Maintenance of Constitutive and Inactive X Heterochromatin in Cancer and a Link to BRCA1: A Dissertation

The development of cancer is a multi-step process which involves a series of events, including activation of oncogenes and loss of tumor suppressor function, leading to cell immortalization and misregulated proliferation. In the last few years, the importance of epigenetic defects in cancer development has become increasingly recognized. While most epigenetic studies focus on silencing of tumor suppressors, this thesis addresses defects in the maintenance of silenced heterochromatin in cancer, particularly breast cancer. Breast cancer is a leading cause of cancer in women and many familial cases have been linked to mutations in the breast cancer susceptibility genes, BRCA1 and BRCA2. BRCA1 has been linked to DNA repair as well as multiple other cellular processes, including cell cycle checkpoints, ubiquitination, centrosome function, and meiotic silencing of the XY body. This work began with a particular interest in the report that BRCA1 was linked to the failed maintenance of random X-inactivation in female somatic cells, via a role in supporting XIST RNA localization to the inactive X chromosome (Xi). XIST RNA is a non-coding RNA that fully coats or “paints” the Xi and induces its silencing. Work presented in Chapter II substantially clarifies the relationship of BRCA1 to XIST RNA, based on several lines of experimentation. Loss of BRCA1 does not lead to loss of XIST RNA in these studies, nor did reconstitution of HCC1937 BRCA1-/- tumor cells with BRCA1 lead to XIST RNA localization on Xi, although an effect on XIST RNA transcription is possible. Studies of BRCA1 localization with Xi showed that BRCA1 has a limited association with the Xi in ~3-10% of cells, it rarely colocalizes with XIST RNA to a significant extent, but rather is in close apposition to a small part of the XIST RNA/Xi territory. Additionally, analysis of several breast cancer cell lines revealed mislocalization of XIST RNA in some breast cancer cell lines. Many studies have examined BRCA1 foci that form following DNA damage and demonstrated that these are sites of repair. However, whether the numerous large foci consistently present in normal S-phase nuclei were storage sites or had any function was unknown. In Chapter III, I demonstrate that the BRCA1 foci in normal S-phase nuclei associate overwhelmingly with specific heterochromatic regions of the genome. More specifically, BRCA1 foci often associate with centromeric or pericentromeric regions in both human and mouse cells. In human cells BRCA1 foci often appear juxtaposed to centromeric signal, whereas in mouse, BRCA1 often rings or paints the large chromocenters, clusters of DAPI-dense pericentric and centric heterochromatin. Using PCNA and BrdU as markers of replication, I demonstrate that BRCA1 preferentially associates with the chromocenters during their replication, although high-resolution analysis indicates that BRCA1 and PCNA foci rarely directly overlap. Interestingly, cells with defects in BRCA1 were found to have lagging chromosomes and DNA bridges which nearly always contained satellite DNA, which is consistent with the possibility that BRCA1 deficit contributes to failed separation of sister chromatids at the centromere. This is consistent with other recent reports that BRCA1 is necessary for DNA decatenation by topoisomerase II during routine replication and with my demonstration that topoisomerase II also accumulates on pericentric heterochromatin (PCH) during replication. Chapter IV presents recent work which reveals that RNA is commonly expressed from the centric/pericentric heterochromatin and appears to be linked to its replication. In mouse cells RNA from heterochromatic sequences is readily detected using a broad molecular cytological assay for repeat transcription (the COT-1 RNA assay). In addition to a more dispersed nucleoplasmic signal from euchromatic nuclear regions, distinct localized foci of repeat RNA are detected with COT1 probe or pancentromeric probe. Further analysis with the minor satellite (centromere proper) and the major satellite (comprising the larger pericentric heterochromatin) reveals that the large RNA foci often contain these satellite sequences, long thought to be essentially silent. These foci generally associate with the PCH of chromocenters, and produce various patterns similar to BRCA1- including a larger signal partially painting or ringing the chromocenter in a fraction of cells. In conjunction again with PCNA staining, it was possible to determine that the major satellite RNAs associate with the chromocenters during replication. While the satellite RNA co-localizes precisely with PCNA, neither of these co-localizes at high resolution with BRCA1, although they all are present on replicating chromocenters contemporaneously. These findings show that satellite RNAs are more widely expressed in normal cells than previously thought and link their expression to replication of centromere-linked heterochromatin. Finally, Chapter V presents three lines of recent results to support a major concept forwarded in this manuscript: that loss of Xi heterochromatin may reflect defects in the broader heterochromatic compartment, which may be manifest at multiple levels. I provide evidence using two new assays that both the peripheral heterochromatic compartment and the expression and silencing of satellite repeats is commonly compromised in cancer, although this appears to vary among cancer lines or types. The final results connect back to the question with which I began: what maintains XIST RNA localization to the chromosome in normal cells. These results demonstrate for the first time that Aurora B Kinase activity, mediated by Protein Phosphatase 1 (PP1) during interphase, controls the interphase retention and mitotic release of XIST RNA from the chromosome, likely linked to chromatin modifications such as H3Ser10 phosphorylation. As Aurora B Kinase is commonly over-expressed in cancer and is linked to chromatin changes, this exemplifies one type of mechanism whereby broad epigenetic changes in cancer may impact XIST RNA localization and the maintenance of heterochromatin more generally. This thesis represents a melding of cancer biology with the study of X inactivation and heterochromatin, with findings of fundamental interest to both of these fields

BRCA1 does not paint the inactive X to localize XIST RNA but may contribute to broad changes in cancer that impact XIST and Xi heterochromatin

The BRCA1 tumor suppressor involved in breast and ovarian cancer is linked to several fundamental cell regulatory processes. Recently, it was reported that BRCA1 supports localization of XIST RNA to the inactive X chromosome (Xi) in women. The apparent cytological overlap between BRCA1 and XIST RNA across the Xi raised the possibility a direct role of BRCA1 in localizing XIST. We report here that BRCA1 does not paint the Xi or XIST territory, as do markers of Xi facultative heterochromatin. A smaller BRCA1 accumulation abuts Xi, although this is not exclusive to Xi. In BRCA1 depleted normal and tumor cells, or BRCA1 reconstituted cells, BRCA1 status does not closely correlate with XIST localization, however in a BRCA1 inducible system over-expression correlated strongly with enhanced XIST expression. We confirm frequent loss of an Xi in tumor cells. In addition to mitotic loss of Xi, we find XIST RNA expression or localization frequently become compromised in cultured breast cancer cells, suggesting Xi heterochromatin may not be fully maintained. We demonstrate that complex epigenetic differences between tumor cell subpopulations can have striking effects on XIST transcription, accumulation, and localization, but this does not strictly correlate with BRCA1. Although BRCA1 can have indirect effects that impact XIST, our results do not indicate a direct and specific role in XIST RNA regulation. Rather, regulatory factors such as BRCA1 that have broad effects on chromatin or gene regulation can impact XIST RNA and the Xi. We provide preliminary evidence that this may occur as part of a wider failure of heterochromatin maintenance in some cancers

The disappearing Barr body in breast and ovarian cancers

Interest has recently reawakened in whether loss of the heterochromatic X chromosome (Barr body) is prevalent in certain breast and ovarian cancers, and new insights into the mechanisms involved have emerged. Mitotic segregation errors commonly explain the loss of the inactive X chromosome (Xi), but compromise of Xi heterochromatin in some cancers may signal broader deficits of nuclear heterochromatin. The debated link between BRCA1 and Xi might reflect a general relationship between BRCA1 and heterochromatin, which could connect BRCA1 to both epigenetic and genetic instability. We suggest that heterochromatic instability is a common but largely unexplored mechanism, leading to widespread genomic misregulation and the evolution of some cancers

Aberrant silencing of cancer-related genes by CpG hypermethylation occurs independently of their spatial organization in the nucleus

Aberrant promoter DNA-hypermethylation and repressive chromatin constitutes a frequent mechanism of gene inactivation in cancer. There is great interest in dissecting the mechanisms underlying this abnormal silencing. Studies have shown changes in the nuclear organization of chromatin in tumor cells as well as the association of aberrant methylation with long-range silencing of neighboring genes. Furthermore, certain tumors show a high incidence of promoter methylation termed as the CpG island methylator phenotype. Here, we have analyzed the role of nuclear chromatin architecture for genes in hypermethylated inactive versus nonmethylated active states and its relation with long-range silencing and CpG island methylator phenotype. Using combined immunostaining for active/repressive chromatin marks and fluorescence in situ hybridization in colorectal cancer cell lines, we show that aberrant silencing of these genes occurs without requirement for their being positioned at heterochromatic domains. Importantly, hypermethylation, even when associated with long-range epigenetic silencing of neighboring genes, occurs independent of their euchromatic or heterochromatic location. Together, these results indicate that, in cancer, extensive changes around promoter chromatin of individual genes or gene clusters could potentially occur locally without preference for nuclear position and/or causing repositioning. These findings have important implications for understanding relationships between nuclear organization and gene expression patterns in cancer