Yesu Jeon

A p53-Dependent Checkpoint Pathway Prevents Rereplication

Eukaryotic cells control the initiation of DNA replication so that origins that have fired once in S phase do not fire a second time within the same cell cycle. Failure to exert this control leads to genetic instability. Here we investigate how rereplication is prevented in normal mammalian cells and how these mechanisms might be overcome during tumor progression. Overexpression of the replication initiation factors Cdt1 and Cdc6 along with cyclin A-cdk2 promotes rereplication in human cancer cells with inactive p53 but not in cells with functional p53. A subset of origins distributed throughout the genome refire within 2-4 hr of the first cycle of replication. Induction of rereplication activates p53 through the ATM/ATR/Chk2 DNA damage checkpoint pathways. p53 inhibits rereplication through the induction of the cdk2 inhibitor p21. Therefore, a p53-dependent checkpoint pathway is activated to suppress rereplication and promote genetic stability.

Jpx RNA Activates Xist by Evicting CTCF

In mammals, dosage compensation between XX and XY individuals occurs through X chromosome inactivation (XCI). The noncoding Xist RNA is expressed and initiates XCI only when more than one X chromosome is present. Current models invoke a dependency on the X-to-autosome ratio (X:A), but molecular factors remain poorly defined. Here, we demonstrate that molecular titration between an X-encoded RNA and an autosomally encoded protein dictates Xist induction. In pre-XCI cells, CTCF protein represses Xist transcription. At the onset of XCI, Jpx RNA is upregulated, binds CTCF, and extricates CTCF from one Xist allele. We demonstrate that CTCF is an RNA-binding protein and is titrated away from the Xist promoter by Jpx RNA. Thus, Jpx activates Xist by evicting CTCF. The functional antagonism via molecular titration reveals a role for long noncoding RNA in epigenetic regulation.

New and Xisting regulatory mechanisms of X chromosome inactivation.

Equalization of X linked gene expression is necessary in mammalian cells due to the presence of two X chromosomes in females and one in males. To achieve this, all female cells inactivate one of the two X chromosomes during development. This process, termed X chromosome inactivation (XCI), is a quintessential epigenetic phenomenon and involves a complex interplay between noncoding RNAs and protein factors. Progress in this area of study has consequently resulted in new approaches to study epigenetics and regulatory RNA function. Here we will discuss recent developments in the field that have advanced our understanding of XCI and its regulatory mechanisms.

Human TopBP1 Participates in Cyclin E/CDK2 Activation and Preinitiation Complex Assembly during G1/S Transition

Human TopBP1 with eight BRCA1 C terminus domains has been mainly reported to be involved in DNA damage response pathways. Here we show that TopBP1 is also required for G1 to S progression in a normal cell cycle. TopBP1 deficiency inhibited cells from entering S phase by up-regulating p21 and p27, resulting in down-regulation of cyclin E/CDK2. Although co-depletion of p21 and p27 with TopBP1 restored the cyclin E/CDK2 kinase activity, however, cells remained arrested at the G1/S boundary, showing defective chromatin-loading of replication components. Based on these results, we suggest a dual role of TopBP1 necessary for the G1/S transition: one for activating cyclin E/CDK2 kinase and the other for loading replication components onto chromatin to initiate DNA synthesis.

Repeat E anchors Xist RNA to the inactive X chromosomal compartment through CDKN1A-interacting protein (CIZ1)

Significance The long noncoding Xist RNA coats and silences one X chromosome in female cells. How Xist localizes in cis to the inactive X compartment is not clear. Here, we reveal a required interaction between CIZ1 protein and Xist Repeat E motifs. Stochastic optical reconstruction microscopy (STORM) shows a tight association of CIZ1 with Xist RNA at the single-molecule level. Deletion of either CIZ1 or Repeat E causes dispersal of Xist RNA throughout the nucleoplasm, as well as loss of the heterochromatin mark H3K27me3 from the inactive X chromosome. We have thus identified a critical factor for stable association of Xist RNA with the inactive X chromosome. X chromosome inactivation is an epigenetic dosage compensation mechanism in female mammals driven by the long noncoding RNA, Xist. Although recent genomic and proteomic approaches have provided a more global view of Xist’s function, how Xist RNA localizes to the inactive X chromosome (Xi) and spreads in cis remains unclear. Here, we report that the CDKN1-interacting zinc finger protein CIZ1 is critical for localization of Xist RNA to the Xi chromosome territory. Stochastic optical reconstruction microscopy (STORM) shows a tight association of CIZ1 with Xist RNA at the single-molecule level. CIZ1 interacts with a specific region within Xist exon 7–namely, the highly repetitive Repeat E motif. Using genetic analysis, we show that loss of CIZ1 or deletion of Repeat E in female cells phenocopies one another in causing Xist RNA to delocalize from the Xi and disperse into the nucleoplasm. Interestingly, this interaction is exquisitely sensitive to CIZ1 levels, as overexpression of CIZ1 likewise results in Xist delocalization. As a consequence, this delocalization is accompanied by a decrease in H3K27me3 on the Xi. Our data reveal that CIZ1 plays a major role in ensuring stable association of Xist RNA within the Xi territory.

Comment on “Xist recruits the X chromosome to the nuclear lamina to enable chromosome-wide silencing”

Chen et al. (Reports, 28 October 2016, p. 468) proposed that an interaction between Xist RNA and Lamin B receptor (LBR) is necessary and sufficient for Xist spreading during X-chromosome inactivation. We reanalyzed their data and found that reported genotypes of mutants are not supported by the sequencing data. These inconsistencies preclude assessment of the role of LBR in Xist spreading.

Genome-wide identification of autosomal genes with allelic imbalance of chromatin state

In mammals, monoallelic gene expression can result from X-chromosome inactivation, genomic imprinting, and random monoallelic expression (RMAE). Epigenetic regulation of RMAE is not fully understood. Here we analyze allelic imbalance in chromatin state of autosomal genes using ChIP-seq in a clonal cell line. We identify approximately 3.7% of autosomal genes that show significant differences between chromatin states of two alleles. Allelic regulation is represented among several functional gene categories including histones, chromatin modifiers, and multiple early developmental regulators. Most cases of allelic skew are produced by quantitative differences between two allelic chromatic states that belong to the same gross type (active, silent, or bivalent). Combinations of allelic states of different types are possible but less frequent. When different chromatin marks are skewed on the same gene, their skew is coordinated as a result of quantitative relationships between these marks on each individual allele. Finally, combination of allele-specific densities of chromatin marks is a quantitative predictor of allelic skew in gene expression.

Abstract IA10: Spreading of X-chromosome inactivation via a hierarchy of defined Polycomb stations

Abstract X-chromosome inactivation (XCI) achieves dosage balance in mammals by repressing one of two X-chromosomes in females. During XCI, the long noncoding Xist RNA and Polycomb proteins spread along the inactive X (Xi) to initiate chromosome-wide silencing. Although inactivation is known to commence at the X-inactivation center (Xic), how it propagates remains unknown. We have examined allele-specific binding of Polycomb repressive complex 2 (PRC2) and chromatin composition during XCI, and generate a chromosome-wide profile of Xi and Xa (active X) at nucleosome-resolution. Initially, Polycomb proteins are localized to a limited number of sites along the X. As Xist RNA spreads in cis during the XCI process, thousands of additional sites are recruited. PRC2 and H3K27 methylation spread along a gradient in an Xi-specific manner not seen on autosomes. Our findings suggest that XCI is governed by a hierarchy of defined Polycomb stations that spread H3K27 methylation in cis. Citation Format: Stefan F. Pinter, Yesu Jeon, Toshiro K. Ohsumi, Mark Borowsky, Jeannie T. Lee, Ruslan I. Sadreyev, Eda Yildirim. Spreading of X-chromosome inactivation via a hierarchy of defined Polycomb stations [abstract]. In: Proceedings of the AACR Special Conference on Noncoding RNAs and Cancer; 2012 Jan 8-11; Miami Beach, FL. Philadelphia (PA): AACR; Cancer Res 2012;72(2 Suppl):Abstract nr IA10.