinate regulation of hundreds of genes, it provides a powerful biological system to elucidate the molecular strategies that underlie signal- and time-dependent transitions of gene expression. Using a combination of genome-wide and gene-specific approaches, we provide evidence that rather than representing off/on transitions, Toll-like receptor 4 (TLR4)-dependent activation of nearly all immediate/early (I/E) and late response genes results from a sequential process in which signal-independent factors, exemplified by Gabpa, initially establish basal levels of gene expression that are then amplified by signal-dependent transcription factors. Promoters of I/E genes are distinguished from those of late genes by the use of distinct sets of signal-dependent transcription factors, preferential binding of TBP and basal enrichment for RNA Pol II immediately downstream of transcriptional start sites. Global nuclear run-on (GRO) sequencing and total RNA sequencing further indicates that TLR4 signaling markedly increases efficiency of transcriptional elongation of nearly all I/E genes, while RNA splicing is largely unaffected. NCoR/SMRT co-repressor complexes are unexpectedly found to be associated with H3K4me3-positive promoters of TLR4-responsive genes that exhibit a broad range of basal expression levels, implying a dynamic, rather than static role in regulation of gene expression. Collectively, these findings reveal mechanisms underlying temporally distinct patterns of TLR4-dependent gene activation required for homeostasis and effective immune responses. ChIP-Seq, Total RNA-Seq, Gro-Seq, and gene expression profiling was performed in macrophages treated with Kdo2 Lipid A. Control samples for H3K4me3 in Macrophages and B cells, in addition to control microarray data are included in GEO accession# GSE21512. FASTA files are available for older experiments that lack quality read information (i.e. no FASTQ file)

Dataset Information

Genome-wide binding profiles of KLF3 and KLF3 mutants in MEF cells

Publications

Regions outside the DNA-binding domain are critical for proper in vivo specificity of an archetypal zinc finger transcription factor.

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