RESUMO
R-loops are three-stranded nucleic acid structures that form during transcription, especially over unmethylated CpG-rich promoters of active genes. In mouse embryonic stem cells (mESCs), CpG-rich developmental regulator genes are repressed by the Polycomb complexes PRC1 and PRC2. Here, we show that R-loops form at a subset of Polycomb target genes, and we investigate their contribution to Polycomb repression. At R-loop-positive genes, R-loop removal leads to decreased PRC1 and PRC2 recruitment and Pol II activation into a productive elongation state, accompanied by gene derepression at nascent and processed transcript levels. Stable removal of PRC2 derepresses R-loop-negative genes, as expected, but does not affect R-loops, PRC1 recruitment, or transcriptional repression of R-loop-positive genes. Our results highlight that Polycomb repression does not occur via one mechanism but consists of different layers of repression, some of which are gene specific. We uncover that one such mechanism is mediated by an interplay between R-loops and RING1B recruitment.
Assuntos
Ilhas de CpG , Regulação da Expressão Gênica no Desenvolvimento , Células-Tronco Embrionárias Murinas/fisiologia , Complexo Repressor Polycomb 1/metabolismo , Regiões Promotoras Genéticas , Transcrição Gênica , Ubiquitina-Proteína Ligases/metabolismo , Animais , Sítios de Ligação , Linhagem Celular , Regulação para Baixo , Proteína Potenciadora do Homólogo 2 de Zeste/genética , Proteína Potenciadora do Homólogo 2 de Zeste/metabolismo , Camundongos , Células-Tronco Embrionárias Murinas/metabolismo , Conformação de Ácido Nucleico , Complexo Repressor Polycomb 1/genética , Ligação Proteica , RNA Polimerase II/genética , RNA Polimerase II/metabolismo , Relação Estrutura-Atividade , Ubiquitina-Proteína Ligases/genéticaRESUMO
The proteasome has pronounced preferences for the amino acid sequence of its substrates at the site where it initiates degradation. Here, we report that modulating these sequences can tune the steady-state abundance of proteins over 2 orders of magnitude in cells. This is the same dynamic range as seen for inducing ubiquitination through a classic N-end rule degron. The stability and abundance of His3 constructs dictated by the initiation site affect survival of yeast cells and show that variation in proteasomal initiation can affect fitness. The proteasome's sequence preferences are linked directly to the affinity of the initiation sites to their receptor on the proteasome and are conserved between Saccharomyces cerevisiae, Schizosaccharomyces pombe, and human cells. These findings establish that the sequence composition of unstructured initiation sites influences protein abundance in vivo in an evolutionarily conserved manner and can affect phenotype and fitness.