Although genetic information can flow from parent to child, or from parental cells to progeny cells, via the sequence of nucleotides in DNA, it can also flow via mechanisms that are independent of DNA sequence and yet stably inherited. This form of inheritance is referred to as epigenetic regulation of gene expression. We have already introduced one form of epigenetic gene regulation, which is the separation of chromatin into transcriptionally active euchromatin and transcriptionally inactive heterochromatin (see p. 76). Although shifting between euchromatin and heterochromatin does not change the DNA sequence, the progeny cells maintain this distinction. Thus, the formation of heterochromatin and euchromatin is an example of epigenetic gene regulation.
Epigenetic regulation can result in long-term gene silencing
Epigenetic regulation can have a long-term influence on gene expression. The following are three examples of long-term regulation of gene expression:
1. X-inactivation. Females carry two X chromosomes (see p. 1074), whereas males carry only one copy. To express X-linked genes at the same levels as males, females during development permanently inactivate one of the X chromosomes by globally converting one X chromosome from euchromatin to heterochromatin.
2. Imprinting. Cells contain two copies of every autosomal gene, one inherited from each parent, and usually express each copy identically. In a few cases, however, genes are differentially expressed, depending on whether they are inherited from the mother or the father. This phenomenon is called genomic imprinting. N4-13 For example, the insulin-like growth factor 2 gene (IGF2; see pp. 996–997) is maternally imprinted—only the copy inherited from the father is expressed; the maternal copy is silenced (i.e., not expressed).
Contributed by Emile Boulpaep, Walter Boron, Sam Mesiano
Genomic imprinting is the process by which certain genes—about 1% of the genome—are silenced; particular genes are silenced only in female gametes, and others, only in male gametes. Thus, these genes are expressed in a manner specific to the parent of origin.
Note that a female diploid oogonium has some paternal genes imprinted or silenced (i.e., only the maternal gene is active) and some maternal genes silenced (i.e., only the paternal gene is active). When the 2N genome splits into two 1N genomes, it is important that all genes in the oocyte have the female pattern of imprinting, which occurs during oocyte maturation.
Failure of proper genomic imprinting causes aberrant gene expression and is associated with several human diseases, including Beckwith-Wiedemann, Prader-Willi, and Angelman syndromes. For example, the IGF2 gene is normally maternally imprinted (i.e., silenced). In Beckwith-Wiedemann syndrome, the maternal IGF2 gene becomes reactivated (by removal of methyl tags) during oocyte formation in the mother or early embryonic development. The result is that the offspring has two (rather than one) active copies of IGF2 and thus excess IGF-2 protein. The most obvious sign is macrosomia (large body size) in the newborn.
Wikipedia. s.v. Genomic imprinting. http://en.wikipedia.org/wiki/Genomic_imprinting [Accessed March 20, 2015].
3. Tissue-specific gene silencing. Many tissue-specific genes are globally inactivated during embryonic development, later to be reactivated only in particular tissues. For example, globin genes are silenced except in erythroid cells. The silencing of genes in nonexpressing tissues is associated with chromatin modifications that are similar to those found in heterochromatin.
X-inactivation, imprinting, and tissue-specific silencing require long-term inactivation of gene expression and the maintenance of this inactivation during DNA replication and cell division. For example, the inactivated X chromosome remains inactivated in the two progeny cells after mitosis. Similarly, genes silenced by imprinting or by tissue-specific silencing remain inactive in progeny cells.
Alterations in chromatin structure may mediate epigenetic regulation, stimulating or inhibiting gene transcription
DNA in the nucleus is organized into a higher-order structure called chromatin, which consists of DNA and associated histones (see p. 75–76). The building block of chromatin, the nucleosome, is composed of DNA wrapped twice around core histones to form a solenoid-like structure (see Fig. 4-3). Nucleosomes present a physical barrier to gene transcription by blocking access of transcription factors to their binding sites in DNA and by interfering with elongation of nascent RNA transcripts. In general, a high density of tightly packed nucleosomes, or a nucleosome positioned at the transcription start site, is associated with gene inactivation. Conversely, repositioning or removal of nucleosomes, creating a nucleosome-free region at the promoter, can lead to gene activation. ATP-dependent chromatin-remodeling enzymes—belonging to the SWI/SNF, ISWI, IO80, and CHD families—can alter the density and location of nucleosomes. Because chromatin remodeling does not change the underlying DNA sequence, it represents a form of epigenetic gene regulation.
Covalent modifications of histones or DNA can also produce alterations in chromatin structure that mediate epigenetic gene regulation. These chemical modifications are sometimes referred to as epigenetic marks. We have already introduced one type of epigenetic mark, histone acetylation (see p. 84). The N termini of core histone proteins contain many lysine residues that impart a highly positive charge. These positively charged domains can bind tightly to the negatively charged DNA via electrostatic interactions (Fig. 4-17). Tight binding between DNA and histones is associated with gene inactivity. However, if the ε-amino groups of lysine side chains are chemically modified by acetylation, the positive charge is neutralized and the interaction with DNA is relaxed, which facilitates the binding of transcription factors and the basal transcriptional machinery. We have already seen that many transcriptional coactivators have intrinsic histone acetylase activity, whereas transcriptional corepressors may function as histone deacetylases (see pp. 85–86). In general, euchromatin is characterized by high levels of histone acetylation, whereas heterochromatin is characterized by low levels of acetylation.
FIGURE 4-17 Effect of histone acetylation on the interaction between histone proteins and DNA. When the histone octamer is deacetylated (top), positively charged lysine groups on the histone strongly attract a strand of DNA. When the histone octamer becomes acetylated (bottom), the acetyl groups neutralize the positive charge on the histone and allow the DNA strand to loosen.
Histone methylation may stimulate or inhibit gene expression
In addition to acetylation, histones are subject to other post-translational modifications that influence epigenetic gene regulation. Especially important is methylation, in which a methyltransferase covalently attaches methyl groups to arginine residues and, most significantly, to specific lysine residues in the core histones. Histone methylation is associated with gene activation or repression, depending on the specific amino-acid residue that is modified. Methylation of histone H3 at Lys-4, Lys-36, and Lys-79 on histone H3 correlates with transcriptionally active chromatin. Demethylation of these H3 residues—but methylation of Lys-9 and Lys-27 on histone H3—correlates with transcriptionally inactive chromatin (i.e., repression). This pattern of differential methylation in transcriptionally active and inactive chromatin is referred to as a histone code, inasmuch as it can be inherited just as the genetic code in DNA.
Methylation of histones affects gene expression by recruiting proteins that alter chromatin structure. For example, trimethylation of Lys-4 on histone H3, which is often found at active gene promoters, is specifically recognized by a subunit of the nucleosome-remodeling factor (NURF) chromatin-remodeling complex. Recruitment of the NURF complex results in the displacement of nucleosomes in the region and promotes gene transcription. Conversely, trimethylation of histone H3 at Lys-9 (H3-K9) is found in heterochromatin and is also characteristic of gene silencing by X-inactivation, imprinting, and tissue-specific silencing. Trimethylated H3-K9 recruits heterochromatin protein 1 (HP1), which then self-dimerizes to produce higher-order structures (Fig. 4-18A). In addition, HP1 recruits HDAC (see pp. 85–86), which promotes nucleosome assembly. Together, these modifications produce a closed chromatin conformation. Cells maintain this H3-K9 methylation during division, possibly by using the HP1. After it binds to methylated histones, HP1 recruits a histone methyltransferase (HMT) that methylates other K9 residues on other H3 histones (see Fig. 4-18B); this provides a mechanism for propagation of histone methylation. During DNA replication, the HMT recruited to a silenced gene on a parental strand of chromatin adds methyl groups to histones on the daughter strands, which maintains gene silencing in the progeny.
FIGURE 4-18 Gene silencing by chromatin modification. A, HP1 binds to methylated Lys-9 in histone H3. Because the HP1 self-dimerizes, the result is chromatin condensation. B, HP1 recruits an HMT that promotes further lysine methylation; this leads to recruitment of additional HP1, propagating histone methylation. C, MBD1 binds to methylated cytosine groups in DNA and can also recruit HMT.
DNA methylation is associated with gene inactivation
Methylation of cytosine residues at its 5 position is the only well-documented postsynthetic modification of DNA in higher eukaryotes. Approximately 5% of cytosine residues are 5-methylcytosines in mammalian DNA. Methylation usually occurs on a cytosine residue that is immediately upstream from a guanosine (i.e., a CpG dinucleotide on one DNA strand).
Several lines of evidence implicate DNA methylation in the control of gene expression.
1. Although CpG dinucleotides are relatively underrepresented in mammalian genomes, they are frequently clustered near the 5′ ends of genes, forming so-called CpG islands. Moreover, methylation of cytosines in these locations is associated with inhibition of gene expression. For example, the inactivated X chromosome in females contains heavily methylated genes.
2. Methylation/demethylation may explain tissue-specific and stage-dependent gene expression. For example, globin genes are methylated in nonexpressing tissues but hypomethylated in erythroid cells. During fetal development, fetal globin genes are demethylated and then become methylated in the adult.
3. Foreign genes that are introduced into cells are transcriptionally inactive if they are methylated but active if demethylated at the 5′ end.
4. Chemical demethylating agents, such as 5-azacytidine, can activate previously inactive genes.
How does DNA methylation cause gene inactivation? One simple mechanism is that methylation inhibits the binding of an essential transcriptional activator. For example, methylation of CpG dinucleotides within the GFAP (glial fibrillary acidic protein) promoter prevents STAT3 binding. A more common mechanism is that methylation produces binding sites for proteins that promote gene inactivation. Cells contain a protein called MeCP2 that binds specifically to methylated CpG dinucleotides as well as to the HDAC (see pp. 85–86). Thus, DNA methylation may silence genes by promoting histone deacetylation. In addition, methylated DNA binds to methyl-CpG–binding protein 1 (MBD1), a protein that complexes with HMT (see Fig. 4-18C). These last two interactions provide mechanisms coupling DNA methylation to histone modifications that promote heterochromatin formation and gene silencing.
The cell maintains patterns of DNA methylation during division. To illustrate, imagine that the DNA at a particular locus is initially methylated on both strands. After replication, the two double strands of daughter DNA are each methylated on only one of the two strands. This hemimethylated DNA recruits proteins that in turn recruit DNA methyltransferase 1 (DNMT1) to copy the methylation pattern onto the newly synthesized DNA strand. This mechanism ensures that epigenetic marks in a parent cell are maintained in the progeny cells.