Distal and proximal cis-regulatory elements sense X chromosome dosage and developmental state at the Xist locus

SUMMARYThe esterase 6 (Est-6) locus in Drosophila melanogaster is located on the third chromosome and is the structural gene for a carboxylesterase (E.C.3.1.1.1) and is polymorphic for two major electromorphs (slow and fast). Isogenic lines containing X chromosomes extracted from natural populations and substituted into a common genetic background were used to detect unlinked factors that affect the activity of the Est-6 locus. Twofold activity differences of esterase 6 (EST 6) were found among males from these derived lines, which differ only in their X chromosome. These unlinked activity modifiers identify possible regulatory elements. Immunoelectrophoresis was used to estimate quantitatively the levels of specific cross-reacting material in the derived lines. The results show that the variation in activity is due to differences in the amount of EST 6 present. The data are consistent with the hypothesis that there is at least one locus on the X chromosome that regulates the synthesis of EST 6 and that this regulatory locus may be polymorphic in natural populations.

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A Functional Chromatin Domain Does Not Resist X Chromosome Inactivation: Silencing of cLys Correlates with Methylation of a Dual Promoter-Replication Origin

Molecular and Cellular Biology ◽

10.1128/mcb.22.13.4667-4676.2002 ◽

2002 ◽

Vol 22 (13) ◽

pp. 4667-4676 ◽

Cited By ~ 11

Author(s):

Suyinn Chong ◽

Joanna Kontaraki ◽

Constanze Bonifer ◽

Arthur D. Riggs

Keyword(s):

X Chromosome ◽

Copy Number ◽

Cpg Island ◽

Regulatory Elements ◽

X Chromosome Inactivation ◽

Chromosome Inactivation ◽

Chromatin Domain ◽

Specific Expression ◽

Position Effects ◽

Chicken Lysozyme

ABSTRACT To investigate the molecular mechanism(s) involved in the propagation and maintenance of X chromosome inactivation (XCI), the 21.4-kb chicken lysozyme (cLys) chromatin domain was inserted into the Hprt locus on the mouse X chromosome. The inserted fragment includes flanking matrix attachment regions (MARs), an origin of bidirectional replication (OBR), and all the cis-regulatory elements required for correct tissue-specific expression of cLys. It also contains a recently identified and widely expressed second gene, cGas41. The cLys domain is known to function as an autonomous unit resistant to chromosomal position effects, as evidenced by numerous transgenic mouse lines showing copy-number-dependent and development-specific expression of cLys in the myeloid lineage. We asked the questions whether this functional chromatin domain was resistant to XCI and whether the X inactivation signal could spread across an extended region of avian DNA. A generally useful method was devised to generate pure populations of macrophages with the transgene either on the active (Xa) or the inactive (Xi) chromosome. We found that (i) cLys and cGas41 are expressed normally from the Xa; (ii) the cLys chromatin domain, even when bracketed by MARs, is not resistant to XCI; (iii) transcription factors are excluded from lysozyme enhancers on the Xi; and (iv) inactivation correlates with methylation of a CpG island that is both an OBR and a promoter of the cGas41 gene.

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Getting to the center of X-chromosome inactivation: the role of transgenesThis paper is one of a selection of papers published in this Special Issue, entitled 30th Annual International Asilomar Chromatin and Chromosomes Conference, and has undergone the Journal’s usual peer review process.

Biochemistry and Cell Biology ◽

10.1139/o09-040 ◽

2009 ◽

Vol 87 (5) ◽

pp. 759-766 ◽

Cited By ~ 3

Author(s):

Jakub Minks ◽

Carolyn J. Brown

Keyword(s):

X Chromosome ◽

Peer Review Process ◽

Regulatory Elements ◽

X Chromosome Inactivation ◽

X Inactivation ◽

Chromosome Inactivation ◽

Xist Expression ◽

Inactivation Center ◽

Xist Rna ◽

Epigenetic Phenomenon

X-chromosome inactivation is a fascinating epigenetic phenomenon that is initiated by expression of a noncoding (nc)RNA, XIST, and results in transcriptional silencing of 1 female X. The process requires a series of events that begins even before XIST expression, and culminates in an active and a silent X within the same nucleus. We will focus on the role that transgenic systems have served in the current understanding of the process of X-chromosome inactivation, both in the initial delineation of an active and inactive X, and in the function of the XIST RNA. X inactivation is strictly cis-limited; recent studies have revealed elements within the X-inactivation center, the region required for inactivation, that are critical for the initial regulation of Xist expression and chromosome pairing. It has been revealed that the X-inactivation center contains a remarkable compendium of cis-regulatory elements, ncRNAs, and trans-acting pairing regions. We review the functional componentry of the X-inactivation center and discuss experiments that helped to dissect the XIST/Xist RNA and its involvement in the establishment of facultative heterochromatin.

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Functional Analysis of the DXPas34Locus, a 3′ Regulator of Xist Expression

Molecular and Cellular Biology ◽

10.1128/mcb.19.12.8513 ◽

1999 ◽

Vol 19 (12) ◽

pp. 8513-8525 ◽

Cited By ~ 72

Author(s):

E. Debrand ◽

C. Chureau ◽

D. Arnaud ◽

P. Avner ◽

E. Heard

Keyword(s):

X Chromosome ◽

Yeast Artificial Chromosome ◽

Es Cells ◽

Artificial Chromosome ◽

Antisense Transcription ◽

Regulatory Elements ◽

X Inactivation ◽

X Chromosomes ◽

Inactivation Center ◽

Controlling Element

ABSTRACT X inactivation in female mammals is controlled by a key locus on the X chromosome, the X-inactivation center (Xic). The Xic controls the initiation and propagation of inactivation in cis. It also ensures that the correct number of X chromosomes undergo inactivation (counting) and determines which X chromosome becomes inactivated (choice). The Xist gene maps to the Xic region and is essential for the initiation of X inactivation in cis. Regulatory elements of X inactivation have been proposed to lie 3′ toXist. One such element, lying 15 kb downstream ofXist, is the DXPas34 locus, which was first identified as a result of its hypermethylation on the active X chromosome and the correlation of its methylation level with allelism at the X-controlling element (Xce), a locus known to affect choice. In this study, we have tested the potential function of theDXPas34 locus in Xist regulation and X-inactivation initiation by deleting it in the context of largeXist-containing yeast artificial chromosome transgenes. Deletion of DXPas34 eliminates both Xistexpression and antisense transcription present in this region in undifferentiated ES cells. It also leads to nonrandom inactivation of the deleted transgene upon differentiation. DXPas34 thus appears to be a critical regulator of Xist activity and X inactivation. The expression pattern of DXPas34 during early embryonic development, which we report here, further suggests that it could be implicated in the regulation of imprintedXist expression.

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Skewing X chromosome choice by modulating sense transcription across the Xist locus

Genes & Development ◽

10.1101/gad.271203 ◽

2003 ◽

Vol 17 (17) ◽

pp. 2177-2190 ◽

Cited By ~ 44

Author(s):

T. B. Nesterova

Keyword(s):

X Chromosome ◽

Xist Locus

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Elements at the 5′ end of Xist harbor SPEN-independent transcriptional antiterminator activity

Nucleic Acids Research ◽

10.1093/nar/gkaa789 ◽

2020 ◽

Vol 48 (18) ◽

pp. 10500-10517

Author(s):

Jackson B Trotman ◽

David M Lee ◽

Rachel E Cherney ◽

Susan O Kim ◽

Kaoru Inoue ◽

...

Keyword(s):

Stem Cells ◽

Embryonic Stem Cells ◽

Gene Silencing ◽

X Chromosome ◽

Embryonic Stem ◽

X Chromosome Inactivation ◽

Xist Rna ◽

Sequence Elements ◽

Cleavage And Polyadenylation ◽

Xist Locus

Abstract The Xist lncRNA requires Repeat A, a conserved RNA element located in its 5′ end, to induce gene silencing during X-chromosome inactivation. Intriguingly, Repeat A is also required for production of Xist. While silencing by Repeat A requires the protein SPEN, how Repeat A promotes Xist production remains unclear. We report that in mouse embryonic stem cells, expression of a transgene comprising the first two kilobases of Xist (Xist-2kb) causes transcriptional readthrough of downstream polyadenylation sequences. Readthrough required Repeat A and the ∼750 nucleotides downstream, did not require SPEN, and was attenuated by splicing. Despite associating with SPEN and chromatin, Xist-2kb did not robustly silence transcription, whereas a 5.5-kb Xist transgene robustly silenced transcription and read through its polyadenylation sequence. Longer, spliced Xist transgenes also induced robust silencing yet terminated efficiently. Thus, in contexts examined here, Xist requires sequence elements beyond its first two kilobases to robustly silence transcription, and the 5′ end of Xist harbors SPEN-independent transcriptional antiterminator activity that can repress proximal cleavage and polyadenylation. In endogenous contexts, this antiterminator activity may help produce full-length Xist RNA while rendering the Xist locus resistant to silencing by the same repressive complexes that the lncRNA recruits to other genes.

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Identification of Developmentally Specific Enhancers for Tsix in the Regulation of X Chromosome Inactivation

Molecular and Cellular Biology ◽

10.1128/mcb.25.7.2757-2769.2005 ◽

2005 ◽

Vol 25 (7) ◽

pp. 2757-2769 ◽

Cited By ~ 55

Author(s):

Nicholas Stavropoulos ◽

Rebecca K. Rowntree ◽

Jeannie T. Lee

Keyword(s):

X Chromosome ◽

Regulatory Elements ◽

X Chromosome Inactivation ◽

Chromosome Inactivation ◽

X Chromosomes ◽

Binary Switch ◽

Inactive X Chromosome ◽

The Future ◽

Hypersensitive Sites ◽

Mammalian Female

ABSTRACT X chromosome inactivation silences one of two X chromosomes in the mammalian female cell and is controlled by a binary switch that involves interactions between Xist and Tsix, a sense-antisense pair of noncoding genes. On the future active X chromosome, Tsix expression suppresses Xist upregulation, while on the future inactive X chromosome, Tsix repression is required for Xist-mediated chromosome silencing. Thus, understanding the binary switch mechanism depends on ascertaining how Tsix expression is regulated. Here we have taken an unbiased approach toward identifying Tsix regulatory elements within the X chromosome inactivation center. First, we defined the major Tsix promoter and found that it cannot fully recapitulate the developmental dynamics of Tsix expression, indicating a requirement for additional regulatory elements. We then delineated two enhancers, one classical enhancer mapping upstream of Tsix and a bipartite enhancer that flanks the major Tsix promoter. These experiments revealed the intergenic transcription element Xite as an enhancer of Tsix and the repeat element DXPas34 as a component of the bipartite enhancer. Each enhancer contains DNase I-hypersensitive sites and appears to confer developmental specificity to Tsix expression. Characterization of these enhancers will facilitate the identification of trans-acting regulatory factors for X chromosome counting and choice.

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Physical Mapping of 2000 kb of the Mouse X Chromosome in the Vicinity of the Xist Locus

Genomics ◽

10.1006/geno.1993.1109 ◽

1993 ◽

Vol 15 (3) ◽

pp. 570-575 ◽

Cited By ~ 9

Author(s):

Penny Cooper ◽

Jacquie T. Keer ◽

Veronica M. McCabe ◽

Renata M.J. Hamvas ◽

Stephen D.M. Brown ◽

...

Keyword(s):

X Chromosome ◽

Physical Mapping ◽

Xist Locus

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Distal and proximal cis-regulatory elements sense X-chromosomal dosage and developmental state at the Xist locus

10.1101/2021.03.29.437476 ◽

2021 ◽

Author(s):

Rutger A.F. Gjaltema ◽

Till Schwämmle ◽

Pauline Kautz ◽

Michael Robson ◽

Robert Schöpflin ◽

...

Keyword(s):

Expression Patterns ◽

Embryonic Stem ◽

Developmental State ◽

Regulatory Elements ◽

Developmental Time ◽

Proximal Region ◽

Spatial Proximity ◽

Enhancer Activity ◽

Regulatory Regions ◽

Spatio Temporal

AbstractDevelopmental genes such as Xist, the master regulator of X-chromosome inactivation (XCI), are controlled by complex cis-regulatory landscapes, which decode multiple signals to establish specific spatio-temporal expression patterns. Xist integrates information on X-chromosomal dosage and developmental stage to trigger XCI at the primed pluripotent state in females only. Through a pooled CRISPR interference screen in differentiating mouse embryonic stem cells, we identify functional enhancer elements of Xist during the onset of random XCI. By quantifying how enhancer activity is modulated by X-dosage and differentiation, we find that X-dosage controls the promoter-proximal region in a binary switch-like manner. By contrast, differentiation cues activate a series of distal elements and bring them into closer spatial proximity of the Xist promoter. The strongest distal element is part of an enhancer cluster ∼200 kb upstream of the Xist gene which is associated with a previously unannotated Xist-enhancing regulatory transcript, we named Xert. Developmental cues and X-dosage are thus decoded by distinct regulatory regions, which cooperate to ensure female-specific Xist upregulation at the correct developmental time. Our study is the first step to disentangle how multiple, functionally distinct regulatory regions interact to generate complex expression patterns in mammals.

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Chromosome X-wide Analysis of Positive Selection in Human Populations: Common and Private Signals of Selection and its Impact on Inactivated Genes and Enhancers

Frontiers in Genetics ◽

10.3389/fgene.2021.714491 ◽

2021 ◽

Vol 12 ◽

Author(s):

Pablo Villegas-Mirón ◽

Sandra Acosta ◽

Jessica Nye ◽

Jaume Bertranpetit ◽

Hafid Laayouni

Keyword(s):

Positive Selection ◽

X Chromosome ◽

Genetic Basis ◽

Target Genes ◽

Regulatory Elements ◽

Human Populations ◽

Third Phase ◽

Genome Wide ◽

Private Signals ◽

Sub Saharan

The ability of detecting adaptive (positive) selection in the genome has opened the possibility of understanding the genetic basis of population-specific adaptations genome-wide. Here, we present the analysis of recent selective sweeps, specifically in the X chromosome, in human populations from the third phase of the 1,000 Genomes Project using three different haplotype-based statistics. We describe instances of recent positive selection that fit the criteria of hard or soft sweeps, and detect a higher number of events among sub-Saharan Africans than non-Africans (Europe and East Asia). A global enrichment of neural-related processes is observed and numerous genes related to fertility appear among the top candidates, reflecting the importance of reproduction in human evolution. Commonalities with previously reported genes under positive selection are found, while particularly strong new signals are reported in specific populations or shared across different continental groups. We report an enrichment of signals in genes that escape X chromosome inactivation, which may contribute to the differentiation between sexes. We also provide evidence of a widespread presence of soft-sweep-like signatures across the chromosome and a global enrichment of highly scoring regions that overlap potential regulatory elements. Among these, enhancers-like signatures seem to present putative signals of positive selection which might be in concordance with selection in their target genes. Also, particularly strong signals appear in regulatory regions that show differential activities, which might point to population-specific regulatory adaptations.

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