Intrinsic Nucleic Acid-Binding Activity of Chp1 Chromodomain Is Required for Heterochromatic Gene Silencing

Abstract

Centromeric heterochromatin assembly in fission yeast requires the RNAi pathway. Chp1, a chromodomain (CD) protein, forms the Ago1-containing RNA-induced transcriptional silencing (RITS) complex and recruits siRNA-bound RITS to methylated histone H3 lysine 9 (H3K9me) via its CD. Here, we show that the CD of Chp1 (Chp1-CD) possesses unique nucleic acid-binding activities that are essential for heterochromatic gene silencing. Detailed electrophoretic-mobility shift analyses demonstrated that Chp1 binds to RNA via the CD in addition to its central RNA-recognition motif. Interestingly, robust RNA- and DNA-binding activity of Chp1-CD was strongly enhanced when it was bound to H3K9me, which was revealed to involve a positively charged domain within the Chp1-CD by structural analyses. These results demonstrate a role for the CD that provides a link between RNA, DNA, and methylated histone tails to ensure heterochromatic gene silencing.

RNAs — Physical and functional modulators of chromatin reader proteins

“…Direct interplay between methyl-H3K9 binding and RNA-binding activities of chromodomains has also been demonstrated for the Chp1 and Clr4 proteins in S. pombe [24]. Interestingly, this organism does not have any Pc like factors.…”

“…The chromodomain of Clr4 alone does not bind to RNAs as tested by EMSA. However, in the presence of 0.5-1× molar excess of methyl-H3K9me3, RNA-binding capacity was observed [24]. The findings indicate that the binding of the chromodomains of Chp1 and Clr4 to their target histone mark leads to conformational changes exposing (additional) RNA-interacting surfaces.…”

“…1A), only the Pc chromodomains possess RNA-binding capacity [7,[23][24][25]33,34]. Besides Cbx2, all other Pc chromodomains showed RNA-binding activity in electrophoretic mobility shift assay (EMSA) using fragments of in vitro transcribed cyclin E RNA as bait [7].…”

“…For both Chp1 and Clr4, the interaction with RNA is partly mediated by a stretch of 5-6 basic amino acids located at the C-terminus of the α-helix within their chromodomains (Fig. 1A, indicated in orange) [24]. In the case of Chp1, aromatic amino acids (WY) and an asparagine (N) of the chromodomain also contribute to RNA binding (Fig.…”

“…Several recent overview articles have summarized the biology of interactions between RNAs and chromatin modifying enzymes [15,[19][20][21]. However, RNA-binding activities of a subset of 'reader' proteins of chromatin modifications have also been described in different experimental systems [7,13,16,[22][23][24][25]. Here we will summarize the current knowledge on these interactions with particular emphasis on chromodomain proteins, whose RNA-binding activities have been …”