X inactivation

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Nucleus of a female human cell made from amniotic fluid . Above: Both X chromosomes are shown by fluorescence in situ hybridization . A single optical section is shown, which was produced with a confocal laser scanning microscope . Below: the same core with DAPI staining, recorded with a CCD camera . The Barr body can be clearly seen here (arrow) and identifies the inactive X chromosome (Xi).

In epigenetics, X inactivation or X chromosome inactivation , also known as lyonization , is a process in which an X chromosome is completely or largely shut down so that no more gene products are created from this chromosome .

X inactivation is best known in mammals . With these, there are two X chromosomes per cell in normal female individuals, whereas in normal male individuals there is one X chromosome and one Y chromosome (see also sex chromosome ). In order to compensate for the higher number of X-linked genes, all but one of them are inactivated by packaging in heterochromatin during embryonic development in cells with more than one X-chromosome , resulting in Barr bodies . This process was first postulated in 1961 by the English geneticist Mary Frances Lyon and accordingly referred to as the Lyon hypothesis . The X-inactivation has now been investigated and documented in detail, so that the designation as a hypothesis only has historical significance.

Once an X chromosome has been inactivated, it remains continuously inactive through epigenetic regulation and is thus passed on from one cell to all daughter cells. It is a regulation of gene expression , not a change in the DNA sequence. Therefore, the X-inactivation is in principle reversible. In fact, it is abolished at certain stages of the germline development . Cancellation does not take place in somatic cells .

In humans, mice and presumably all higher mammals , it is a matter of chance which of the two X chromosomes present is inactivated in a cell during embryonic development and then remains inactive in all daughter cells. This is a possible cause of phenotypic differences in identical sisters . In marsupials , however, the paternal X chromosome is always inactive. In some animal species, X-inactivation also results in differences in color design between the two sexes.

X-inactivation was also observed in the roundworm Caenorhabditis elegans . Unless expressly stated otherwise, the information in this article relates to X inactivation in marsupials and higher mammals (placenta animals) or to test results in human and mouse cells.

Benefit of inactivation: dose compensation

see also: sex chromosome

In mammals, the sex is determined by the presence of so-called sex chromosomes (gonosomes). Females have two X chromosomes, males one X and one Y chromosome . It is now assumed that sex chromosomes evolved from a pair of autosomes (see Y chromosome). According to this view, the Y chromosome has steadily shrunk, so that ultimately many genes only appeared on the X chromosome. In this way, increased reading of the X chromosome in males was evolutionarily promoted, which counteracts a reduction in the dose of gene products. As a consequence, a mechanism developed that shuts down one of the two female X chromosomes, X inactivation. As a result of X inactivation, there are no gender-specific differences for most of the gene products of X-linked genes, as would otherwise be expected.

The inactivation of the X chromosome is not complete, a number of genes are not inactivated. This applies to genes of the identical pseudoautosomal regions on the X and Y chromosomes . Outside of the pseudoautosomal region, some genes occur on both the X and the Y chromosome, so that dose compensation does not offer any selection advantage. The Xist gene, which itself plays an important role in inactivation (see below), on the other hand, is only read ( transcribed ) on the inactive X chromosome .

Types of inactivation

Male meiosis

Meiotic sex chromosome inactivation (MSCI) has been observed in very different organisms. In addition to the mouse, in which detailed investigations were carried out, the shrew pouch rat Monodelphis domestica , the mold Neurospora crassa and C. elegans should be mentioned here (see below). Since X and Y chromosomes are equally affected, there is no specific X inactivation. This inactivation occurs during pachytan, the stage of meiosis in which the homologous chromosomes are paired. Unpaired chromosomes or chromosomal sections are basically shut down here. Perhaps the original use of this mechanism for the organism is to halt meiosis while there are still unpaired segments, or to prevent gene expression of foreign DNA such as transposons or retroviruses .

In male mammals, pairing of the X and Y chromosomes in meiosis is only possible in the pseudoautosomal regions that are at the ends of the chromosomes, but not in the regions in between. In the mouse, these unpaired sections are recognized by proteins that are otherwise responsible for recognizing DNA breaks. These in turn store markings that stop gene expression. A heterochromatic , microscopically detectable structure is created, the 'XY-Body'. The inactivated state is retained for the remainder of spermatogenesis , with the exception of some genes that are needed for spermatogenesis itself. Marsupials do not have pseudo-autosomal regions. A meiotic pairing of the X and Y chromosomes via homologous sequences is therefore not possible. The two inactivated chromosomes come together late in the pachytan and then also remain inactive for the rest of the spermatogenesis.

Paternal X chromosome

In marsupials, the paternal X chromosome is always inactivated. Its recognition is controlled by imprinting (English: imprinted X-Inactivation): In the female germline, different markings are attached to the chromosomes than in the male germline, so that the organism that is developing can recognize from which parent a certain chromosome was inherited . The exact mechanism of inactivation in marsupials is still not understood. The inactivation markers attached to the X chromosome in the male germ line may be decisive. Also, not all genes are shut down on the inactive X.

In placentations, a non-random, specific inactivation of the paternal X chromosome takes place during early embryonic development. The following sequence has been described for mice: In the 2-cell stage, when the gene expression starts for the first time in mice, the Xist gene is read on the paternal X chromosome . It is known that the Xist RNA that is produced in this way plays an important role at least in the later random X inactivation. Now it initially remains in the immediate vicinity of the Xist gene, but during the next cell divisions its range expands increasingly until it finally covers the paternal X chromosome. This is accompanied by an increasing inactivation of the chromosome. From the morula stage (spherical cell cluster), the Xist expression decreases again, so that in the subsequent blastocyst stage (embryo already separated from trophoblast from which the placenta emerges) both X chromosomes are active in the embryo itself. In the extraembryonic tissue, however, the paternal X chromosome remains inactive throughout. This inactivation is in turn regulated by Xist .

Marsupials do not have an Xist gene. Since the non-random inactivation of the paternal X is found in marsupials and placental animals, it is believed that this is the original process from which random selection in higher mammals evolved.

Random selection

The black and red tortoiseshell pattern in some female cats is caused by X inactivation: The responsible gene is located on the X chromosome and occurs in various forms ( alleles ). Depending on which X chromosome is active in the corresponding cells , either the red or black coat color occurs. Since the inactivated X chromosome is selected at random in the early embryonic development and is then passed on to all daughter cells, patches of fur with the same color are created.

In humans and the higher mammals (placenta animals), the permanent selection of the X chromosome to be inactivated in the cells of the embryo is made randomly and independently in each cell. This type of selection is seen as an advantage, since in the female organism a damaging mutation on one of the X chromosomes only affects about half of the cells and the healthy cells can largely compensate for this in many cases. The underlying mechanism is shown in the following section.

Inactivation process in higher mammals

The processes presented below have been well studied in humans and mice. It is assumed that they are similar in other mammals.

Xist RNA

In embryonic development , X inactivation takes place around the time the pluripotent cells differentiate into different cell layers, i.e. during the blastocyst stage . The Xist gene plays an important role in this.

X inactivation occurs through the binding of a special ribonucleic acid (RNA) to the X chromosome in question. This RNA is known as X inactive specific transcript or Xist-RNA for short , the corresponding gene is located in a section of the X chromosome called the X Inactivation Center (XIC). It is assumed that there is no protein corresponding to the Xist gene , but that this gene acts exclusively via the corresponding RNA. It is therefore subject to transcription from DNA to RNA, but not to translation from RNA to protein (see Non-coding RNA ).

By binding the Xist RNA to the X chromosome to be inactivated, many of the genes on this chromosome are inactivated. Exceptions are the Xist gene itself and around a quarter of the remaining genes, especially in the areas of the sections called pseudoautosomal regions at both ends of the X chromosome, areas with corresponding homologous sections on the Y chromosome.

Before X inactivation, small amounts of Xist RNA are produced by both chromosomes . Not then done on the later active X chromosome (Xa) expression of Xist gene more, while it counts on the inactivated X chromosome (Xi) among the few active genes.

The Xist gene on the X chromosome of the mother is initially methylated in the egg cell . H. inactive, the X chromosome is accordingly active. If a Y chromosome is added with the sperm , the Xist gene remains methylated. If, however, another X chromosome is added, the Xist gene is not methylated (the Xist gene is only expressed during spermatogenesis ). The Xist RNA is created and the second X chromosome is also inactivated. However, this state is not stable, as the EED protein is required for maintenance and this is also encoded on the X chromosome. As a result, both X chromosomes and thus the Xist gene become active again in the morula stage . This time, however, only one X chromosome is inactivated, with either the paternal or maternal X chromosome being switched off. Since this only takes place in the embryo lasting several days (in the mouse after the seventh day, in humans after 16 days), female mammals are so-called genetic X-mosaics. This can be seen, for example, in the color of the fur of cats (see illustration).

Heterochromatinization and Barr bodies

see also: Gender Chromatin
The nucleus of a human, female fibroblast was stained with the blue fluorescent DNA dye DAPI in order to represent the Barr body, i.e. the inactive X chromosome (arrow). In addition, a special form of a histone (macroH2A) with antibodies coupled to a green fluorescent dye was detected in the same nucleus . This special histone form is enriched in the Barr body.

The binding of the Xist RNA together with other factors triggers hypoacetylation of the histones , which is associated with special inactivating methylation patterns of the histones. Finally, the normally present histone H2A is replaced by the variant macroH2A (see figure). This leads to DNA methylation and thus to inactivation of the promoters . This goes hand in hand with the compression of chromatin in the form of heterochromatin, which replicates late and is mostly found in peripheral areas of the cell nucleus. Correspondingly inactivated and compacted X chromosomes are referred to as Barr bodies or sex chromatin , the formation of Barr bodies as Lyonization .

The inactivation of the X chromosome results in the following molecular changes:

  • many promoters (especially GC blocks ) are methylated and thus the genes are switched off
  • the histones H3 are methylated and the histones H4 are deacetylated . This binds the DNA more tightly to the histones, making it difficult to read. The inactivated X chromosome thus becomes the optically denser heterochromatin and is called the Barr body .

The Heterochromatinisierung happens when people do not complete and identical, so in heterozygous present alleles of X-chromosomally recessive they do not need to break inherited diseases. This explains why men are more likely to develop diseases such as hemophilia . You are missing the second X for possible compensation.

The microscopic detection of Barr bodies is used to determine the genetic sex of a person, for example in sports medicine .

Different number of X chromosomes and Barr bodies

The presence of additional X chromosomes and thus deviations from the karyotype XX in women or XY in men usually leads to mild and easily treatable symptoms or even to symptoms due to X inactivation, in contrast to the often serious consequences of additional autosomes a symptom-free course. This applies, for example, to Triplo-X syndrome (47, XXX) in women or Klinefelter syndrome (47, XXY) in men.

Inactivation by XIST can also inactivate genes on other chromosomes if the XIST gene was transferred to another chromosome by a chromosome mutation or artificially. For example, an XIST gene was transferred to an excess chromosome 21 in human cells, which was then inactivated. An extra chromosome 21 is the cause of Down syndrome . However, it is unclear whether this discovery will be of therapeutic importance.

Discovery story

Barr bodies were first described in 1949 by Murray Llewellyn Barr and Ewart George Bertram for female cat neurons. They found a chromatin -Körperchen on the inside of nuclear membrane , which she as a sex chromatin ( Barr body designated). They were able to show that a determination of the sex was possible through the proof.

The English geneticist Mary Frances Lyon postulated in 1961 that one of the two X chromosomes present in a female cell was inactive, that inactivation would take place early in embryonic development and that the selection of the X chromosome in question was random. This hypothesis was based on observations made when different strains of mice were crossed and the resulting differences in coat color and hair shape in the offspring. Mary Frances Lyon also recognized the sex chromatin described by Barr and Bertram as inactivated X chromosomes and coined the term Barr body, which is used today, mostly translated as Barr body, less often than Barr body.

The breakthrough in understanding the mechanism of X inactivation came in 1991 with the description of the Xist genes in humans and mice. A year later, the RNA transcripts of the two genes were characterized in several further papers and the expression and its connection with X-inactivation were documented.

In 1993 it was possible for the first time to show a connection between disorders of Xist expression and a rare clinical picture based on the presence of X-ring chromosomes . A year later, the Xist gene was first used for diagnostic purposes to detect Klinefelter syndrome . In the following years, the mechanisms underlying X inactivation were elucidated in detail through further work.

X inactivation in C. elegans

In the nematode Caenorhabditis elegans , inactivation of X chromosomes has also been described. However, this is not present in somatic cells, but in a stage of the germ line . In C. elegans , there are two sexes: hermaphrodites with two X chromosomes and males with only one X chromosome (X0, see also the sex chromosomes ). The gene expression on the X chromosome is downregulated during meiosis in both sexes, in the female on both X chromosomes. Here too, certain modifications of the histones are associated with the inactivation. Corresponding differences in the histone modifications were also made in meiotic cells of other nematode species, both in hermaphroditic species and in species with males and females.

literature

Individual evidence

  1. ↑ a b c d e f g h Payer B, Lee JT: X Chromosome Dosage Compensation: How Mammals Keep the Balance . In: Annual review of genetics . August 2008. doi : 10.1146 / annurev.genet.42.110807.091711 . PMID 18729722 .
  2. ↑ Jun Jiang, Yuanchun Jing, Gregory J. Cost, Jen-Chieh Chiang, Heather J. Kolpa, Allison M. Cotton, Dawn M. Carone, Benjamin R. Carone, David A. Shivak, Dmitry Y. Guschin, Jocelynn R. Pearl, Edward J. Rebar, Meg Byron, Philip D. Gregory, Carolyn J. Brown, Fyodor D. Urnov, Lisa L. Hall, Jeanne B. Lawrence: Translating dosage compensation to trisomy 21. In: Nature. 2013, S., doi: 10.1038 / nature12394 .
  3. ↑ ML Barr and EG Bertram: A morphological distinction between neurones of the male and female and the behavior of the nuclear satellite during accelerated nucleoprotein synthesis. In: Nature . 163/1949. Nature Publishing Group, pp. 676-677, ISSN  0028-0836
  4. ^ Mary F. Lyon: Gene Action in the X-chromosome of the Mouse (Mus musculus L.) . In: Nature . tape 190 , no. 4773 , April 1961, p. 372-373 , doi : 10.1038 / 190372a0 .
  5. ↑ CJ Brown, A. Ballabio, JL Rupert, RG Lafreniere, M. Grompe, R. Tonlorenzi, HF Willard: A gene from the region of the human X inactivation center is expressed exclusively from the inactive X chromosome . In: Nature . tape 349 , no. 6304 , January 1991, p. 38-44 , doi : 10.1038 / 349038a0 , PMID 1985261 .
  6. ↑ G. Borsani, R. Tonlorenzi, M.-C. Simmler, L. Dandolo, D. Arnaud, V. Capra, M. Grompe, A. Piizzuti, D. Muzny, C. Lawrence, HF Willard, P. Avner, A. Ballabio: Characterization of a murine gene expressed from the inactive X chromosomes . In: Nature . tape 351 , no. 6324 , May 1991, pp. 325-329 , doi : 10.1038 / 351325a0 .
  7. ^ William G. Kelly, Christine E. Schaner, Abby F. Dernburg, Min-Ho Lee, Stuart K. Kim, Anne M. Villeneuve and Valerie Reinke: X-chromosome silencing in the germline of C. elegans . Development. 2002 Jan; 129 (2): 479-92. PMID 11807039 .

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