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E Heard

Publications and source records attributed to E Heard.

At least 19 recordsLinked to original sources
PubMed2006

The dynamics of imprinted X inactivation during preimplantation development in mice.

In the mouse, there are two forms of X chromosome inactivation (XCI), random XCI in the fetus and imprinted paternal XCI, which is limited to the extraembryonic tissues. While the mechanism of random XCI has been studied extensively using the in vitro XX ES cell differentiation system, imprinted XCI during early embryonic development has been less well characterized. Recent studies of early embryos have reported unexpected findings for the paternal X chromosome (Xp). Imprinted XCI may not be linked to meiotic silencing in the male germ line but rather to the imprinted status of the Xist gene. Furthermore, the Xp becomes inactivated in all cells of cleavage-stage embryos and then reactivated in the cells of the inner cell mass (ICM) that form the epiblast, where random XCI ensues.

Animals↗
PubMed2002

Integrated kinetics of X chromosome inactivation in differentiating embryonic stem cells.

Inactivation of the X chromosome during early female development and the subsequent maintenance of this transcriptionally inert state through countless cell divisions remain a paradigm for epigenetic regulation in mammals. Nevertheless, the exact mechanisms underlying this chromosome-wide silencing process remain unclear. Using differentiating female embryonic stem (ES) cells as a model system, we recently found that histone H3 tail modifications are among the earliest known chromatin changes in the X inactivation process, appearing as soon as Xist RNA accumulates on the X chromosome, but prior to transcriptional silencing of X-linked genes (Heard et al., 2001). In this report we present an integrated analysis of the sequence of early events and chromatin modifications underlying X inactivation in differentiating female ES cells. We have extended our previous analysis concerning changes in histone tail modification states. We find that the hypomethylation of Arg-17 and that of Lys-36 on histone H3 also characterize the inactive X chromosome, and that these profiles show a similarly early onset during the initiation of X inactivation. In addition, we have investigated the kinetics of the shift in replication timing of the X chromosome undergoing inactivation. This event occurs slightly later than Xist RNA coating and the chromatin modifications. Finally, from an early stage in the X inactivation process, characteristic histone modification patterns can be found on the X chromosome at mitosis, suggesting that they represent true epigenetic marks of the inactive state.

Acetylation↗
PubMed2001

Methylation of histone H3 at Lys-9 is an early mark on the X chromosome during X inactivation.

Coating of the X chromosome by Xist RNA is an essential trigger for X inactivation. However, little is known about the early chromatin remodeling events that transform this signal into transcriptional silencing. Here we report that methylation of histone H3 lysine 9 on the inactive X chromosome occurs immediately after Xist RNA coating and before transcriptional inactivation of X-linked genes. X-chromosomal H3 Lys-9 methylation occurs during the same window of time as H3 Lys-9 hypoacetylation and H3 Lys-4 hypomethylation. Histone H3 modifications thus represent the earliest known chromatin changes during X inactivation. We also identify a unique "hotspot" of H3 Lys-9 methylation 5' to Xist, and we propose that this acts as a nucleation center for Xist RNA-dependent spread of inactivation along the X chromosome via H3 Lys-9 methylation.

A Kinase Anchor Proteins↗
PubMed2001

X-chromosome inactivation: counting, choice and initiation.

In many sexually dimorphic species, a mechanism is required to ensure equivalent levels of gene expression from the sex chromosomes. In mammals, such dosage compensation is achieved by X-chromosome inactivation, a process that presents a unique medley of biological puzzles: how to silence one but not the other X chromosome in the same nucleus; how to count the number of X's and keep only one active; how to choose which X chromosome is inactivated; and how to establish this silent state rapidly and efficiently during early development. The key to most of these puzzles lies in a unique locus, the X-inactivation centre and a remarkable RNA--Xist--that it encodes.

Animals↗
PubMed2001

Improvement of FISH mapping resolution on combed DNA molecules by iterative constrained deconvolution: a quantitative study.

Image restoration approaches, such as digital deconvolution, are becoming widely used for improving the quality of microscopic images. However, no quantification of the gain in resolution of fluorescence images is available. We show that, after iterative constrained deconvolution, fluorescent cosmid signals appear to be 25% smaller, and 1.2-kb fragment signals on combed molecules faithfully display the expected length.

Chromosomes, Artificial, Yeast↗
PubMed1999

Human XIST yeast artificial chromosome transgenes show partial X inactivation center function in mouse embryonic stem cells.

Initiation of X chromosome inactivation requires the presence, in cis, of the X inactivation center (XIC). The Xist gene, which lies within the XIC region in both human and mouse and has the unique property of being expressed only from the inactive X chromosome in female somatic cells, is known to be essential for X inactivation based on targeted deletions in the mouse. Although our understanding of the developmental regulation and function of the mouse Xist gene has progressed rapidly, less is known about its human homolog. To address this and to assess the cross-species conservation of X inactivation, a 480-kb yeast artificial chromosome containing the human XIST gene was introduced into mouse embryonic stem (ES) cells. The human XIST transcript was expressed and could coat the mouse autosome from which it was transcribed, indicating that the factors required for cis association are conserved in mouse ES cells. Cis inactivation as a result of human XIST expression was found in only a proportion of differentiated cells, suggesting that the events downstream of XIST RNA coating that culminate in stable inactivation may require species-specific factors. Human XIST RNA appears to coat mouse autosomes in ES cells before in vitro differentiation, in contrast to the behavior of the mouse Xist gene in undifferentiated ES cells, where an unstable transcript and no chromosome coating are found. This may not only reflect important species differences in Xist regulation but also provides evidence that factors implicated in Xist RNA chromosome coating may already be present in undifferentiated ES cells.

Animals↗
PubMed1999

A developmental switch in H4 acetylation upstream of Xist plays a role in X chromosome inactivation.

We have investigated the role of histone acetylation in X chromosome inactivation, focusing on its possible involvement in the regulation of Xist, an essential gene expressed only from the inactive X (Xi). We have identified a region of H4 hyperacetylation extending up to 120 kb upstream from the Xist somatic promoter P1. This domain includes the promoter P0, which gives rise to the unstable Xist transcript in undifferentiated cells. The hyperacetylated domain was not seen in male cells or in female XT67E1 cells, a mutant cell line heterozygous for a partially deleted Xist allele and in which an increased number of cells fail to undergo X inactivation. The hyperacetylation upstream of Xist was lost by day 7 of differentiation, when X inactivation was essentially complete. Wild-type cells differentiated in the presence of the histone deacetylase inhibitor Trichostatin A were prevented from forming a normally inactivated X, as judged by the frequency of underacetylated X chromosomes detected by immunofluorescence microscopy. Mutant XT67E1 cells, lacking hyperacetylation upstream of Xist, were less affected. We propose that (i) hyperacetylation of chromatin upstream of Xist facilitates the promoter switch that leads to stabilization of the Xist transcript and (ii) that the subsequent deacetylation of this region is essential for the further progression of X inactivation.

Acetylation↗
PubMed1999

Functional analysis of the DXPas34 locus, a 3' regulator of Xist expression.

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' to Xist. One such element, lying 15 kb downstream of Xist, 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 the DXPas34 locus in Xist regulation and X-inactivation initiation by deleting it in the context of large Xist-containing yeast artificial chromosome transgenes. Deletion of DXPas34 eliminates both Xist expression 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 imprinted Xist expression.

Animals↗
PubMed1999

Xist yeast artificial chromosome transgenes function as X-inactivation centers only in multicopy arrays and not as single copies.

X-chromosome inactivation in female mammals is controlled by the X-inactivation center (Xic). This locus is required for inactivation in cis and is thought to be involved in the counting process which ensures that only a single X chromosome remains active per diploid cell. The Xist gene maps to the Xic region and has been shown to be essential for inactivation in cis. Transgenesis represents a stringent test for defining the minimal region that can carry out the functions attributed to the Xic. Although YAC and cosmid Xist-containing transgenes have previously been reported to be capable of cis inactivation and counting, the transgenes were all present as multicopy arrays and it was unclear to what extent individual copies are functional. Using two different yeast artificial chromosomes (YACs), we have found that single-copy transgenes, unlike multicopy arrays, can induce neither inactivation in cis nor counting. These results demonstrate that despite their large size and the presence of Xist, the YACs that we have tested lack sequences critical for autonomous function with respect to X inactivation.

Animals↗
PubMed1999

Rapid-fire improvement with short-cycle kaizen.

Continuous improvement is an attractive idea, but it is typically more myth than reality. SCK is no myth. It delivers dramatic improvements in traditional measures quickly. SCK accomplishes this via kaizens: rapid, repeated, time-compressed changes for the better in bite-sized chunks of the business.

Efficiency, Organizational↗
PubMed1998

Cloning and localization of the murine Xpct gene: evidence for complex rearrangements during the evolution of the region around the Xist gene.

The overall organization of the X-inactivation center (XIC/Xic) candidate region seems poorly conserved between human and mouse. The orientation of a region containing the X-inactive-specific transcript (Xist/ XIST) gene and three genes located 3' of Xist/XIST has been shown to be inverted between the two species, although the actual extent of this rearrangement is unknown. We have cloned and mapped the mouse homolog of the human XPCT (X-linked PEST-containing transporter) gene, which encodes a putative transmembrane transporter. Human XPCT is located about 200 kb outside of the XIC candidate region and 600 kb 5' of or telomeric to the XIST gene. The mouse Xpct gene, which lies approximately 300 kb 5' of and centromeric to Xist, displays 85% identity at the nucleotide level with the human gene, and the overall protein structure is conserved. The transcriptional orientation of mouse Xpct with respect to Xist is the opposite of that in human. Consequently, the evolution of the region between human and mouse appears to be highly complex, with structural rearrangements involving a region of up to 600 kb or more around the Xist gene.

Amino Acid Sequence↗
PubMed1998

Molecular correlates of the murine Xce locus.

The murine Xce locus, first identified by Bruce Cattanach, influences the primary choice of the X chromosome to be inactivated. Methylation of a GC-rich region (DXPas34) that includes multiple 34 bp repeats and lies some 15 kb 3' to Xist has been shown to vary with Xce haplotype. The degree of methylation on the active X chromosome at this locus represents one of the few molecular correlates of Xce action currently available. Data relating to the specificity and other characteristics of this association are presented.

Animals↗
PubMed1997

Localization and expression analysis of a novel conserved brain expressed transcript, Brx/BRX, lying within the Xic/XIC candidate region.

The X inactivation center candidate region (Xic/XIC in mouse and human) is poorly characterized for the presence of transcription units. Only two conserved genes have been isolated to date, Xist/XIST and Cdx4/CDX4. The other known gene lying within this region, Tsx, has been identified so far only in rodents by analyzing the complete genomic sequence of a 94-kb region distal to Xist. Here, we report the characterization of an additional gene lying within this 94-kb sequenced region. Brx, for Brain X-linked gene, is a rare transcript preferentially expressed in the brain. It is normally X-inactivated in the mouse. Localisation of BRX, its human homolog has shown the gene to be located within the orthologous but inverted human CDX4-XIST segment. These results suggest that the gene order of the region encompassing the Cdx4-Xist interval in the mouse is similar in human. Comparison of the Xist-Brx and Brx-Cdx4 regions in mouse and human indicates that these intervals are three times longer in human than in mouse. BRX is a new potential candidate for one of the X-linked mental retardation syndromes mapped within the pericentromeric region of the human X Chromosome (Chr).

Amino Acid Sequence↗
PubMed1997

X-chromosome inactivation in mammals.

The inactive X chromosome differs from the active X in a number of ways; some of these, such as allocyclic replication and altered histone acetylation, are associated with all types of epigenetic silencing, whereas others, such as DNA methylation, are of more restricted use. These features are acquired progressively by the inactive X after onset of initiation. Initiation of X-inactivation is controlled by the X-inactivation center (Xic) and influenced by the X chromosome controlling element (Xce), which causes primary nonrandom X-inactivation. Other examples of nonrandom X-inactivation are also presented in this review. The definition of a major role for Xist, a noncoding RNA, in X-inactivation has enabled investigation of the mechanism leading to establishment of the heterochromatinized X-chromosome and also of the interactions between X-inactivation and imprinting as well as between X-inactivation and developmental processes in the early embryo.

Animals↗
PubMed1996

Transgenic mice carrying an Xist-containing YAC.

The initiation of X-chromosome inactivation in female mammals is controlled by a key locus, the X-inactivation centre (Xic). The Xist gene, which maps to the candidate region for Xic and is expressed exclusively from the inactive X chromosome, is thought to be an essential component of the Xic. To test whether sequences spanning several hundred kilobases and including Xist from the Xic region are capable of initiating inactivation, we have created a series of transgenic mice using a 460 kb yeast artificial chromosome (YAC). Analysis in these mice of the expression of Xist, of a LacZ reporter gene and of two genes in the region that are normally silent on the inactive X chromosome, suggests that essential sequences for Xist expression and X-inactivation may be absent in these transgenic animals.

Animals↗