How Do Eukaryotes Coordinate Control Of Multiple Genes That Must Be Turned On Simultaneously?

Although the command of factor expression is far more circuitous in eukaryotes than in bacteria, the same basic principles employ. The expression of eukaryotic genes is controlled primarily at the level of initiation of transcription, although in some cases transcription may exist adulterate and regulated at subsequent steps. Equally in bacteria, transcription in eukaryotic cells is controlled by proteins that demark to specific regulatory sequences and modulate the activity of RNA polymerase. The intricate task of regulating gene expression in the many differentiated cell types of multicellular organisms is accomplished primarily past the combined actions of multiple dissimilar transcriptional regulatory proteins. In addition, the packaging of DNA into chromatin and its modification past methylation impart further levels of complexity to the control of eukaryotic gene expression.

cis-Acting Regulatory Sequences: Promoters and Enhancers

Every bit already discussed, transcription in leaner is regulated past the bounden of proteins to cis-acting sequences (due east.chiliad., the lac operator) that command the transcription of adjacent genes. Similar cis-acting sequences regulate the expression of eukaryotic genes. These sequences have been identified in mammalian cells largely past the use of gene transfer assays to study the activity of suspected regulatory regions of cloned genes (Effigy 6.18). The eukaryotic regulatory sequences are unremarkably ligated to a reporter gene that encodes an hands detectable enzyme. The expression of the reporter gene following its transfer into cultured cells and then provides a sensitive assay for the ability of the cloned regulatory sequences to directly transcription. Biologically agile regulatory regions can thus exist identified, and in vitro mutagenesis tin be used to determine the roles of specific sequences within the region.

Figure 6.18

Identification of eukaryotic regulatory sequences. The regulatory sequence of a cloned eukaryotic gene is ligated to a reporter gene that encodes an easily detectable enzyme. The resulting plasmid is then introduced into cultured recipient cells by transfection. (more...)

Genes transcribed by RNA polymerase 2 take two core promoter elements, the TATA box and the Inr sequence, that serve as specific bounden sites for general transcription factors. Other cis-acting sequences serve every bit bounden sites for a wide variety of regulatory factors that command the expression of private genes. These cis-acting regulatory sequences are often, though non always, located upstream of the TATA box. For example, 2 regulatory sequences that are plant in many eukaryotic genes were identified past studies of the promoter of the herpes simplex virus factor that encodes thymidine kinase (Figure half dozen.nineteen). Both of these sequences are located within 100 base pairs upstream of the TATA box: Their consensus sequences are CCAAT and GGGCGG (called a GC box). Specific proteins that bind to these sequences and stimulate transcription have since been identified.

Figure 6.19

A eukaryotic promoter. The promoter of the thymidine kinase gene of herpes simplex virus contains 3 sequence elements upstream of the TATA box that are required for efficient transcription: a CCAAT box and two GC boxes (consensus sequence GGGCGG). (more...)

In contrast to the relatively simple organization of CCAAT and GC boxes in the herpes thymidine kinase promoter, many genes in mammalian cells are controlled by regulatory sequences located farther away (sometimes more than than x kilobases) from the transcription start site. These sequences, chosen enhancers, were get-go identified by Walter Schaffner in 1981 during studies of the promoter of another virus, SV40 (Effigy 6.xx). In addition to a TATA box and a prepare of six GC boxes, two 72-base of operations-pair repeats located further upstream are required for efficient transcription from this promoter. These sequences were found to stimulate transcription from other promoters as well equally from that of SV40, and, surprisingly, their activity depended on neither their distance nor their orientation with respect to the transcription initiation site (Figure 6.21). They could stimulate transcription when placed either upstream or downstream of the promoter, in either a forrad or backward orientation.

Figure vi.xx

The SV40 enhancer. The SV40 promoter for early on cistron expression contains a TATA box and six GC boxes bundled in three sets of repeated sequences. In addition, efficient transcription requires an upstream enhancer consisting of ii 72-base-pair (bp) repeats. (more...)

Figure half dozen.21

Activity of enhancers. Without an enhancer, the factor is transcribed at a low basal level (A). Addition of an enhancer, E—for example, the SV40 72-base of operations-pair repeats—stimulates transcription. The enhancer is active non just when placed just (more than...)

The ability of enhancers to function even when separated by long distances from transcription initiation sites at first suggested that they work by mechanisms different from those of promoters. However, this has turned out non to be the example: Enhancers, similar promoters, function past bounden transcription factors that then regulate RNA polymerase. This is possible because of Dna looping, which allows a transcription cistron bound to a distant enhancer to interact with RNA polymerase or general transcription factors at the promoter (Figure half dozen.22). Transcription factors bound to distant enhancers can thus work by the same mechanisms as those leap adjacent to promoters, so in that location is no fundamental difference between the deportment of enhancers and those of cis-acting regulatory sequences adjacent to transcription start sites. Interestingly, although enhancers were first identified in mammalian cells, they have subsequently been establish in bacteria—an unusual instance in which studies of eukaryotes served as a model for the simpler prokaryotic systems.

Effigy 6.22

DNA looping. Transcription factors bound at distant enhancers are able to collaborate with general transcription factors at the promoter because the intervening Dna can grade loops. There is therefore no fundamental departure between the activeness of transcription (more than...)

The bounden of specific transcriptional regulatory proteins to enhancers is responsible for the control of factor expression during development and differentiation, also equally during the response of cells to hormones and growth factors. 1 of the most thoroughly studied mammalian enhancers controls the transcription of immunoglobulin genes in B lymphocytes. Gene transfer experiments take established that the immunoglobulin enhancer is active in lymphocytes, but not in other types of cells. Thus, this regulatory sequence is at least partly responsible for tissue-specific expression of the immunoglobulin genes in the appropriate differentiated cell type.

An of import aspect of enhancers is that they usually contain multiple functional sequence elements that bind different transcriptional regulatory proteins. These proteins work together to regulate gene expression. The immunoglobulin heavy-concatenation enhancer, for example, spans approximately 200 base of operations pairs and contains at least nine distinct sequence elements that serve as poly peptide-binding sites (Effigy vi.23). Mutation of any one of these sequences reduces simply does non cancel enhancer action, indicating that the functions of individual proteins that demark to the enhancer are at least partly redundant. Many of the private sequence elements of the immunoglobulin enhancer by themselves stimulate transcription in nonlymphoid cells. The restricted action of the intact enhancer in B lymphocytes therefore does not event from the tissue-specific role of each of its components. Instead, tissue-specific expression results from the combination of the private sequence elements that make upward the complete enhancer. These elements include some cis-interim regulatory sequences that bind transcriptional activators that are expressed specifically in B lymphocytes, as well as other regulatory sequences that bind repressors in nonlymphoid cells. Thus, the immunoglobulin enhancer contains negative regulatory elements that inhibit transcription in inappropriate cell types, also as positive regulatory elements that activate transcription in B lymphocytes. The overall activeness of the enhancer is greater than the sum of its parts, reflecting the combined activeness of the proteins associated with each of its individual sequence elements.

Effigy 6.23

The immunoglobulin enhancer. The immunoglobulin heavy-chain enhancer spans most 200 bases and contains nine functional sequence elements (East, μE1-5, π, μB, and October), which together stimulate transcription in B lymphocytes.

Transcriptional Regulatory Proteins

The isolation of a variety of transcriptional regulatory proteins has been based on their specific bounden to promoter or enhancer sequences. Protein binding to these DNA sequences is ordinarily analyzed past 2 types of experiments. The first, footprinting, was described earlier in connectedness with the binding of RNA polymerase to prokaryotic promoters (run across Effigy 6.3). The second approach is the electrophoretic-mobility shift assay, in which a radiolabeled DNA fragment is incubated with a protein preparation and and then subjected to electrophoresis through a nondenaturing gel (Effigy 6.24). Protein binding is detected as a subtract in the electrophoretic mobility of the DNA fragment, since its migration through the gel is slowed by the bound poly peptide. The combined use of footprinting and electrophoretic-mobility shift assays has led to the correlation of protein-bounden sites with the regulatory elements of enhancers and promoters, indicating that these sequences generally institute the recognition sites of specific DNA-binding proteins.

Figure half-dozen.24

Electrophoretic-mobility shift assay. A sample containing radiolabeled fragments of Dna is divided into two, and one half of the sample is incubated with a poly peptide that binds to a specific Dna sequence. Samples are then analyzed past electrophoresis in (more than...)

One of the prototypes of eukaryotic transcription factors was initially identified by Robert Tjian and his colleagues during studies of the transcription of SV40 Deoxyribonucleic acid. This factor (called Sp1, for specificity protein one) was found to stimulate transcription from the SV40 promoter, just not from several other promoters, in cell-free extracts. Then, stimulation of transcription by Sp1 was institute to depend on the presence of the GC boxes in the SV40 promoter: If these sequences were deleted, stimulation by Sp1 was abolished. Moreover, footprinting experiments established that Sp1 binds specifically to the GC box sequences. Taken together, these results signal that the GC box represents a specific binding site for a transcriptional activator—Sp1. Like experiments take established that many other transcriptional regulatory sequences, including the CCAAT sequence and the various sequence elements of the immunoglobulin enhancer, also represent recognition sites for sequence-specific Dna-binding proteins (Tabular array 6.ii).

Table 6.2

Examples of Transcription Factors and Their DNA-Binding Sites.

The specific bounden of Sp1 to the GC box not only established the action of Sp1 every bit a sequence-specific transcription factor; it also suggested a general approach to the purification of transcription factors. The isolation of these proteins initially presented a formidable claiming considering they are present in very small quantities (e.thou., only 0.001% of total jail cell poly peptide) that are hard to purify by conventional biochemical techniques. This problem was overcome in the purification of Sp1 by Dna-affinity chromatography (Figure six.25). Multiple copies of oligonucleotides respective to the GC box sequence were bound to a solid back up, and prison cell extracts were passed through the oligonucleotide cavalcade. Because Sp1 bound to the GC box with loftier affinity, it was specifically retained on the cavalcade while other proteins were non. Highly purified Sp1 could thus be obtained and used for further studies, including partial decision of its amino acid sequence, which in plow led to cloning of the gene for Sp1.

Effigy 6.25

Purification of Sp1 by Dna-affinity chromatography. A double-stranded oligonucleotide containing repeated GC box sequences is bound to agarose beads, which are poured into a column. A mixture of cell proteins containing Sp1 is and so applied to the column; (more...)

The general approach of Dna-affinity chromatography, first optimized for the purification of Sp1, has been used successfully to isolate a wide multifariousness of sequence-specific DNA-binding proteins from eukaryotic cells. Protein purification has been followed by gene cloning and nucleotide sequencing, leading to the accumulation of a groovy deal of information on the structure and function of these critical regulatory proteins.

Structure and Function of Transcriptional Activators

Because transcription factors are central to the regulation of cistron expression, agreement the mechanisms of their action is a major area of ongoing research in cell and molecular biology. The most thoroughly studied of these proteins are transcriptional activators, which, like Sp1, bind to regulatory DNA sequences and stimulate transcription. In general, these factors have been found to consist of 2 domains: Ane region of the protein specifically binds DNA; the other activates transcription by interacting with other components of the transcriptional machinery (Figure 6.26). Transcriptional activators appear to be modular proteins, in the sense that the Dna binding and activation domains of different factors can often be interchanged using recombinant Deoxyribonucleic acid techniques. Such manipulations result in hybrid transcription factors, which actuate transcription by binding to promoter or enhancer sequences determined past the specificity of their DNA-binding domains. It therefore appears that the basic part of the Dna-bounden domain is to anchor the transcription factor to the proper site on Dna; the activation domain so independently stimulates transcription by interacting with other proteins.

Figure 6.26

Construction of transcriptional activators. Transcriptional activators consist of two independent domains. The Deoxyribonucleic acid-binding domain recognizes a specific Dna sequence, and the activation domain interacts with other components of the transcriptional machinery. (more...)

Many different transcription factors accept now been identified in eukaryotic cells, as might exist expected, given the intricacies of tissue-specific and inducible gene expression in complex multicellular organisms. Molecular characterization has revealed that the Dna-binding domains of many of these proteins are related to one another (Figure 6.27). Zinc finger domains contain repeats of cysteine and histidine residues that demark zinc ions and fold into looped structures ("fingers") that bind Dna. These domains were initially identified in the polymerase Three transcription cistron TFIIIA but are besides common among transcription factors that regulate polymerase Ii promoters, including Sp1. Other examples of transcription factors that contain zinc finger domains are the steroid hormone receptors, which regulate cistron transcription in response to hormones such every bit estrogen and testosterone.

Figure 6.27

Families of DNA-bounden domains. (A) Zinc finger domains consist of loops in which an α helix and a β sheet coordinately bind a zinc ion. (B) Helix-turn-helix domains consist of iii (or in some cases four) helical regions. 1 helix (more...)

The helix-turn-helix motif was first recognized in prokaryotic DNA-bounden proteins, including the Due east. coli catabolite activator poly peptide (CAP). In these proteins, one helix makes virtually of the contacts with DNA, while the other helices lie across the complex to stabilize the interaction. In eukaryotic cells, helix-plow-helix proteins include the homeodomain proteins, which play critical roles in the regulation of factor expression during embryonic evolution. The genes encoding these proteins were offset discovered as developmental mutants in Drosophila. Some of the primeval recognized Drosophila mutants (termed homeotic mutants in 1894) resulted in the evolution of flies in which one body function was transformed into another. For example, in the homeotic mutant called Antennapedia, legs rather than antennae abound out of the head of the fly (Effigy half dozen.28). Genetic assay of these mutants, pioneered past Ed Lewis in the 1940s, has shown that Drosophila contains nine homeotic genes, each of which specifies the identity of a different trunk segment. Molecular cloning and analysis of these genes then indicated that they contain conserved sequences of 180 base of operations pairs (called homeoboxes) that encode the DNA-bounden domains (homeodomains) of transcription factors. A wide variety of additional homeodomain proteins accept since been identified in fungi, plants, and other animals, including humans. Vertebrate homeobox genes are strikingly like to their Drosophila counterparts in both construction and office, demonstrating the highly conserved roles of these transcription factors in animate being development.

Figure 6.28

The Antennapedia mutation. Antennapedia mutant flies take legs growing out of their heads in identify of antennae. (A) Head of a normal wing. (B) Head of an Antennapedia mutant. (Courtesy of F. Rudolf Turner, Indiana University.)

Two other families of Deoxyribonucleic acid-bounden proteins, leucine zipper and helix-loop-helix proteins, contain DNA-bounden domains formed by dimerization of two polypeptide chains. The leucine zipper contains iv or v leucine residues spaced at intervals of seven amino acids, resulting in their hydrophobic side bondage being exposed at one side of a helical region. This region serves as the dimerization domain for the two protein subunits, which are held together by hydrophobic interactions between the leucine side bondage. Immediately following the leucine attachment is a region rich in positively charged amino acids (lysine and arginine) that binds Dna. The helix-loop-helix proteins are similar in structure, except that their dimerization domains are each formed by two helical regions separated past a loop. An of import feature of both leucine zipper and helix-loop-helix transcription factors is that different members of these families tin dimerize with each other. Thus, the combination of distinct poly peptide subunits can form an expanded array of factors that can differ both in DNA sequence recognition and in transcription-stimulating activities. Both leucine attachment and helix-loop-helix proteins play important roles in regulating tissue-specific and inducible gene expression, and the germination of dimers between different members of these families is a disquisitional attribute of the control of their function.

The activation domains of transcription factors are not as well characterized as their DNA-bounden domains. Some, called acidic activation domains, are rich in negatively charged residues (aspartate and glutamate); others are rich in proline or glutamine residues. These activation domains are idea to stimulate transcription past interacting with general transcription factors, such as TFIIB or TFIID, thereby facilitating the associates of a transcription circuitous on the promoter. For example, the activation domains of several transcription factors (including Sp1) have been shown to interact with TFIID by binding to TBP-associated factors (TAFs) (Figure vi.29). An important feature of these interactions is that different activators can bind to different general transcription factors or TAFs, providing a machinery by which the combined action of multiple factors tin can synergistically stimulate transcription—a primal feature of transcriptional regulation in eukaryotic cells.

Figure half-dozen.29

Synergistic activeness of transcriptional activators. Dissimilar transcriptional activators tin interact with the general transcription factor TFIID past binding to different TAFs.

Eukaryotic Repressors

Factor expression in eukaryotic cells is regulated by repressors likewise as past transcriptional activators. Like their prokaryotic counterparts, eukaryotic repressors demark to specific DNA sequences and inhibit transcription. In some cases, eukaryotic repressors simply interfere with the binding of other transcription factors to DNA (Figure 6.30A). For example, the binding of a repressor virtually the transcription start site tin can block the interaction of RNA polymerase or general transcription factors with the promoter, which is like to the activeness of repressors in leaner. Other repressors compete with activators for binding to specific regulatory sequences. Some such repressors comprise the same DNA-bounden domain as the activator but lack its activation domain. Every bit a result, their binding to a promoter or enhancer blocks the binding of the activator, thereby inhibiting transcription.

Effigy 6.30

Activeness of eukaryotic repressors. (A) Some repressors cake the binding of activators to regulatory sequences. (B) Other repressors have agile repression domains that inhibit transcription by interactions with full general transcription factors.

In contrast to repressors that simply interfere with activator binding, many repressors (called active repressors) contain specific functional domains that inhibit transcription via protein-protein interactions (Figure 6.30B). The offset such active repressor was described in 1990 during studies of a factor chosen Krüppel, which is involved in embryonic evolution in Drosophila. Molecular analysis of the Krüppel poly peptide demonstrated that it contains a discrete repression domain, which is linked to a zinc finger Deoxyribonucleic acid-binding domain. The Krüppel repression domain could be interchanged with distinct Deoxyribonucleic acid-binding domains of other transcription factors. These hybrid molecules as well repressed transcription, indicating that the Krüppel repression domain inhibits transcription via protein-poly peptide interactions, irrespective of its site of bounden to Dna.

Many active repressors take since been found to play cardinal roles in the regulation of transcription in animal cells, in many cases serving as critical regulators of jail cell growth and differentiation. Equally with transcriptional activators, several distinct types of repression domains have been identified. For example, the repression domain of Krüppel is rich in alanine residues, whereas other repression domains are rich in proline or acidic residues. The functional targets of repressors are as well diverse. Some repressors inhibit transcription by interacting with general transcription factors, such every bit TFIID; others are thought to collaborate with specific activator proteins.

The regulation of transcription past repressors also as by activators considerably extends the range of mechanisms that command the expression of eukaryotic genes. One important function of repressors may be to inhibit the expression of tissue-specific genes in inappropriate cell types. For instance, as noted before, a repressor-binding site in the immunoglobulin enhancer is thought to contribute to its tissue-specific expression past suppressing transcription in nonlymphoid cell types. Other repressors play central roles in the control of cell proliferation and differentiation in response to hormones and growth factors (see Chapters 13 and 14).

Relationship of Chromatin Structure to Transcription

In the preceding discussion, the transcription of eukaryotic genes was considered as if they were nowadays within the nucleus every bit naked Dna. Notwithstanding, this is not the case. The DNA of all eukaryotic cells is tightly bound to histones, forming chromatin. The basic structural unit of chromatin is the nucleosome, which consists of 146 base pairs of Deoxyribonucleic acid wrapped around two molecules each of histones H2A, H2B, H3, and H4, with i molecule of histone H1 bound to the DNA as it enters the nucleosome core particle (meet Effigy iv.9). The chromatin is and then farther condensed by being coiled into higher-order structures organized into big loops of DNA. This packaging of eukaryotic DNA in chromatin clearly has important consequences in terms of its availability as a template for transcription, and then chromatin structure is a critical aspect of cistron expression in eukaryotic cells. Indeed, both activators and repressors regulate transcription in eukaryotes not only by interacting with general transcription factors and other components of the transcriptional machinery, but too by inducing changes in the construction of chromatin.

The relationship between chromatin structure and transcription is evident at several levels. First, actively transcribed genes are constitute in decondensed chromatin, respective to the extended 10-nm chromatin fibers discussed in Affiliate iv (meet Effigy 4.10). For example, microscopic visualization of the polytene chromosomes of Drosophila indicates that regions of the genome that are actively engaged in RNA synthesis represent to decondensed chromosome regions (Effigy 6.31). Similarly, actively transcribed genes in vertebrate cells are present in a decondensed fraction of chromatin that is more than accessible to transcription factors than is the rest of the genome.

Figure vi.31

Decondensed chromosome regions in Drosophila. A light micrograph showing decondensed regions of polytene chromosomes (arrows), which are agile in RNA synthesis. (Courtesy of Joseph Gall, Carnegie Establish.)

Decondensation of chromatin, however, is not sufficient to brand the DNA an attainable template for transcription. Even in decondensed chromatin, actively transcribed genes remain bound to histones and packaged in nucleosomes, so transcription factors and RNA polymerase are withal faced with the trouble of interacting with chromatin rather than with naked Deoxyribonucleic acid. The tight winding of DNA around the nucleosome core particle is a major obstacle to transcription, affecting both the power of transcription factors to bind DNA and the power of RNA polymerase to transcribe through a chromatin template. This inhibitory upshot of nucleosomes is relieved by acetylation of histones and by the binding of two nonhistone chromosomal proteins (called HMG-14 and HMG-17) to nucleosomes of actively transcribed genes. (HMG stands for loftier-mobility group proteins; these proteins drift quickly during gel electrophoresis.) Additional proteins chosen nucleosome remodeling factors facilitate the binding of transcription factors to chromatin by altering nucleosome structure.

Acetylation of histones has been correlated with transcriptionally agile chromatin in a broad variety of cell types (Effigy 6.32). The cadre histones (H2A, H2B, H3 and H4) have two domains: a histone fold domain, which is involved in interactions with other histones and in wrapping Deoxyribonucleic acid effectually the nucleosome core particle, and an amino-concluding tail domain, which extends outside of the nucleosome. The amino-terminal tail domains are rich in lysine and tin can exist modified by acetylation at specific lysine residues. Acetylation reduces the net positive charge of the histones, and may weaken their bounden to DNA equally well every bit altering their interactions with other proteins. Importantly, recent experiments have provided direct evidence that histone acetylation facilitates the binding of transcription factors to nucleosomal Dna, indicating that histone acetylation increases the accessibility of chromatin to Dna-bounden proteins. In addition, direct links betwixt histone acetylation and transcriptional regulation have come up from experiments showing that transcriptional activators and repressors are associated with histone acetyltransferases and deacetylases, respectively. This association was first revealed past cloning a gene encoding a histone acetyltransferase from Tetrahymena. Unexpectedly, the sequence of this histone acetyltransferase revealed that information technology was closely related to a previously known yeast transcriptional coactivator called Gcn5p, which stimulates transcription in association with several different sequence-specific transcriptional activators. Further experiments revealed that Gcn5p itself has histone acetyltransferase activity, suggesting that transcriptional activation results directly from histone acetylation. These results have been extended past the finding that several mammalian transcriptional coactivators are also histone acetyltransferases, as is a general transcription cistron (TAFII250, a component of TFIID). Conversely, histone deacetylases (which remove the acetyl groups from histone tails) are associated with transcriptional repressors in both yeast and mammalian cells. Histone acetylation is thus regulated by both transcriptional activators and repressors, indicating that information technology plays a key role in eukaryotic cistron expression.

Figure six.32

Histone acetylation. (A) The core histones have histone-fold domains, which collaborate with other histones and with DNA in the nucleosome, and N-last tails, which extend exterior of the nucleosome. The N-terminal tails of the core histones (e.g., H3) (more...)

Nucleosome remodeling factors are poly peptide complexes that facilitate the binding of transcription factors by altering nucleosome structure (Figure half dozen.33). The mechanism of action of nucleosome remodeling factors is not nonetheless clear, just they appear to increment the accessibility of nucleosomal DNA to other proteins (such as transcription factors) without removing the histones. One possibility is that they catalyze the sliding of histone octamers along the Deoxyribonucleic acid molecule, thereby repositioning nucleosomes to facilitate transcription gene binding. The mechanisms by which nucleosome remodeling factors are targeted to actively transcribed genes also remain to be established, although some studies advise that they can be brought to enhancer or promoter sites in association with transcriptional activators or as components of the RNA polymerase II holoenzyme (encounter Effigy 6.14).

Figure 6.33

Nucleosome remodeling factors. Nucleosome remodeling factors facilitate the binding of transcription factors to chromatin by repositioning nucleosomes on the DNA.

Peradventure surprisingly, the packaging of Dna in nucleosomes does not present an impassable bulwark to transcriptional elongation by RNA polymerase, which is able to transcribe through a nucleosome core by disrupting histone-DNA contacts. The ability of RNA polymerase to transcribe chromatin templates is facilitated by acetylation of histones and past the association of the nonhistone chromosomal proteins HMG-14 and HMG-17 with the nucleosomes of actively transcribed genes. The binding sites of these proteins on nucleosomes overlap the binding site of histone H1, and HMG-14 and HMG-17 appear to stimulate transcription by altering the interaction of histone H1 with nucleosomes to maintain a decondensed chromatin structure that facilitates transcription through a nucleosome template. Every bit with nucleosome remodeling factors, the signals that target HMG-14 and HMG-17 to actively transcribed genes remain to be elucidated by time to come research.

DNA Methylation

The methylation of Deoxyribonucleic acid is another general mechanism by which control of transcription in vertebrates is linked to chromatin structure. Cytosine residues in vertebrate DNA can exist modified past the addition of methyl groups at the v-carbon position (Figure 6.34). Dna is methylated specifically at the C's that precede Chiliad'south in the Deoxyribonucleic acid chain (CpG dinucleotides). This methylation is correlated with reduced transcriptional activity of genes that contain high frequencies of CpG dinucleotides in the vicinity of their promoters. Methylation inhibits transcription of these genes via the action of a poly peptide, MeCP2, that specifically binds to methylated DNA and represses transcription. Interestingly, MeCP2 functions as a circuitous with histone deacetylase, linking DNA methylation to alterations in histone acetylation and nucleosome structure.

Figure 6.34

Dna methylation. A methyl group is added to the 5-carbon position of cytosine residues in DNA.

Although DNA methylation is capable of inhibiting transcription, its general significance in factor regulation is unclear. In many cases, methylation of inactive genes is thought to be a consequence, rather than the master cause, of their lack of transcriptional activity. Nonetheless, an of import regulatory function of Deoxyribonucleic acid methylation has been established in the phenomenon known every bit genomic imprinting, which controls the expression of some genes involved in the development of mammalian embryos. In most cases, both the paternal and maternal alleles of a gene are expressed in diploid cells. Withal, at that place are a few imprinted genes (over two dozen have been described in mice and humans) whose expression depends on whether they are inherited from the mother or from the begetter. In some cases, only the paternal allele of an imprinted gene is expressed, and the maternal allele is transcriptionally inactive. For other imprinted genes, the maternal allele is expressed and the paternal allele is inactive.

Although the biological role of genomic imprinting is uncertain, DNA methylation appears to distinguish betwixt the paternal and maternal alleles of imprinted genes. A good case is the gene H19, which is transcribed but from the maternal copy (Figure half dozen.35). The H19 gene is specifically methylated during the development of male, but not female, germ cells. The matrimony of sperm and egg at fertilization therefore yields an embryo containing a methylated paternal allele and an unmethylated maternal allele of the cistron. These differences in methylation are maintained following Dna replication by an enzyme that specifically methylates CpG sequences of a daughter strand that is hydrogen-bonded to a methylated parental strand (Effigy half-dozen.36). The paternal H19 allele therefore remains methylated, and transcriptionally inactive, in embryonic cells and somatic tissues. However, the paternal H19 allele becomes demethylated in the germ line, allowing a new design of methylation to be established for transmittal to the next generation.

Figure vi.35

Genomic imprinting. The H19 factor is specifically methylated during evolution of male germ cells. Therefore, sperm comprise a methylated H19 allele and eggs incorporate an unmethylated allele. Following fertilization, the methylated paternal allele remains (more...)

Effigy 6.36

Maintenance of methylation patterns. In parental Deoxyribonucleic acid, both strands are methylated at complementary CpG sequences. Following replication, just the parental strand of each girl molecule is methylated. The newly synthesized daughter strands are then (more...)

Box

Primal Experiment: Isolation of a Eukaryotic Transcription Gene.

Box

Molecular Medicine: The Pit-1 Transcription Factor and Growth Hormone Deficiency.