Abstract
Active and repressed ribosomal RNA (rRNA) genes are characterised by specific epigenetic marks and differentially positioned nucleosomes at their promoters. Repression of the rRNA genes requires a non-coding RNA (pRNA) and the presence of the nucleolar remodeling complex (NoRC). ATP-dependent chromatin remodeling enzymes are essential regulators of DNA-dependent processes, and this regulation occurs via the modulation of DNA accessibility in chromatin. We have studied the targeting of NoRC to the rRNA gene promoter; its mechanism of nucleosome positioning, in which a nucleosome is placed over the transcription initiation site; and the functional role of the pRNA. We demonstrate that NoRC is capable of recognising and binding to the nucleosomal rRNA gene promoter on its own and binds with higher affinity the nucleosomes positioned at non-repressive positions. NoRC recognises the promoter nucleosome within a chromatin array and positions the nucleosomes, as observed in vivo. NoRC uses the release mechanism of positioning, which is characterised by a reduced affinity for the remodeled substrate. The pRNA specifically binds to NoRC and regulates the enzyme by switching off its ATPase activity. Given the known role of pRNA in tethering NoRC to the rDNA, we propose that pRNA is a key factor that links the chromatin modification activity and scaffolding function of NoRC.
Highlights
Nucleosomes present a major obstacle for the binding of sequence-specific DNA-binding factors, the interaction of positively charged histone tails with DNA and the masking of DNA binding sites that face in towards the histone octamer surface [1,2]
Tumour cells overexpress ribosomal RNA, which is required for ribosome assembly and cell growth. rRNA gene repression is mediated by the chromatin remodeling complex (NoRC) and a non-coding RNA that binds to this enzyme
This study addresses the mechanism of nucleosome positioning by nucleolar remodeling complex (NoRC) and the functional role of the non-coding RNA, which is termed promoter RNA (pRNA) because it corresponds to the promoter sequence
Summary
Nucleosomes present a major obstacle for the binding of sequence-specific DNA-binding factors, the interaction of positively charged histone tails with DNA and the masking of DNA binding sites that face in towards the histone octamer surface [1,2]. All DNA-dependent processes, such as transcription, replication, repair and recombination, are affected by the positioning of nucleosomes on regulatory sites. Due to the high combinatorial complexity, it is estimated that several hundred different chromatin remodeling complexes exist in humans These remodeling enzymes comprise several groups of ATPases classified into the Snf, ISWI, Mi-2, Chd, Ino, ERCC6, ALC1, CHD7, Swr, RAD54 and Lsh subfamilies [9,11]. In addition to their diversity, chromatin remodeling enzymes are highly abundant, with approximately one enzyme for every 10 nucleosomes in yeast and human cells [5,10]
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