It was once assumed that genes present in two copies in the genome would be expressed from both homologues at comparable levels. However, it has become increasingly evident that there can be extensive imbalance between alleles, reflecting both the amount of sequence variation in the genome and the interplay between genome sequence and epigenetic patterns that were just discussed.
In Chapter 2, we introduced the general finding that any individual genome carries two different alleles at a minimum of 3 to 5 million positions around the genome, thus distinguishing by sequence the maternally and paternally inherited copies of that sequence position. Here we explore ways in which those sequence differences reveal allelic imbalance in gene expression, both at autosomal loci and at X chromo some loci in females.
By determining the sequences of all the RNA products—the transcriptome—in a population of cells, one can quantify the relative level of transcription of all the genes (both protein coding and noncoding) that are transcriptionally active in those cells. Consider, for example, the collection of protein-coding genes.
Although an average cell might contain ~300,000 copies of mRNA in total, the abundance of specific mRNAs can differ over many orders of magnitude; among genes that are active, most are expressed at low levels (estimated to be <10 copies of that gene’s mRNA per cell), whereas others are expressed at much higher levels (several hundred to a few thousand copies of that mRNA per cell). Only in highly specialized cell types are particular genes expressed at very high levels (many tens of thousands of copies) that account for a significant proportion of all mRNA in those cells.
Now consider an expressed gene with a sequence variant that allows one to distinguish between the RNA products (whether mRNA or ncRNA) transcribed from each of two alleles, one allele with a T that is transcribed to yield RNA with an A and the other allele with a C that is transcribed to yield RNA with a G (Fig. 1). By sequencing individual RNA molecules and comparing the number of sequences generated that contain an A or G at that position, one can infer the ratio of transcripts from the two alleles in that sample. Although most genes show essentially equivalent levels of biallelic expression, recent analyses of this type have demonstrated wide spread unequal allelic expression for 5% to 20% of autosomal genes in the genome (Table 1). For most of these genes, the extent of imbalance is twofold or less, although up to tenfold differences have been observed for some genes. This allelic imbalance may reflect interactions between genome sequence and gene regulation; for example, sequence changes can alter the relative binding of various transcription factors or other transcriptional regulators to the two alleles or the extent of DNA methylation observed at the two alleles (see Table 1).
Fig1. Allelic expression patterns for a gene sequence with a transcribed DNA variant (here, a C or a T) to distinguish the alleles. As described in the text, the relative abundance of RNA transcripts from the two alleles (here, carrying a G or an A) demonstrates whether the gene shows balanced expression (top), allelic imbalance (center), or exclusively monoallelic expression (bottom). Different underlying mechanisms for allelic imbalance are compared in Table 1. SNP, Single nucleotide polymorphism.
Table1. Allelic Imbalance in Gene Expression
Monoallelic Gene Expression
Some genes, however, show a much more complete form of allelic imbalance, resulting in monoallelic gene expression (see Fig. 1). Several different mechanisms have been shown to account for allelic imbalance of this type for particular subsets of genes in the genome: DNA rearrangement including copy number variation, random monoallelic expression, parent-of-origin imprinting, and, for genes on the X chromosome in females, X chromosome inactivation. Their distinguishing characteristics are summarized in Table 1.
Somatic Rearrangement
A highly specialized form of monoallelic gene expression is observed in the genes encoding immunoglobulins and T-cell receptors, expressed in B cells and T cells, respectively, as part of the immune response. Antibodies are encoded in the germline by a relatively small number of genes that, during B-cell development, undergo a unique process of somatic rearrangement that involves the cutting and pasting of DNA sequences in lymphocyte precursor cells (but not in any other cell lineages) to rearrange genes in somatic cells to generate enormous antibody diversity. The highly orchestrated DNA rearrangements occur across many hundreds of kilobases but involve only one of the two alleles, which is chosen randomly in any given B cell (see Table 1). Thus expression of mature mRNAs for the immunoglobulin heavy or light chain subunits is exclusively monoallelic.
This mechanism of somatic rearrangement and random monoallelic gene expression is also observed at the T-cell receptor genes in the T-cell lineage. However, such behavior is unique to these gene families and cell lineages; the rest of the genome, even DNA segments bearing genomic repeats, remains surprisingly stable throughout development and differentiation.
Random Monoallelic Expression
In contrast to this highly specialized form of DNA rearrangement, monoallelic expression typically results from differential epigenetic regulation of the two alleles. One well-studied example of random monoallelic expression involves the OR gene family described earlier. In this case, only a single allele of one OR gene is expressed in each olfactory sensory neuron; the many hundred other copies of the OR family remain repressed in that cell. Other genes with chemosensory or immune system functions also show random monoallelic expression, suggesting that this mechanism may be a general one for increasing the diversity of responses for cells that interact with the outside world. However, this mechanism is apparently not restricted to the immune and sensory systems because a substantial subset of all human genes (5–10% in different cell types) has been shown to undergo random allelic silencing; these genes are broadly distributed on all autosomes, have a wide range of functions, and vary in terms of the cell types and tissues in which monoallelic expression is observed.
Parent-of-Origin Imprinting
For the examples just described, the choice of which allele is expressed is not dependent on parental origin; either the maternal or paternal copy can be expressed in different cells and their clonal descendants. This distinguishes random forms of monoallelic expression from genomic imprinting, in which the choice of the allele to be expressed is nonrandom and is determined solely by parental origin. Imprinting is a process involving the introduction of epigenetic marks in the germline of one parent, but not the other, at specific locations in the genome. These lead to monoallelic expression of a gene or, in some cases, of multiple genes within the imprinted region.
Imprinting takes place during gametogenesis, before fertilization, and marks certain genes as having come from the mother or father (Fig. 2). After conception, the parent-of-origin imprint is maintained in some or all of the somatic tissues of the embryo and silences gene expression on allele(s) within the imprinted region; whereas some imprinted genes show monoallelic expression throughout the embryo, others show tissue-specific imprinting, especially in the placenta, with biallelic expression in other tissues. The imprinted state persists postnatally into adulthood through hundreds of cell divisions so that only the maternal or paternal copy of the gene is expressed. Yet, imprinting must be reversible: a paternally derived allele, when it is inherited by a female, must be converted in her germline so that she can then pass it on with a maternal imprint to her offspring. Likewise, an imprinted maternally derived allele, when it is inherited by a male, must be converted in his germline so that he can pass it on as a paternally imprinted allele to his offspring (see Fig. 2). Control over this conversion process appears to be governed by specific DNA elements called imprinting control regions or imprinting centers that are located within imprinted regions throughout the genome; although their mechanism of action is not fully known, many appear to involve ncRNAs that initiate the epigenetic change in chromatin, which then spreads outward along the chromosome over the imprinted region. Notably, although the imprinted region can encompass more than a single gene, this form of monoallelic expression is confined to a delimited genomic segment, typically a few hundred kilobase pairs to a few megabases in overall size; this distinguishes genomic imprinting both from the more general form of random monoallelic expression described earlier (which appears to involve individual genes under locus specific control) and from X chromosome inactivation, described in the next section (which involves genes along the entire chromosome).
Fig2. Genomic imprinting and conversion of maternal and paternal imprints during passage through male or female gametogenesis. Within a hypothetical imprinted region on a pair of homologous autosomes, paternally imprinted genes are indicated in blue, whereas a maternally imprinted gene is indicated in red. After fertilization, both male and female embryos have one copy of the chromosome carrying a paternal imprint and one copy carrying a maternal imprint. During oogenesis (top) and spermatogenesis (bottom), the imprints are erased by removal of epigenetic marks, and new imprints determined by the sex of the parent are established within the imprinted region. Gametes thus carry a monoallelic imprint appropriate to the parent of origin, whereas somatic cells in both sexes carry one chromosome of each imprinted type.
To date, ~100 imprinted genes have been identified on many different autosomes. The involvement of these genes in various chromosomal disorders is described more fully in Chapter 6. For clinical conditions due to a single imprinted gene, such as Prader-Willi syndrome and Beckwith-Wiedemann syndrome.
X Chromosome Inactivation
The chromosomal basis for sex determination, introduced in Chapter 2 and discussed in more detail in Chapter 6, results in a dosage difference between typical males and females with respect to genes on the X chromosome. Here we discuss the chromosomal and molecular mechanisms of X chromosome inactivation, the most extensive example of random monoallelic expression in the genome and a mechanism of dosage compensation that results in the epigenetic silencing of most genes on one of the two X chromosomes in females.
In normal female cells, the choice of which X chromosome is to be inactivated is a random one that is then maintained in each clonal lineage. Thus females are mosaic with respect to X-linked gene expression; some cells express alleles on the paternally inherited X but not the maternally inherited X, whereas other cells do the opposite (Fig. 3). This mosaic pattern of gene expression distinguishes most X-linked genes from imprinted genes, whose expression, as we just noted, is determined strictly by parental origin.
Fig3. Random X chromosome inactivation early in female development. Shortly after conception of a female embryo, both the paternally and maternally inherited X chromosomes (pat and mat, respectively) are active. Within the first week of embryogenesis, one or the other X is chosen at random to become the future inactive X, through a series of events involving the X inactivation center (black box). That X then becomes the inactive X (Xi, indicated by the shading) in that cell and its progeny and forms the Barr body in interphase nuclei. The resulting female embryo is thus a clonal mosaic of two epigenetically determined cell types: one expresses alleles from the maternal X (pink cells), whereas the other expresses alleles from the paternal X (blue cells). The ratio of the two cell types is determined randomly but varies among normal females and among females who are carriers of X-linked disease alleles (see Chapters 6 and 7).
Although the inactive X chromosome was first identified cytologically by the presence of a heterochromatic mass (called the Barr body) in interphase cells, many epigenetic features at the molecular level distinguish the active and inactive X chromosomes, including DNA methylation, histone modifications, and a specific his tone variant, macroH2A, that is particularly enriched in chromatin on the inactive X. As well as providing insights into the mechanisms of X inactivation, these features can be useful diagnostically for identifying inactive X chromosomes in clinical material.
Although X inactivation is clearly a chromosomal phenomenon, not all genes on the X chromosome show monoallelic expression in female cells. Extensive analysis of expression of nearly all X-linked genes has demonstrated that at least 15% of the genes show biallelic expression and are expressed from both active and inactive X chromosomes, at least to some extent; a proportion of these show significantly higher levels of mRNA production in female cells compared to male cells and are interesting candidates for a role in explaining sexually dimorphic traits.
A special subset of genes is located in the pseudoautosomal segments, which are essentially identical on the X and Y chromosomes and undergo recombination during spermatogenesis . These genes have two copies in both females (two X-linked copies) and males (one X-linked and one Y-linked copy) and thus do not undergo X inactivation; as expected, these genes show balanced biallelic expression, as one sees for most autosomal genes.
The X Inactivation Center and the XIST Gene. X inactivation occurs very early in female embryonic development, and determination of which X will be designated the inactive X in any given cell in the embryo is a random choice under the control of a complex locus called the X inactivation center. This region contains a unique ncRNA gene, XIST, that appears to be a key master regulatory locus for X inactivation. XIST (an acronym for inactive X [Xi]–specific transcripts) has the novel feature that it is expressed only from the allele on the inactive X; it is transcriptionally silent on the active X in both male and female cells. Although the exact mode of action of XIST is unknown, X inactivation cannot occur in its absence. The product of XIST is a long ncRNA that stays in the nucleus in close association with the inactive X chromosome.
Additional aspects and consequences of X chromosome inactivation will be discussed in Chapter 6, in the context of individuals with structurally abnormal X chromosomes or an abnormal number of X chromosomes, and in Chapter 7, in the case of females carrying deleterious mutant alleles for X-linked disease.
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