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IDENTIFICATION AND CHARACTERIZATION OF HISTONE VARIANT H2A.Z IN THE LONG TERMINAL REPEAT RETROTRANSPOSON INTEGRATION
Author(s) -
Rai Sudhir K.,
Guo Yabin,
Levin Henry L.
Publication year - 2016
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.30.1_supplement.580.1
Subject(s) - retrotransposon , long terminal repeat , biology , genetics , integrase , transposable element , genome , retrovirus , gene , complementary dna , schizosaccharomyces pombe , saccharomyces cerevisiae
Unique integration patterns of Long Terminal Repeat (LTR)‐retrotransposons and the use of retroviruses in gene therapy have generated great interest in target‐site selection. Initially, the RNA transcripts of retroviruses and retrovirus‐like elements are reverse transcribed into full‐length cDNA copies that are subsequently inserted into the host cell genome. Although integrase (IN), encoded by retroviruses and LTR‐retrotransposons, is responsible for catalyzing the insertion of cDNA into the host genome, it is now clear that a variety of host factors are required for targeting and selection of the integration sites. To understand how retroviruses and LTR‐retrotransposons position their sites of integration in the host genome, we study the LTR‐retrotransposon Tf1 of Schizosaccharomyces pombe . Tf1 integrates with a strong preference for the promoters of RNA pol II transcribed genes. A genetic assay that measures cDNA present in the nucleus allows us to determine whether mutations that reduce transposition frequencies have a direct impact on the process of integration. We utilized these assays to screen a collection of 3,004 S. pombe deletion strains to identify genes that contribute to integration. We found that 181 deletion strains had reduced transposition and 98 of these likely have specific defects in integration. All 98 deletion strains were tested for Tf1 IN and Gag expression using immunoblots. A total of 73 strains had normal levels of IN and GAG proteins, suggesting the deleted genes played a direct role in various steps of Tf1 integration. Candidates that may contribute to integration include factors responsible for DNA repair, transcription activation, signal transduction, and nucleosome structure. We characterized the role of candidate protein H2A.Z (pht1) because this histone variant assembles into the nucleosomes at the promoters of RNA pol II transcribed genes. We found that the reduced transposition of the strain lacking pht1 could be complemented with a plasmid encoding a wild type copy of pht1. In addition, bioinformatics analysis of 73,125 unique Tf1 integration sites demonstrated that the nucleosome‐free regions (NFRs) of promoters were the preferred targets of integration. Nucleosomes with H2A.Z are adjacent the NFR suggesting the possibility that H2A.Z may contribute directly to targeting integration. Support or Funding Information NICHD/NIH Intramural Research Funding