X-Chromosome Inactivation

Stanley M Gartler, University of Washington, Seattle, Washington, USA Michael A Goldman, San Francisco State University, San Francisco, California, USA
In female mammals, one of the X-chromosomes is transcriptionally silenced by heterochromatin formation to bring about equal expression of X-linked genes in XX females and XY males.

Secondary article
Article Contents
. Introduction . X Inactivation in Somatic Cells . X Inactivation in the Germline . Random and Nonrandom X Inactivation . Stability of X Inactivation . Genes that Escape Inactivation

Introduction
In mammals, the female has two large, gene-rich X chromosomes, whereas the male has a single X chromosome and a Y chromosome, which harbours few genes. If the X-linked genes were expressed equally in both sexes, females would produce approximately twice the level of Xlinked gene products as would males. Since the great majority of genes on the X are not concerned with sex determination and reproduction, this dierence would disturb metabolic balance in one sex or the other. In mammals and some other groups with XY sex determination, a system has evolved to bring about the equivalence in expression of X-linked genes in females and males. H. J. Muller, in 1931, dubbed this phenomenon dosage compensation (Muller, 1932). Dierent groups of organisms have evolved distinct strategies to achieve dosage compensation. In Drosophila, the transcription rate of the single X in males is higher than that of the two Xs in females. In Caenorhabditis elegans, the transcription rate is halved in the females two Xs. In mammals, most of the genes on one of the Xs in the female are transcriptionally silenced in a process called X-chromosome inactivation (XCI), the focus of this entry. Muller described dosage compensation in Drosophila. It was not until 30 years after that the phenomenon was clearly recognized in mammals. Based on observations by several workers, Mary Lyon, in 1961, proposed that each cell of a mammal maintained one and only one active X chromosome, inactivating the other to produce a darklystaining mass called the sex chromatin or Barr body (Lyon, 1961). Morphological evidence for X-chromosome inactivation could be seen in the coat colours of various mammals, including the calico cat a female cat in which one X chromosome encodes one coat colour, the other a dierent one, and the cats fur is a mosaic of patches having one coat colour or the other. This early model was, of course, incomplete, but the basic idea of a single active X chromosome in female cells has stood the test of time.

. XIST and the X-Inactivation Centre . DNA Methylation . Replication Timing . Histone Acetylation . Inactive-X-Specific Proteins . A Model for X-Chromosome Inactivation . Perspective

X Inactivation in Somatic Cells

In the mouse, the two X chromosomes in the early female embryo are not dierentiated, either cytologically or functionally; both X chromosomes are active. In the late morula or early blastocyst stages, the earliest evidence of XCI are seen: asynchrony of deoxyribonucleic acid (DNA) replication between the two Xs, dierential gene expression and sex chromatin formation. These dierences are rst observed in the extraembryonic cell lineages that form the membranes surrounding the embryo. In these cells the paternal X is preferentially inactivated. At about the time of gastrulation, random X inactivation occurs in the epiblast, which contains the cells that will give rise to the embryo itself. When inactivation occurs in these cells, the paternal or maternal X is randomly chosen to be inactivated. However, once inactivation has occurred, all of the descendants of a cell will have the same X silent. This results in patches of cells having an inactive maternal X, and in patches having an inactive paternal X, explaining the mosaic pattern of allelic expression seen, for example, in the calico cat.

X Inactivation in the Germline

In contrast to somatic cells, in which X inactivation is extremely stable, inactivation in the germline is cyclic. In the female, one X is inactive during the oogonial or mitotic phase, while both X chromosomes are active in oogenesis. Having both Xs active may be necessary to permit pairing and recombination in meiosis, which may not be possible if one X is heterochromatic and highly condensed and the other is not. In the male, with a single X, inactivation does not occur in somatic cells; however, early in
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X-Chromosome Inactivation

spermatogenesis, the single X is condensed and inactivated, possibly to restrict pairing and recombination between the nonhomologous parts of the X and Y chromosomes.

Random and Nonrandom X Inactivation

X inactivation in somatic cells is generally random, with the maternal and paternal Xs having an equal probability of inactivation. Thus, in females heterozygous for X-linked traits, such as haemophilia, about half the cells of the liver should, on average, produce normal clotting factor. This is typically enough to ensure normal blood clotting. If the trait aects fur colour, as in the calico cat, about half the patches in the cat should exhibit one phenotype. Statistically, sometimes one X will be inactivated more frequently than the other, just as a series of coin tosses may sometimes yield something other than a 50:50 ratio of heads to tails. Thus, chance could lead to a female heterozygous for haemophilia in which little or no clotting factor is produced and the typical symptoms of this disease are observed. Such statistical extremes are uncommon. Aside from statistical uctuation, preferential inactivation may occur, as in extraembryonic cells or in marsupials, where nonrandom inactivation of the paternal X is the rule (Graves, 1996). Genetic dierences on the chromosome may render one X more likely to undergo inactivation. In addition, if one X has a mutant allele whose expression renders a cell inviable or inhibits its growth, cells inactivating the mutant allele will have a selective advantage over cells in which the normal allele is inactivated. Thus, although inactivation may occur randomly at rst, cells that inactivate the wrong allele will be overgrown, and the outcome will be an organism in which all, or nearly all, cells have the same X inactivated.

related reactivation typically do not utilize DNA methylation, while those genes that clearly utilize DNA methylation as a regulatory mechanism are not reactivated with age. In transformed cell lines, reactivation of some inactive X-linked genes can be induced with 5-azacytidine (5AC), a drug known to reverse DNA methylation. The same drug treatment does not reactivate X-linked genes in normal somatic cells. The reactivation of the X in transformed cells probably occurs because some of the redundant mechanisms maintaining the inactive state are not functioning in these cells, leaving methylation as one of the only repressive inuences on X-linked gene expression (Hansen et al., 1998). Embryonal carcinoma (EC) cells are derived from gonadal tumours or embryonic tissues, and maintain their Xs in an active state as long as they remain undierentiated. However, upon chemical-induced dierentiation, the cells will go through X inactivation and one of the two X chromosomes will be silenced (Rastan and Robertson, 1985). When female mouse lymphocytes, having one active and one silent X, are fused with EC cells, the inactive X reactivates. This suggests that there are specic factors present in the EC cells that maintain or permit the active state of both X chromosomes.

Genes that Escape Inactivation

Once, every gene on the inactive X chromosome was believed to be transcriptionally silenced. We now know that a fair number of genes on human and mouse inactive Xs are not repressed (Disteche, 1995). One group of genes escaping X inactivation is found in the pseudoautosomal region (PAR) at the tip of the short arm of the human X, a region of homology between the X and Y chromosomes. Fertility in mammalian males requires pairing and recombination between the X and Y chromosomes within the PAR. Genes in the PAR are present on both the X and Y chromosomes, so no gene dosage dierence between males and females exists; compensation is therefore not necessary and genes in the PAR escape X inactivation. In addition to the PAR at the tip of the X short arm, a second, smaller, PAR is present at the tip of the long arm in humans. Several genes have been detected there, although, surprisingly, one of these is inactivated. Several genes in humans and mice outside the PAR have functional Ylinked alleles and, as expected, their X-linked counterparts escape inactivation. In these latter cases the X- and Ylinked alleles may dier from one another, as no recombination between them occurs. Some X-linked genes that escape inactivation have nonfunctional Y-linked copies (pseudogenes), presumably due to accumulated inactivating mutations. Lastly, there are several reports of

Stability of X Inactivation
In placental mammals, the inactive state of the X is generally highly stable. As we discuss below, a number of factors collaborate to repress alleles on the inactive X. If all factors ensuring X inactivation operated independently and were functional throughout life, reactivation would probably not be possible. In the germline, for instance, the inactivation state is cyclic and DNA methylation, an important repressive factor in somatic cells (see below), is absent. DNA methylation also seems to be absent from the inactivation system in marsupials, where, even in somatic cells, the inactivation of the X is relatively unstable. Even in mouse somatic cells, some genes on the inactive X are reactivated at low, but signicant levels as the animals age. We think that DNA methylation stabilizes the inactive state. Consistent with this notion, genes that exhibit age2

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X-Chromosome Inactivation

X-linked genes in humans that have no Y-chromosome counterparts, but still escape inactivation. Whether in the PAR or not, most genes that escape inactivation are organized in clusters or functional domains in which all of the genes escape inactivation. Such regulatory domains may contain sequence information specifying the spread or inhibition of an inactivation signal. Some that escape inactivation may have been repressed when X inactivation rst occurs, but are reactivated later. Thus the entire X chromosome may be inactivated initially, but selected inactive domains may be inherently unstable and rapidly reversed. The mouse X appears to be more completely inactivated than is the human X, perhaps reecting the occurrence of a longer evolutionary time for X inactivation in the mouse. Escape from inactivation of the SHOX gene in the human short arm PAR is apparently responsible for the short stature characteristic of Turner syndrome (XO) females. In mice, where XO is not associated with the phenotypic problems typical of Turner syndrome in humans, the SHOX homologue is not X linked.

XIST and the X-Inactivation Centre

A central feature of mammalian X inactivation is its chromosomal nature. With one exception (XIST, described below), all the inactivated genes are on one X, while the homologue contains the active alleles. This pattern is most easily explained by a single initiation site from which inactivation spreads along the rest of the chromosome. The evidence for the existence and location of an X-inactivation centre came from studies of X-autosomal translocations and deletions which interfered with the manifestation of X inactivation. Analysis of such translocations allows one to identify a short segment of the X, termed the Xinactivation centre or XIC, essential for X inactivation. Moreover, in mice, some Xs are more prone to inactivation than are others. The dierence between them resides in a locus called Xce (or X-controlling element), which maps to about the same position as the XIC identied in translocation experiments. A single gene that maps to the XIC and that has properties suggesting that it might be the locus essential for the initiation of X inactivation has been designated Xinactive specic transcript or XIST. (Human genes are designated entirely in capital letters, such as XIST, whereas mouse genes are designated in capitals and lower-case letters, as in Xist. For convenience here we use a single acronym, XIST.) XIST is expressed only from the inactive X (Brown et al., 1991). The ribonucleic acid (RNA) transcript has no signicant open reading frame and the product remains in the nucleus, coating the inactive X. This suggests that XIST is among those loci that produce a functional RNA molecule that is never translated into a

protein. XIST expression is detected early in preimplantation development, often from both Xs, but well before any sign of X inactivation. However, this early expression is probably not functional, as the transcript is not stable, is unprocessed, and does not coat the X. In the mouse, the paternal X begins to accumulate transcripts in the early morula, apparently as a result of an imprint in the gametes that leads to the paternal nonrandom inactivation found in extraembryonic cells. Later, in the inner cell mass, unstable XIST is transcribed from both Xs, but the XIST transcript from one or the other X becomes stabilized and accumulates on that X. Random X inactivation follows and XIST transcription from the active X is silenced. Recent evidence suggests that alernative promoter utilization or an antisense transcript may be involved in XIST regulation. Although the properties and map location of XIST strongly suggest that it should be a key element in the Xinactivation process, further experimental evidence was required to show that this locus is necessary for inactivation of the X chromosome. This was shown through the use of various deletions of the promoter or exon region of the XIST gene that prevent production of full-sized XIST RNA. In these cases the chromosome bearing the deletion is not inactivated, showing that the XIST gene is required in cis for inactivation to take place. Further support for a critical role of XIST comes from experiments in which the XIST gene and varying amounts of surrounding sequence have been incorporated into an autosome in male embryonic stem cells. In some cases, where multiple copies of the XIST transgene have been tandemly incorporated, XIST RNA coats the autosome and represses transcription. Although other factors will probably be involved, these experiments indicate that XIST RNA appears to propagate inactivation by binding to the chromosome from which it is expressed. They also imply that specic Xlinked sequences are not required for XIST RNA to coat a chromosome.

DNA Methylation
Methylation of the base cytosine occurs enzymatically after DNA synthesis and in mammals is restricted to the dinucleotide 5-CpG-3 (CpG). About 7% of CpGs are present in clusters called CpG islands, which are usually located at the 5 ends of genes and have a relatively high density of CpGs. The remaining CpGs are dispersed throughout the genome, usually as singlets. Most CpG islands are unmethylated, but those near inactivated genes on the X chromosome, and those near some imprinted genes on autosomes, are methylated. Methylated CpG islands repress transcription, and the great majority of them in the genome are found on the inactive X in normal cells. They are important in maintaining the repressed state
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X-Chromosome Inactivation

of inactive X-linked genes and may also play a role in establishing X inactivation, as indicated by the fact that dierential CpG island methylation occurs close to the time of inactivation of two X-linked genes that have been studied in detail. Interestingly, most of the dispersed CpGs throughout the genome tend to be methylated. There is limited evidence that the dispersed CpGs on the inactive X may be hypomethylated relative to the active X, suggesting a similarity in overall methylation levels on the active and inactive Xs. No evidence exists that the dispersed CpGs, methylated or unmethylated, play a role in gene silencing. A crucial role of methylation may be in the control of XIST expression, and therefore the initiation of X inactivation itself. Methylation analysis of a small cluster of CpGs in the XIST promoter shows that the silent allele on the active X becomes methylated while the expressed allele on the inactive X is hypomethylated. Developmental studies show that the sperm and oocyte are dierentially methylated in part of the promoter region (sperm hypermethylated, oocyte hypomethylated) but these differences in methylation do not persist into preimplantation development (McDonald et al., 1998). Methylation may not be the factor responsible for turning XIST on or o in the rst place, but it may function as an imprint and probably is crucial for keeping the silent copy of XIST stably repressed.

replication of the inactive X may play a role in the incorporation of the inactive X into a subnuclear compartment inhibiting transcription.

Histone Acetylation
Acetylation of histones occurs in all plant and animal forms. It occurs at specic lysine residues and is catalysed by histone acetyltransferases (HATs) and deacetylases (HDACs). An association between histone acetylation and gene expression has been considered for decades, and has attracted considerable attention recently with the discovery that HATs and HDACs are often identical to, or associated with, known regulators of transcription. In general, hyperacetylation is associated with gene expression, and hypoacetylation is associated with gene silencing. Using uorescent antisera for individual acetylated lysine residues, cytological studies showed that the inactive X, with the exception of regions containing genes that escape inactivation, has a low level of the acetylated isoforms of histones H2A, H3 and H4 in humans, mice and marsupials (Keohane et al., 1998). That histone deacetylation is not an initiating event in X inactivation was shown by a study of the development of H4 deacetylation in female embryonic stem cells undergoing X inactivation. Increase in XIST expression, late replication onset and initial gene silencing occur some 2 days before H4 deacetylation of an X chromosome. Deacetylation of histones on the inactive X may be a consequence of other events associated with X inactivation. It seems possible that other histone modications may also be associated with the activity state of the inactive X. For example, recently it has been shown that phosphorylation of histone H3 is associated with chromosome condensation in Tetrahymena.

Replication Timing
The inactive X chromosome begins and ends DNA replication later than its active homologue. Replication timing studies of inactivated genes indicate that they replicate in the last half of S phase. The appearance of a late-replicating X is observed in early embryogenesis. In female embryonic stem cells, which can undergo X inactivation in culture, a late-replicating X is detectable 2 days after the onset of dierentiation, well before the silencing of X-linked genes. Replication timing does not simply reect whether a chromosome is active or inactive. For example, the blood clotting factor IX gene on the active X in nonexpressing cells replicates somewhat earlier than its allele on the inactive X and replicates even earlier in expressing cells. Thus, the time of replication appears to be related to the potential for transcription, whether determined by the chromosome activation state or the cells dierentiation phase. Studies with the demethylating agent 5AC suggest that methylation at controlling regions may play a role in altering timing of replication over domains of a megabase or more. Although it is possible that late replication may be merely an eect of the complicated inactivation process, its early appearance suggests a more signicant role. There is evidence that late-replicating domains are compartmentalized and it is possible that late
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Inactive-X-Specific Proteins
Although transcriptional proteins bind to the promoter regions of transcribed genes on the active X, all in vivo footprinting attempts to detect transcription inhibiting factors bound to promoter regions on the inactive X have failed. Recently, an in vitro binding assay identied a nuclear protein that recognizes the methylated region of the mouse XIST promoter on the active X. Although much work remains to be done, these results suggest that silencing proteins involved in repressing genes on the inactive X are more likely to function at a chromosomal level rather than at the level of individual genes. Probably XIST RNA, which coats the inactive X, does so by interacting with a DNA-binding protein, as suggested by a recent in vitro study showing signicant interactions between heteronuclear proteins and XIST.

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X-Chromosome Inactivation

The most exciting result in this area is the recent nding of a new family of histones, called macroH2A. One of these is macro histone 2A1(mH2A1) whose N-terminal third of the molecule is similar to H2A1, but the remainder of which is unrelated to any known histone (Costanzi and Pehrson, 1998). The unique part of mH2A1 was found to preferentially localize with the inactive X-chromosome during metaphase and interphase in a number of mammals, including humans. Surprisingly, other studies indicate that the nonhistone part of the protein is widely conserved, and that the gene may have originated before the appearance of eukaryotes. Although mH2A1 is enriched on the inactive X, it is found throughout the genome, in both males and females. It is conceivable that mH2A1 may interact with XIST RNA in the process of X inactivation.

A Model for X-Chromosome Inactivation

The study of X-chromosome inactivation is at a juncture in which models, grounded in recent data, can be used to direct future research. We can conveniently divide the Xinactivation process into three phases: initiation, spreading and maintenance. One of the key features of the initiation of XCI is the choice of which X is to remain active. In murine extraembryonic cells, where the paternal X is preferentially inactivated, choice may follow from the methylation imprint in the sperm XIST gene, the comparable oocyte region being unmethylated. In the epiblast where random X inactivation occurs, the earlier methylation imprint of the XIST gene has disappeared and both Xs are transcribing unstable XIST RNA. The popular idea has been that choice should involve selecting an X to remain active rather than the X or Xs to become inactive. This reasoning follows from the presumed diculty of choice in cells with more than two Xs, where it should be simpler to choose one to be active rather than choosing two or more to become inactive. One proposal assumes a limiting amount of an autosomally produced blocking factor for which the XICs compete to remain active. A recent study has identied the 3 end of the XIST gene as a candidate region for the binding site of the blocking factor (Clerc and Avner, 1998). The requirement for stabilization of the XIST transcript has led to the proposal of XIST interacting factors aecting stability. A further complexity is the fact that mutations in dierent parts of the XIST gene can alter susceptibility to inactivation in dierent ways. It should not be surprising that there might be two or more mechanisms for ensuring that only one X remains active, as the presence of two active Xs is apparently lethal (Carrel and Willard, 1998; Panning and Jaenisch, 1998).

XIST is certainly one of the keys to the initiation of X inactivation. How does it come to act only in cis, never aecting the other X-chromosome or the autosomes? One possibility is that the inactive X is compartmentalized within the nucleus. The inactive X appears isolated in a cell compartment in the form of sex chromatin, generally occupying a position near the periphery of the nucleus. A number of years ago it was proposed that compartmentalization might be an early event in X inactivation, helping target various repressive factors that modify the inactive X. This idea was based on the observation that the inactive X in somatic cell hybrids is relatively unstable and does not form a normal sex chromatin structure, suggesting that the inactive X does not occupy its traditional compartment within the nucleus. More recently it has been shown by high voltage electron microscopy and microscopic analysis of a protein (perichromin) normally covering chromosomes at mitosis, but present on the inactive X at interphase, that the sex chromatin has unique structural properties (Gartler et al., 1992). As mentioned earlier, late replication might signal this compartmentalization. How the inactivation signal spreads must also involve XIST. Since XIST RNA does not contact DNA, it seems likely that an RNAprotein interaction might be involved in stabilization and propagation of the signal. It has been proposed that specic sequences along the X-chromosome could play an important role in signal spread, and, recently, long interspersed repetitive sequence elements (LINEs), which are present in an unusually high concentration on the X, have been proposed as a candidate (Lyon, 1998). LINEs occur throughout the genome, perhaps accounting for the fact that autosomal genes can be inactivated in X:autosome translocations. Moreover, LINEs vary subtly in sequence from one locus to another, possibly accounting for the fact that some loci on the Xchromosome escape X inactivation; such regions may simply lack the appropriate LINE sequence. However, the RNAprotein interaction may be all that is necessary for the accumulation and spread of XIST RNA along the chromosome. How is the X-inactivation pattern maintained? Several observations on transformed cells indicate that if the XIST locus is deleted after X inactivation has occurred, the Xinactivation pattern is maintained. This is not surprising, as we know that a complex of repressive factors distinguish the inactive from the active X, and that promoter methylation, for one, can maintain the X-inactivation pattern in the absence of functioning XIST RNA. What we do not know is whether XIST is able to maintain inactivation in the absence of methylation or any combination of the other repressive factors.

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Perspective
During the rst 20 years after the X-inactivation model was proposed, late replication and sex chromatin were the only physical features known to distinguish the two Xs. Methylation dierences were detected in the 1980s and for a time it was believed to be the key process. XIST as a single explanation of X inactivation promised a solution in the 1990s, only to be modied by the more recent reports on histone acetylation dierences between the two Xs. As this entry was being written, the rst report of a protein (mH2a1) preferentially associated with the inactive X appeared. It is likely that that report is merely the rst associating proteins with the inactivation process.

References
Brown CJ, Ballabio A, Rupert JL et al. (1991) A gene from the region of the human inactivation center is expressed exclusively from the inactive X chromosome. Nature 349: 3844. Carrel L and Willard HF (1998) Counting on Xist. Nature Genetics 19: 211212. Clerc P and Avner P (1998) Role of the region 3 to Xist exon 6 in the counting process of X-chromosome inactivation. Nature Genetics 19: 249253. Costanzi C and Pehrson JR (1998) Histone macroH2Al is concentrated in the inactive X chromosome of female mammals. Nature 393: 599 601. Disteche CM (1995) Escape from X inactivation in human and mouse. Trends in Genetics 11: 1722. Gartler SM, Dyer KA and Goldman MA (1992) Mammalian X chromosome inactivation. Molecular Genetic Medicine 2: 121160. Graves JAM (1996) Mammals that break the rules: genetics of marsupials and monotremes. Annual Review of Genetics. 30: 233260. Hansen RS, Caneld TK, Stanek AM, Keitges EA and Gartler SM (1998) Reactivation of XIST in normal broblasts and a somatic cell

hybrid: abnormal localization of XIST RNA in hybrid cells. Proceedings of the National Academy of Sciences of the USA 95: 51335138. Keohane AM, Lavender JS, ONeill LP and Turner BM (1998) Histone acetylation and X inactivation. Developmental Genetics 22: 6573. Lyon MF (1961) Gene action in the X-chromosome of the mouse (Mus musculus L). Nature 190: 372373. Lyon MF (1998) X-chromosome inactivation: a repeat hypothesis. Cytogenetics and Cell Genetics 80: 133140. McDonald LE, Paterson CA and Kay GF (1998) Bisulte genomic sequencing-derived methylation prole of the Xist gene throughout early mouse development. Genomics 54: 379386. Muller HJ (1932) Further studies on the nature and causes of gene mutation. Proceedings of the 6th Congress of Genetics 1: 213255. Panning B and Jaenisch R (1998) RNA and the epigenetic regulation of X chromosome inactivation. Cell 93: 305308. Rastan S and Robertson EJ (1985) X-chromosome deletions in embryoderived (EK) cell lines associated with lack of X-chromosome inactivation. I. Embryology and Experimental Morphology 90: 379 388.

Further Reading
Brockdor N and Duthie SM (1998) X-chromosome inactivation and the Xist gene. Cellular and Molecular Life Sciences 54: 104112. Carrel L and Willard HF (1998) Counting on Xist. Nature Genetics 19: 211212. Goto T and Monk M (1998) Regulation of X-chromosome inactivation in development in mice and humans. Microbiology and Molecular Biology Reviews 62: 362378. Heard C, Clerc P and Avner P (1997) X-chromosome inactivation in mammals. Annual Review of Genetics 31: 571610. Panning B and Jaenisch R (1998) RNA and the epigenetic regulation of X chromosome inactivation. Cell 93: 305308. Spusta SC and Goldman MA (1999) XISTential wanderings: the role of XIST RNA in X-chromosome inactivation. Current Science 77: 530 537.

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