Sharon A. White

RNAi-Mediated Chromatin Silencing in Fission Yeast

In the fission yeast Schizosaccharomyces pombe, the RNAi pathway plays an important role in the formation and maintenance of heterochromatin. Heterochromatin, or silent chromatin, is an epigenetically inherited attribute of eukaryotic chromosomes which is required for gene regulation, chromosome segregation and maintenance of genome stability. In S. pombe, heterochromatin forms on related repetitive DNA sequences at specific loci. These repetitive sequences, in concert with the RNAi machinery, are thought to attract several proteins including chromatin-modifying enzymes which act to promote heterochromatin formation. The purification of complexes participating in heterochromatin formation has allowed us to begin to analyse in detail the processes involved. In the future this will help us to understand how the RNAi machinery acts to induce the chromatin modifications which lead to heterochromatin assembly in fission yeast.

RNA interference is required for normal centromere function in fission yeast

In plants, animals and fungi, active centromeres are associated with arrays of repetitive DNA sequences. The outer repeats at fission yeast (Schizosaccharomyces pombe) centromeres are heterochromatic and are required for the assembly of an active centromere. Components of the RNA interference (RNAi) machinery process transcripts derived from these repeats and mediate the formation of silent chromatin. A subfragment of the repeat (dg) is known to induce silencing of marker genes at euchromatic sites and is required for centromere formation. We show that the RNAi components, Argonaute (Ago1), Dicer (Dcr1) and RNA-dependent RNA polymerase (Rdp1), are required to maintain silencing, lysine 9 methylation of histone H3 and association of Swi6 via this dg ectopic silencer. Deletion of Ago1, Dcr1 or Rdp1 disrupts chromosome segregation leading to a high incidence of lagging chromosomes on late anaphase spindles and sensitivity to a microtubule poison. Analysis of dg transcription indicates that csp mutants, previously shown to abrogate centromere silencing and chromosome segregation, are also defective in the regulation of non-coding centromeric RNAs. In addition, histone H3 lysine 9 methylation at, and recruitment of Swi6 and cohesin to, centromeric repeats is disrupted in these mutants. Thus the formation of silent chromatin on dg repeats and the development of a fully functional centromere is dependent on RNAi.

Estrogen-Independent Proliferation Is Present in Estrogen-Receptor HER2-Positive Primary Breast Cancer After Neoadjuvant Letrozole

PURPOSE To investigate the impact of human epidermal growth factor receptor (HER) 1 and HER2 gene amplification on endocrine therapy responsiveness, a fluorescence in situ hybridization (FISH) study was conducted on tumor samples from 305 postmenopausal patients with stage II and III estrogen receptor (ER) -positive (ER > or = 10%) breast cancers treated on two independent neoadjuvant endocrine therapy trials. PATIENTS AND METHODS FISH analysis focused on HER1 and/or HER2 immunohistochemistry (IHC) -positive patients and a random selection of HER1/2 IHC-negative patients. HER2 FISH status was correlated with response and changes in the proliferation marker Ki67. RESULTS HER1 was rarely amplified (< 1%), and HER2 amplification was observed in 9.2% of patients. Letrozole response by clinical measurement (71% HER2 FISH positive v 71% HER2 FISH negative), mammogram (44% HER2 FISH positive v 47% HER2 FISH negative), or ultrasound (47% HER2 FISH positive v 54% HER2 FISH negative) was not impaired by HER2 FISH-positive status. In contrast, HER2 FISH-positive tumors showed higher histologic grade (P = .009), higher pretreatment Ki67 (P = .005), and less Ki67 suppression after letrozole when compared with HER2 FISH-negative tumors (P = .0001). Similar observations regarding Ki67 were made in a smaller cohort of tamoxifen-treated tumors. CONCLUSION Neoadjuvant letrozole is clinically effective in ER-positive HER2 FISH-positive tumors, indicating sensitivity to short-term estrogen deprivation. However, continued proliferation despite ongoing letrozole or tamoxifen treatment in the majority of ER-positive HER2 FISH-positive samples (88%) could imply therapeutic resistance that may manifest later in the clinical course of the disease. Discordance between clinical and biomarker findings in this study serves to emphasize the need for surrogate end point validation in neoadjuvant endocrine trials through correlation with information on long-term outcomes.

Stc1: A Critical Link between RNAi and Chromatin Modification Required for Heterochromatin Integrity

Summary In fission yeast, RNAi directs heterochromatin formation at centromeres, telomeres, and the mating type locus. Noncoding RNAs transcribed from repeat elements generate siRNAs that are incorporated into the Argonaute-containing RITS complex and direct it to nascent homologous transcripts. This leads to recruitment of the CLRC complex, including the histone methyltransferase Clr4, promoting H3K9 methylation and heterochromatin formation. A key question is what mediates the recruitment of Clr4/CLRC to transcript-bound RITS. We have identified a LIM domain protein, Stc1, that is required for centromeric heterochromatin integrity. Our analyses show that Stc1 is specifically required to establish H3K9 methylation via RNAi, and interacts both with the RNAi effector Ago1, and with the chromatin-modifying CLRC complex. Moreover, tethering Stc1 to a euchromatic locus is sufficient to induce silencing and heterochromatin formation independently of RNAi. We conclude that Stc1 associates with RITS on centromeric transcripts and recruits CLRC, thereby coupling RNAi to chromatin modification.

Changes in breast cancer transcriptional profiles after treatment with the aromatase inhibitor, letrozole.

Objective The aim of the study was to identify changes in tumour expression profiling associated with short-term therapy of breast cancer patients with letrozole. Experimental design Microarray analysis was performed on RNA extracted from paired tumour core biopsies taken before and after 14 days of treatment with letrozole (2.5 mg/daily) in 58 patients. Changes in expression profile were identified by three different approaches on the basis of frequency of changes, magnitude of changes and significance analysis of microarray. Results No single gene was consistently changed by therapy in all cases. Fifty-two genes, however, were downregulated and 36 upregulated in at least 45 of the 58 cases. In terms of quantitative change, 46 genes showed at least a median 1.5-fold change in expression. Significance analysis of microarray identified 62 genes that were significantly changed by therapy (P<0.0001, 56 downregulated and six upregulated). All three approaches showed that greater numbers of genes were downregulated rather than upregulated. Merging data produced a total of 143 genes, which were subject to gene ontology and cluster analysis. The ontology of the 91 downregulated genes showed that they were functionally associated with cell cycle progression, particularly mitosis. In contrast, upregulated genes were associated with organ development, connective tissue extracellular matrix regulation and inflammatory response. Cluster analysis segregated the patients into four groups differing in patterns of gene expression. Conclusion Genes have been identified which either change markedly or consistently in breast cancer after 14 days treatment with letrozole. These are new important data in understanding letrozoles molecular mechanism of action in breast cancers.

Aromatase inhibitors—Gene discovery

Microarray analysis of tumour RNA is an extremely powerful tool which allows global gene expression to be measured. When used in combination with neoadjuvant treatment protocols in which therapy is given with the primary tumour within the breast, sequential biopsies may be analysed and results correlated with clinical and pathological response. In the present study, a neoadjuvant protocol has been used, administering the third generation inhibitor, letrozole, for 3 months and subjecting RNA extracted from biopsies taken before and after 10-14 days of treatment to microarray analysis. The objectives were to discover: (i) genes that change with estrogen deprivation (the only known biological effect of letrozole is to inhibit aromatase activity and reduce endogenous estrogens in postmenopausal women) and (ii) genes whose basal, on treatment or change in expression differ between tumours which are either responsive or resistant to treatment (so that predictive indices of response/resistance may be developed). Early changes in gene expression were identified by comparing paired tumour core biopsies taken before and after 14 days treatment in 58 patients using three different approaches based on frequency of changes, magnitude of changes and SAM analysis. All three approaches showed a greater number of genes were down-regulated than up-regulated. Merging of the data produced a total of 143 genes which were subject to gene ontology and cluster analysis. The ontology of the 91 down-regulated genes showed that they were functionally associated with cell cycle progression, particularly mitosis. In contrast, up-regulated genes were associated with organ development and extra-cellular matrix turnover and regulation. Clinical response was assessable in 52 patients; 37 (71%) tumours were classified as clinical responders (>50% reduction in volume at 3 months). Microarray analysis of pre- and 14-day biopsies identified 291 covariates (84 baselines, 72 14-day and 135 changes) highly predictive of response status. A similarity matrix using the covariates showed responding tumours have a similar genetic profile which was dissimilar to non-responding cancers whereas non-responsive cases were distinctive from each other. Changed genes predicting for response showed no concordance with those changed significantly by treatment in the overall group.

Raf1 is a DCAF for the Rik1 DDB1-like protein and has separable roles in siRNA generation and chromatin modification

Non-coding transcription can trigger histone post-translational modifications forming specialized chromatin. In fission yeast, heterochromatin formation requires RNAi and the histone H3K9 methyltransferase complex CLRC, composed of Clr4, Raf1, Raf2, Cul4, and Rik1. CLRC mediates H3K9 methylation and siRNA production; it also displays E3-ubiquitin ligase activity in vitro. DCAFs act as substrate receptors for E3 ligases and may couple ubiquitination with histone methylation. Here, structural alignment and mutation of signature WDxR motifs in Raf1 indicate that it is a DCAF for CLRC. We demonstrate that Raf1 promotes H3K9 methylation and siRNA amplification via two distinct, separable functions. The association of the DCAF Raf1 with Cul4-Rik1 is critical for H3K9 methylation, but dispensable for processing of centromeric transcripts into siRNAs. Thus the association of a DCAF, Raf1, with its adaptor, Rik1, is required for histone methylation and to allow RNAi to signal to chromatin.

Loss of Dicer fowls up centromeres

Centromeres, specialized regions on chromosomes, are essential for accurate chromosome segregation during cell division. In fission yeast, the RNA interference machinery has a pivotal function in the assembly of centromeric heterochromatin, which mediates sister centromere cohesion. Studies in vertebrate cells now suggest that many aspects of this process are conserved.

A systematic genetic screen identifies new factors influencing centromeric heterochromatin integrity in fission yeast.

BackgroundHeterochromatin plays important roles in the regulation and stability of eukaryotic genomes. Both heterochromatin components and pathways that promote heterochromatin assembly, including RNA interference, RNAi, are broadly conserved between the fission yeast Schizosaccharomyces pombe and humans. As a result, fission yeast has emerged as an important model system for dissecting mechanisms governing heterochromatin integrity. Thus far, over 50 proteins have been found to contribute to heterochromatin assembly at fission yeast centromeres. However, previous studies have not been exhaustive, and it is therefore likely that further factors remain to be identified.ResultsTo gain a more complete understanding of heterochromatin assembly pathways, we have performed a systematic genetic screen for factors required for centromeric heterochromatin integrity. In addition to known RNAi and chromatin modification components, we identified several proteins with previously undescribed roles in heterochromatin regulation. These included both known and newly characterised splicing-associated proteins, which are required for proper processing of centromeric transcripts by the RNAi pathway, and COP9 signalosome components Csn1 and Csn2, whose role in heterochromatin assembly can be explained at least in part by a role in the Ddb1-dependent degradation of the heterochromatin regulator Epe1.ConclusionsThis work has revealed new factors involved in RNAi-directed heterochromatin assembly in fission yeast. Our findings support and extend previous observations that implicate components of the splicing machinery as a platform for RNAi, and demonstrate a novel role for the COP9 signalosome in heterochromatin regulation.

DegrAAAded into Silence

In fission yeast, RNA interference (RNAi)-dependent heterochromatin formation silences transgenes inserted at centromeres. In this issue, Bühler et al. (2007) demonstrate that the RNAi machinery directly targets transgene transcripts. Furthermore, they link transgene silencing to a protein complex resembling the TRAMP complex of budding yeast, which promotes transcript degradation via the exosome. Thus, RNAi-independent transcript degradation may also contribute to heterochromatin gene silencing.