RESUMEN
Histone chaperones and chromatin remodelers control nucleosome dynamics, which are essential for transcription, replication, and DNA repair. The histone chaperone Anti-Silencing Factor 1 (ASF1) plays a central role in facilitating CAF-1-mediated replication-dependent H3.1 deposition and HIRA-mediated replication-independent H3.3 deposition in yeast and metazoans. Whether ASF1 function is evolutionarily conserved in plants is unknown. Here, we show that Arabidopsis ASF1 proteins display a preference for the HIRA complex. Simultaneous mutation of both Arabidopsis ASF1 genes caused a decrease in chromatin density and ectopic H3.1 occupancy at loci typically enriched with H3.3. Genetic, transcriptomic, and proteomic data indicate that ASF1 proteins strongly prefers the HIRA complex over CAF-1. asf1 mutants also displayed an increase in spurious Pol II transcriptional initiation and showed defects in the maintenance of gene body CG DNA methylation and in the distribution of histone modifications. Furthermore, ectopic targeting of ASF1 caused excessive histone deposition, less accessible chromatin, and gene silencing. These findings reveal the importance of ASF1-mediated histone deposition for proper epigenetic regulation of the genome.
Asunto(s)
Arabidopsis , Arabidopsis/genética , Arabidopsis/metabolismo , Proteínas de Ciclo Celular/metabolismo , Cromatina/genética , Factor 1 de Ensamblaje de la Cromatina/genética , Factor 1 de Ensamblaje de la Cromatina/metabolismo , Epigénesis Genética , Chaperonas de Histonas/genética , Chaperonas de Histonas/metabolismo , Histonas/genética , Histonas/metabolismo , Chaperonas Moleculares/genética , Chaperonas Moleculares/metabolismo , Proteómica , Proteínas de ArabidopsisRESUMEN
RNA polymerase II (RNAPII) transcription converts the DNA sequence of a single gene into multiple transcript isoforms that may carry alternative functions. Gene isoforms result from variable transcription start sites (TSSs) at the beginning and polyadenylation sites (PASs) at the end of transcripts. How alternative TSSs relate to variable PASs is poorly understood. Here, we identify both ends of RNA molecules in Arabidopsis thaliana by transcription isoform sequencing (TIF-seq) and report four transcript isoforms per expressed gene. While intragenic initiation represents a large source of regulated isoform diversity, we observe that ~14% of expressed genes generate relatively unstable short promoter-proximal RNAs (sppRNAs) from nascent transcript cleavage and polyadenylation shortly after initiation. The location of sppRNAs correlates with the position of promoter-proximal RNAPII stalling, indicating that large pools of promoter-stalled RNAPII may engage in transcriptional termination. We propose that promoter-proximal RNAPII stalling-linked to premature transcriptional termination may represent a checkpoint that governs plant gene expression.