X-chromosome inactivation in XX androgenetic mouse embryos surviving implantation

Development ◽  
2000 ◽  
Vol 127 (19) ◽  
pp. 4137-4145 ◽  
Author(s):  
I. Okamoto ◽  
S. Tan ◽  
N. Takagi
Keyword(s):  
X Chromosome ◽  
Ectopic Expression ◽  
Blastocyst Stage ◽  
X Chromosome Inactivation ◽  
X Inactivation ◽  
Chromosome Inactivation ◽  
Current View ◽  
Replication Banding ◽  
Xist Rna ◽  
Morula Stage

Using genetic and cytogenetic markers, we assessed early development and X-chromosome inactivation (X-inactivation) in XX mouse androgenones produced by pronuclear transfer. Contrary to the current view, XX androgenones are capable of surviving to embryonic day 7.5, achieving basically random X-inactivation in all tissues including those derived from the trophectoderm and primitive endoderm that are characterized by paternal X-activation in fertilized embryos. This finding supports the hypothesis that in fertilized female embryos, the maternal X chromosome remains active until the blastocyst stage because of a rigid imprint that prevents inactivation, whereas the paternal X chromosome is preferentially inactivated in extra-embryonic tissues owing to lack of such imprint. In spite of random X-inactivation in XX androgenones, FISH analyses revealed expression of stable Xist RNA from every X chromosome in XX and XY androgenonetic embryos from the four-cell to morula stage. Although the occurrence of inappropriate X-inactivation was further suggested by the finding that Xist continues ectopic expression in a proportion of cells from XX and XY androgenones at the blastocyst and the early egg cylinder stage, a replication banding study failed to provide positive evidence for inappropriate X-inactivation at E6. 5.

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.

2009 ◽  
Vol 87 (5) ◽  
pp. 759-766 ◽  
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.

Investigation of variability among mouse aggregation chimaeras and X-chromosome inactivation mosaics

Development ◽  
1984 ◽  
Vol 84 (1) ◽  
pp. 309-329
Author(s):  
John D. West ◽  
Theodor Bücher ◽  
Ingrid M. Linke ◽  
Manfred Dünnwald
Keyword(s):  
X Chromosome ◽  
Phosphoglycerate Kinase ◽  
X Chromosome Inactivation ◽  
Yolk Sac ◽  
X Inactivation ◽  
Chromosome Inactivation ◽  
Cell Populations ◽  
Primitive Endoderm ◽  
Female Progeny ◽  
Positive Correlations

Mouse aggregation chimaeras were produced by aggregating C3H/HeH and C3H/HeHa—Pgk-1a/Ws embryos. At mid-term the proportions of the two cell populations in these conceptuses and the X-inactivation mosaic female progeny of C3H/HeH ♀ × C3H/HeHa—Pgk-1a/Ws ♂ matings were estimated using quantitative electrophoresis of phosphoglycerate kinase (PGK-1) allozymes. The percentage of PGK-1B was more variable in the foetus, amnion and yolk sac mesoderm of the chimaeras than in the corresponding tissues of the mosaic conceptuses. Positive correlations were found for the percentage of PGK-1B between these three primitive ectoderm tissues in both chimaeras and mosaics and between the two primitive endoderm tissues (yolk sac endoderm and parietal endoderm) of the chimaeras. There was no significant correlation between the primitive ectoderm and primitive endoderm tissues of the chimaeras. The results suggest that unequal allocation of cell populations to the primitive ectoderm and primitive endoderm considerably increases the variability among chimaeras but variation probably exists before this segregation occurs. The variation that arises before and at this allocation event is present before X-chromosome inactivation occurs in the primitive ectoderm lineage and explains why the proportions of the two cell populations are more variable among chimaeras than mosaics. Additional variation arises within the primitive ectoderm lineage, after X-inactivation. This variation may be greater in chimaeras than mosaics but the evidence is inconclusive. The results also have some bearing on the nature of the allocation of cells to the primitive ectoderm and primitive endoderm lineages and the timing of X-chromosome inactivation in the primitive ectoderm lineage.

Regulation of imprinted X-chromosome inactivation in mice by Tsix

Development ◽  
2001 ◽  
Vol 128 (8) ◽  
pp. 1275-1286 ◽  
Author(s):  
T. Sado ◽  
Z. Wang ◽  
H. Sasaki ◽  
E. Li
Keyword(s):  
X Chromosome ◽  
X Chromosome Inactivation ◽  
X Inactivation ◽  
Chromosome Inactivation ◽  
Maternal Allele ◽  
Xist Expression ◽  
Developmental Defects ◽  
X Chromosomes ◽  
Embryonic Tissues ◽  
Early Embryonic Lethality

In mammals, X-chromosome inactivation is imprinted in the extra-embryonic lineages with paternal X chromosome being preferentially inactivated. In this study, we investigate the role of Tsix, the antisense transcript from the Xist locus, in regulation of Xist expression and X-inactivation. We show that Tsix is transcribed from two putative promoters and its transcripts are processed. Expression of Tsix is first detected in blastocysts and is imprinted with only the maternal allele transcribed. The imprinted expression of Tsix persists in the extra-embryonic tissues after implantation, but is erased in embryonic tissues. To investigate the function of Tsix in X-inactivation, we disrupted Tsix by insertion of an IRES(β)geo cassette in the second exon, which blocked transcripts from both promoters. While disruption of the paternal Tsix allele has no adverse effects on embryonic development, inheritance of a disrupted maternal allele results in ectopic Xist expression and early embryonic lethality, owing to inactivation of both X chromosomes in females and single X chromosome in males. Further, early developmental defects of female embryos with maternal transmission of Tsix mutation can be rescued by paternal inheritance of the Xist deletion. These results provide genetic evidence that Tsix plays a crucial role in maintaining Xist silencing in cis and in regulation of imprinted X-inactivation in the extra-embryonic tissues.

scholarly journals Localized accumulation of Xist RNA in X chromosome inactivation

Open Biology ◽  
2019 ◽  
Vol 9 (12) ◽  
pp. 190213 ◽  
Author(s):  
Neil Brockdorff
Keyword(s):  
X Chromosome ◽  
X Chromosome Inactivation ◽  
Chromosome Inactivation ◽  
X Chromosomes ◽  
Non Coding Rna ◽  
Xist Rna ◽  
Analogous Manner ◽  
Mammalian Embryos ◽  
Non Coding Rnas ◽  
In Cis

The non-coding RNA Xist regulates the process of X chromosome inactivation, in which one of the two X chromosomes present in cells of early female mammalian embryos is selectively and coordinately shut down. Remarkably Xist RNA functions in cis , affecting only the chromosome from which it is transcribed. This feature is attributable to the unique propensity of Xist RNA to accumulate over the territory of the chromosome on which it is synthesized, contrasting with the majority of RNAs that are rapidly exported out of the cell nucleus. In this review I provide an overview of the progress that has been made towards understanding localized accumulation of Xist RNA, drawing attention to evidence that some other non-coding RNAs probably function in a highly analogous manner. I describe a simple model for localized accumulation of Xist RNA and discuss key unresolved questions that need to be addressed in future studies.

139 SEX-SPECIFIC GENE EXPRESSION IN PORCINE PRE-IMPLANTATION EMBRYOS

2016 ◽  
Vol 28 (2) ◽  
pp. 199
Author(s):  
D. Kradolfer ◽  
J. Knubben ◽  
V. Flöter ◽  
J. Bick ◽  
S. Bauersachs ◽  
...  
Keyword(s):  
Gene Expression ◽  
X Chromosome ◽  
Current Knowledge ◽  
Critical Time ◽  
Blastocyst Stage ◽  
X Chromosome Inactivation ◽  
Chromosome Inactivation ◽  
Specific Gene ◽  
Embryo Survival ◽  
Specific Gene Expression

X-Chromosome inactivation in female mammals starts during early blastocyst stage with expression of the X-inactive specific transcript (XIST), which coats and silences the inactive X chromosome. However, this compensation is not complete in blastocysts, as a large number of X-linked transcripts are more highly expressed in female embryos than in males. Furthermore, the process of X chromosome inactivation is altered in IVF and cloned porcine embryos, possibly explaining problems of embryo survival with these techniques. The aim of this study was to gain more insights into the transcriptional dynamics of the porcine pre-implantation embryo, with a particular focus on sex-specific differences. RNA sequencing (RNA-Seq) was performed for individual blastocysts at 8, 10, and 12 days after ovulation, and the temporal development of sex-specific transcripts was analysed. German Landrace sows were cycle synchronized and inseminated with sperm of the same Pietrain boar. On Days 8, 10, and 12 post-insemination, sows were slaughtered and embryos were removed from the uterus using 10 mL of PBS (pH 7.4) per horn. Single embryos were shock frozen in liquid nitrogen and stored at –80°C until the extraction of RNA and DNA (AllPrep DNA/RNA Micro Kit, Qiagen, Valencia, CA, USA). Using the isolated DNA, the sex of the embryos was determined and 5 female and male embryos, respectively, were analysed per stage. Illumina TruSeq Stranded mRNA libraries (Illumina Inc., San Diego, CA, USA) were sequenced on a HiSEqn 2500 (Illumina Inc.), and 15 to 25 million 100-bp single-end reads were generated per sample. Reads were filtered and processed using Trimmomatic and mapped to the porcine genome assembly Sscrofa10.2 with TopHat2. Mapped reads were counted by the use of QuasR qCount based on the current National Center for Biotechnology Information (http://www.ncbi.nlm.nih.gov/) GFF3 annotation file. Statistical analysis of count data was performed with the BioConductor R (https://www.bioconductor.org/) package DESEqn 2. At all 3 stages, we found 7 Y-linked transcripts that were highly expressed in male embryos (EIF2S3, EIF1AY, LOC100624590, LOC100625207, LOC100624329, LOC102162178, LOC100624937). On the other hand, 47 X-linked transcripts showed increased expression in female blastocysts, most of them at all 3 time points. However, a small number of genes (DDX3X, LAMP2, and RPS6KA3) were more highly expressed in females at Days 8 and 10 but more highly expressed in males at Day 12. Three X-linked genes (OFD1, KAL1, and LOC100525092) were more highly expressed in male embryos, although only at a low fold change of 1.2 to 1.4. Furthermore, expression of 8 transcripts located on autosomes was higher in females. In conclusion, our study expands the current knowledge of sex-specific gene expression in 8- to 12-day-old porcine blastocysts, a critical time period during pre-implantation embryo development.

scholarly journals Initiation of X Chromosome Inactivation during Bovine Embryo Development

Cells ◽  
2020 ◽  
Vol 9 (4) ◽  
pp. 1016 ◽  
Author(s):  
Bo Yu ◽  
Helena T. A. van Tol ◽  
Tom A.E. Stout ◽  
Bernard A. J. Roelen
Keyword(s):  
Embryo Development ◽  
X Chromosome ◽  
Developmental Process ◽  
Inner Cell Mass ◽  
Cell Mass ◽  
X Chromosome Inactivation ◽  
Chromosome Inactivation ◽  
Bovine Embryo ◽  
Cell Stage ◽  
Morula Stage

X-chromosome inactivation (XCI) is a developmental process that aims to equalize the dosage of X-linked gene products between XY males and XX females in eutherian mammals. In female mouse embryos, paternal XCI is initiated at the 4-cell stage; however, the X chromosome is reactivated in the inner cell mass cells of blastocysts, and random XCI is subsequently initiated in epiblast cells. However, recent findings show that the patterns of XCI are not conserved among mammals. In this study, we used quantitative RT-PCR and RNA in situ hybridization combined with immunofluorescence to investigate the pattern of XCI during bovine embryo development. Expression of XIST (X-inactive specific transcript) RNA was significantly upregulated at the morula stage. For the first time, we demonstrate that XIST accumulation in bovine embryos starts in nuclei of female morulae, but its colocalization with histone H3 lysine 27 trimethylation was first detected in day 7 blastocysts. Both in the inner cell mass and in putative epiblast precursors, we observed a proportion of cells with XIST RNA and H3K27me3 colocalization. Surprisingly, the onset of XCI did not lead to a global downregulation of X-linked genes, even in day 9 blastocysts. Together, our findings confirm that diverse patterns of XCI initiation exist among developing mammalian embryos.

Monoallelic Methylation of the Human Androgen Receptor Gene as Detected by the HUMARA Clonality Assay Does Not Consistently Reflect X-Chromosome Inactivation and Is Therefore Unreliable for Assessment of Clonal Hematopoiesis.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 4566-4566
Author(s):  
Sabina Swierczek ◽  
Jaroslav Jelinek ◽  
Neeraj Agarwal ◽  
Andrew Wilson ◽  
Kimberly Hickman ◽  
...  
Keyword(s):  
Gene Expression ◽  
X Chromosome ◽  
X Chromosome Inactivation ◽  
X Inactivation ◽  
Chromosome Inactivation ◽  
Cag Repeats ◽  
Clonal Hematopoiesis ◽  
Healthy Elderly ◽  
Polymorphic Genes ◽  
Exon 1

Abstract Abstract 4566 Even in the absence of a disease specific chromosomal marker, clonality can be assessed in somatic tissues of female origin by using assays based on the pattern of X-chromosome inactivation. The most widely used technique for quantifying X-chromosome inactivation is the HUMARA method that is based on the putative role of DNA methylation at CpG sites close to trinucleotide repeats in exon 1 in silencing of the AR locus. However, using the HUMARA method, we and others have observed that approximately 30% of healthy elderly volunteers appear to have clonal hematopoiesis. This observation is at odds with the concept that normal hematopoiesis is a polyclonal process, suggesting that the finding of monoclonality or oligoclonality in a high percentage of healthy volunteers by using the HUMARA method, is a technical artifact. To address this issue, we developed a clonality assay based on gene expression of five X-linked polymorphic genes and found no evidence of clonal hematopoiesis in healthy elderly volunteers, although we confirmed extreme skewing of X inactivation (consistent with monoallelic methylation of AR) in 30% of the study subject when analyzed by HUMARA (Swierczek et al., Blood 2008). In the present studies, we have validated the accuracy and reproducibility of our quantitative transcription-based clonality assay (qTCA) using two different methods for quantifying gene expression and compared the results with those obtained using the HUMARA method. DNA and RNA were extracted from peripheral blood samples from 31 healthy female volunteers (age in years as follows: range, 22-55; mean, 35; median, 34). RNA was reverse transcribed (RT) and analyzed by using our qTCA in which expression of three polymorphic genes (MPP1, IDS and FHL1), that are subject to X inactivation, is quantified by allele-specific, real-time RT-PCR. Based on DNA analysis, 25 of the 31 (80%) volunteers were polymorphic for at least one of the test genes. Results are reported as the percentage of each of the two single nucleotide polymorphisms (SNPs) that is present in the sample (e. g., 60% A; 40% G). PCR primers are designed to provide maximum discrimination between SNPs with >13 PCR cycles (i. e., 13 log2) separating true-positive from false-positive amplification. Aliquots of the isolated RNA from the test samples were sent to an independent investigator (JJ), at a separate institution, who was blinded to the results of our qTCA, and the allele ratio was determined by using a different technique (quantitative pyrosequencing). Comparison of the results, confirmed the accuracy and reproducibility of the two methods with coefficients of correlation for each gene as follows: (MPP1, r=0.9385; IDS, r=0.8565; FHL1, r=0.8657). One of the 25 informative females (4%) showed extreme skewing (SNP ratio >75%:25%) of X inactivation by both methods. Based on allelic differences in the number of CAG repeats, 29/31 participants were informative in the HUMARA. Among most of the samples, a good correlation was observed between the pattern of X chromosome inactivation as determined by HUMARA and that determined by both qTCA and quantitative pyrosequencing, however, 8/29 (27%) samples analyzed by the HUMARA showed extreme skewing of allele methylation (ratio >75%:25%). Of the 8 subjects with extreme skewing, 3 were homozygous (i. e., non-informative) for all of the X-chromosome polymorphic genes used in the qTCA. Samples from the 5 informative participants were analyzed by using the qTCA, and, in contrast to the HUMARA results, only one subject showed extreme skewing of the SNP ratio (the same subject as identified in the original qTCA). We also quantified HUMARA gene expression using the difference in the number of exon 1 CAG repeats between the two AR alleles as the polymorphic marker. These experiments showed that, of the 8 volunteers with skewing of X inactivation based on HUMARA, 5 had skewing of AR allele expression and 3 had expression of both AR alleles, indicating that the correlation between DNA methylation at the AR locus and AR mRNA transcription is inconsistent. In conclusion, we found a good correlation between the HUMARA and qTCA in some females; however, this was not the case in many healthy females both elderly and young. These experiments demonstrate the accuracy and reproducibility of the qTCA and confirmed that this technique is not subject to the artifact of aberrant skewing of X-inactivation due to monoallelic methylation of AR that limits the applicability and value of the HUMARA. Disclosures: No relevant conflicts of interest to declare.

Impact of Xist RNA on chromatin modifications and transcriptional silencing maintenance at different stages of imprinted X chromosome inactivation in vole Microtus levis

Chromosoma ◽  
2017 ◽  
Vol 127 (1) ◽  
pp. 129-139 ◽  
Author(s):  
Alexander I. Shevchenko ◽  
Elena V. Grigor’eva ◽  
Sergey P. Medvedev ◽  
Irina S. Zakharova ◽  
Elena V. Dementyeva ◽  
...  
Keyword(s):  
X Chromosome ◽  
Transcriptional Silencing ◽  
X Chromosome Inactivation ◽  
Chromosome Inactivation ◽  
Chromatin Modifications ◽  
Xist Rna ◽  
Microtus Levis

scholarly journals The search for the mouse X-chromosome inactivation centre

1990 ◽  
Vol 56 (2-3) ◽  
pp. 99-106 ◽  
Author(s):  
S. Rastan ◽  
S. D. M. Brown
Keyword(s):  
X Chromosome ◽  
Strong Evidence ◽  
Molecular Level ◽  
Molecular Genetic ◽  
X Chromosome Inactivation ◽  
X Inactivation ◽  
Chromosome Inactivation ◽  
Chromosome Identification ◽  
Female Embryo ◽  
Mary Lyon

SummaryThe phenomenon of X-chromosome inactivation in female mammals, whereby one of the two X chromosome present in each cell of the female embryo is inactivated early in development, was first described by Mary Lyon in 1961. Nearly 30 years later, the mechanism of X-chromosome inactivation remains unknown. Strong evidence has accumulated over the years, however, for the involvement of a major switch or inactivation centre on the mouse X chromosome. Identification of the inactivation centre at the molecular level would be an important step in understanding the mechanism of X-inactivation. In this paper we review the evidence for the existence and location of the X-inactivation centre on the mouse X-chromosome, present data on the molecular genetic mapping of this region, and describe ongoing strategies we are using to attempt to identify the inactivation centre at the molecular level.

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