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Transcriptional gene silencing (TGS) play important roles in regulating transposon, chromosomal repeats, transgenes as well as protein-coding genes during plant growth and development, but the molecular mechanisms that regulate TGS is not well understood in plants.We are usingtwo T-DNA insertion Arabidopsis lines, one contains an active 35S-NPTII and another one contains a silencing 35S-NPTII, to characterize the TGS maintaining mechanisms in Arabidopsis.Previous study identified REPRESSOR OF SILENCING 1 (ROS1), a DNA demethylation enzyme that plays a crucial roles in maintaining the expression of both RD29A-LUC and 35S-NPTII transgenes on the active T-DNA construct.By screening for second-site suppressors of rosl using silenced 35S-NPTII, we have identified genes that are mainly involved in DNA replication and maintenance of transcriptional silencing.By screening 35S-NPTII silenced mutants, we identified two new genes, including ROS4 encoding a putative histone acetyltransferase, and ROS5 encoding a small heat shock protein.We also found that the silencing of 35S-NPTII in the RdDM pathway mutants is due to the reduced expression of ROS1 in the mutants.We used another silenced 35S-NPTII line carrying a hypermethylated, silenced 35S-NTPII that is independent on RdDM pathway to identify the releasing silencing mutants.From the mutated population, we identified several kanamycin resistant mutants, including different mutant alleles of ddml, metl and hda6, and two new mutant alleles, mcm10-1 and mcml0-2.Mutations in MCM10 released the TGS of 35S-NPTII and the targeted genes including two maternal imprinting genes with decreasing the level of H3K9me2.Our study demonstrates a complex regulation mechanism of TGS in Arabidopsis.