Early Loss of Xist RNA Expression and Inactive X Chromosome Associated Chromatin Modification in Developing Primordial Germ Cells

The inactive X chromosome characteristic of female somatic lineages is reactivated during development of the female germ cell lineage. In mouse, analysis of protein products of X-linked genes and/or transgenes located on the X chromosome has indicated that reactivation occurs after primordial germ cells reach the genital ridges.We present evidence that the epigenetic reprogramming of the inactive X-chromosome is initiated earlier than was previously thought, around the time that primordial germ cells (PGCs) migrate through the hindgut. Specifically, we find that Xist RNA expression, the primary signal for establishment of chromosome silencing, is extinguished in migrating PGCs. This is accompanied by displacement of Polycomb-group repressor proteins Eed and Suz(12), and loss of the inactive X associated histone modification, methylation of histone H3 lysine 27.We conclude that X reactivation in primordial germ cells occurs progressively, initiated by extinction of Xist RNA around the time that germ cells migrate through the hindgut to the genital ridges. The events that we observe are reminiscent of X reactivation of the paternal X chromosome in inner cell mass cells of mouse pre-implantation embryos and suggest a unified model in which execution of the pluripotency program represses Xist RNA thereby triggering progressive reversal of epigenetic silencing of the X chromosome

Epigenetic modifications during X-chromosome inactivation and reactivation

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Xist RNA expression in XX PGCs.

<p>(A) Example of FACS plot showing GFP-positive and GFP-negative cell populations collected for analysis from dissociated genital ridges of female Oct4-GFP embryos. GFP intensity is shown on the X axis. (B) Example of RNA FISH analysis detecting Xist RNA in the Oct4-GFP negative cells shown in (A). (C) Example of RNA FISH analysis showing Xist RNA domains are absent in the majority of the Oct4-GFP positive PGCs shown in (A). (D) Scoring results showing proportion of cells from XX embryos with Xist RNA domains in Oct4-GFP positive cell (PGCs) compared to unsorted dissociated cell (control) populations. For 9.5 dpc 105 FACS sorted and 405 control cells obtained in two independent experiments were scored. For 11.5 dpc data 140 FACS sorted and 262 control cells were scored.</p

Elevated PRC2 and H3K27me3 levels in developing PGCs.

<p>(A) Indirect IF on the hind gut of 9.5 dpc XX embryos and whole genital ridges of 10.5 dpc and 11.5 dpc XX embryos using anti-Eed (in green) and TG-1 (in red/yellow) antibodies. PGCs (TG-1 positive cells) can be easily distinguished from neighbouring cells by their stronger Eed nuclear staining. Both primary antibodies are mouse monoclonals but after sequential detection the signals can be distinguished because TG1 signal is restricted to the cell membrane/cytoplasm and Eed protein is present only in the nucleus. (B) Indirect IF on the hind gut of 9.5 dpc XX embryos and whole genital ridges of 10.5 dpc and 11.5 dpc XX embryos detecting Oct4 (in green) and H3K27me3 (in red). At 9.5 dpc PGCs and neighbouring somatic cells present similar nuclear levels of H3K27me3. At 10.5 dpc and 11.5 dpc, PGCs show much higher levels of this modification than neighbouring somatic cells. The bottom panels show an example where DNA was counterstained with DAPI (blue) to reveal the nuclei of all cells in the section. Images correspond to a single confocal section at 630× original magnification.</p

Polycomb group proteins ring1A/B link ubiquitylation of histone H2A to heritable gene silencing and X inactivation

In many higher organisms, 5%-15% of histone H2A is ubiquitylated at lysine 119 (uH2A). The function of this modification and the factors involved in its establishment, however, are unknown. Here we demonstrate that uH2A occurs on the inactive X chromosome in female mammals and that this correlates with recruitment of Polycomb group (PcG) proteins belonging to Polycomb repressor complex 1 (PRC1). Based on our observations, we tested the role of the PRC1 protein Ring1B and its closely related homolog Ring1A in H2A ubiquitylation. Analysis of Ring1B null embryonic stem (ES) cells revealed extensive depletion of global uH2A levels. On the inactive X chromosome, uH2A was maintained in Ring1A or Ring1B null cells, but not in double knockout cells, demonstrating an overlapping function for these proteins in development. These observations link H2A ubiquitylation, X inactivation, and PRC1 PcG function, suggesting an unanticipated and novel mechanism for chromatin-mediated heritable gene silencing. Copyright © 2004 by Cell Press.This work was supported by the Medical Research Council, UK, by Special Coordination Funds for Promoting Science and Technology from the Japanese Government, and by the DGI, Spanish Ministry of Education and Science (SAF2001-2211-CO2-01). M.d.N. is supported by Programa Gulbenkian de Doutoramento em Biologia e Medicina, PortugalPeer Reviewe

International Nosocomial Infection Control Consortiu (INICC) report, data summary of 43 countries for 2007-2012. Device-associated module

We report the results of an International Nosocomial Infection Control Consortium (INICC) surveillance study from January 2007-December 2012 in 503 intensive care units (ICUs) in Latin America, Asia, Africa, and Europe. During the 6-year study using the Centers for Disease Control and Prevention's (CDC) U.S. National Healthcare Safety Network (NHSN) definitions for device-associated health care–associated infection (DA-HAI), we collected prospective data from 605,310 patients hospitalized in the INICC's ICUs for an aggregate of 3,338,396 days. Although device utilization in the INICC's ICUs was similar to that reported from ICUs in the U.S. in the CDC's NHSN, rates of device-associated nosocomial infection were higher in the ICUs of the INICC hospitals: the pooled rate of central line–associated bloodstream infection in the INICC's ICUs, 4.9 per 1,000 central line days, is nearly 5-fold higher than the 0.9 per 1,000 central line days reported from comparable U.S. ICUs. The overall rate of ventilator-associated pneumonia was also higher (16.8 vs 1.1 per 1,000 ventilator days) as was the rate of catheter-associated urinary tract infection (5.5 vs 1.3 per 1,000 catheter days). Frequencies of resistance of Pseudomonas isolates to amikacin (42.8% vs 10%) and imipenem (42.4% vs 26.1%) and Klebsiella pneumoniae isolates to ceftazidime (71.2% vs 28.8%) and imipenem (19.6% vs 12.8%) were also higher in the INICC's ICUs compared with the ICUs of the CDC's NHSN