Differential Gene Transcription (2024)

Anatomy of the gene: Exons and introns

There are two fundamental differences distinguishing most eukaryotic genes from most prokaryotic genes. First, eukaryotic genes are contained within a complex of DNA and protein called chromatin. The protein component constitutes about half the weight of chromatin and is composed largely of nucleosomes. The nucleosome is the basic unit of chromatin structure. It is composed of an octamer of histone proteins (two molecules each of histones H2A-H2B and histones H3-H4) wrapped with two loops containing approximately 140 base pairs of DNA (Figure 5.1; Kornberg and Thomas 1974). Chromatin can thus be visualized as a string of nucleosome beads linked by ribbons of DNA. While classic geneticists have likened genes to “beads on a string,” molecular geneticists liken genes to “string on the beads.” Most of the time, the nucleosomes are themselves wound into tight “solenoids” that are stabilized by histone H1. Histone H1 is found in the 60 or so base pairs of “linker” DNA between the nucleosomes (Weintraub 1984). This H1-dependent conformation of nucleosomes inhibits the transcription of genes in somatic cells by packing adjacent nucleosomes together into tight arrays that prohibit the access of transcription factors and RNA polymerases to the genes (Thoma et al. 1979; Schlissel and Brown 1984). It is generally thought, then, that the “default” condition of chromatin is a repressed state, and that tissue-specific genes become activated by local interruption of this repression (Weintraub 1985).

The second difference is that eukaryotic genes are not co-linear with their peptide products. Rather, the single nucleic acid strand of eukaryotic mRNA comes from noncontiguous regions on the chromosome. Between the regions of DNA coding for a protein—exons—are intervening sequences—introns—that have nothing whatsoever to do with the amino acid sequence of the protein.^* The structure of a typical eukaryotic gene can be represented by the human β-globin gene, shown in Figure 5.2. This gene consists of the following elements:

1.

A promoter region, which is responsible for the binding of RNA polymerase and for the subsequent initiation of transcription. The promoter region of the human β-globin gene has three distinct units and extends from 95 to 26 base pairs before (“upstream from”)^† the transcription initiation site (i.e., from -95 to -26).

2.

The transcription initiation site, which for human β-globin is ACATTTG. This site is often called the cap sequence because it represents the 5´ end of the RNA, which will receive a “cap” of modified nucleotides soon after it is transcribed. The specific cap sequence varies among genes.

3.

The translation initiation site, ATG. This codon (which becomes AUG in the mRNA) is located 50 base pairs after the transcription initiation site in the human β-globin gene (although this distance differs greatly among different genes). The intervening sequence of 50 base pairs between the initiation points of transcription and translation is the 5´ untranslated region, often called the 5´ UTR or leader sequence. The 5´ UTR can determine the rate at which translation is initiated.

4.

The first exon, which contains 90 base pairs coding for amino acids 1–30 of human β-globin.

5.

An intron containing 130 base pairs with no coding sequences for the globin protein. The structure of this intron is important in enabling the RNA to be processed into messenger RNA and exit from the nucleus.

6.

An exon containing 222 base pairs coding for amino acids 31–104.

7.

A large intron—850 base pairs—having nothing to do with the globin protein structure.

8.

An exon containing 126 base pairs coding for amino acids 105–146.

9.

A translation termination codon, TAA. This codon becomes UAA in the mRNA. The ribosome dissociates at this codon, and the protein is released.

10.

A 3´ untranslated region that, (3´ UTR) although transcribed, is not translated into protein. This region includes the sequence AATAAA, which is needed for polyadenylation: the placement of a “tail” of some 200 to 300 adenylate residues on the RNA transcript. This poly(A) tail (1) confers stability on the mRNA, (2) allows the mRNA to exit the nucleus, and (3) permits the mRNA to be translated into protein. The poly(A) tail is inserted into the RNA about 20 bases downstream of the AAUAAA sequence. Transcription continues beyond the AATAAA site for about 1000 nucleotides before being terminated.

The original nuclear RNA transcript for such a gene contains the capping sequence, the 5´ untranslated region, the exons, the introns, and the 3´ untranslated region (Figure 5.3). In addition, both its ends become modified. A cap consisting of methylated guanosine is placed on the 5´ end of the RNA in opposite polarity to the RNA itself. This means that there is no free 5´ phosphate group on the nuclear RNA. The 5´ cap is necessary for the binding of mRNA to the ribosome and for subsequent translation (Shatkin 1976). The 3´ terminus is usually modified in the nucleus by the addition of a poly(A) tail. These adenylate residues are put together enzymatically and are added to the transcript; they are not part of the gene sequence. Both the 5´ and 3´ modifications may protect the RNA from exonucleases that would otherwise digest the mRNA (Sheiness and Darnell 1973; Gedamu and Dixon 1978). The modifications thus stabilize the message and its precursor.

Anatomy of the gene: Promoters and enhancers

In addition to the protein-encoding region of the gene, there are regulatory sequences that can be on either end of the gene (or even within it). These sequences—the promoters and enhancers—are necessary for controlling where and when a particular gene is transcribed.

Promoters are the sites where RNA polymerase binds to the DNA to initiate transcription. Promoters of genes that synthesize messenger RNAs (i.e., genes that encode proteins^‡) are typically located immediately upstream from the site where the RNA polymerase initiates transcription. Most of these promoters contain the sequence TATA, where RNA polymerase will be bound (Figure 5.4). This site, known as the TATA box, is usually about 30 base pairs upstream from the site where the first base is transcribed. Eukaryotic RNA polymerases, however, will not bind to this naked DNA sequence. Rather, they require additional protein factors to bind efficiently to the promoter. The protein-encoding genes are transcribed by RNA polymerase II, and at least six nuclear proteins have been shown to be necessary for the proper initiation of transcription by RNA polymerase II (Buratowski et al. 1989; Sopta et al. 1989). These proteins are called basal transcription factors. The first of these, TFIID,^§ recognizes the TATA box through one of its subunits, TATA-binding protein (TBP). TFIID serves as the foundation of the transcription initiation complex, and it also serves to keep nucleosomes from forming in this region. Once TFIID is stabilized by TFIIA, it becomes able to bind TFIIB. Once TFIIB is in place, RNA polymerase can bind to this complex. Other transcription factors (TFIIE, F, and H) are then used to release RNA polymerase from the complex so that it can transcribe the gene, and to unwind the DNA helix so that the RNA polymerase will have a free template from which to transcribe.

In addition to these basal transcription factors, which are found in each nucleus, there is also a set of transcription factors called TBP-associated factors, or TAFs (Figure 5.5; Buratowski 1997; Lee and Young 1998), which can stabilize the TBP. This function is critical for gene transcription, for if the TBP is not stabilized, it can fall off the small TATA sequence. The TAFs are bound by upstream promoter elements on the DNA. These DNA sequences are near the TATA sequence, and usually upstream from it. These TAFs need not be in every cell of the body, however. Cell-specific transcription factors (such as the Pax6 and microphthalmia proteins mentioned in Chapter 4) can also activate the gene by stabilizing the transcription initiation complex. They can do so by binding to the TAFs, by binding directly to other factors such as TFIIB, or (as we will see soon) by destabilizing nucleosomes.

An enhancer is a DNA sequence that can activate the utilization of a promoter, controlling the efficiency and rate of transcription from that particular promoter. Enhancers can activate only cis-linked promoters (i.e., promoters on the same chromosome^¶), but they can do so at great distances (some as great as 50 kilobases away from the promoter). Moreover, enhancers do not need to be on the 5´ (upstream) side of the gene. They can also be at the 3´ end, in the introns, or even on the complementary DNA strand (Maniatis et al. 1987). The human β-globin gene has an enhancer in its 3´ UTR, roughly 700 base pairs downstream from the AATAAA site. This sequence is necessary for the temporal- and tissue-specific expression of the β-globin gene in adult red blood cell precursors (Trudel and Constantini 1987). Like promoters, enhancers function by binding specific regulatory proteins called transcription factors.

Enhancers can regulate the temporal and tissue-specific expression of any differentially regulated gene, but different types of genes normally have different enhancers. In the pancreas, for instance, the exocrine protein genes (for the digestive proteins chymotrypsin, amylase, and trypsin) have enhancers different from that of the gene for the endocrine protein insulin. These enhancers both lie in the 5´ flanking sequences of their genes. Walker and colleagues et al. 1983 created transgenes by placing flanking regions from the genes for chymotrypsin and insulin onto the gene for bacterial chloramphenicol acetyltransferase (CAT), an enzyme that is not found in mammalian cells. CAT activity is easy to assay in mammalian cells, so the bacterial CAT gene can be used as a reporter gene to tell investigators whether a particular enhancer is functioning. The researchers then transfected the transgenes into (1) ovary cells (which do not secrete either insulin or chymotrypsin), (2) an insulin-secreting cell line, and (3) a chymotrypsin-secreting cell line, and measured the activity of CAT in each of these cells. As shown in Figure 5.6, neither enhancer sequence caused the enzyme to be made in the ovarian cells. In the insulin-secreting cell, however, the 5´ flanking region from the insulin gene enabled the CAT gene to be expressed, but the 5´ flanking region of the chymotrypsin gene did not. Conversely, when the clones were placed into the exocrine pancreatic cell line, the chymotrypsin 5´ flanking sequence allowed CAT expression, while the insulin enhancer did not. The enhancers for 10 different exocrine proteins share a 20-base-pair consensus sequence, suggesting that these similar sequences play a role in activating an entire set of genes specifically expressed in the exocrine cells of the pancreas (Boulet et al. 1986). Thus, the expression of genes in exocrine and in endocrine cells of the pancreas appears to be controlled by different enhancers.

By taking an enhancer from one gene and fusing it to another gene, it has been shown that enhancers can direct the expression of any gene sequence. For instance, the β-galactosidase gene from E. coli (the lacZ gene) can be used as a reporter gene and placed onto an enhancer that normally directs a particular mouse gene to become expressed in muscles. If the resulting transgene is injected into a newly fertilized mouse egg and gets incorporated into its DNA, the β-galactosidase gene will be expressed in the muscle cells. By staining for the presence of β-galactosidase, the expression pattern of that muscle-specific gene can be seen (Figure 5.7A). Similarly, if the gene for green fluorescent protein (GFP, a reporter protein that is usually made only in jellyfish) is placed on the enhancer of genes encoding the crystallin proteins of the eye lens, GFP expression is seen solely in the lens (Figure 5.7B).

Enhancers are critical in the regulation of normal development. Over the past decade, six generalizations that emphasize their importance for differential gene expression have been made:

1.

Most genes require enhancers for their transcription.

2.

Enhancers are the major determinant of differential transcription in space (cell type) and time.

3.

The ability of an enhancer to function while far from the promoter means that there can be multiple signals to determine whether a given gene is transcribed. A given gene can have several enhancer sites linked to it, and each enhancer can be bound by more than one transcription factor.

4.

The interaction between the proteins bound to the enhancer sites and the transcription initiation complex assembled at the promoter is thought to regulate transcription. The mechanism of this association is not fully known, nor do we comprehend how the promoter integrates all these signals.

5.

Enhancers are modular. There are various DNA elements that regulate temporal and spatial gene expression, and these can be mixed and matched. As we will see, the enhancers for endocrine hormones such as insulin and for lens-specific proteins such as crystallins both have sites that bind Pax6 protein. But Pax6 doesn't tell the lens to make insulin or the pancreas to make crystallins, because there are other transcription factor proteins that also must bind. It is the combination of transcription factors that causes particular genes to be transcribed.

6.

A gene can have several enhancer elements, each turning it on in a different set of cells

7.

Enhancers can also be used to inhibit transcription. In some cases, the same transcription factors that activate the transcription of one gene can be used to repress the transcription of other genes. These “negative enhancers “ are also called silencers.

Transcription factors

Transcription factors are proteins that bind to enhancer or promoter regions and interact to activate or repress the transcription of a particular gene. Most transcription factors can bind to specific DNA sequences. These proteins can be grouped together in families based on similarities in structure (Table 5.1). The transcription factors within such a family share a common framework structure in their DNA-binding sites, and slight differences in the amino acids at the binding site can alter the sequence of the DNA to which the factor binds.

Transcription factors have three major domains. The first is a DNA-binding domain that recognizes a particular DNA sequence. The second is a trans-activating domain that activates or suppresses the transcription of the gene whose promoter or enhancer it has bound. Usually, this trans-activating domain enables the transcription factor to interact with proteins involved in binding RNA polymerase (such as TFIIB or TFIIE; see Sauer et al. 1995). In addition, there may be a protein-protein interaction domain that allows the transcription factor's activity to be modulated by TAFs or other transcription factors.

Silencers

Some sequences act specifically to block transcription. These silencer domains are useful in restricting the transcription of a particular gene to a particular group of cells or for regulating the timing of the gene's expression. For example, the fetal mouse liver makes serum albumin, but only after a certain stage of gut development. At first, the endodermal cells that will form the liver do not transcribe this albumin gene. However, when the endodermal tube contacts the cardiac mesoderm (which is in the process of forming a heart), the heart precursors are able to instruct the endodermal tube to begin forming the liver and to start transcribing liver-specific genes (Le Douarin 1964; Gualdi et al. 1996). This contact is thought to release a transcription factor bound to a silencer region in the serum albumin gene. This silencer site is occupied prior to the contact of the endoderm with the cardiac mesoderm, but it becomes vacant in the endodermal tube immediately after contact with the heart-forming cells (Figure 5.16; Gualdi et al. 1996).

Another example of a silencer is found in certain neural genes. There is a sequence found in certain promoters that prevents the promoter's activation in any tissue except neurons. This sequence was given the name neural restrictive silencer element (NRSE), and it has been found in several mouse genes whose expression is limited to the nervous system: those for synapsin I, sodium channel type II, brain-derived neurotrophic factor, Ng-CAM, and L1. The protein that binds to the NRSE is a zinc finger transcription factor called neural restrictive silencer factor (NRSF). NRSF appears to be expressed in every cell that is not a mature neuron (Chong et al. 1995; Schoenherr and Anderson 1995). Thus, the down-regulation of NRSF seems to be a key event in allowing the expression of several genes that are critical to neural function.

To test the hypothesis that NRSE was necessary in the normal repression of neural genes in non-neural cells, lacZ transgenes were made by fusing a β-galactosidase gene (lacZ) with part of the L1 neural cell adhesion gene. (L1 is a protein whose function is critical for brain development, as we will see in later chapters.) In one case, the L1 gene, from its promoter through the fourth exon, was fused to the lacZ sequences. A second transgene was made just like the first, except that the NRSE had been deleted from the L1 promoter. The two transgenes were separately inserted into the pronuclei of fertilized oocytes, and the resulting transgenic mice were analyzed for the expression of β-galactosidase (Kallunki et al. 1995, 1997). In the embryos receiving the complete transgene (which included the NSRE), expression was seen only in the nervous system (Figure 5.17A). However, in those mice whose transgene lacked the NRSE, expression was seen in the heart, limb mesenchyme and limb ectoderm, kidney mesoderm, ventral body wall, and cephalic mesenchyme (Figure 5.17B).

Locus control regions in globin genes

There are some regions of DNA called locus control regions (LCRs), which function as “super-enhancers.” These LCRs establish an “open” chromatin configuration, inhibiting the normal repression of transcription over an area spanning several genes. The mechanism by which the LCR opens up the chromatin is not yet known.

One of the best-studied LCRs is that regulating the tissue-specific expression of the β-globin family of genes in humans, mice, and chicks. In many species, including chicks and humans, the embryonic or fetal hemoglobin differs from that found in adult red blood cells (Figure 5.18). Human hemoglobin consists largely of four globin chains of two different types and four molecules of heme. Human embryonic hemoglobin has two zeta (ζ) globin chains and two epsilon (ϵ) globin chains (and four molecules of heme). During the second month of human gestation, ζ- and ϵ-globin synthesis abruptly ceases, while alpha (α) and gamma (γ) globin synthesis increases. The association of two γ-globin chains with two α-globin chains produces fetal hemoglobin (α₂γ₂). (The physiological importance of the γ-globin chain in fetal hemoglobin is examined in Chapter 14.) At 3 months gestation, the beta (β) globin and delta (δ) globin genes begin to be active, and their products slowly increase, while γ-globin levels gradually decline. This switchover is greatly accelerated after birth, and fetal hemoglobin is replaced by adult hemoglobin (α₂β₂). The normal adult hemoglobin profile is 97% α₂β₂, 2–3% α₂δ₂, and 1% α₂γ₂.

A schematic diagram of human hemoglobin types and the genes that code for them is shown in Figure 5.19. In humans, the ζ- and α-globin genes are located on chromosome 16, but the ϵ-, γ-, δ-, and β-globin genes (known as the β-globin gene family) are linked together, in order of appearance, on chromosome 11. It appears, then, that there is a mechanism that directs the sequential switching of the chromosome 11 genes from embryonic, to fetal, to adult globins.

The discovery of the human globin LCR came from studies of the genetic disease β-thalassemia. This anemic condition results from a lack of β-globin and can be caused in several ways. The usual causes of β-thalassemia involve deletions or mutations in either the coding region of the β-globin gene or its promoter. However, in some patients, there is a deletion in a region upstream from the β-globin gene family, while the genes themselves are normal. Moreover, without this upstream region, the β-globin family DNA was found to be DNase I-insensitive (van der Ploeg et al. 1980; Kioussis et al. 1983). DNase I treatment is used to see whether the DNA in chromatin is accessible to transcription factors. If the DNA in chromatin is not digested by DNase I, it means that DNase I cannot reach it, and therefore, transcription factors couldn't reach it either. Promoters are usually DNase I-sensitive in the cells where they function; and they are usually DNase I-insensitive in those cells where they are not active (Weintraub and Groudine 1976; Stalder et al. 1980). So it appeared that there was a region of DNA upstream from the β-globin gene cluster that was responsible for “opening up” the chromatin of the genes, making them accessible to transcription factors. This region of DNA was termed the β-globin locus control region.

The locus control region for the β-globin gene complex is located far upstream from its most 5´ member (ϵ). This LCR contains four sites that are DNase I-hypersensitive only in erythroid precursor cells. Sites are said to be DNase-hypersensitive when the DNA in the chromatin can be digested there with only small amounts of DNase I. In most instances, a site is thought to be DNase I hypersensitive when it lacks nucleosomes (Elgin 1988). These sites in the LCR are therefore within nucleosome coils in most cells' nuclei, but in the precursors of the red blood cells, this DNA is exposed. The entire LCR is necessary for activating high levels of erythroid cell-specific transcription of the entire β-globin gene family on human chromosome 11 (Grosveld et al. 1987). Deletion or mutation of the LCR causes the silencing of all these genes. Conversely, if the LCR is placed adjacent to a gene that is not usually expressed in red blood cells (such as the T cell-specific thy-1 gene) and then transfected into erythroid precursor cells, the additional gene will be expressed in the red blood cells. This effect is specific for red blood cell precursors, since only they have the appropriate transcription factors to bind to the LCR (Blom van Assendelft et al. 1989; Fiering et al. 1993). If any of the globin genes are separated from the LCR, they are repressed, even in the erythroid cells that would normally be transcribing them. The locus control region is crammed with binding sites for transcription factors. As Gary Felsenfeld (1992) observed, “The domains look as though they were put together by an overenthusiastic student determined to construct a powerful cis-acting element.” He suggested that one of the functions of the LCR is to loop around to one of the globin promoter regions during DNA replication and bind to it in a manner that prevents nucleosomes from forming on that promoter. Indeed, the globin promoters are not DNase I-hypersensitive (i.e., accessible to transcription factors) except in the presence of the LCR.

It is not known why the genes closest to the LCR are transcribed earlier than the genes farther from it. Interestingly, experiments have shown that the distance between the LCR and the globin genes does affect their activation (Hanscombe et al. 1991; Dillon et al. 1997). When linked closely to the LCR, the human β-globin gene becomes expressed in transgenic mouse embryonic cells. Its correct activation (in adult red cells only) is restored only when it is placed farther away from the LCR. Similarly, the human γ-globin gene is repressed earlier (like the normal β-globin gene) when it is placed farther from the LCR. These findings suggest that the interaction between the LCR and the globin genes is polarized: those globin genes closest to the LCR are turned on earliest, while those genes more distal are turned on later. Presumably, there is physical contact between the LCR and the gene-specific promoters and enhancers.

LCRs have been found for other genes, such as the human growth hormone locus, the macrophage-specific lysozyme gene, and the CD2 and CD4 genes expressed in T lymphocytes (see Kioussis and Festenstein 1997; Fraser and Grosveld 1998).

*

The term exon refers to a nucleotide sequence whose RNA “exits” the nucleus. It has taken on the functional definition of a protein-encoding nucleotide sequence. Leader sequences and 3´ untranslated sequences are also derived from exons, even though they are not translated into protein.

†

By convention, upstream, downstream, 5´, and 3´ directions are specified in relation to the RNA. Thus, the promoter is upstream of the gene, near its 5´ end.

‡

There are several types of RNA that do not encode proteins. These include the ribosomal RNAs and transfer RNAs (which are used in protein synthesis) and the small nuclear RNAs (which are used in RNA processing). In addition, there are regulatory RNAs (such as Xist and lin-4, which we will discuss later in this chapter) that are involved in regulating gene expression (and which do not encode proteins).

§

TF stands for transcription factor; II indicates that the factor was first found to be needed for RNA polymerase II; and the letter designations refer to the active fractions from the phosphocellulose columns used to purify these proteins.

¶

Cis- and trans-regulatory elements are so named by analogy with E. coli genetics. There, cis-factors are regulatory elements that reside on the same strand of DNA (cis-, meaning “on the same side as”), while trans-elements are those that could be supplied from another chromosome (trans-, meaning “on the other side of”). Cis-regulatory elements now refers to those DNA sequences that regulate a gene on the same stretch of DNA (i.e., the promoters and enhancers). Trans-regulatory factors are soluble molecules whose genes are located elsewhere in the genome and which bind to the cis-regulatory elements. They are usually transcription factors.