Abstract
BackgroundLong Interspersed Nuclear Element-1 (L1) is an oncogenic mammalian retroelement silenced early in development via tightly controlled epigenetic mechanisms. We have previously shown that the regulatory region of human and murine L1s interact with retinoblastoma (RB) proteins to effect retroelement silencing. The present studies were conducted to identify the corepressor complex responsible for RB-mediated silencing of L1.MethodsChromatin immunoprecipitation and silencing RNA technology were used to identify the repressor complex that silences L1 in human and murine cells.ResultsComponents of the Nucleosomal and Remodeling Deacetylase (NuRD) multiprotein complex specifically enriched the L1 5′-untranslated DNA sequence in human and murine cells. Genetic ablation of RB proteins in murine cells destabilized interactions within the NuRD macromolecular complex and mediated nuclear rearrangement of Mi2-β, an ATP-dependent helicase subunit with nucleosome remodeling activity. Depletion of Mi2-β, RbAP46 and HDAC2 reduced the repressor activity of the NuRD complex and reactivated a synthetic L1 reporter in human cells. Epigenetic reactivation of L1 in RB-null cells by DNA damage was markedly enhanced compared to wild type cells.ConclusionsRB proteins stabilize interactions of the NuRD corepressor complex within the L1 promoter to effect L1 silencing. L1 retroelements may serve as a scaffold on which RB builds heterochromatic regions that regulate chromatin function.
Highlights
Long Interspersed Nuclear Element-1 (L1) is an oncogenic mammalian retroelement silenced early in development via tightly controlled epigenetic mechanisms
We have previously shown that recruitment of E2F/RB (E2F-retinoblastoma) to the L1 promoter, along with histone deacetylases 1 and 2 (HDAC1 and HDAC2, respectively) [4, 5] and methyl binding protein 2 (MBD2) [5] are critical to L1 silencing
The Nucleosomal and Remodeling Deacetylase (NuRD) corepressor complex is recruited to the L1 promoter The NuRD multiprotein complex includes the dermatomyositis-specific Mi2 autoantigen (Mi2-β), RB-associated proteins 46 and 48 (RbAp46 and RbAp48), MBD2 and MBD3, and metastasis-associated proteins 2 and 3 (MTA2 and MTA3) [9,10,11]
Summary
Long Interspersed Nuclear Element-1 (L1) is an oncogenic mammalian retroelement silenced early in development via tightly controlled epigenetic mechanisms. We have previously shown that the regulatory region of human and murine L1s interact with retinoblastoma (RB) proteins to effect retroelement silencing. The present studies were conducted to identify the corepressor complex responsible for RB-mediated silencing of L1. Human L1s are non-LTR mammalian retrotransposons that consist of an internal bidirectional promoter, two open reading frames encoding for ORF1p (RNA-binding protein) and ORF2p (reverse transcriptase and endonuclease activities), a 3′- untranslated region (UTR), and a polyA tail [1]. We have previously shown that recruitment of E2F/RB (E2F-retinoblastoma) to the L1 promoter, along with histone deacetylases 1 and 2 (HDAC1 and HDAC2, respectively) [4, 5] and methyl binding protein 2 (MBD2) [5] are critical to L1 silencing. Cells were visualized in a CARL ZEISS AXIOVERT 200 inverted microscope at a magnification of 63X
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