Human factors and pathways essential for mediating epigenetic gene silencing

Cellular identity in both normal and disease processes is determined by programmed epigenetic activation or silencing of specific gene subsets. Here, we have used human cells harboring epigenetically silent GFP-reporter genes to perform a genome-wide siRNA knockdown screen for the identification of cellular factors that are required to maintain epigenetic gene silencing. This unbiased screen interrogated 21,121 genes, and we identified and validated a set of 128 protein factors. This set showed enrichment for functional categories, and protein-protein interactions. Among this set were known epigenetic silencing factors, factors with no previously identified role in epigenetic gene silencing, as well as unstudied factors. The set included non-nuclear factors, for example, components of the integrin-adhesome. A key finding was that the E1 and E2 enzymes of the small ubiquitin-like modifier (SUMO) pathway (SAE1, SAE2/UBA2, UBC9/UBE2I) are essential for maintenance of epigenetic silencing. This work provides the first genome-wide functional view of human factors that mediate epigenetic gene silencing. The screen output identifies novel epigenetic factors, networks, and mechanisms, and provides a set of candidate targets for epigenetic therapy and cellular reprogramming.     

Human factors and pathways essential for mediating epigenetic gene silencing

Cellular identity in both normal and disease processes is determined by programmed epigenetic activation or silencing of specific gene subsets. Here, we have used human cells harboring epigenetically silent GFP-reporter genes to perform a genome-wide siRNA knockdown screen for the identification of cellular factors that are required to maintain epigenetic gene silencing. This unbiased screen interrogated 21,121 genes, and we identified and validated a set of 128 protein factors. This set showed enrichment for functional categories, and protein-protein interactions. Among this set were known epigenetic silencing factors, factors with no previously identified role in epigenetic gene silencing, as well as unstudied factors. The set included non-nuclear factors, for example, components of the integrin-adhesome. A key finding was that the E1 and E2 enzymes of the small ubiquitin-like modifier (SUMO) pathway (SAE1, SAE2/UBA2, UBC9/UBE2I) are essential for maintenance of epigenetic silencing. This work provides the first genome-wide functional view of human factors that mediate epigenetic gene silencing. The screen output identifies novel epigenetic factors, networks, and mechanisms, and provides a set of candidate targets for epigenetic therapy and cellular reprogramming.     

Dynamic and reversibility of heterochromatic gene silencing in human disease

MOLECULAR MECHANISM OF DNA METH- YLATION REACTION Among all epigenetic mechanisms involved in gene expression regulation, DNA methylation has been the most widely studied subject. DNA methylation results from the transfer of a methyl group from a methyl d…     

Emerging similarities in epigenetic gene silencing by long noncoding RNAs

Long noncoding RNAs (lncRNAs) such as Xist, Air, and Kcnq1ot1 are required for epigenetic silencing of multiple genes in cis within large chromosomal domains, including distant genes located hundreds of kilobase pairs away. Recent evidence suggests that all three of these lncRNAs are functional and that they silence gene expression, in part, through an intimate interaction with chromatin. Here we provide an overview of lncRNA-dependent gene silencing, focusing on recent findings for the Air and Kcnq1ot1 lncRNAs. We review molecular evidence indicating that these lncRNAs interact with chromatin and correlate their presence with specific histone modifications associated with gene silencing. A general model for a lncRNA-dependent gene-silencing mechanism is presented based on the apparent ability of lncRNAs to recruit histone-modifying activities to chromatin. However, alternate mechanisms may be required to explain the silencing of some lncRNA-dependent genes. Finally, we discuss unanswered questions and future perspectives associated with these enigmatic lncRNA molecules.     

Cyclin-dependent kinases regulate epigenetic gene silencing through phosphorylation of EZH2

The Polycomb group (PcG) protein, enhancer of zeste homologue 2 (EZH2), has an essential role in promoting histone H3 lysine 27 trimethylation (H3K27me3) and epigenetic gene silencing. This function of EZH2 is important for cell proliferation and inhibition of cell differentiation, and is implicated in cancer progression. Here, we demonstrate that under physiological conditions, cyclin-dependent kinase 1 (CDK1) and cyclin-dependent kinase 2 (CDK2) phosphorylate EZH2 at Thr 350 in an evolutionarily conserved motif. Phosphorylation of Thr 350 is important for recruitment of EZH2 and maintenance of H3K27me3 levels at EZH2-target loci. Blockage of Thr 350 phosphorylation not only diminishes the global effect of EZH2 on gene silencing, it also mitigates EZH2-mediated cell proliferation and migration. These results demonstrate that CDK-mediated phosphorylation is a key mechanism governing EZH2 function and that there is a link between the cell-cycle machinery and epigenetic gene silencing.     

Inheritance of epigenetic chromatin silencing

Maintenance of alternative chromatin states through cell divisions pose some fundamental constraints on the dynamics of histone modifications. In this paper, we study the systems biology of epigenetic inheritance by defining and analyzing general classes of mathematical models. We discuss how the number of modification states involved plays an essential role in the stability of epigenetic states. In addition, DNA duplication and the consequent dilution of marked histones act as a large perturbation for a stable state of histone modifications. The requirement that this large perturbation falls into the basin of attraction of the original state sometimes leads to additional constraints on effective models. Two such models, inspired by two different biological systems, are compared in their fulfilling the requirements of multistability and of recovery after DNA duplication. We conclude that in the presence of multiple histone modifications that characterize alternative epigenetic stable states, these requirements are more easily fulfilled.     

tRNA genes protect a reporter gene from epigenetic silencing in mouse cells

It is a well-established fact that the tRNA genes in yeast can function as chromatin barrier elements. However, so far there is no experimental evidence that tRNA and other Pol III-transcribed genes exhibit barrier activity in mammals. This study utilizes a recently developed reporter gene assay to test a set of Pol III-transcribed genes and gene clusters with variable promoter and intergenic regions for their ability to prevent heterochromatin-mediated reporter gene silencing in mouse cells. The results show that functional copies of mouse tRNA genes are effective barrier elements. The number of tRNA genes as well as their orientation influence barrier function. Furthermore, the DNA sequence composition of intervening and flanking regions affects barrier activity of tRNA genes. Barrier activity was maintained for much longer time when the intervening and flanking regions of tRNA genes were replaced by AT-rich sequences, suggesting a negative role of DNA methylation in the establishment of a functional barrier. Thus, our results suggest that tRNA genes are essential elements in establishment and maintenance of chromatin domain architecture in mammalian cells.Key words: barrier elements, tRNA genes, Pol III-transcribed genes     

tRNA genes protect a reporter gene from epigenetic silencing in mouse cells

It is a well-established fact that the tRNA genes in yeast can function as chromatin barrier elements. However, so far there is no experimental evidence that tRNA and other Pol III-transcribed genes exhibit barrier activity in mammals. This study utilizes a recently developed reporter gene assay to test a set of Pol III-transcribed genes and gene clusters with variable promoter and intergenic regions for their ability to prevent heterochromatin-mediated reporter gene silencing in mouse cells. The results show that functional copies of mouse tRNA genes are effective barrier elements. The number of tRNA genes as well as their orientation influence barrier function. Furthermore, the DNA sequence composition of intervening and flanking regions affects barrier activity of tRNA genes. Barrier activity was maintained for much longer time when the intervening and flanking regions of tRNA genes were replaced by AT-rich sequences, suggesting a negative role of DNA methylation in the establishment of a functional barrier. Thus, our results suggest that tRNA genes are essential elements in establishment and maintenance of chromatin domain architecture in mammalian cells.