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1.
Stem Cell Reports ; 18(11): 2154-2173, 2023 11 14.
Article in English | MEDLINE | ID: mdl-37802073

ABSTRACT

Our understanding of how STAG proteins contribute to cell identity and disease have largely been studied from the perspective of chromosome topology and protein-coding gene expression. Here, we show that STAG1 is the dominant paralog in mouse embryonic stem cells (mESCs) and is required for pluripotency. mESCs express a wide diversity of naturally occurring Stag1 isoforms, resulting in complex regulation of both the levels of STAG paralogs and the proportion of their unique terminal ends. Skewing the balance of these isoforms impacts cell identity. We define a novel role for STAG1, in particular its N-terminus, in regulating repeat expression, nucleolar integrity, and repression of the two-cell (2C) state to maintain mESC identity. Our results move beyond protein-coding gene regulation via chromatin loops to new roles for STAG1 in nucleolar structure and function, and offer fresh perspectives on how STAG proteins, known to be cancer targets, contribute to cell identity and disease.


Subject(s)
Mouse Embryonic Stem Cells , Neoplasms , Animals , Mice , Cell Differentiation , Chromatin/genetics , Chromatin/metabolism , Mouse Embryonic Stem Cells/metabolism , Neoplasms/metabolism , Protein Isoforms/genetics , Protein Isoforms/metabolism
2.
Elife ; 122023 04 03.
Article in English | MEDLINE | ID: mdl-37010886

ABSTRACT

Most studies of cohesin function consider the Stromalin Antigen (STAG/SA) proteins as core complex members given their ubiquitous interaction with the cohesin ring. Here, we provide functional data to support the notion that the SA subunit is not a mere passenger in this structure, but instead plays a key role in the localization of cohesin to diverse biological processes and promotes loading of the complex at these sites. We show that in cells acutely depleted for RAD21, SA proteins remain bound to chromatin, cluster in 3D and interact with CTCF, as well as with a wide range of RNA binding proteins involved in multiple RNA processing mechanisms. Accordingly, SA proteins interact with RNA, and R-loops, even in the absence of cohesin. Our results place SA1 on chromatin upstream of the cohesin ring and reveal a role for SA1 in cohesin loading which is independent of NIPBL, the canonical cohesin loader. We propose that SA1 takes advantage of structural R-loop platforms to link cohesin loading and chromatin structure with diverse functions. Since SA proteins are pan-cancer targets, and R-loops play an increasingly prevalent role in cancer biology, our results have important implications for the mechanistic understanding of SA proteins in cancer and disease.


Subject(s)
R-Loop Structures , RNA , RNA/metabolism , Chromosomal Proteins, Non-Histone/metabolism , Cell Cycle Proteins/metabolism , Chromatin , CCCTC-Binding Factor/metabolism , Cohesins
3.
Nat Commun ; 10(1): 2908, 2019 07 02.
Article in English | MEDLINE | ID: mdl-31266948

ABSTRACT

Cohesin and CTCF are master regulators of genome topology. How these ubiquitous proteins contribute to cell-type specific genome structure is poorly understood. Here, we explore quantitative aspects of topologically associated domains (TAD) between pluripotent embryonic stem cells (ESC) and lineage-committed cells. ESCs exhibit permissive topological configurations which manifest themselves as increased inter- TAD interactions, weaker intra-TAD interactions, and a unique intra-TAD connectivity whereby one border makes pervasive interactions throughout the domain. Such 'stripe' domains are associated with both poised and active chromatin landscapes and transcription is not a key determinant of their structure. By tracking the developmental dynamics of stripe domains, we show that stripe formation is linked to the functional state of the cell through cohesin loading at lineage-specific enhancers and developmental control of CTCF binding site occupancy. We propose that the unique topological configuration of stripe domains represents a permissive landscape facilitating both productive and opportunistic gene regulation and is important for cellular identity.


Subject(s)
CCCTC-Binding Factor/chemistry , CCCTC-Binding Factor/metabolism , Enhancer Elements, Genetic , Pluripotent Stem Cells/metabolism , CCCTC-Binding Factor/genetics , Cell Cycle Proteins/chemistry , Cell Cycle Proteins/genetics , Cell Cycle Proteins/metabolism , Cell Lineage , Chromatin/chemistry , Chromatin/genetics , Chromatin/metabolism , Chromosomal Proteins, Non-Histone/chemistry , Chromosomal Proteins, Non-Histone/genetics , Chromosomal Proteins, Non-Histone/metabolism , Pluripotent Stem Cells/chemistry , Protein Binding , Protein Domains , Species Specificity , Cohesins
4.
Nat Genet ; 50(10): 1388-1398, 2018 10.
Article in English | MEDLINE | ID: mdl-30202056

ABSTRACT

Structural variants (SVs) can contribute to oncogenesis through a variety of mechanisms. Despite their importance, the identification of SVs in cancer genomes remains challenging. Here, we present a framework that integrates optical mapping, high-throughput chromosome conformation capture (Hi-C), and whole-genome sequencing to systematically detect SVs in a variety of normal or cancer samples and cell lines. We identify the unique strengths of each method and demonstrate that only integrative approaches can comprehensively identify SVs in the genome. By combining Hi-C and optical mapping, we resolve complex SVs and phase multiple SV events to a single haplotype. Furthermore, we observe widespread structural variation events affecting the functions of noncoding sequences, including the deletion of distal regulatory sequences, alteration of DNA replication timing, and the creation of novel three-dimensional chromatin structural domains. Our results indicate that noncoding SVs may be underappreciated mutational drivers in cancer genomes.


Subject(s)
Genome, Human , Genomic Structural Variation , Neoplasms/genetics , Systems Biology/methods , A549 Cells , Cell Line, Tumor , Chromosome Mapping , DNA, Neoplasm/analysis , DNA, Neoplasm/genetics , Genes, Neoplasm , Genetic Variation , High-Throughput Nucleotide Sequencing/methods , Humans , K562 Cells , Linkage Disequilibrium , Sequence Analysis, DNA/methods , Systems Integration
6.
Curr Opin Genet Dev ; 43: 93-100, 2017 Apr.
Article in English | MEDLINE | ID: mdl-28189962

ABSTRACT

Recent years have witnessed a dramatic expansion in our understanding of gene control. It is now widely appreciated that the spatial organization of the genome and the manner in which genes and regulatory elements are embedded therein has a critical role in facilitating the regulation of gene expression. The loop structures that underlie chromosome organization are anchored by cohesin complexes. Several components of the cohesin complex have multiple paralogs, leading to different levels of cohesin complex variants in cells. Here we review the current literature around cohesin variants and their known functions. We further discuss how variation in cohesin complex composition can result in functional differences that can impact genome organization and determine cell fate.


Subject(s)
Cell Cycle Proteins/genetics , Cell Differentiation/genetics , Chromosomal Proteins, Non-Histone/genetics , Chromosomes/genetics , Gene Expression Regulation/genetics , Cell Lineage/genetics , Genetic Variation , Genome, Human , Humans , Cohesins
7.
Adv Exp Med Biol ; 886: 51-77, 2016.
Article in English | MEDLINE | ID: mdl-26659487

ABSTRACT

Transposable elements (TEs) have the capacity to replicate and insert into new genomic locations. This contributs significantly to evolution of genomes, but can also result in DNA breaks and illegitimate recombination, and therefore poses a significant threat to genomic integrity. Excess damage to the germ cell genome results in sterility. A specific RNA silencing pathway, termed the piRNA pathway operates in germ cells of animals to control TE activity. At the core of the piRNA pathway is a ribonucleoprotein complex consisting of a small RNA, called piRNA, and a protein from the PIWI subfamily of Argonaute nucleases. The piRNA pathway relies on the specificity provided by the piRNA sequence to recognize complementary TE targets, while effector functions are provided by the PIWI protein. PIWI-piRNA complexes silence TEs both at the transcriptional level - by attracting repressive chromatin modifications to genomic targets - and at the posttranscriptional level - by cleaving TE transcripts in the cytoplasm. Impairment of the piRNA pathway leads to overexpression of TEs, significantly compromised genome structure and, invariably, germ cell death and sterility.The piRNA pathway is best understood in the fruit fly, Drosophila melanogaster, and in mouse. This Chapter gives an overview of current knowledge on piRNA biogenesis, and mechanistic details of both transcriptional and posttranscriptional TE silencing by the piRNA pathway. It further focuses on the importance of post-translational modifications and subcellular localization of the piRNA machinery. Finally, it provides a brief description of analogous pathways in other systems.


Subject(s)
DNA Transposable Elements , Genome, Human/physiology , Genome, Insect/physiology , Genomic Instability , RNA Interference/physiology , RNA, Small Interfering/metabolism , Animals , Drosophila melanogaster , Humans , Mice , RNA, Small Interfering/genetics
8.
Cell Rep ; 12(8): 1234-43, 2015 Aug 25.
Article in English | MEDLINE | ID: mdl-26279574

ABSTRACT

In developing male germ cells, prospermatogonia, two Piwi proteins, MILI and MIWI2, use Piwi-interacting RNA (piRNA) guides to repress transposable element (TE) expression and ensure genome stability and proper gametogenesis. In addition to their roles in post-transcriptional TE repression, both proteins are required for DNA methylation of TE sequences. Here, we analyzed the effect of Miwi2 deficiency on piRNA biogenesis and transposon repression. Miwi2 deficiency had only a minor impact on piRNA biogenesis; however, the piRNA profile of Miwi2-knockout mice indicated overexpression of several LINE1 TE families that led to activation of the ping-pong piRNA cycle. Furthermore, we found that MILI and MIWI2 have distinct functions in TE repression in the nucleus. MILI is responsible for DNA methylation of a larger subset of TE families than MIWI2 is, suggesting that the proteins have independent roles in establishing DNA methylation patterns.


Subject(s)
Argonaute Proteins/metabolism , DNA Methylation , RNA, Small Interfering/genetics , Animals , Argonaute Proteins/genetics , Base Sequence , DNA Transposable Elements/genetics , Long Interspersed Nucleotide Elements/genetics , Male , Mice , Molecular Sequence Data , Spermatogonia/metabolism
10.
Nat Commun ; 5: 5795, 2014 Dec 12.
Article in English | MEDLINE | ID: mdl-25503965

ABSTRACT

The Microrchidia (Morc) family of GHKL ATPases are present in a wide variety of prokaryotic and eukaryotic organisms but are of largely unknown function. Genetic screens in Arabidopsis thaliana have identified Morc genes as important repressors of transposons and other DNA-methylated and silent genes. MORC1-deficient mice were previously found to display male-specific germ cell loss and infertility. Here we show that MORC1 is responsible for transposon repression in the male germline in a pattern that is similar to that observed for germ cells deficient for the DNA methyltransferase homologue DNMT3L. Morc1 mutants show highly localized defects in the establishment of DNA methylation at specific classes of transposons, and this is associated with failed transposon silencing at these sites. Our results identify MORC1 as an important new regulator of the epigenetic landscape of male germ cells during the period of global de novo methylation.


Subject(s)
DNA Transposable Elements , Epigenesis, Genetic , Nuclear Proteins/genetics , Spermatozoa/metabolism , Animals , Cell Nucleus/metabolism , Cell Nucleus/ultrastructure , DNA (Cytosine-5-)-Methyltransferases/genetics , DNA (Cytosine-5-)-Methyltransferases/metabolism , DNA Methylation , Embryo, Mammalian , Male , Mice , Mice, Transgenic , Nuclear Proteins/metabolism , RNA, Small Interfering/genetics , RNA, Small Interfering/metabolism , Spermatozoa/cytology , Spermatozoa/growth & development , Time Factors
11.
Genes Dev ; 28(13): 1410-28, 2014 Jul 01.
Article in English | MEDLINE | ID: mdl-24939875

ABSTRACT

Transposable elements (TEs) occupy a large fraction of metazoan genomes and pose a constant threat to genomic integrity. This threat is particularly critical in germ cells, as changes in the genome that are induced by TEs will be transmitted to the next generation. Small noncoding piwi-interacting RNAs (piRNAs) recognize and silence a diverse set of TEs in germ cells. In mice, piRNA-guided transposon repression correlates with establishment of CpG DNA methylation on their sequences, yet the mechanism and the spectrum of genomic targets of piRNA silencing are unknown. Here we show that in addition to DNA methylation, the piRNA pathway is required to maintain a high level of the repressive H3K9me3 histone modification on long interspersed nuclear elements (LINEs) in germ cells. piRNA-dependent chromatin repression targets exclusively full-length elements of actively transposing LINE families, demonstrating the remarkable ability of the piRNA pathway to recognize active elements among the large number of genomic transposon fragments.


Subject(s)
Germ Cells/metabolism , Long Interspersed Nucleotide Elements/physiology , RNA, Small Interfering/metabolism , Animals , Chromatin/metabolism , CpG Islands , DNA Methylation , Gene Expression Regulation , Gene Silencing , Genome/genetics , Histones/metabolism , Long Interspersed Nucleotide Elements/genetics , Male , Mice , Mutation , RNA, Small Interfering/genetics
12.
Nat Rev Mol Cell Biol ; 12(4): 246-58, 2011 Apr.
Article in English | MEDLINE | ID: mdl-21427766

ABSTRACT

PIWI-interacting RNAs (piRNAs) are a distinct class of small non-coding RNAs that form the piRNA-induced silencing complex (piRISC) in the germ line of many animal species. The piRISC protects the integrity of the genome from invasion by 'genomic parasites'--transposable elements--by silencing them. Owing to their limited expression in gonads and their sequence diversity, piRNAs have been the most mysterious class of small non-coding RNAs regulating RNA silencing. Now, much progress is being made into our understanding of their biogenesis and molecular functions, including the specific subcellular compartmentalization of the piRNA pathway in granular cytoplasmic bodies.


Subject(s)
Genome/genetics , RNA Interference , RNA, Small Interfering/genetics , RNA, Small Untranslated/genetics , Animals , DNA Transposable Elements/genetics , Models, Genetic , Mutagenesis, Insertional , RNA, Small Interfering/metabolism , RNA, Small Untranslated/metabolism
13.
Mol Cell ; 31(6): 785-99, 2008 Sep 26.
Article in English | MEDLINE | ID: mdl-18922463

ABSTRACT

piRNAs and Piwi proteins have been implicated in transposon control and are linked to transposon methylation in mammals. Here we examined the construction of the piRNA system in the restricted developmental window in which methylation patterns are set during mammalian embryogenesis. We find robust expression of two Piwi family proteins, MIWI2 and MILI. Their associated piRNA profiles reveal differences from Drosophila wherein large piRNA clusters act as master regulators of silencing. Instead, in mammals, dispersed transposon copies initiate the pathway, producing primary piRNAs, which predominantly join MILI in the cytoplasm. MIWI2, whose nuclear localization and association with piRNAs depend upon MILI, is enriched for secondary piRNAs antisense to the elements that it controls. The Piwi pathway lies upstream of known mediators of DNA methylation, since piRNAs are still produced in dnmt3L mutants, which fail to methylate transposons. This implicates piRNAs as specificity determinants of DNA methylation in germ cells.


Subject(s)
DNA Methylation , DNA Transposable Elements/genetics , RNA, Small Interfering/metabolism , Animals , Argonaute Proteins , Base Sequence , Drosophila melanogaster/genetics , Embryonic Development , Genome/genetics , Germ Cells/metabolism , Long Interspersed Nucleotide Elements/genetics , Mice , Models, Genetic , Molecular Sequence Data , Protein Binding , Proteins/metabolism , Short Interspersed Nucleotide Elements/genetics
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