Hepatoma-derived growth factor binds DNA through the N-terminal PWWP domain.
BACKGROUND
Hepatoma Derived Growth Factor (HDGF) is a nuclear protein with nuclear targeting required for mitogenic activity. Recently we demonstrated that HDGF is a transcriptional repressor, but whether HDGF binds DNA, the specificity of DNA binding and what protein domain is required are still unknown. In this study, we aimed to identify if HDGF is a DNA binding protein, map the functional DNA binding domain and DNA binding element for HDGF.


RESULTS
Using chromatin immunoprecipitation (ChIP) of human DNA, we isolated 10 DNA sequences sharing a conserved ~200 bp element. Homology analysis identified the binding sequences as a motif within the promoter of the SMYD1 gene, a HDGF target gene. Electrophoretic Mobility Shift Assays (EMSA) confirmed the binding of HDGF to this conserved sequence. As a result, an 80 bp conserved sequence located in the SMYD1 promoter bound GST-HDGF tightly. The binding core sequence for HDGF was narrowed down to 37 bp using a deletion mapping strategy from both the 5' and 3' ends. Moreover, ChIP and DNase I footprinting analysis revealed that HDGF binds this 80 bp DNA fragment specifically. Functionally overexpression of HDGF represses a reporter gene which is controlled by an SV-40 promoter containing the 80 bp DNA element. Using serial truncations of GST-HDGF, we mapped the DNA binding domain of HDGF to the N-terminal PWWP domain.


CONCLUSIONS
HDGF is a DNA binding protein, binds DNA specifically, and prefers a minimum of 37 bp long DNA fragment. The N-terminal PWWP domain of HDGF is required for DNA binding. HDGF exerts its transcription repressive effect through binding to a conserved DNA element in the promoter of target genes.
Yang
Jun
J
Department of Pediatrics, Division of Pediatric Cardiology, Johns Hopkins University, Baltimore, MD 21205, USA. jyang31@jhmi.edu
Everett
Allen D
AD
D001483:Base Sequence; D002460:Cell Line / Q000378:metabolism; D047369:Chromatin Immunoprecipitation; D002874:Chromosome Mapping; D016384:Consensus Sequence; D004247:DNA / Q000378:metabolism; D018983:DNA Footprinting; D017930:Genes, Reporter; D006801:Humans; D036341:Intercellular Signaling Peptides and Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D032446:Myoblasts / Q000378:metabolism; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins / Q000502:physiology; D016415:Sequence Alignment; D012689:Sequence Homology, Nucleic Acid; D013379:Substrate Specificity; D014158:Transcription, Genetic
D036341:Intercellular Signaling Peptides and Proteins; D011993:Recombinant Fusion Proteins; C051610:hepatoma-derived growth factor; D004247:DNA