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Eukaryotic Cell, November 2005, p. 1775-1784, Vol. 4, No. 11
1535-9778/05/$08.00+0     doi:10.1128/EC.4.11.1775-1784.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

Involvement of a Short Interspersed Element in Epigenetic Transcriptional Silencing of the Amoebapore Gene in Entamoeba histolytica

Michael Anbar, Rivka Bracha, Yael Nuchamowitz, Yan Li, Anat Florentin, and David Mirelman^*

Department of Biological Chemistry, Weizmann Institute of Science, Rehovot 76100, Israel

Received 22 June 2005/ Accepted 8 September 2005

Transcriptional silencing of an amoebapore (ap-a) gene occurred in Entamoeba histolytica following the transfection of plasmids containing a DNA segment (473 bp) homologous to the 5' upstream region of the gene (R. Bracha, Y. Nuchamowitz, and D. Mirelman, Eukaryot. Cell 2:295-305, 2003). This segment contains the promoter region of the ap-a gene, a T-rich stretch, followed by a truncated SINE1 (short interspersed element 1) that is transcribed from the antisense strand. Transfection of plasmids containing truncated SINE1 sequences which lack their 3' regulatory elements upstream of the ap-a gene was essential for the downstream silencing of the ap-a gene while transfection with plasmids containing the entire SINE1 sequence or without the T-rich stretch promoted the overexpression of the ap-a gene. Both the T-rich stretch and sequences of the 5' SINE1 were essential for the transcription of SINE1. RNA extracts from gene-silenced cultures showed small amounts of short (140-nucleotide), single-stranded molecules with homology to SINE1 but no short interfering RNA. Chromatin immunoprecipitation analysis with an antibody against methylated K4 of histone H3 showed a demethylation of K4 at the domain of the ap-a gene, indicating transcriptional inactivation. These results suggest the involvement of SINE1 in triggering the gene silencing and the role of histone modification in its epigenetic maintenance.

M.A. and R.B. contributed equally to this work.

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