Evidence for feasibility of fetal trophoblastic cell-based noninvasive prenatal testing.

OBJECTIVE: The goal was to develop methods for detection of chromosomal and subchromosomal abnormalities in fetal cells in the mother's circulation at 10-16 weeks' gestation using analysis by array comparative genomic hybridization (CGH) and/or next-generation sequencing (NGS). METHOD: Nucleated cells from 30 mL of blood collected at 10-16 weeks' gestation were separated from red cells by density fractionation and then immunostained to identify cytokeratin positive and CD45 negative trophoblasts. Individual cells were picked and subjected to whole genome amplification, genotyping, and analysis by array CGH and NGS. RESULTS: Fetal cells were recovered from most samples as documented by Y chromosome PCR, short tandem repeat analysis, array CGH, and NGS including over 30 normal male cells, one 47,XXY cell from an affected fetus, one trisomy 18 cell from an affected fetus, nine cells from a trisomy 21 case, three normal cells and one trisomy 13 cell from a case with confined placental mosaicism, and two chromosome 15 deletion cells from a case known by CVS to have a 2.7 Mb de novo deletion. CONCLUSION: We believe that this is the first report of using array CGH and NGS whole genome sequencing to detect chromosomal abnormalities in fetal trophoblastic cells from maternal blood. © 2016 The Authors. Prenatal Diagnosis published by John Wiley & Sons, Ltd.

US Military Child and Adolescent Psychiatry Training Programs and Careers of Military Child Psychiatrists.

Military child and adolescent psychiatry (CAP) fellowship programs offer educational experiences universal to all civilian training programs in the USA. They also offer unique training opportunities not found in civilian CAP fellowships in order to prepare graduates to serve the needs of military families. Military-specific curricula and exposures prepare trainees to address various issues faced by military families, in contending with frequent military moves, parental deployments, and disrupted social ties. Curricula are also designed to provide the psychiatrist with a greater understanding of the rigors of military service. CAP training and subsequent assignments prepare military psychiatrists for diverse career paths in the military environment. CAP military careers often include duties in addition to treating patients. Administrative roles, academic teaching positions, as well as school consultation positions are all career options available to military CAP.

Unusual chromatin status and organization of the inactive X chromosome in murine trophoblast giant cells.

Mammalian X-chromosome inactivation (XCI) enables dosage compensation between XX females and XY males. It is an essential process and its absence in XX individuals results in early lethality due primarily to extra-embryonic defects. This sensitivity to X-linked gene dosage in extra-embryonic tissues is difficult to reconcile with the reported tendency of escape from XCI in these tissues. The precise transcriptional status of the inactive X chromosome in different lineages has mainly been examined using transgenes or in in vitro differentiated stem cells and the degree to which endogenous X-linked genes are silenced in embryonic and extra-embryonic lineages during early postimplantation stages is unclear. Here we investigate the precise temporal and lineage-specific X-inactivation status of several genes in postimplantation mouse embryos. We find stable gene silencing in most lineages, with significant levels of escape from XCI mainly in one extra-embryonic cell type: trophoblast giant cells (TGCs). To investigate the basis of this epigenetic instability, we examined the chromatin structure and organization of the inactive X chromosome in TGCs obtained from ectoplacental cone explants. We find that the Xist RNA-coated X chromosome has a highly unusual chromatin content in TGCs, presenting both heterochromatic marks such as H3K27me3 and euchromatic marks such as histone H4 acetylation and H3K4 methylation. Strikingly, Xist RNA does not form an overt silent nuclear compartment or Cot1 hole in these cells. This unusual combination of silent and active features is likely to reflect, and might underlie, the partial activity of the X chromosome in TGCs.

Nuclear organization and dosage compensation.

Dosage compensation is a strategy to deal with the imbalance of sex chromosomal gene products relative to autosomes and also between the sexes. The mechanisms that ensure dosage compensation for X-chromosome activity have been extensively studied in mammals, worms, and flies. Although each entails very different mechanisms to equalize the dose of X-linked genes between the sexes, they all involve the co-ordinate regulation of hundreds of genes specifically on the sex chromosomes and not the autosomes. In addition to chromatin modifications and changes in higher order chromatin structure, nuclear organization is emerging as an important component of these chromosome-wide processes and in the specific targeting of dosage compensation complexes to the sex chromosomes. Preferential localization within the nucleus and 3D organization are thought to contribute to the differential treatment of two identical homologs within the same nucleus, as well as to the chromosome-wide spread and stable maintenance of heterochromatin.

LINE-1 activity in facultative heterochromatin formation during X chromosome inactivation.

During X chromosome inactivation (XCI), Xist RNA coats and silences one of the two X chromosomes in female cells. Little is known about how XCI spreads across the chromosome, although LINE-1 elements have been proposed to play a role. Here we show that LINEs participate in creating a silent nuclear compartment into which genes become recruited. A subset of young LINE-1 elements, however, is expressed during XCI, rather than being silenced. We demonstrate that such LINE expression requires the specific heterochromatic state induced by Xist. These LINEs often lie within escape-prone regions of the X chromosome, but close to genes that are subject to XCI, and are associated with putative endo-siRNAs. LINEs may thus facilitate XCI at different levels, with silent LINEs participating in assembly of a heterochromatic nuclear compartment induced by Xist, and active LINEs participating in local propagation of XCI into regions that would otherwise be prone to escape.

High-resolution analysis of epigenetic changes associated with X inactivation.

Differentiation of female murine ES cells triggers silencing of one X chromosome through X-chromosome inactivation (XCI). Immunofluorescence studies showed that soon after Xist RNA coating the inactive X (Xi) undergoes many heterochromatic changes, including the acquisition of H3K27me3. However, the mechanisms that lead to the establishment of heterochromatin remain unclear. We first analyze chromatin changes by ChIP-chip, as well as RNA expression, around the X-inactivation center (Xic) in female and male ES cells, and their day 4 and 10 differentiated derivatives. A dynamic epigenetic landscape is observed within the Xic locus. Tsix repression is accompanied by deposition of H3K27me3 at its promoter during differentiation of both female and male cells. However, only in female cells does an active epigenetic landscape emerge at the Xist locus, concomitant with high Xist expression. Several regions within and around the Xic show unsuspected chromatin changes, and we define a series of unusual loci containing highly enriched H3K27me3. Genome-wide ChIP-seq analyses show a female-specific quantitative increase of H3K27me3 across the X chromosome as XCI proceeds in differentiating female ES cells. Using female ES cells with nonrandom XCI and polymorphic X chromosomes, we demonstrate that this increase is specific to the Xi by allele-specific SNP mapping of the ChIP-seq tags. H3K27me3 becomes evenly associated with the Xi in a chromosome-wide fashion. A selective and robust increase of H3K27me3 and concomitant decrease in H3K4me3 is observed over active genes. This indicates that deposition of H3K27me3 during XCI is tightly associated with the act of silencing of individual genes across the Xi.

Combined immunofluorescence, RNA fluorescent in situ hybridization, and DNA fluorescent in situ hybridization to study chromatin changes, transcriptional activity, nuclear organization, and X-chromosome inactivation.

Epigenetic mechanisms lead to the stable regulation of gene expression without alteration of DNA and trigger initiation and/or maintenance of cell-type-specific transcriptional profiles. Indeed, modulation of chromatin structure and the global 3D organization of the genome and nuclear architecture participate in the precise control of transcription. Thus, dissection of these epigenetic mechanisms is essential for our understanding of gene regulation. In this chapter, we describe challenging combinations of immunofluorescence, and RNA and DNA fluorescent in situ hybridization and their application to our studies of a remarkable example of epigenetic control of gene expression in female mammals, the process of X chromosome inactivation.

A phosphorylated subpopulation of the histone variant macroH2A1 is excluded from the inactive X chromosome and enriched during mitosis.

Histone variants play an important role in numerous biological processes through changes in nucleosome structure and stability and possibly through mechanisms influenced by posttranslational modifications unique to a histone variant. The family of histone H2A variants includes members such as H2A.Z, the DNA damage-associated H2A.X, macroH2A (mH2A), and H2ABbd (Barr body-deficient). Here, we have undertaken the challenge to decipher the posttranslational modification-mediated "histone code" of mH2A, a variant generally associated with certain forms of condensed chromatin such as the inactive X chromosome in female mammals. By using female human cells as a source of mH2A, endogenous mH2A was purified and analyzed by mass spectrometry. Although mH2A is in low abundance compared with conventional histones, we identified a phosphorylation site, S137ph, which resides within the "hinge" region of mH2A. This lysine-rich hinge is an approximately 30-aa stretch between the H2A and macro domains, proposed to bind nucleic acids. A specific antibody to S137ph was raised; by using this reagent, S137 phosphorylation was found to be present in both male and female cells and on both splice variants of the mH2A1 gene. Although mH2A is generally enriched on the inactive X chromosome in female cells, mH2AS137ph is excluded from this heterochromatic structure. Thus, a phosphorylated subpopulation of mH2A appears to play a unique role in chromatin regulation beyond X inactivation. We provide evidence that S137ph is enriched in mitosis, suggestive of a role in the regulation of mH2A posttranslational modifications throughout the cell cycle.

Inducible XIST-dependent X-chromosome inactivation in human somatic cells is reversible.

During embryogenesis, the XIST RNA is expressed from and localizes to one X chromosome in females and induces chromosome-wide silencing. Although many changes to inactive X heterochromatin are known, the functional relationships between different modifications are not well understood, and studies of the initiation of X-inactivation have been largely confined to mouse. We now present a model system for human XIST RNA function in which induction of an XIST cDNA in somatic cells results in localized XIST RNA and transcriptional silencing. Chromatin immunoprecipitation and immunohistochemistry shows that this silencing need only be accompanied by a subset of heterochromatic marks and that these can differ between integration sites. Surprisingly, silencing is XIST-dependent, remaining reversible over extended periods. Deletion analysis demonstrates that the first exon of human XIST is sufficient for both transcript localization and the induction of silencing and that, unlike the situation in mice, the conserved repeat region is essential for both functions. In addition to providing mechanistic insights into chromosome regulation and formation of facultative heterochromatin, this work provides a tractable model system for the study of chromosome silencing and suggests key differences from mouse embryonic X-inactivation.

Silencing of the mammalian X chromosome.

Mammalian X chromosome inactivation is one of the most striking examples of epigenetic gene regulation. Early in development one of the pair of approximately 160-Mb X chromosomes is chosen to be silenced, and this silencing is then stably inherited through subsequent somatic cell divisions. Recent advances have revealed many of the chromatin changes that underlie this stable silencing of an entire chromosome. The key initiator of these changes is a functional RNA, XIST, which is transcribed from, and associates with, the inactive X chromosome, although the mechanism of association with the inactive X and recruitment of facultative heterochromatin remain to be elucidated. This review describes the unique evolutionary history and resulting genomic structure of the X chromosome as well as the current understanding of the factors and events involved in silencing an X chromosome in mammals.

Characterization of expression at the human XIST locus in somatic, embryonal carcinoma, and transgenic cell lines.

X inactivation requires XIST, a functional RNA that is expressed exclusively from, and localizes to, the inactive X in female somatic cells. In mouse, low-level unstable transcription of Xist is observed prior to the time of inactivation, and an antisense transcript, Tsix, is a critical regulator of early Xist expression. To examine the presence and impact of an antisense transcript in humans we have characterized the extent of sense and antisense transcription in human somatic, transgenic, and embryonal carcinoma (EC) cell lines. Downstream antisense expression at the human XIST locus was not detected in somatic cells, but was detected in the EC line N-Tera2D1 and in somatic cells with an ectopic XIST locus. Presence of the antisense did not disrupt the stability or localization of the sense transcript. We have also identified additional sense transcripts in EC and female somatic cells and demonstrate that the 5' flanking JPX/ENOX gene is expressed from both the active and the inactive X chromosome in somatic cell hybrids, delimiting the extent of inactive X-specific transcriptional control in somatic cells. These analyses reveal similarities to and differences from the murine Xist and Tsix transcripts and generate a complex picture of developmentally regulated transcription through the region.

Beyond sense: the role of antisense RNA in controlling Xist expression.

The Xist RNA is a critical component of X inactivation, and Tsix is a non-coding antisense RNA to the Xist gene. We review the data from mouse that demonstrates that Tsix serves to regulate Xist expression. TSIX antisense transcripts have also been detected in humans, but without a manipulatable system to study the inactivation process in humans it remains unknown whether these antisense transcripts are functional in regulating human XIST. After a review of the differences between the human and mouse antisense, we discuss how the question of whether or not the human TSIX is functional impacts models of Tsix function.