DamID, a new tool for studying plant chromatin profiling in vivo, and its use to identify putative LHP1 target loci.
We show here that the in vivo methylation-based tagging technique DamID (DNA adenine methyltransferase identification) can be used for studies of DNA-protein interactions or chromatin profiling in plants. We have demonstrated the feasibility, reproducibility and sensitivity of the method in Arabidopsis thaliana, using the well-known yeast GAL4 transcription factor, for which DNA-binding sites (UAS(G)) were introduced into the plant genome. We monitored the methylation resulting from the activity of DNA adenine methyltransferase fused to the protein of interest, by combining digestion with methylation-sensitive restriction enzymes and quantitative PCR. We then used DamID to identify genomic targets of LHP1, a protein mostly associated with euchromatin. We showed that LHP1 was targeted to the promoter and transcribed regions of four genes: AG, AP3, FT and PI. Our data also demonstrate that LHP1, like its animal homologues, has a high binding affinity for A/T-rich regions, binding particularly strongly to the large regulatory introns of AG and PI. We identified three major characteristics of LHP1 binding, highlighting the similarities between plant LHP1 and animal HP1 proteins.
Germann
Sophie
S
Laboratoire de Biologie Cellulaire, IJPB, INRA, Route de Saint-Cyr, 78026 Versailles Cedex, France.
Juul-Jensen
Trine
T
Letarnec
Bruno
B
Gaudin
Valérie
V
D017360:Arabidopsis / Q000235:genetics* / Q000378:metabolism; D029681:Arabidopsis Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D001665:Binding Sites; D002843:Chromatin / Q000378:metabolism*; D002868:Chromosomal Proteins, Non-Histone / Q000032:analysis* / Q000235:genetics / Q000378:metabolism; D019175:DNA Methylation; D004268:DNA-Binding Proteins / Q000032:analysis* / Q000235:genetics / Q000378:metabolism; D018390:Gene Targeting; D007438:Introns; D030821:Plants, Genetically Modified / Q000378:metabolism; D016133:Polymerase Chain Reaction / Q000379:methods; D011401:Promoter Regions, Genetic; D011993:Recombinant Fusion Proteins / Q000032:analysis / Q000378:metabolism; D015265:Site-Specific DNA-Methyltransferase (Adenine-Specific) / Q000032:analysis*
D029681:Arabidopsis Proteins; D002843:Chromatin; D002868:Chromosomal Proteins, Non-Histone; D004268:DNA-Binding Proteins; D011993:Recombinant Fusion Proteins; C443901:like heterochromatin protein 1; D015265:Site-Specific DNA-Methyltransferase (Adenine-Specific)