The centromere is a complex structure, the components and assembly pathway of which remain inadequately defined. Here, we demonstrate that centromeric ␣-satellite RNA and proteins CENPC1 and INCENP accumulate in the human interphase nucleolus in an RNA polymerase I-dependent manner. The nucleolar targeting of CENPC1 and INCENP requires ␣-satellite RNA, as evident from the delocalization of both proteins from the nucleolus in RNase-treated cells, and the nucleolar relocalization of these proteins following ␣-satellite RNA replenishment in these cells. Using protein truncation and in vitro mutagenesis, we have identified the nucleolar localization sequences on CENPC1 and INCENP. We present evidence that CENPC1 is an RNA-associating protein that binds ␣-satellite RNA by an in vitro binding assay. Using chromatin immunoprecipitation, RNase treatment, and "RNA replenishment" experiments, we show that ␣-satellite RNA is a key component in the assembly of CENPC1, INCENP, and survivin (an INCENP-interacting protein) at the metaphase centromere. Our data suggest that centromere satellite RNA directly facilitates the accumulation and assembly of centromere-specific nucleoprotein components at the nucleolus and mitotic centromere, and that the sequestration of these components in the interphase nucleolus provides a regulatory mechanism for their timely release into the nucleoplasm for kinetochore assembly at the onset of mitosis.[Supplemental material is available online at www.genome.org.]The centromere is a specialized structure on chromosomes for microtubule attachment to ensure the equal partitioning of chromosomes during cell division. This structure comprises two defined domains: the central core for the assembly of the kinetochore and the flanking pericentric heterochromatin for centromere cohesion. In Schizosaccharomyces pombe, the outer centromeric repeat sequences give rise to small interfering RNAs (siRNA) that participate in chromatin repression (Volpe et al. 2002). The depletion of Dicer (a nuclease required for the processing of siRNAs) in a chicken cell line leads to the disruption of heterochromatin assembly and cohesion (Fukagawa et al. 2004). However, Dicer depletion has no observable effect on the binding of the core kinetochore proteins CENPA and CENPC1 (Fukagawa et al. 2004), indicating that while the evolutionarily conserved RNA interference (RNAi) machinery is crucial for the establishment of the pericentric heterochromatin, it may not be essential for the core kinetochore region. A recent study in maize has further described the association of single-stranded centromeric transposable element and repeat RNA with the core kinetochore complex that is distinct from those at pericentric heterochromatin (Topp et al. 2004); however, the functional significance of the observed centromere RNA transcripts is unclear. Furthermore, little is known about the subnuclear distribution of centromere RNA, and the pathway and significance, if any, of such RNA in kinetochore formation and function.The nucleolus is a sp...

We have previously identified and characterized the phenomenon of ectopic human centromeres, known as neocentromeres. Human neocentromeres form epigenetically at euchromatic chromosomal sites and are structurally and functionally similar to normal human centromeres. Recent studies have indicated that neocentromere formation provides a major mechanism for centromere repositioning, karyotype evolution, and speciation. Using a marker chromosome mardel(10) containing a neocentromere formed at the normal chromosomal 10q25 region, we have previously mapped a 330-kb CENP-A–binding domain and described an increased prevalence of L1 retrotransposons in the underlying DNA sequences of the CENP-A–binding clusters. Here, we investigated the potential role of the L1 retrotransposons in the regulation of neocentromere activity. Determination of the transcriptional activity of a panel of full-length L1s (FL-L1s) across a 6-Mb region spanning the 10q25 neocentromere chromatin identified one of the FL-L1 retrotransposons, designated FL-L1b and residing centrally within the CENP-A–binding clusters, to be transcriptionally active. We demonstrated the direct incorporation of the FL-L1b RNA transcripts into the CENP-A–associated chromatin. RNAi-mediated knockdown of the FL-L1b RNA transcripts led to a reduction in CENP-A binding and an impaired mitotic function of the 10q25 neocentromere. These results indicate that LINE retrotransposon RNA is a previously undescribed essential structural and functional component of the neocentromeric chromatin and that retrotransposable elements may serve as a critical epigenetic determinant in the chromatin remodelling events leading to neocentromere formation.

Purpose: Patients presenting with locally advanced rectal cancer currently receive preoperative radiotherapy with or without chemotherapy. Although pathologic complete response is achieved for approximately 10% to 30% of patients, a proportion of patients derive no benefit from this therapy while being exposed to toxic side effects of treatment. Therefore, there is a strong need to identify patients who are unlikely to benefit from neoadjuvant therapy to help direct them toward alternate and ultimately more successful treatment options.Experimental Design: In this study, we obtained expression profiles from pretreatment biopsies for 51 rectal cancer patients. All patients underwent preoperative chemoradiotherapy, followed by resection of the tumor 6 to 8 weeks posttreatment. Gene expression and response to treatment were correlated, and a supervised learning algorithm was used to generate an original predictive classifier and validate previously published classifiers.Results: Novel predictive classifiers based on Mandard's tumor regression grade, metabolic response, TNM (tumor node metastasis) downstaging, and normal tissue expression profiles were generated. Because there were only 7 patients who had minimal treatment response (>80% residual tumor), expression profiles were used to predict good tumor response and outcome. These classifiers peaked at 82% sensitivity and 89% specificity; however, classifiers with the highest sensitivity had poor specificity, and vice versa. Validation of predictive classifiers from previously published reports was attempted using this cohort; however, sensitivity and specificity ranged from 21% to 70%.Conclusions: These results show that the clinical utility of microarrays in predictive medicine is not yet within reach for rectal cancer and alternatives to microarrays should be considered for predictive studies in rectal adenocarcinoma. Clin Cancer Res; 17(9); 3039-47. Ó2011 AACR.

Granulocyte-macrophage colony stimulating factor (GM-CSF) plays a key role in myeloid cell function and is rapidly and transiently expressed in T cells in response to immune or inflammatory stimuli. Induction of GM-CSF gene expression is accompanied by changes in chromatin structure across the proximal promoter region of the gene. We show that the promoter remodelling and subsequent gene transcription occurs with distinct signal and transcription factor requirements. Activation of the protein kinase C (PKC) signalling pathway is sufficient to induce changes in chromatin structure across the promoter, but both the PKC and calcium signalling pathways are required for efficient gene transcription. Although NFAT transcription factors contribute to GM-CSF gene transcription, they are not required for promoter remodelling. However, the presence of the nuclear factor-κB transcription factor, c-Rel, in the nucleus is strongly correlated with and required for the events of chromatin remodelling.

BackgroundRadiotherapy is a chosen treatment option for prostate cancer patients and while some tumours respond well, up to 50% of patients may experience tumour recurrence. Identification of functionally relevant predictive biomarkers for radioresponse in prostate cancer would enable radioresistant patients to be directed to more appropriate treatment options, avoiding the side-effects of radiotherapy.MethodsUsing an in vitro model to screen for novel biomarkers of radioresistance, transcriptome analysis of a radioresistant (PC-3) and radiosensitive (LNCaP) prostate cancer cell line was performed. Following pathway analysis candidate genes were validated using qRT-PCR. The DNA repair pathway in radioresistant PC-3 cells was then targeted for radiation sensitization using the PARP inhibitor, niacinimide.ResultsOpposing regulation of a DNA repair and replication pathway was observed between PC-3 and LNCaP cells from RNA-seq analysis. Candidate genes BRCA1, RAD51, FANCG, MCM7, CDC6 and ORC1 were identified as being significantly differentially regulated post-irradiation. qRT-PCR validation confirmed BRCA1, RAD51 and FANCG as being significantly differentially regulated at 24 hours post radiotherapy (p-value =0.003, 0.045 and 0.003 respectively). While the radiosensitive LNCaP cells down-regulated BRCA1, FANCG and RAD51, the radioresistant PC-3 cell line up-regulated these candidates to promote cell survival post-radiotherapy and a similar trend was observed for MCM7, CDC6 and ORC1. Inhibition of DNA repair using niacinamide sensitised the radioresistant cells to irradiation, reducing cell survival at 2 Gy from 66% to 44.3% (p-value =0.02).ConclusionsThese findings suggest that the DNA repair candidates identified via RNA-seq hold potential as both targets for radiation sensitization and predictive biomarkers in prostate cancer.Electronic supplementary materialThe online version of this article (doi:10.1186/1471-2407-14-808) contains supplementary material, which is available to authorized users.

The genome has the ability to respond in a precise and co-ordinated manner to cellular signals. It achieves this through the concerted actions of transcription factors and the chromatin platform, which are targets of the signaling pathways. Our understanding of the molecular mechanisms through which transcription factors and the chromatin landscape each control gene activity has expanded dramatically over recent years, and attention has now turned to understanding the complex, multifaceted interplay between these regulatory layers in normal and disease states. It has become apparent that transcription factors as well as the components and modifiers of the epigenetic machinery are frequent targets of genomic alterations in cancer cells. Through the study of these factors, we can gain unique insight into the dynamic interplay between transcription factors and the epigenome, and how their dysregulation leads to aberrant gene expression programs in cancer. Here, we will highlight how these factors normally co-operate to establish and maintain the transcriptional and epigenetic landscape of cells, and how this is reprogramed in cancer, focusing on the RUNX1 transcription factor and oncogenic derivative RUNX1–ETO in leukemia as paradigms of transcriptional and epigenetic reprograming.

Cancer treatment is now moving toward a personalized approach, promising improved rates of response and survival. A number of studies have employed the use of microarrays to investigate the predictive potential of expression profiling in gastrointestinal (GI) cancer patients. However while many robust predictive classifiers relating to response and prognosis have been generated for GI cancer patients, these have yet to make the transition to the clinic. The main obstacle is the limited cross validation between predictive gene lists identified for the same tumor type and outcome. Differences in the experimental design, analysis, and interpretation of results all contribute to this variation, with numerous factors influencing which genes are highlighted as predictive. While predictive genomics shows immense potential, it is still a relatively new field and the validation of predictive gene lists derived from microarray data remains a challenge. Future studies must carefully consider all aspects of experimental design to ensure a clinically applicable predictive test can be developed. With this in mind, more extensive and collaborative research must be undertaken before microarray-based platforms can be used routinely in tailoring GI cancer treatment and change clinical practice. Larger cohorts and consistency in methodology will enable the findings from this research to make the transition to the clinic.

Granulocyte macrophage-colony stimulating factor (GM-CSF) is produced by T cells, but not B cells, in response to immune signals. GM-CSF gene activation in response to T-cell stimulation requires remodelling of chromatin associated with the gene promoter, and these changes do not occur in B cells. While the CpG methylation status of the murine GM-CSF promoter shows no correlation with the ability of the gene to respond to activation, we find that the basal chromatin environment of the gene promoter influences its ability to respond to immune signals. In unstimulated T cells but not B cells, the GM-CSF promoter is selectively marked by enrichment of histone acetylation, and association of the chromatin-remodelling protein BRG1. BRG1 is removed from the promoter upon activation concomitant with histone depletion and BRG1 is required for efficient chromatin remodelling and transcription. Increasing histone acetylation at the promoter in T cells is paralleled by increased BRG1 recruitment, resulting in more rapid chromatin remodelling, and an associated increase in GM-CSF mRNA levels. Furthermore, increasing histone acetylation in B cells removes the block in chromatin remodelling and transcriptional activation of the GM-CSF gene. These data are consistent with a model in which histone hyperacetylation and BRG1 enrichment at the GM-CSF promoter, generate a chromatin environment competent to respond to immune signals resulting in gene activation.