Chapter 8:  Human gene expression

8.2.1. Ubiquitous transcription factors are required for transcription of RNA polymerases I and III

RNA polymerases I and III in eukaryotic cells are dedicated to transcribing genes to give RNA molecules (rRNA, tRNA, etc.) which assist in expression of the polypeptide-encoding genes. The transcribed genes are housekeeping genes since rRNA and tRNA are required in essentially all cells to assist in protein synthesis. As a result, ubiquitous transcription factors are required to assist RNA polymerases I and III.

Transcription by RNA polymerase I

Figure 8.1

The major human rRNA species are synthesized by cleavage (more...)

Figure 8.1

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   The major human rRNA species are synthesized by cleavage from a common 13 kb transcription unit which is part of a 40 kb tandemly repeated unit

Small arrows indicated by letters A-D signify positions of endonuclease cleavage of RNA precursors. Cleavage of the 41S precursor at B generates two products: 20S + 32S. Following cleavage of the 32S precursor at D, and excision of the small 5.8S rRNA, hydrogen bonding takes place between the 5.8S rRNA and a complementary central segment of the 28S rRNA. The approximately 6 kb of RNA sequence originating from the external and internal transcribed spacer units (ETS, ITS1 and ITS2) are degraded in the nucleus. S is the sedimentation coefficient, a measure of size.

RNA polymerase I is confined to the nucleolus and is devoted to transcription of the 18S, 5.8S and 28S rRNA genes. The latter are consecutively organized on a common 13 kb transcription unit (Figure 8.1). A compound unit of the 13-kb transcription unit and an adjacent 27 kb nontranscribed spacer is tandemly repeated about 50–60 times on the short arms of each of the five human acrocentric chromosomes, at the nucleolar organizer regions (see Figure 2.18). The resulting five clusters of rRNA genes, each about 2 Mb long, are referred to as ribosomal DNA or rDNA.

Figure 8.2

Initiation of transcription by RNA polymerase I

Figure 8.2

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   Initiation of transcription by RNA polymerase I

One possible model envisages initial binding of the two identical subunits of the upstream binding factor to the upstream control element and the core promoter element, and forcing these two sequences to come into close proximity, enabling their subsequent binding by the selectivity factor 1 (SL1) which consists of four subunits. The stabilized structure permits subsequent binding of other factors (not shown) and subsequently RNA polymerase I.

Initiation of transcription of the 28S, 5.8S and 18S rRNA genes is initiated following binding of two transcription factors to a core promoter element at the transcription initiation site and an upstream control element located over 100 nucleotides upstream. One of the transcription factors, UBF (upstream binding factor), is a homodimer and its identical subunits may bind first to the core promoter element and upstream control element, bringing them together so that they can be bound by the second factor, SL1 (selectivity factor 1; known in mouse as TFI-1B; Figure 8.2). The bound transcription factors subsequently recruit RNA polymerase I to form an initiation complex.

The primary transcript expressed from the single 13 kb transcription unit is a 45S precursor rRNA which undergoes a variety of cleavage reactions and base-specific modifications (carried out by a large number of different types of small nucleolar RNA (snoRNA)) to generate the mature 28S, 5.8S and 18S rRNA species (see Figure 8.1). Thus, these genes differ from the vast majority of nuclear genes, which are individually transcribed. Instead, rDNA transcription resembles mtDNA transcription (see Section 7.1.1 and Figure 7.2): both result in multigenic transcripts which yield functionally related products. This unusual use of polygenic primary transcripts is no different in principle, however, from the way in which a single primary translation product is occasionally cleaved to generate two or more functionally related polypeptides (see the example of human insulin in Figure 1.23).

Transcription by RNA polymerase III

Figure 8.3

tRNA and 5S rRNA genes have promoters located within (more...)

Figure 8.3

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   tRNA and 5S rRNA genes have promoters located within the coding sequence

(A) Positions of promoter elements in tRNA and 5S rRNA genes. The promoter elements A and B in the tRNA genes are located in the sequences specifying the D loop and the TwCG loops respectively (see tRNA structure in Figure 1.7B). (B) Initiation of transcription of a tRNA gene. Binding of the TFIIIC transcription factor to the promoter elements permits subsequent binding of the trimeric TFIIIB factor to the sequence immediately upstream of the transcription start site. In response to binding of the TFIIIB factor, RNA polymerase III binds and initiates transcription. In the case of the 5S rRNA genes, a similar mechanism occurs but in this case an additional transcription factor TFIIIA is required to bind to the C box, and the bound TFIIIA factor permits subsequent binding of TFIIIB followed by recruitment of TFIIIC and RNA polymerase III as in the case of tRNA genes.

RNA polymerase III is also involved in transcription of a variety of housekeeping genes, encoding various small stable RNA molecules such as 5S rRNA, tRNA molecules, 7SL RNA and some of the snRNA molecules needed for RNA splicing. These genes are characterized by promoters that lie within the coding sequence of the gene, rather than upstream of it. In tRNA genes, the promoter is bipartite, consisting of two well conserved sequences, the A box and the B box, while in the 5S rRNA gene, a single promoter element is present, the C box. In each case, transcription by RNA polymerase III is thought to proceed by binding of ubiquitous transcription factors to the promoter elements, followed by subsequent binding of other factors and finally recruitment of the polymerase (Figure 8.3).

8.2.2. Transcription of polypeptide-encoding genes often requires complex sets of cis-acting transcriptional control sequences and tissue-specific transcription factors

RNA polymerase II is responsible for transcribing all genes which encode polypeptides and also certain species of snRNA gene. Like RNA polymerases I and III, RNA polymerase II is dependent on auxiliary general transcription factors (the usual nomenclature has a common prefix TF to denote transcription factor followed by a Roman numeral to denote the associated RNA polymerase). In the case of RNA polymerase II, there are a variety of auxiliary transcription factors such as TFIIA, TFIIB, TFIID, TFIIE, TFIIF, TFIIH, etc, which can be complex in structure (Nikolov and Burley, 1997). For example, the TATA box-binding protein (TBP), is only one of the multiple protein subunits that make up TFIID and the associated proteins are known as TBP-associated factors, or TAF proteins. The complex of polymerase and general transcription factors is known as the basal transcription apparatus; it constitutes all that is required to initiate transcription. Genes are constitutively expressed at a minimum rate determined by the core promoter (see below) unless the rate of transcription is increased or switched off by additional positive or negative regulatory elements (which may be located some distance away or by intrinsic components of the promoter itself).

Some of the genes which encode polypeptides are housekeeping genes, but unlike the products of genes transcribed by RNA polymerases I and III, a large percentage of genes transcribed by RNA polymerase II show tissue-restricted or tissue-specific expression patterns. Since the DNA in different nucleated cells of an individual is essentially identical, the identity of a cell, whether it be a hepatocyte or a T lymphocyte for instance, is defined by the proteins made by the cell. In addition to general ubiquitous transcription factors, therefore, tissue-specific or tissue-restricted transcription factors regulate the expression of many genes which encode polypeptides, by recognizing and binding specific cis-acting sequence elements.

Figure 8.6

The HS-40 α-globin regulatory site contains (more...)

Figure 8.6

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   The HS-40 α-globin regulatory site contains many recognition elements for erythroid-specific transcription factors

Note that the HS-40 site appears to be a locus control region for the α-globin gene cluster (see Section 8.5.2).

Figure 8.6

In addition to actively promoting tissue-specific transcription, some cis-acting silencer elements confer tissue or developmental stage specificity by blocking expression in all but the desired tissue. For example, the neural restrictive silencer element (NRSE) represses expression of several genes in all tissues other than neural tissues (Schoenherr et al., 1996). A transcription factor that binds to the NRSE and which is variously called the neural restrictive silencer factor (NRSF) or the RE-1 silencing transcription factor (REST) is ubiquitously expressed in non-neural tissue and neuronal precursors during early development but subsequently it is specifically not expressed in more mature (postmitotic) neurons.

8.2.3. Transcription factors contain conserved structural motifs that permit DNA binding

Transcription factors recognize and bind a short nucleotide sequence, usually as a result of extensive complementarity between the surface of the protein and surface features of the double helix in the region of binding. Although the individual interactions between the amino acids and nucleotides are weak (usually hydrogen bonds, ionic bonds and hydrophobic interactions), the region of DNA-protein binding is typically characterized by about 20 such contacts, which collectively ensure that the binding is strong and specific. In human and other eukaryotic transcription factors, two distinct functions can often be identified and located in different parts of the protein:

• An activation domain. As the name suggests, this type of domain functions in activating transcription of the target genes once the transcription factor has bound to it. Activation domains are thought to stimulate transcription by interacting with basal transcription factors so as to assist the formation of the transcription complex on the promoter. Although not so well-studied as DNA-binding domains, some are known to be rich in aspartate and glutamate residues (acidic activation domains); others are rich in proline or glutamate.

• A DNA-binding domain. This type of domain is necessary to permit specific binding of the transcription factor to its target genes. In contrast to activation domains, DNA-binding domains of transcription factors have been well-studied. A number of conserved structural motifs have been identified which are common to many different transcription factors with quite different specificities, including the leucine zipper, helix-loop-helix, helix-turn-helix, and zinc finger motifs which are described below. Each of the motifs uses α-helices (or occasionally β-sheets; see Figure 1.24) to bind to the major groove of DNA. Clearly, although the motifs in general provide the basis for DNA binding, the precise collection of sequence elements in the DNA-binding domain will provide the basis for the required sequence-specific recognition. Most transcription factors bind to DNA as homodimers, with the DNA-binding region of the protein usually distinct from the region responsible for forming dimers.

The leucine zipper motif

Figure 8.7

Structural motifs commonly found in transcription factors (more...)

Figure 8.7

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   Structural motifs commonly found in transcription factors and DNA-binding proteins

Abbreviations: HTH, helix-turn-helix; HLH, helix-loop-helix. Note that the leucine zipper monomer is amphipathic [i.e. has hydrophobic residues (leucines) consistently on one face of the helix]. Two such helices can align with their hydrophobic faces in opposition to form a coiled-coil structure.

Figure 8.8

Binding of conserved structural motifs in transcription (more...)

Figure 8.8

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   Binding of conserved structural motifs in transcription factors to the double helix

Note that the individual monomers of the helix-loop-helix (HLH) dimer and the leucine zipper dimer are colored differently to permit distinction, but may be identical (homodimers). HLH heterodimers and leucine zipper heterodimers may provide a higher level of regulation (see text).

The leucine zipper is a helical stretch of amino acids rich in leucine residues (typically occurring once every seven amino acid residues, i.e. once every two turns of the helix - see Figure 8.7), which readily forms a dimer. Each monomer unit consists of an amphipathic a-helix (hydrophobic side groups of the constituent amino acids face one way; polar groups face the other way, see Figure 1.24). The two α-helices of the individual monomers join together over a short distance to form a coiled-coil (see Section 1.5.5) with the predominant interactions occurring between opposed hydrophobic amino acids of the individual monomers. Beyond this region the two α-helices separate, so that the overall dimer is a Y-shaped structure. The dimer is thought to grip the double helix much like a clothes peg grips a clothes line (Figure 8.8). In addition to forming homodimers, leucine zipper proteins can occasionally form heterodimers depending on the compatibility of the hydrophobic surfaces of the two different monomers. Such heterodimer formation provides an important combinatorial control mechanism in gene regulation.

The helix-loop-helix motif

The helix-loop-helix (HLH) motif is related to the leucine zipper and should be distinguished from the helix- turn-helix (HTH) motif described in the next section. It consists of two α-helices, one short and one long, connected by a flexible loop. Unlike the short turn in the HTH motif, the loop in the HLH motif is flexible enough to permit folding back so that the two helices can pack against each other; that is, the two helices lie in planes that are parallel to each other, in contrast to the two helices in the HTH motif (Figure 8.7). The HLH motif mediates both DNA binding and protein dimer formation (Figure 8.8) and it permits occasional heterodimer formation. In the latter case, however, heterodimers form between a full-length HLH protein and a truncated HLH protein which lacks the full length of the α-helix necessary to bind to the DNA. The resulting heterodimer is unable to bind DNA tightly. As a result, HLH dimers are thought to act as a control mechanism, by enabling inactivation of specific gene regulatory proteins.

The helix-turn-helix motif

The HTH motif is a common motif found in homeoboxes, and a number of other transcription factors. It consists of two short α-helices separated by a short amino acid sequence which induces a turn, so that the two α-helices are orientated differently (i.e. the two helices do not lie in the same plane, unlike those in the HLH motif; Figure 8.7). The structure is very similar to the DNA-binding motif of several bacteriophage regulatory proteins such as the λ cro protein whose binding to DNA has been intensively studied by X-ray crystallography. In the case of both the λ cro protein and eukaryotic HTH motifs, it is thought that while the HTH motif in general mediates DNA binding, the more C-terminal helix acts as a specific recognition helix because it fits into the major groove of the DNA (Figure 8.8), controlling the precise DNA sequence which is recognized.

The zinc finger motif

The zinc finger motif involves binding a zinc ion by four conserved amino acids to form a loop (finger), a structure which is often tandemly repeated. Although several different forms exist, common forms involve binding of a Zn²⁺ ion by two conserved cysteine residues and two conserved histidine residues, or by four conserved cysteine residues. The resulting structure may then consist of an α-helix and a β-sheet held together by coordination with the Zn²⁺ ion, or of two α-helices. In either case, the primary contact with the DNA is made by an α-helix binding to the major groove. The so-called Cys₂/His₂ finger typically comprises about 23 amino acids with neighboring fingers separated by a stretch of about seven or eight amino acids (Figure 8.7).

8.2.4. A variety of mechanisms permit transcriptional regulation of gene expression in response to external stimuli

Table 8.3

Different modes of cell signaling

Table 8.3

Different modes of cell signaling

Mode	Characteristics	Examples
Direct cell to cell signaling	A signal on the surface of one cell is bound by a specific receptor on another cell	Membrane-anchored growth factors and their receptors
Endocrine signaling	Hormones are secreted by specialized endocrine cells and carried through the circulation to bind to receptors in target cells at distant locations in the body	Release of glucocorticoid hormones by the adrenal glands
Paracrine signaling	A molecule released from one cell acts locally to affect nearby target cells	Neurotransmitters and receptors; nitric oxide in the immune system, nervous system etc.
Autocrine signaling	A cell produces a signaling molecule to which it also responds	T lymphocytes can respond to antigenic stimulation by synthesizing factors that drive their own proliferation

Ligand-inducible transcription factors

Small hydrophobic hormones and morphogens such as steroid hormones, thyroxine and retinoic acid are able to diffuse through the plasma membrane of the target cell and bind intracellular receptors in the cytoplasm or nucleus. These receptors (often called hormone nuclear receptors) are inducible transcription factors. Following binding of the homologous ligand, the receptor protein associates with a specific DNA response element located in the promoter regions of perhaps 50–100 target genes and activates their transcription.

Figure 8.10

Transcriptional regulation by glucocorticoids

Figure 8.10

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   Transcriptional regulation by glucocorticoids

The glucocorticoid receptor is normally inactivated by being bound to an inhibitor protein, Hsp90. Binding of glucocorticoids to the glucocorticoid receptor releases Hsp90, the receptor dimerizes and then activates selected genes which have a glucocorticoid response element in their promoter (see Figure 8.9).

Although thyroxine and retinoic acid are structurally and biosynthetically unrelated to the steroid hormones, their receptors belong to a common nuclear receptor superfamily. Two conserved domains characterize the family: a centrally located DNA binding domain of about 68 amino acids, and a ~240 amino acid ligand-binding domain located close to the C terminus (Figure 8.9). The DNA binding domain contains zinc fingers and binds as a dimer with each monomer recognizing one of two hexanucleotides in the response element. The two hexanucleotides are either inverted repeats or direct repeats which are typically separated by three or five nucleotides (Figure 8.9). In the absence of the ligand, the receptor is inactivated by direct repression of the DNA binding domain function by the ligand-binding domain, or by binding to an inhibitory protein, as in the case of the glucocorticoid receptor (Figure 8.10).

Activation of transcription factors by signal transduction

Table 8.5

Major classes of cell surface receptor

Table 8.5

Major classes of cell surface receptor

Receptor class	Characteristics	Activation by
G protein-coupled	Activate heterotrimeric G-proteins (GTP-binding regulatory proteins). The latter consist of three subunits, α, β and γ. Upon activation, the α subunit translocates to activate a target protein (see Figure 8.11A for an example)	Various molecules, including peptides, hormones, e.g. epinephrine, etc.
Serine-threonine kinase	Activate intracellular proteins by phosphorylating serine or threonine residues on target proteins	Hormones, growth factors
Tyrosine kinase	Activate intracellular proteins by phosphorylating tyrosine on target proteins, e.g. PDGF receptor, insulin receptor, etc (see Figure 8.11B)	Hormones, growth factors
Tyrosine kinase-associated	Receptors do not phosphorylate target proteins directly, but instead rely on an associated tyrosine kinase. Examples include cytokine receptors with associated JAK (janus protein kinase) activity involved in JAK-STAT signaling	Hormones, growth factors
Ion channel-linked	Involved in rapid synaptic signaling	Neurotransmitters

Unlike lipid-soluble hormones or morphogens, hydrophilic signaling molecules such as polypeptide hormones, cannot diffuse through the plasma membrane. Instead, they bind to a specific receptor on the cell surface. After binding of the ligand molecule, the receptor undergoes a conformational change and becomes activated in such a way that it passes on the signal via other molecules within the cell (signal transduction). Various classes of cell surface receptor are known but many of them have a kinase activity or can activate intracellular kinases (see Table 8.5). Signal transduction pathways are often characterized by complex regulatory interplay between kinases and phosphatases which can activate or repress intermediates by phosphorylation/dephosphorylation. In many cases, the phosphorylation or dephosphorylation induces an altered conformation. In the case of activation of a signaling molecule, the altered conformation often means that a signaling factor is no longer inhibited by some repressor sequence present in an inhibitory protein to which it is bound, or in a domain or sequence motif within its own structure.

In terms of transcriptional activation, two general mechanisms permit rapid transmission of signals from cell-surface receptors to the nucleus, both involving protein phosphorylation:

• protein kinases are activated and then translocated from the cytoplasm to the nucleus where they phosphorylate target transcription factors;

• inactive transcription factors stored in the cytoplasm are activated by phosphorylation and translocated into the nucleus.

The following two sections provide examples to illustrate these two mechanisms (see also Karin and Hunter, 1995).

Hormonal signaling through the cyclic AMP pathway

Table 8.6

Examples of secondary messengers in cell signaling

Table 8.6

Examples of secondary messengers in cell signaling

Secondary messenger	Characteristics	Examples
Cyclic AMP (cAMP)	Produced from ATP by adenylate cyclase. Effects are usually mediated through protein kinase A	Activation of CREB transcription factor (see Figure 8.11A)
Cyclic GMP (cGMP)	Produced from GTP by guanylate cyclase. Best characterized role is in visual reception in the vertebrate eye
Phospholipids/Ca2+	Activated downstream of G protein-coupled receptors and protein tyrosine kinases. Hydrolysis of phosphatidylinositol 4,5-bis-phosphate (PIP2) yields diacylglycerol and inositol 1,4,5-trisphosphate (IP3) which activate protein kinase C and mobilize Ca from intracellular stores	See Figure 8.11B

Cyclic AMP is an important second messenger (see Table 8.6) which acts in response to a variety of hormones and other signaling molecules. It is synthesized from ATP by a membrane bound enzyme, adenylate cyclase. Hormones which activate adenylate cyclase bind to a cell surface receptor which is of the G protein-coupled receptor class. Binding of the hormone to the receptor promotes the interaction of the receptor with a G protein which consists of three subunits, α, β and γ. Following this interaction the α subunit of the G protein is activated, causing it to dissociate and stimulate adenylate cyclase.

The increase in intracellular cAMP produced by activated adenylate cyclase can then activate the transcription of specific target sequences that contain a cAMP response element or CRE. This function of cAMP is mediated by the enzyme protein kinase A. Cyclic AMP binds to protein kinase and activates it by permitting release of the two catalytically active subunits which then enter the nucleus and phosphorylate a specific transcription factor, CREB (CRE-binding protein). Activated CREB then activates transcription of genes with the cAMP response element (Figure 8.11B).

Activation of NF-κB via protein kinase C signaling

NF-κB is a transcription factor which is involved in a variety of aspects of the immune response. In its inactive state, NF-κB is retained in the cytoplasm where it is complexed with an inhibitory subunit, IκB. However, the latter can be targeted for degradation following phosphorylation by protein kinase C. The consequent destruction of IκB permits NF-κB to translocate to the nucleus and activate its various target genes. Protein kinase C is activated by diacylglycerol. The latter is produced when binding of various growth factors and hormones to specific cell surface receptors triggers activation of receptor-linked phospholipase C activity. The activated enzyme converts PIP₂ (phosphatidylinositol 4,5 bisphosphate) to IP₃ (inositol 1,4,5-trisphosphate) and diacylglycerol (Figure 8.11B).

8.2.5. Translational control of gene expression can involve specific recognition by RNA-binding proteins of regulatory sequences within the untranslated sequences of mRNA

Different forms of translational control of gene expression are evident and an increasing number of eukaryotic and mammalian mRNA species have been shown to contain regulatory sequences in their untranslated sequences (most frequently at the 3′ end; see Wickens et al., 1997; Day and Tuite, 1998). Several eukaryotic and mammalian RNA-binding proteins have also been identified and shown to bind to specific regulatory sequences present in untranslated sequences, thereby providing the basis for translational control of gene expression (Siomi and Dreyfuss, 1997). A variety of different RNA-binding domains have been identified and they include elements which have previously been associated with DNA-binding properties of transcription factors such as zinc fingers and homeodomains (see Section 8.2.3 and Siomi and Dreyfuss, 1997).

Translational control of gene expression in response to external stimuli

Translational control of gene expression can permit a more rapid response to altered environmental stimuli than the alternative of activating transcription. Iron metabolism provides two useful examples. Increased iron levels stimulate the synthesis of the iron-binding protein, ferritin, without any corresponding increase in the amount of ferritin mRNA. Conversely, decreased iron levels stimulate the production of transferrin receptor (TfR) without any effect on the production of transferrin receptor mRNA. The 5′-UTR of both ferritin heavy chain mRNA and light chain mRNA contain a single iron-response element (IRE), a specific cis-acting regulatory sequence which forms a hairpin structure. Several such IRE sequences are also found in the 3′ UTR of the transferrin receptor mRNA (see Klausner et al., 1993). Regulation is exerted by binding of IREs by a specific IRE-binding protein which is activated at low iron levels (Figure 8.12).

8.3.1. Transcription of a single human gene can be initiated from a variety of alternative promoters and can result in a variety of tissue-specific isoforms

Several human and mammalian genes are known to have two or more alternative promoters, which can result in different isoforms with different properties (see Ayoubi and van de Ven, 1996). The isoforms can provide:

• tissue-specificity (a frequent occurrence; see the example of the human dystrophin gene below);

• developmental stage-specificity (e.g. the insulin-like growth factor II gene);

• differential subcellular localization (e.g. soluble and membrane-bound isoforms);

• differential functional capacity (as in the case of the progesterone receptor);

• sex-specific gene regulation (see the case of the Dnmt1 methyltransferase gene in Section 8.4.2 and Figure 8.20).

Figure 8.13

Figure 8.13

8.3.2. Human genes often encode more than one product as a result of alternative splicing and alternative polyadenylation events

In addition to differential use of promoters which can enforce alternative use of exons, a variety of alternative RNA processing events can also result in alternative isoforms. The primary mechanisms are alternative splicing events (that is, distinct from those induced by differential promoter usage), and alternative polyadenylation events. In many cases a combination of these mechanisms can result during the processing of a single gene. Together with the additional possibility of differential promoter usage, these mechanisms can result in very large numbers of isoforms for a single gene.

Alternative polyadenylation

The usage of alternative polyadenylation signals is also quite common in human mRNA, and different types of alternative polyadenylation have been identified (see Edwards-Gilbert et al., 1997). In many genes, two or more polyadenylation signals are found in the 3′ UTR and the alternatively polyadenylated transcripts can show tissue specificity; in other cases, alternative polyadenylation signals may be brought into play following alternative splicing. As an example of the latter, a combination of alternative splicing and alternative polyadenylation of the calcitonin gene (CALC) results in tissue-specific expression of two isoforms. Calcitonin, a circulating Ca²⁺ homeostatic hormone, is produced in the thyroid; the calcitonin gene-related peptide (CGRP), which may have both neuromodulatory and trophic activities, is synthesized in the hypothalamus (Figure 8.15).

8.3.3. RNA editing is a rare form of post-transcriptional processing whereby base-specific changes are enzymatically introduced at the RNA level

RNA editing is a form of post-transcriptional processing which can involve enzyme-mediated insertion or deletion of nucleotides or substitution of single nucleotides at the RNA level. Insertion or deletion RNA editing appears to be a peculiar property of gene expression in mitochondria of kinetoplastid protozoa and slime molds. Substitution RNA editing is frequently employed in some systems, such as the mitochondria and chloroplasts of vascular plants where individual mRNAs may undergo multiple C → U or U → C editing events, and has also been observed in a few mammalian genes (Ashkenas, 1997). At least four different classes of RNA editing are known to occur in human cells:

• C U editing. Human APOB lipoprotein mRNA editing has been well-studied. In the liver the APOB gene encodes a 14.1 kb mRNA transcript and a 4536 amino acid product, apoB100. However, in the intestine the same gene encodes a 7 kb mRNA which contains a premature stop codon not present in the gene and encodes a product, apoB48, which is identical in sequence to the first 2152 amino acids of apoB100. A specific cytosine deaminase, Apobec1, converts a single cytosine at nucleotide 6666 in the intestinal APOB mRNA to uridine, thereby generating a stop codon (Figure 8.16).

• A I editing. Genes encoding some ligand-gated ion channels including glutamate receptors and related proteins are subject to this type of mRNA editing. An adenosine is deaminated to give inosine (I), a base not normally present in mRNA (the amino group at carbon 6 of adenosine is replaced by a C=O carbonyl group). Inosine base pairs preferentially with cytosine and also interacts with ribosomes during translation as if it were a G. In the case of the glutamate receptor B gene, for example, RNA editing replaces a CAG (glutamine) codon by CIG which is translated as if it were the CGG codon (arginine). This type of editing which brings about a Gln Arg is often referred to as Q/R editing after the single letter code for the two amino acids involved.

• Other classes of RNA editing. Two other documented forms of editing in human mRNA are the U C editing in mRNA from the WT1 Wilms' tumor gene and U A editing in α-galactosidase mRNA (Ashkenas, 1997).

8.4.1. Selective gene expression in cells of mammalian embryos most likely develops in response to short range cell-cell signaling events

In order to explain subsequent tissue-, cell- and developmental stage-specific patterns of expression, some mechanism is required to set up an asymmetry or axis in the fertilized egg cell or in very early development. In Drosophila, the egg is inherently asymmetrical because of transfer of gene products from asymmetrically sited nurse cells. The embryo develops initially as a multinucleate syncytium (effectively one big cell) and regionalization depends on the response of individual nuclei to long-range gradients of regulatory molecules. In mammals, however, the egg cell is relatively small and early embryonic development creates an apparently symmetrical aggregate of individual cells. Nevertheless, development becomes asymmetric.

The generation of asymmetry in mammalian cells could derive from early positional clues. Some aspects of early development are inherently asymmetrical including the point of entry of the sperm during fertilization, the attachment of the embryo to the uterine wall during implantation and the location of cells with respect to their neighbors. As the embryo develops into a ball of cells, and later on as more complex structures develop, individual cells will vary in the number of cell neighbors available. Short range intercellular signaling events (by direct cell-cell signaling or short-range intercellular signaling events) can provide a means of identifying cell position, and triggering differential gene expression. For example, if an intercellular signaling molecule has a range of, say, one cell diameter, then the cells at the outside of the blastula will receive different signals from those surrounded by neighbors on all sides, and the different positional cues may be translated into differential gene expression. As particular cell systems develop during, for example, organogenesis (mostly accomplished between the 4th and 9th embryonic weeks), particular cell type growth or differentiation factors may then induce the expression of developmental stage-specific and/or tissue-specific transcription factors.

8.4.2. Vertebrate DNA methylation is very largely confined to CpG dinucleotides and patterns of DNA methylation can be inherited when cells divide

Once differential expression patterns have been set up, epigenetic mechanisms can ensure that differential expression patterns are stably inherited when cells divide. DNA methylation is thought to play a major role in this respect, permitting the stable transmission from a diploid cell to daughter cells of chromatin states which repress gene expression. However, the precise function of DNA methylation in eukaryotes is still imperfectly understood and clearly shows species differences. Some organisms, for example, have no detectable DNA methlyation, as in the case of Drosophila, C. elegans and the yeast Saccharomyces cerevisiae. In those organisms where DNA methylation does occur, the patterns and functions of DNA methylation may differ.

Patterns of DNA methylation in vertebrates

The pattern of vertebrate DNA methylation differs from that in bacterial cells. In the latter, adenine and cytosine can both be methylated but in vertebrates methylation of DNA is restricted to cytosine residues. Only about 3% of the cytosines in human DNA are methylated, but most that are methylated are found in the CpG dinucleotide (that is, the methylated cytosines are almost always ones whose 3′ carbon atom is linked by a phosphodiester bond to the 5′ carbon atom of a guanine). In addition, a much smaller percentage of methylated cytosines occur within the sequence CpNpG.

Figure 8.17

The CpG dinucleotide is underrepresented in vertebrate (more...)

Figure 8.17

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   The CpG dinucleotide is underrepresented in vertebrate DNA because it is prone to methylation and deaminated 5-methylcytosine is subject to ineffective DNA repair

(A) Cytosine occurring in the sequence 5′-CpG-3′ is a target for methylation at the 5′ carbon atom. The deaminated products of cytosine and its methylated derivative 5-methylcytosine are differentially recognized by DNA repair enzymes. (B) The methylation pattern of CpGs is perpetuated by a requirement for the specific methylase to recognize a hemimethylated target sequence. The sequence CpG has dyad symmetry. Following methylation of a hemimethylated target (i.e. methylated on one strand only), the two methylated strands will separate at DNA duplication and act as templates for the synthesis of two unmethylated daughter strands. The resulting daughter duplexes will now provide new hemimethylated targets for continuing the same pattern of methylation. (C) Deamination of 5-methylcytosine in the sequence CpG results in conversion of CpG dinucleotides to TpG and CpA dinucleotides.

Box 8.5

CpG islands

Cytosine residues occurring in CpG dinucleotides in vertebrate DNA are targets for methylation by a specific cytosine methyltransferase. Methylation occurs at carbon atom 5 of the cytosine to generate 5-methylcytosine, which is chemically unstable and can spontaneously deaminate to give thymine (Figure 8.17A). Over long periods of evolutionary time, the number of CpGs in vertebrate DNA has gradually been eroded, although regions of the normal (expected) CpG frequency are known and often mark transcriptionally active sequences (CpG islands, see Box 8.5).

Maintenance and de novo methylation during development

Unlike bacterial methylases, vertebrate cytosine methyltransferases show a strong preference for recognizing a hemi-methylated DNA target (i.e. one that is already methylated on one strand only). The sequence CpG shows dyad symmetry and so, following DNA replication, the newly synthesized DNA strands will receive the same CpG methylation pattern as the parental DNA (Figure 8.17B). As a result, the CpG methylation pattern can be stably transmitted to daughter cells. The perpetuation of a pre-existing methylation pattern is sometimes known as maintenance methylation and is carried out in mammalian cells by the product of the Dnmt1 gene.

Figure 8.19

Changes in DNA methylation during mammalian (more...)

Figure 8.19

.

   Changes in DNA methylation during mammalian development

Developmental stages for gametogenesis and early embryo development are expanded for clarity; those for later development are contracted, as indicated by double slashes. Note the very rapid changes in DNA methylation during: (i) gametogenesis - de novo methylation gives rise to substantially methylated genomes in the sperm and egg (albeit with differences in both the overall level of methylation and the pattern of methylation in these genomes - see text), and in (ii) the early embryo where a wave of genome-wide demethylation occurs at the preimplantation stage (morula and early blastula), and is succeeded shortly afterwards by large-scale de novo methylation beginning at the pregastrulation stage. The latter is particularly pronounced in somatic lineages, and to a lesser extent in trophoblast lineages giving rise to placenta and yolk sac, but does not occur in the primordial germ cells (the cells of the embryo which will eventually give rise to sperm and egg cells).

The pattern of 5-methlycytosine distribution in the genome of differentiated somatic cells varies according to cell type but maintenance methylation ensures that methylation patterns in individual somatic cell lineages are quite stable. During gametogenesis and in the developing embryo, however, there are dramatic changes in methylation (Razin and Kafri, 1994). The genomes of the primordial germ cells of the embryo are not methylated to any extent. After gonadal differentiation and as the germ cells begin to develop, de novo methylation occurs leading to substantial methylation of the DNA of mammalian sperm and egg cells (Figure 8.19). The sperm genome is more heavily methylated than the egg's genome, and sex-specific differences in methylation patterns are found, notably at imprinted loci (see Mertineit et al., 1998 for references). The Dnmt1 methyltransferase gene, in addition to being the predominant maintenance DNA methyltransferase in mammalian cells may also be the major de novo methyltransferase. It is highly expressed in male germ cells, mature oocytes and in the early embryo. Dnmt1 gene expression has been shown to be subject to sex-specific regulation with oocyte- and spermatocyte-specific promoters introducing oocyte- and spermatocyte-specific exons leading to different gene products (Figure 8.20; Mertineit et al., 1998).

The genome of the fertilized oocyte is an aggregate of the sperm and egg genomes and so it and the very early embryo are substantially methylated with methylation differences at paternal and maternal alleles of many genes. Later on, at the morula and early blastula stages in the preimplantation embryo, genome-wide demethylation occurs (Figure 8.19). Later still, at the pregastrulation stage, there is widespread de novo methylation. However, the extent of this methylation varies in different cell lineages:

• the somatic cell lineage is heavily methylated;

• trophoblast-derived lineages which give rise to the placenta, yolk sac, etc., are undermethylated;

• early primordial germ cells are spared; their genomic DNA remains very largely unmethylated until after gonadal differentiation and as the germ cells develop whereupon widespread de novo methylation occurs.

8.4.3. DNA methylation in animals has been thought to act as a form of host defense against transposons as well as a way of perpetuating patterns of transcriptional repression

Although not all eukaryotes appear to be subject to DNA methylation, its function in animal cells does appear to be critically important, and targeted mutagenesis of the cytosine methyltransferase gene in mice results in embryonic lethality. The precise function of DNA methylation in animal cells, however, remains unclear. Current views have focused in particular on two aspects of animal cells: the genome size (animals have comparatively large genomes with large numbers of genes, and also large numbers of highly repetitive DNA families belonging to the transposon class); and the mode of development (especially the variation in terms of lifespan and rate of cell turnover). Two quite contrasting views regarding the primary function of DNA methylation in animal cells have been the subject of much controversy: the host defense model and the gene regulation model.

DNA methylation and gene expression

Table 8.7

Features associated with transcriptionally active and (more...)

Table 8.7

Features associated with transcriptionally active and inactive chromatin

Feature	Transcriptionally active chromatin	Transcriptionally inactive chromatin
Chromatin conformation	Open, extended conformation	Highly condensed conformation; particularly apparent in heterochromatin (both facultative and constitutive; see Section 3.5)
DNA methylation	Relatively unmethylated, especially at promoter regions	Methylated, including at promoter regions
Histone acetylation	Acetylated histones	Deacetylated histones

The DNA of transcriptionally active and inactive chromatin differs in a number of features including the degree of compaction and the extent of its methylation (Table 8.7). While methylation of CpG islands downstream of promoters does not block continued transcription through these regions (Jones, 1999), there is no doubt that methylated promoter regions are correlated with transcriptional silencing. In addition, the extent of histone acetylation is an important factor. Specific histone acetyltransferases add acetyl groups to lysine residues close to the N terminus of histone proteins. The acetylated N termini then form tails that protrude from the nucleosome core. As the acetylated histones are thought to have a reduced affinity for the DNA, and possibly for each other, the chromatin may be able to adopt a more open structure that is more suited to gene expression. Deacetylation of the histones, however, promotes repression of gene expression presumably because the chromatin can become more condensed.

Figure 8.21

Transcriptional repression by histone deacetylation (more...)

Figure 8.21

.

   Transcriptional repression by histone deacetylation may be mediated by DNA methylation

CpG dinucleotides are targets for DNA methylation and, in turn, methylated CpGs are targets for specific binding by proteins such as MeCP2, which acts as a transcriptional repressor and recruits a corepressor complex consisting of the transcription factor repressor mSin3A and histone deacetylases. The latter removes acetyl groups from histones. The reverse process involves sequential histone acetylation then DNA demethylation (see Ng and Bird, 1999).

Recently, the processes of DNA methylation and histone deacetylation have been shown to be linked. Repression at methlyated CpG sequences in promoter regions appears to be mediated by proteins which specifically bind to methylated CpG. Two of these proteins have been identified, MeCP1 and MeCP2 (methylated CpG binding proteins 1 and 2), and the latter has been shown to be essential for embryonic development and to function as a transcriptional repressor. The ability of MeCP2 to repress gene expression has been shown to involve a histone deacetylase complex (Ng and Bird, 1999). One possible model envisages that an initial signal for transcriptional repression is the binding of MeCP2 to methylated CpG promoter sequences. The bound MeCP2 protein is then recognized by a complex consisting of a transcriptional repressor and a histone deacetylase which removes the acetyl groups from the N termini of the histones, so that the chromatin becomes more condensed (Figure 8.21).

8.5.1. Chromatin structure may exert long-range control over gene expression

A predominant theme in eukaryotic gene expression, distinguishing it from bacterial gene expression, has been that genes are individually transcribed. Promoters and related upstream elements typically control expression of a single gene with a transcription start point located within 1 kb of the control element. Some cis-acting elements, however, exert long-range control over a much larger chromosomal region and there is increasing evidence for coordinate regulation of gene clusters. Studies where genes are repositioned elsewhere in the genome have also suggested that chromosomes are organized into functional domains of gene expression (chromatin domains). For example, when genes are translocated to new chromosomal regions (either as a result of spontaneous chromosome breakage events, or by genetically manipulating model organisms - see Section 21.3), aberrant gene expression may often occur, even although the entire gene and the required control sequences in its immediate flanking sequences are preserved intact. Neighboring chromosome domains are envisaged to be separated by boundary elements (also called insulators) which act as barriers to the effects of distal enhancers and silencers (Geyer, 1997).

8.5.2. Expression of individual genes in gene clusters may be coordinated by a common locus control region

Figure 8.22

Human hemoglobin switching occurs at two distinct developmental (more...)

Figure 8.22

.

   Human hemoglobin switching occurs at two distinct developmental stages

Figure 8.22

Figure 8.23

Gene expression in the α- and β-globin (more...)

Figure 8.23

.

   Gene expression in the α- and β-globin gene clusters is controlled by common locus control regions

(A) Organization of the human α- and β-globin gene clusters.The locus control regions (LCRs) consist of one or more erythroid-specific DNase I-hypersensitive sites (HS-40, etc.) located upstream of the cluster. Arrows mark the direction of transcription of expressed genes. The functional status of the θ-globin gene is uncertain: it is expressed, but may be an expressed pseudogene (see Box 7.3). (B) Regulation of gene expression by the β-globin LCR. The strong blue arrows indicate a powerful enhancer effect by the LCR on the indicated genes, resulting in a high expression level; dotted blue arrows indicate correspondingly weak effects.

Figure 8.23

Figure 8.6

Figure 8.23

In addition to the human globin LCRs a number of different additional LCRs have been identified (see Kioussis and Festenstein, 1997). However, the role of the human β-globin LCR (which has relied on analysis of human transgenes) has been challenged by gene targeting studies in mice which show that the β-globin LCR has a contributory function rather than a dominant one, and that it is not required for initiation of DNase sensitivity and expression of the genes in the mouse β-globin cluster. These contradictory findings may possibly mean that there is a functional difference between the human and murine LCRs (see Grosveld, 1999 for a review).

8.5.3. Some human genes show selective expression of only one of the two parental alleles

X-linked genes in females and all autosomal genes are biallelic because both father and mother normally contribute one allele each. In males possessing one X chromosome and one Y chromosome, the great majority of sex-linked genes are monoallelic: most of the many genes on the X do not have a functional homolog on the Y chromosome; and some of the few genes on the Y chromosome are known to be Y-specific, for example SRY, the major male sex-determining locus. A few genes on the Y chromosome do have functional homologs on the X chromosome and so are biallelic. In some cases of X-Y homologous loci, both homologs are normally functional (see Section 14.3.1 and Figure 14.9).

We are accustomed to assuming that both the paternal and maternal alleles of biallelic genes are expressed, unless one or both copies have sustained mutations which affect expression. Clearly the expression can be tissue-specific so that in some cells both parental alleles are strongly expressed; in others, both gene copies are not apparently expressed. Thus, although there may be cell type-specific differences in expression, there is no discrimination between the capacity of the two parental alleles to be expressed, other than that due to genetic (mutational) differences between them. However, in humans and other mammals, several biallelic genes are known where the expression of one parental allele, either the paternal or the maternal allele but not both, is normally repressed in some cells (allelic exclusion). In such cells the relevant gene is said to exhibit functional hemizygosity: only one half of the maximum gene product is normally obtained even although the sequences of both parental alleles are perfectly consistent with normal gene expression and may even be identical. In some cases the allelic exclusion may be a property of select cells or tissues while in other cells of the same individual both alleles may be expressed normally.

Although initially considered a rarity, monoallelic expression of biallelic genes has been demonstrated for a growing number of human genes. A variety of different expression mechanisms can be involved and two broad classes of mechanism are involved (see Chess, 1998; Ohlsson et al., 1998):

• Allelic exclusion according to parent of origin (imprinting). In some cases, the choice of which of the two inherited copies is expressed is not random. This means that for some genes the allele whose expression is repressed is always the paternally inherited allele; in others it is always the maternally inherited allele (see Section 8.5.4).

• Allelic exclusion independent of parent of origin. Here the decision as to which of the two alleles is repressed is initially made randomly, but afterwards that pattern of allelic exclusion is transmitted stably to daughter cells following cell division. A variety of different mechanisms may be involved (see Box 8.6). In some cases, complex gene regulation may be required. For example, olfactory receptor genes are found in large clusters or arrays in mammalian genes. In an individual olfactory neuron only one allelic array of olfactory receptor genes is active (see Chess, 1998). A unique form of control is the programed DNA rearrangements which are required for individual cell-specific expression of the immunoglobulin genes in B cells and the T cell receptor genes in T lymphocytes. Because of the complexity of the latter mechanisms they are discussed separately in Section 8.6.

8.5.4. Genomic imprinting involves differences in the expression of alleles according to parent of origin

Prevalence and evolution of imprinting

Most human genes are not subject to imprinting, otherwise we would not see so many simple mendelian characters. Systematic surveys have been made to identify imprinted chromosomal regions in the mouse. Unlike in humans, all mouse chromosomes are acrocentric and Robertsonian translocations can permit crosses to be set up which produce offspring having both copies of one particular chromosome derived from a single parent (uniparental disomy, UPD, see Section 2.6.4). These reveal that UPD for some chromosomes has no phenotypic effect; for others it produces abnormal phenotypes. The abnormal phenotypes are sometimes complementary for different parental origins, e.g. overgrowth is often seen in maternal UPD and growth retardation in paternal UPD. For some chromosomes, UPD is lethal.

Figure 8.24

Imprinted gene clusters in 11p15.5 and 15q11-q13

Figure 8.24

.

   Imprinted gene clusters in 11p15.5 and 15q11-q13

Genes not known to be imprinted are shown in black; imprinted genes are in blue. Genes shown as solid blue boxes, e.g. KCNQ1, UBE3A, show preferential repression of paternal alleles (so that in some tissues only the maternal allele is expressed). Genes shown in open blue boxes, e.g. IGF2, ZNF127, have the opposite pattern: preferential repression of maternal alleles. Arrows indicate direction of transcription. IC denotes imprinting center (see text). The 15q11-q13 region has been less well studied and other genes are likely to be found there. Note that some of the genes have other names in the literature, e.g. KCNQ1 (KvLQT1), CDKN1C (p57 or KIP2), CD81 (TAPA1). Data from Lee et al. (1999) and Schweizer et al. (1999).

Further dissection at the chromosomal and genetic levels shows that imprinting is a property of a limited number of individual genes or small chromosomal regions. Currently, a total of over 30 genes are known to be imprinted in humans and mice (electronic reference 1), but the list can be expected to grow. Thus far, two major clusters of imprinted genes are known in the human genome: a 1 Mb region at 11p15 (encompassing the Beckwith- Wiedemann region) contains at least seven imprinted genes which may be arranged in two clusters (Lee et al., 1999); a 2.3 Mb cluster at 15q11-q13 region (encompassing the Prader-Willi and Angelman syndrome loci) also contains at least seven imprinted genes (Schweizer et al., 1999; see Figure 8.24). The imprinted gene clusters contain examples of neighboring genes with different parental imprints e.g. the H19 gene is expressed only from the maternal chromosome 11 whilst the adjacent IGF2 gene is expressed only from the paternal chromosome.

The great majority of known imprinted genes are autosomal. However, the XIST gene which has a major role in establishing X chromosome inactivation (see next section) may be considered an example of an imprinted X-linked gene since expression of the maternally inherited allele is preferentially repressed in trophoblast. An imprinted X-linked gene which affects cognitive function has also been suggested from differential behavior patterns in Turner syndrome. Girls with Turner syndrome lack a Y chromosome but have only one X chromosome. If the X chromosome is inherited from the mother, socially disruptive behavior is common, but if inherited from the father, the girl shows behavior closer to normal for that of a girl (Skuse et al., 1997).

Imprinting is known to occur in seed plants, some insects and mammals. No major imprinting effect, as judged by phenotype, has been observed in some model organisms such as Drosophila, C. elegans and the zebrafish, although the potential for imprinting may exist in Drosophila. Mammals are unusual in the way in which embryos are totally dependent on flow of nutrients from the maternal placenta. As many imprinted genes are involved in regulating fetal growth, one explanation envisages conflict between the parental genomes: the paternal genome propagates itself best by creating an embryo which aggressively removes nutrients from the mother; the maternal genome suppresses this to protect the mother and spare some resources for future offspring. As seen in cases of uniparental diploidy (see Box 8.7), paternal genes are preferentially expressed in the trophoblast and extraembryonic membranes, while maternal genes are preferentially expressed in the embryo.

8.5.5. The mechanism of genomic imprinting is unclear but a key component appears to be DNA methylation

Table 8.8

Examples of tissue and developmental stage regulation (more...)

Table 8.8

Examples of tissue and developmental stage regulation of imprinted genes in mammals

Gene and location	Repressed allele	Differences in expression patterns
IGF2 (insulin-like growth factor type 2)	Maternal	Imprinted in many tissues but biallelic expression in brain, adult liver and chondrocytes etc.
PEG1/MEST	Maternal	Imprinted in fetal tissue but biallelically expressed in adult blood
UBE3A (ubiquitin protein ligase 3)	Paternal	Imprinted exclusively in brain; biallelically expressed in other tissues
KCNQ1 (potassium channel involved)	Paternal	Imprinted in several tissues but biallelically expressed in heart
WT1 (Wilms' tumor gene)	Paternal	Frequently imprinted in cells of placenta and brain but biallelic expression in kidney

The above observations suggest that some mechanism must be able to distinguish between maternally and paternally inherited alleles: as chromosomes pass through the male and female germlines they must acquire some imprint to signal a difference between paternal and maternal alleles in the developing organism. A key component, at least in maintaining the imprinted status, is allele-specific DNA methylation (Brannan and Bartolomei, 1999; Tilghman, 1999). The imprinting of several imprinted genes has been shown to be disrupted in mutant mice that are deficient in the Dnmt1 cytosine methyltransferase gene and all imprinted genes are characterized by CG-rich regions of differential methylation.

Figure 8.25

Genomic (gametic) imprinting requires erasure of the (more...)

Figure 8.25

.

   Genomic (gametic) imprinting requires erasure of the imprint in the germline

The diagram illustrates the fate of a chromosome carrying two genes, A and B, which are subject to imprinting: A is imprinted in the female germline, B is imprinted in the male germline, as indicated by asterisks. As a result, in diploid somatic cells A is imprinted when present on a maternally inherited chromosome and B is imprinted when present on a paternally inherited chromosome. An individual chromosome may pass through the male and female germlines in successive generations: a man may transmit a chromosome inherited from his mother and a woman can transmit a chromosome inherited from her father, as indicated by the gametes in the left panel. As a result, there must be a mechanism whereby the old imprint is erased from the germline prior to establishing a new sex-specific imprint.

Figure 8.20

Figure 8.25

Figure 8.19

The timing of imprinting has become more clear recently. In the female germline, the maternal imprint, including the maternal pattern of methylation, is likely to be established during oocyte maturation which is consistent with the finding that the Dmnt1 protein is not detectable in nongrowing oocytes but is produced abundantly in growing oocytes. In the male germline, the functional paternal imprint is likely to be established prior to meiosis, possibly in the postmitotic primary spermatocyte (Brannan and Bartolomei, 1999).

Figure 8.24

Figure 8.24,

8.5.6. X chromosome inactivation in mammals involves very long-range cis-acting repression of gene expression

8.6.1. Ig and TCR genes exhibit a unique organization: multiple gene segments can encode each of several different regions of the polypeptide

Polypeptide structure

Table 8.9

Ig classes and subclasses

Table 8.9

Ig classes and subclasses

Class (and subclass)	Type of heavy chain	Location
IgA (IgA1,IgA2)	α (α1, α2)	Predominant Ig in seromucous secretions, e.g. saliva, milk, etc.
IgD	δ	Low in serum but present in large quantities on surface of many circulating B cells
IgE	ε	Especially on surface membrane of basophils and mast cells
IgG (IgG1, IgG2, IgG3, IgG4)	γ (γ1, γ2, γ3, γ4)	Major serum Ig
IgM	μ	Predominant ‘early’ antibody

An Ig molecule consists of four polypeptide chains, two identical heavy chains and two identical light chains (see Figure 7.10). The light chains fall into two classes: kappa (κ) and lambda (λ) light chains, which are functionally equivalent. At the N-terminal segments of each type of chain are the so-called variable (V) regions, which need to bind foreign antigen; the remaining C-terminal segments are constant (C) regions. In the case of the heavy chains, there are different alternatives for the constant region which specify the tissues in which the Ig will be expressed and dictate the immunoglobulin class (Table 8.9). Similarly, TCRs, which provide cell-mediated immune responses to foreign antigens, consist of two types of chain. Each such chain has Ig-like variable regions which bind foreign antigen, and constant regions which anchor the molecule to the cell surface (see Figure 7.10). The most frequently occurring TCRs have a β and a γ chain; a minor population consists of an α chain and a δ chain.

Gene structure

Table 8.10

Functional human Ig and TCR loci

Table 8.10

Functional human Ig and TCR loci

Locus	Location	Number of gene segments
		V	D	J	C
IGH	14q32.3	86	30	9	11
IGK	2p12	76	0	5	1
IGL	22q11	52	0	7	7
TCRA	14q11-12	60	0	75	1
TCRB	7q32-33	70–100	2	13	2
TCRG	7p15	8	0	5	2
TCRD	14q11-12	6	3	3	1

Figure 8.26

The Ig heavy chain locus on 14q32 contains about 86 (more...)

Figure 8.26

.

   The Ig heavy chain locus on 14q32 contains about 86 variable (V) region sequences, 30 diversity (D) segments, nine joining (J) segments and 11 constant region (C) sequences

The entire locus spans about 1200 kb of 14q32.3 and, for clarification, is shown as three segments of 400 kb from the telomeric end (top) to the centromeric end (bottom). Although the DH segments are mostly located in a few clusters separating the V_H and J_H segments, at least one such segment is located in the J_H segment region. Segments indicated by open circles have the required open reading frames but have not been observed in productive rearrangements and so their functional status is unknown. Segments which are known to be nonfunctional are indicated in blue, and account for approximately one third of all the segments. Note that although this is the only functional human heavy chain locus, small clusters of V_H and D segments are also located on 15q11.2 and 16p11.2. Adapted from data in Cook et al. (1994) Nature Genet., 7, pp. 162–168, with permission from Nature America Inc.

The genes which encode the different types of chain in Igs and TCRs are located on different chromosomes and are organized as clusters of numerous gene segments (Table 8.10). Each such cluster is unusual in that the coding sequences for specific segments of each chain are often present in numerous different copies that are sequentially repeated. For example, although the constant region of human κ light chain Ig is encoded by a single C_κ sequence, the variable regions are encoded by a combination of a V_κ segment (which encodes most of the variable region) and a short J_κ segment (joining segment; encodes a small part at the C-terminal end of the variable region) which are selected from a total of about 76 alternative V_κ segments and five alternative J_κ segments. Although the λ light chain is similarly encoded by V_λ, J_γ and C_γ segments, the heavy chain Ig locus shows some differences. The variable region is encoded by a combination of a V_H gene segment, a J_H gene segment and also a D_H gene segment (encoding a diversity segment), each selected from many repeated gene segments. Additionally, there are a variety of different C_H sequences which specify the class of the Ig (see above). In total this cluster comprises about 140 gene segments, of which about one-third are known to be incapable of expression, and spans about 1200 kb (Figure 8.26).

As each Ig gene cluster or TCR gene cluster in an individual B or T lymphocyte only ever gives rise to at most one Ig or TCR polypeptide, an entire cluster can functionally be regarded as a single, albeit unusual, type of gene. However, the individual gene segments cannot be regarded as the functional equivalent of classical exons. This is so because individual gene segments in these clusters are sometimes composed of coding DNA and noncoding DNA and may consist of several exons. For example, each of the human C_H sequences is itself composed of three or four classical exons separated by introns: after transcription into RNA, the intronic sequences are discarded, and only the exonic sequences are retained in the mRNA.

8.6.2. Programmed DNA rearrangements at the Ig and TCR loci occur during the maturation of B and T lymphocytes, respectively

The unique arrangement of gene segments in the Ig and TCR gene clusters reflects the very unusual way in which somatic recombinations are required in B and T lymphocytes before functional Ig and TCR genes can be assembled and then expressed (see below). Such somatic recombinations result in bringing together different combinations of the different gene segments in different individual lymphocytes. Consequently, they can be regarded as both tissue-specific (confined to B and T lymphocytes) and cell-specific events which involve alternative DNA splicing (as opposed to alternative RNA splicing which brings about different combinations of exons at the RNA level - Section 8.3.2). As a result, the original germline gene organization is altered: gene segments that were distant in the germline are spliced together at the DNA level. Because the choice of which of the many repeated gene segments are recombined to give a functional V-J or V-D-J unit is cell specific, individual B and T cells produce different Igs and TCRs. This means that, in a sense, every individual is a mosaic with respect to the organization of the Ig and TCR genes in B and T lymphocytes; even identical twins will diverge genetically.

The rearrangements which lead to the production of functional light chains and heavy chains of Igs are slightly different.

• 

  

  Figure 8.27

  Igs are synthesized following somatic recombination (more...)

  

  Figure 8.27

  .

     Igs are synthesized following somatic recombination of V and J, or V, D and J segments and subsequent RNA splicing to C sequences

  (A) Light chain synthesis. Somatic recombination (DNA splicing) results in joining of a specific variable (V) segment to a specific joining (J) segment; the example shows a V₃-J₂ joining which is only one of many possibilities. The VJ unit is then spliced to the constant region (C) sequence by RNA splicing. (B) Heavy chain synthesis. Two sequential somatic recombinations produce first D-J joining, then a VDJ unit. Subsequent RNA splicing results in splicing of the VDJ sequence to the C_μ sequence. As the B cell matures, however, subsequent somatic recombinations result in joining of the previously selected VDJ unit to different C genes (heavy chain switch, see text and Figure 8.29).

  Making a light chain. In order to generate a functional κ light chain Ig, for example, a somatic recombination event brings together a specific combination of one of the V_κ gene segments and one of the J_κ gene segments (V-J joining). Thereafter, splicing to the single C_κ sequence occurs at the RNA level (Figure 8.27A).

• Making a heavy chain. Two successive somatic recombinations are required, resulting first in D_H-J_H joining, and then V_H-D_H-J_H joining. Subsequently the resulting V_H-D_H-J_H coding sequence is spliced at the RNA level to the nearest C_H sequence, initially C_μ (Figure 8.27B).

Because there are three types of functional Ig gene loci in human cells (heavy chain, κ light chain and λ light chain), and because these occur on both maternal and paternal homologs, there are six chromosomal segments in which DNA rearrangments can result in production of an Ig chain. However, an individual B cell is monospecific: it produces only one type of Ig molecule with a single type of heavy chain and a single type of light chain. This is so for two reasons:

• Allelic exclusion. A light chain or a heavy chain can be synthesized from a maternal chromosome or a paternal chromosome in any one B cell, but not from both parental homologs. As a result, there is monoallelic expression at the heavy chain gene locus in B cells. This phenomenon also applies to TCR gene clusters.

• Light chain exclusion. A light chain synthesized in a single B cell may be a κ chain, or a λ chain, but never both. As a result of this requirement, plus that of allelic exclusion, there is monoallelic expression at one of the two functional light chain gene clusters and no expression at the other. The decision to choose which of the two heavy chain alleles and which of the four possible segments to make a light chain appears to be random. Most likely, in each B-cell precursor, productive DNA rearrangements are attempted at all six Ig alleles but the chances of productive arrangements in more than one light chain cluster or more than one heavy chain allele may not be high. Additionally, however, there appears to be some kind of negative feedback regulation: a functional rearrangement at one of the heavy chain alleles suppresses rearrangements occurring in the other allele, and a functional rearrangement at any one of the four regions capable of encoding a light chain suppresses rearrangements occurring in the other three.

8.6.3. V-J and V-D-J joining is often achieved by intrachromatid deletions, and also by megabase inversions in the former case

Figure 8.28

Inversion or deletion results in V-J splicing to produce (more...)

Figure 8.28

.

   Inversion or deletion results in V-J splicing to produce functional Ig κ light chain genes

The human κ light chain gene cluster contains about 76 V_κ segments arranged in two large clusters, in opposite orientations. V segments in the distal cluster have the opposite orientation to the J_κ segments and the single C_κ sequence. As a result, the DNA rearrangements used to splice distal V_κ segments to a J_κ segment are megabase inversions (Weichhold et al., 1990). Those in the proximal cluster can undergo V-J joining by a somatic recombination resulting in a deletion of the intervening chromosomal segment, most likely through an intrachromatid recombination event such as that used in class switching (see Figure 8.29).

Figure 8.28

• V segments in the distal cluster are joined to J segments by inversions.

• V segments in the proximal cluster are joined to J segments by deletions (Figure 8.28).

Note that the joining process is imprecise, and so can also introduce a measure of variability in the sequence at the junctions of joined segments.

8.6.4. Class switching of heavy chains involves differential joining of a single VDJ unit to alternative DNA segments encoding constant regions

Figure 8.27B

• initial synthesis of IgM only by immature B cells. This occurs because the VDJ unit is spliced at the RNA level to a C_μ sequence (Figure 8.27B).

• 

  

  Figure 8.29

  Ig heavy chain class switching is mediated by intrachromatid (more...)

  

  Figure 8.29

  .

     Ig heavy chain class switching is mediated by intrachromatid recombination

  Note that joining of the same VDJ unit to a C_μ or a C_δ sequence occurs at the level of RNA splicing to generate heavy chains for IgM and IgD respectively. In contrast, class switching to generate IgA, IgE or IgG involves joining of the same VDJ unit at the DNA level to, respectively, a C_α, C_ε or, as illustrated in the figure, a C_γ sequence.

  Later synthesis of both IgM and IgD by immature B cells. The partial switch to making IgD occurs because the VDJ unit can be spliced at the RNA level to a C_δ sequence, as a result of alternative RNA splicing (Figure 8.29).

• Synthesis of IgG, IgE or IgA by mature B cells. Class switching events involve splicing the same VDJ unit to a C_γ, C_ε or C_α sequence, respectively, at the DNA level as a result of a somatic recombination event (VDJ-C joining). The mechanism involves deletion of the intervening sequence by intrachromatid recombination (Figure 8.29).