A zinc-finger protein Moc3 functions as a transcription activator to promote RNAi-dependent constitutive heterochromatin establishment in fission yeast.

In fission yeast, Schizosaccharomyces pombe, constitutive heterochromatin defined by methylation of histone H3 lysine 9 (H3K9me) and its binding protein Swi6/HP1 localizes at the telomere, centromere, and mating-type loci. These loci contain DNA sequences called dg and dh, and the RNA interference (RNAi)-dependent system establishes and maintains heterochromatin at dg/dh. Bi-directional transcription at dg/dh induced by RNA polymerase II is critical in RNAi-dependent heterochromatin formation because the transcribed RNAs provide substrates for siRNA synthesis and a platform for assembling RNAi factors. However, a regulator of dg/dh transcription during the establishment of heterochromatin is not known. Here, we found that a zinc-finger protein Moc3 localizes dh and activates dh-forward transcription in its zinc-finger-dependent manner when heterochromatin structure or heterochromatin-dependent silencing is compromised. However, Moc3 does not localize at normal heterochromatin and does not activate the dh-forward transcription. Notably, the loss of Moc3 caused a retarded heterochromatin establishment, showing that Moc3-dependent dh-forward transcription is critical for RNAi-dependent heterochromatin establishment. Therefore, Moc3 is a transcriptional activator that induces RNAi to establish heterochromatin.

A novel tracking and analysis system for time-lapse cellular imaging of Schizosaccharomyces pombe.

The importance of the parent-progeny relationship tracking technique in single-cell analysis has grown with the passage of time. In this study, fundamental image-processing techniques were combined to develop software capable of inferring cell cycle alterations in fission yeast cells, which exhibit equipartition during division. These methods, exclusively relying on bright-field images as input, could track parent-progeny relationships after cell division by assessing the temporal morphological transformation of these cells. In the application of this technique, the software was employed for calculating intracellular fluorescent dots during every stage of the cell cycle, using a yeast strain expressing EGFP-fused Swi6, which binds to chromatin. The results obtained with this software were consistent with those of previous studies. This software facilitated single-cell-level tracking of parent-progeny relationships in cells exhibiting equipartition during division and enabled the monitoring of spatial fluctuations in a cell cycle-dependent protein. This method, expediting the analysis of extensive datasets, may also empower large-scale screening experiments that cannot be conducted manually.

RNA aptamer capture of macromolecular complexes for mass spectrometry analysis.

Specific genomic functions are dictated by macromolecular complexes (MCs) containing multiple proteins. Affinity purification of these complexes, often using antibodies, followed by mass spectrometry (MS) has revolutionized our ability to identify the composition of MCs. However, conventional immunoprecipitations suffer from contaminating antibody/serum-derived peptides that limit the sensitivity of detection for low-abundant interacting partners using MS. Here, we present AptA-MS (aptamer affinity-mass spectrometry), a robust strategy primarily using a specific, high-affinity RNA aptamer against Green Fluorescent Protein (GFP) to identify interactors of a GFP-tagged protein of interest by high-resolution MS. Utilizing this approach, we have identified the known molecular chaperones that interact with human Heat Shock Factor 1 (HSF1), and observed an increased association with several proteins upon heat shock, including translation elongation factors and histones. HSF1 is known to be regulated by multiple post-translational modifications (PTMs), and we observe both known and new sites of modifications on HSF1. We show that AptA-MS provides a dramatic target enrichment and detection sensitivity in evolutionarily diverse organisms and allows identification of PTMs without the need for modification-specific enrichments. In combination with the expanding libraries of GFP-tagged cell lines, this strategy offers a general, inexpensive, and high-resolution alternative to conventional approaches for studying MCs.

Heterochromatin suppresses gross chromosomal rearrangements at centromeres by repressing Tfs1/TFIIS-dependent transcription.

Heterochromatin, characterized by histone H3 lysine 9 (H3K9) methylation, assembles on repetitive regions including centromeres. Although centromeric heterochromatin is important for correct segregation of chromosomes, its exact role in maintaining centromere integrity remains elusive. Here, we found in fission yeast that heterochromatin suppresses gross chromosomal rearrangements (GCRs) at centromeres. Mutations in Clr4/Suv39 methyltransferase increased the formation of isochromosomes, whose breakpoints were located in centromere repeats. H3K9A and H3K9R mutations also increased GCRs, suggesting that Clr4 suppresses centromeric GCRs via H3K9 methylation. HP1 homologs Swi6 and Chp2 and the RNAi component Chp1 were the chromodomain proteins essential for full suppression of GCRs. Remarkably, mutations in RNA polymerase II (RNAPII) or Tfs1/TFIIS, the transcription factor that facilitates restart of RNAPII after backtracking, specifically bypassed the requirement of Clr4 for suppressing GCRs. These results demonstrate that heterochromatin suppresses GCRs by repressing Tfs1-dependent transcription of centromere repeats.

Ser7 of RNAPII-CTD facilitates heterochromatin formation by linking ncRNA to RNAi.

Some long noncoding RNAs (ncRNAs) transcribed by RNA polymerase II (RNAPII) are retained on chromatin, where they regulate RNAi and chromatin structure. The molecular basis of this retention remains unknown. We show that in fission yeast serine 7 (Ser7) of the C-terminal domain (CTD) of RNAPII is required for efficient siRNA generation for RNAi-dependent heterochromatin formation. Surprisingly, Ser7 facilitates chromatin retention of nascent heterochromatic RNAs (hRNAs). Chromatin retention of hRNAs and siRNA generation requires both Ser7 and an RNA-binding activity of the chromodomain of Chp1, a subunit of the RNA-induced transcriptional silencing (RITS) complex. Furthermore, RITS associates with RNAPII in a Ser7-dependent manner. We propose that Ser7 promotes cotranscriptional chromatin retention of hRNA by recruiting the RNA-chromatin connector protein Chp1, which facilitates RNAi-dependent heterochromatin formation. Our findings reveal a function of the CTD code: linking ncRNA transcription to RNAi for heterochromatin formation.

DNA-RNA hybrid formation mediates RNAi-directed heterochromatin formation.

Certain noncoding RNAs (ncRNAs) implicated in the regulation of chromatin structure associate with chromatin. During the formation of RNAi-directed heterochromatin in fission yeast, ncRNAs transcribed from heterochromatin are thought to recruit the RNAi machinery to chromatin for the formation of heterochromatin; however, the molecular details of this association are not clear. Here, using RNA immunoprecipitation assay, we showed that the heterochromatic ncRNA was associated with chromatin via the formation of a DNA-RNA hybrid and bound to the RNA-induced transcriptional silencing (RITS) complex. The presence of DNA-RNA hybrid in the cell was also confirmed by immunofluorescence analysis using anti-DNA-RNA hybrid antibody. Over-expression and depletion of RNase H in vivo decreased and increased the amount of DNA-RNA hybrid formed, respectively, and both disturbed heterochromatin. Moreover, DNA-RNA hybrid was formed on, and over-expression of RNase H inhibited the formation of, artificial heterochromatin induced by tethering of RITS to mRNA. These results indicate that heterochromatic ncRNAs are retained on chromatin via the formation of DNA-RNA hybrids and provide a platform for the RNAi-directed heterochromatin assembly and suggest that DNA-RNA hybrid formation plays a role in chromatic ncRNA function.

Deubiquitylation of histone H2A activates transcriptional initiation via trans-histone cross-talk with H3K4 di- and trimethylation.

Transcriptional initiation is a key step in the control of mRNA synthesis and is intimately related to chromatin structure and histone modification. Here, we show that the ubiquitylation of H2A (ubH2A) correlates with silent chromatin and regulates transcriptional initiation. The levels of ubH2A vary during hepatocyte regeneration, and based on microarray expression data from regenerating liver, we identified USP21, a ubiquitin-specific protease that catalyzes the hydrolysis of ubH2A. When chromatin is assembled in vitro, ubH2A, but not H2A, specifically represses the di- and trimethylation of H3K4. USP21 relieves this ubH2A-specific repression. In addition, in vitro transcription analysis revealed that ubH2A represses transcriptional initiation, but not transcriptional elongation, by inhibiting H3K4 methylation. Notably, ubH2A-mediated repression was not observed when H3 Lys 4 was changed to arginine. Furthermore, overexpression of USP21 in the liver up-regulates a gene that is normally down-regulated during hepatocyte regeneration. Our studies revealed a novel mode of trans-histone cross-talk, in which H2A ubiquitylation controls the di- and trimethylation of H3K4, resulting in regulation of transcriptional initiation.