1.1.1

Nucleic Acids

The molecular definition of a eukaryotic gene is complex, but in the simplest terms, it is a nucleic acid sequence that encodes one polypeptide and one messenger ribonucleic acid molecule (mRNA). Genes are comprised of “two intertwining polymers” of deoxyribonucleic acids (DNAs) that are noncovalently attached to a variety of proteins, including histones and specialized proteins (e.g., polymerases and various accessory proteins). The association of DNA, histones, and specialized nuclear proteins collectively is called chromatin. Chromosomes are comprised of continuous strands of chromatin that have been compacted by supercoiling and looping so as to fit into the nucleus ( Fig.1.1 ). The steps governing the compacting and location of chromatin are now an area of intense investigation and will be discussed in Section 1.2 . Chromosomes are the basic heritable unit in the mammalian cell. In humans, there are 46 individual chromosomes or 23 chromosome pairs. The smallest unit of the DNA polymer is a nucleotide—a base attached to the first carbon of a five-carbon sugar phosphorylated at its fifth carbon ( Fig. 1.2 ). Nucleosides do not contain phosphates linked to the pentose sugar, thus differing from nucleotides, which contain one, two, or three phosphate groups. The type of base distinguishes the 4 nt found in DNA: adenine (A), thymine (T), cytosine (C), or guanine (G). They are bases because of the nitrogen groups contained within their single-ring (thymine, cytosine, or uracil) or double-ring structures (adenine or guanine). DNA contains the sugar deoxy ribose, whereas RNA contains the sugar ribose and the base uracil (U) instead of thymine. CpG islands are dinucleotides consisting of a deoxycytidine in the 5′ position adjacent to deoxyguanosine. These dinucleotides are “hot spots” for enzymes (e.g., DNMTs = DNA methyl transferases), which add a methyl group to the 5th carbon of the cytosine ring. The “p” indicates that one phosphate group separates these two nucleosides. This epigenetic mark blocks the expression of DNA and is a mechanism used frequently by gastrointestinal (GI) cancers to silence genes that block their ability to proliferate.

Chromatin structure and organization. Each chromosome exists in a haploid (germ cells) or diploid/tetraploid state depending on their stage in the cell cycle. The short arm of the chromosome relative to the centromere is the “p” arm and the long arm is the “q” arm. Chromosomes represent compressed, compacted DNA double strand helix wrapped around core histones.

(From the Language of medicine. 4th ed.)

Nucleic acid structure. A nucleoside consists of a purine or pyrimidine base covalently linked to the firs carbon of the pentose ring. The addition of one, two, or three phosphate groups is a nucleotide mono-, di-, or triphosphate. The type of sugar determines the type of nucleic acid: ribose in ribonucleic acids (RNAs) and deoxyribose in deoxyribonucleic acids (DNAs).

(Reprinted from Physiology of the gastrointestinal tract. 4th ed. 2006.)

1.1.2

Nucleic Acid Polymers: DNA, RNA

Polymers of nucleotides or nucleic acids (also called nucleoside mono-, di-, or triphosphates) are formed when the free phosphate group attached to the fifth carbon of an adjacent nucleotide of the pentose sugar condenses with the hydroxyl group on the third pentose carbon to produce two ester bonds and water (phosphodiester bond). Accordingly, the proximal end of each DNA strand (5′ end) contains a phosphate group at the fifth carbon of the deoxyribose sugar residue. The terminal nucleic acid at the 3′ end of each DNA strand contains a free hydroxyl group at the third carbon of the deoxyribose ring. By convention, nucleotide sequences are written from 5′ to 3′ reading from left to right with the sense strand presented as the upper strand. The antisense strand, written on the bottom, is antiparallel and complementary to the sense strand so that the 5′ to 3′ direction proceeds from right to left. Each nucleotide within the polymer is base paired with a particular nucleotide on the opposing strand by hydrogen bonds: adenine with thymine and guanine with cytosine. The DNA strand containing the same sequence as the messenger RNA (mRNA) is designated the sense strand , and the strand that it pairs with is designated the antisense strand . The antisense strand becomes the template sequence that will be transcribed by RNA polymerase II (Pol II) into mRNA and subsequently translated into amino acids.

Most of the studies on transcriptional control focus on genes transcribed by the 12-subunit enzyme Pol II and thus are designated as class II genes. It is Pol II that is responsible for transcribing gene sequences into protein-encoding messenger RNA (mRNA). Less than 2% of total RNA in the cell is mRNA. Many of these initial primary transcripts (hnRNA for heterogeneous nuclear RNA) are further processed as discussed below. Therefore, 98% of the nucleotides in the human genome do not reside in exons (sequences that encode proteins). Nevertheless, at least 50% of the noncoding RNA is transcribed and serves a function. Nine percent of cellular RNA is hnRNA, the bulk of which are small nuclear RNAs (snRNA, e.g., U2 involved in RNA splicing, 4%) and small nucleolar RNAs, for example, U22 snoRNA, comprising 1%. The other 4% of hnRNA is mRNA. An additional 1% of total cell RNA is microRNA (miRNA), previously called guide RNA (gRNA), which edits mature mRNA transcripts. RNA polymerase I (Pol I) transcribes all of the ribosomal genes except for the 5S gene. Ribosomal RNA represents about 75% of the RNA in the cell and is therefore essential for translation. RNA polymerase III (Pol III) transcribes the 5S ribosomal gene and the genes-encoding transfer RNA (tRNA). Transfer RNA represents about 15% of the total RNA in the cell. Pol I and III transcribe genes that will not be further translated into peptides and noncoding RNA transcripts, although their primary transcripts are also processed before reaching the cytoplasm. Since Pol II transcribes genes-encoding proteins, peptides, long noncoding RNA (lncRNA), and miRNAs, Pol II-regulated genes will be the primary focus of this chapter.

1.1.3

Gene Composition

A gene is analogous to a long sentence read from left to right and comprised of letters organized into words separated by spaces and punctuations. Specific DNA sequences “punctuate” the gene with important start and stop signals for transcription and translation. Several hundred to several thousand DNA base pairs (bp) may comprise one gene. These bp (the alphabet) are organized into functional groups (phrases) on the basis of whether a particular sequence is untranscribed, only transcribed (RNA), or both transcribed and translated (RNA and protein) ( Fig. 1.3 ). Exons are DNA sequences that are transcribed into mRNA by Pol II and exit the nucleus. Within the cytoplasm, exons may or may not be translated into peptides. Those exons that are transcribed and translated form the coding sequences (coding exon). In general, the term intron is used to describe the intervening DNA sequence that is transcribed but is subsequently removed from the primary transcript by RNA splicing (RNA processing) before exiting the nucleus as a mature transcript. However, it is now clear that many transcribed DNA sequences generate small noncoding RNA transcripts such as miRNAs or lncRNAs that can inhibit or modulate protein-coding genes in “cis or trans.” LncRNAs are commonly defined as transcripts that are > 200 nt that do not encode a protein compared to the significantly shorter miRNAs.

Gene structure, transcription, and posttranscriptional processing. A gene is comprised of several hundred to several thousand bp, subdivided into functional elements. The locations of 5′ and 3′ untranslated sequences, exons, and introns are shown. The 5′ flanking sequences contain specific DNA elements (e.g., TATA box). RNA polymerase II transcribes DNA into heterogeneous nuclear RNA (hnRNA) during transcription. Twenty bp after the sequence AATAAA is transcribed to AAUAAA, mRNA is cleaved and the polyadenylate (poly(A)) tail is added to the 3′ end. A methylated guanylate residue is added to the 5′ end of the mRNA through a triphosphate linkage. Prior to exiting the nucleus, intron segments are removed by splicing factors during posttranscriptional processing.

(Reprinted from Physiology of the gastrointestinal tract. 4th ed. 2006.)

DNA sequences or elements that regulate transcription and are not transcribed into mRNA usually reside in the 5′ portion of a gene upstream (to the left) of the promoter. The promoter is a cluster of DNA sequences that binds Pol II in concert with accessory proteins to initiate the synthesis of mRNA. Accessory proteins control the accuracy and rate of polymerase binding. The first nucleotide transcribed into mRNA is assigned the number 1 with subsequent nucleotides (downstream or to the right of the promoter) assigned positive numbers as transcription proceeds toward the 3′ end. Nucleotides preceding the promoter (upstream or 5′) are assigned negative numbers. DNA sequences that encode a polypeptide (open reading frame) begin with the translational start site codon ATG (encoding methionine) and end with one of the three stop codons: TAA, TAG, and TGA. Thus, the translational start and three stop codons, respectively, are transcribed into mRNA as AUG, UAA, UAG, and UGA. Since there are four different DNA bases and it takes only three bases (a triplet) to encode an amino acid. There are 4 ³ = 64 possible codons for 20 amino acids. In this way, the nucleotide code for proteins is considered “degenerate.” The redundant genetic code protects against the deleterious effects of mutations as detailed in the next paragraph. In addition, two or three peptides can be encoded by overlapping codons simply by shifting the reading frame by 1 or 2 nt. Regulatory sequences that are transcribed but not translated reside at both the 5′ and 3′ ends of the mature RNA transcript. Both 5′ and 3′ untranslated regulatory sequences, which range from 10 to several thousand nucleotides, participate in the fidelity of translation and mRNA stabilization or destabilization.

The degeneracy of the genetic code (several codon triplets encoding one amino acid) is what makes some bp changes (mutations) within an exon exhibit no deleterious phenotype. The bp change is designated synonomous if the same amino acid is substituted (also known as a silent mutation) or nonsynonomous if a different amino acid is substituted. Strictly speaking, mutations mean that there has been a bp change whether or not the change affects the type of amino acid inserted into a peptide. Despite a nonsynonomous mutation in the coding sequence, the amino acid substitution might not exhibit a change in the physical characteristics (phenotype) of the organism nor render phenotypic advantages or disadvantages to the organism. Changes in the genetic code that put an organism at a disadvantage and contribute to disease are what we commonly call “mutations.” BP changes in DNA that are neutral or impart a positive or negative advantage to the organism are also known as single nucleotide polymorphisms (SNPs). These SNPs can render subtle differences in the way an organism responds to its environment or other genetic influences ( Fig. 1.4 ). SNPs are a focus of intense investigation due to their use in genome-wide scans to identify genes contributing to common multigene disorders, for example, diabetes, hypertension, etc.

Single nucleotide polymorphism (SNP). Schematic diagram of a SNP in which a protein encoding gene sequence differs between two individuals by one nucleotide.

1.1.4

RNA Species

RNA molecules that encode proteins (except most histone proteins) are distinguished from ribosomal and transfer RNA by the series of adenosines added to the 3′ end of the molecule commonly referred to as the poly(A) RNA tail ( Fig. 1.3 ). This feature is a useful means to isolate mRNA from more abundant RNA species (transfer and ribosomal RNA) and also designates the functional termination of the protein-encoding portion of the gene. During transcription, the primary RNA transcript is cleaved 20 bp downstream of the AAUAAA site at the 3′ end, and ~ 150–200 adenine nucleotides are added to form the poly(A) tail. The 5′ end of the mRNA transcript receives a protective “cap” after synthesis of the first 30 nt that consists of a guanylate residue methylated at the seventh position and linked to the first nucleotide of RNA by three phosphates. The RNA cap is a high-affinity binding site for ribosomes. It should be noted that the element AATAA indicates the site of the poly A tail, but is not necessarily the functional end of the gene. Rather, the 3′ untranslated region (3′UTR) and 3′ untranscribed regions may still contain regulatory elements that modulate gene expression. In fact, most mRNAs bind sequences in the 3′UTR. Therefore, like the 5′ end of a gene, the 3′ end of the gene must be determined empirically.

Two classes of noncoding RNAs transcribed by Pol II have motivated the current expanded interest in RNA biology—mRNAs and long noncoding RNAs. mRNAs (miRNAs) are a class of noncoding RNAs generated primarily from DNA sequences between genes (intergenic) within introns or at the 3′ end of the gene. They were originally identified in plants and worms as posttranscriptional regulators of gene silencing. Pol II and sometimes Pol III transcribe DNA to produce primary miRNA transcripts. In addition, transcription factors modulate the expression of these mRNAs as for protein-encoding genes. For instance, extracellular signaling via typical signal transduction pathways and epigenetic mechanisms regulate the expression of mRNAs. The gene product is RNA rather than protein and exerts its effect on its own locus as well as multiple loci due to their small size and less stringent binding requirements. In this way, miRNAs are thought to regulate at least one-third of all human genes.

miRNAs are synthesized in the nucleus as a primary transcript (pri-miRNA) capable of forming several hairpin structures through internal complementarity ( Fig. 1.5 ). The microprocessing complex containing a nuclear RNase III endonuclease called Drosha and the DiGeorge syndrome critical region 8 protein (DGCR8) cleaves the pri-miRNA transcript. The Drosha protein complex removes flanking segments and an ~ 11 bp stem region. This step converts the pri-miRNA to precursor miRNAs (pre-miRNAs). Pre-miRNAs are typically 60–70-nt long hairpin RNAs with 2-nt overhangs at the 3′ end. The nuclear export receptor exportin-5 and RanGTP transport the pre-miRNA into the cytoplasm where it is further processed by a complex containing another RNase III endonuclease called Dicer. Dicer partners with RNA-binding proteins to cleave the pre-miRNA into 21–25 nt duplexes. The miRNA/miRNA* duplex consists of a guide RNA strand and a passenger strand indicated by an asterisk (miRNA*) that is discarded upon assembly of the R NA- i nduced s ilencing c omplex (RISC). Loading the miRNA/miRNA* duplex into RISC is a four-step process requiring ATP hydrolysis and the major RISC protein component called Argonaute (Ago proteins). Upon unwinding of the duplex, the miRNA* strand is discarded leaving a single strand 21–25 nt RNA molecule available for silencing specific clusters of genes by hybridizing to their 3′UTRs. Ago protein coat miRNAs and along with exosomes protect miRNAs from degradation in biofluids such as blood and urine rendering them potential biomarkers.

Synthesis of microRNAs (miRNA). miRNAs are synthesized from the primary miRNA (pri-miRNA), which are then edited to the pre-miRNA. The RAN-GTP/Exportin 5 complex transports the Pre-RNA to the nucleus where the pre-miRNA is further processed to the miRNA/miRNA* duplex. *miRNA indicates the passenger strand that is discarded upon assembly of the R NA- i nduced s ilencing c omplex (RISC). The Argonaute (Ago) protein are the major protein component of the RISC. TRBP = TAR RNA-binding protein (aka PACT).

(Reproduced from Kwak PB, Iwasaki S, Tomari Y. The microRNA pathway and cancer. Cancer Sci 2010; 101 (11):2309–15. doi: 10.1111/j.1349-7006.2010.01683.x)

Long noncoding RNAs are nucleic acids that do not encode a protein and are at least 200-nt long or greater. They are distinguished from miRNAs by their size (lncRNA > 200 nt versus miRNAs ~ 22 nt) and the ability to exhibit more diverse functions. miRNAs typically suppress multiple gene targets, whereas lncRNAs typically regulate the gene from which they are transcribed, albeit by multiple mechanisms. The advent of whole genome sequencing has identified more noncoding transcripts than coding complicating our ability to define their function. lncRNAs can function in “cis” or “trans,” can circularize or remain linear. Moreover, lncRNAs can function as protein scaffolds by recruiting regulatory complexes to genes, or behave as decoys, signaling molecules or as antisense interference transcripts. Therefore, through these diverse behaviors, lncRNAs exhibit pleomorphic functions such as genomic imprinting, chromosome shaping, and allosterically enzyme regulation. The function of most lncRNAs is unknown and thus the transcripts have simply been named numerically. Those lncRNAs that have been assigned a function include XIST (X chromosome inactivation), HOTAIR (Hox transcript antisense RNA), and TERC (telomerase elongation).

1.1.5

Linking Gene Structure to Function

Previously the 5′ border of a gene was identified by the promoter region (functionally determined) and by the first nucleotide transcribed into mRNA (cap site) determined empirically by various reverse transcriptase methods—for example, primer extension analysis or anchored polymerase chain reaction (PCR and DNAse1 hypersensitivity sites). These techniques used reverse transcriptase to synthesize complementary or copy DNA (cDNA) ( Fig. 1.6 ). Radiolabeled primers complementary to the 5′ end of the DNA sequence to be copied were allowed to anneal to mRNA. Reverse transcriptase then adds deoxynucleotides to the primer in the 3′ to 5′ direction. Synthesis of the cDNA terminates when the 5′ end of the mRNA is reached. Template mRNA molecules were removed by ribonucleases (RNases), and the synthesis of a double-stranded cDNA was completed through the action of DNA polymerase. Because the newly synthesized cDNA was radiolabeled at the 5′ end, the length of the cDNA (and hence the transcriptional start site) was determined by resolving the fragments on a denaturing polyacrylamide gel and comparing the length observed in bp to the known cDNA sequence.

Complementary DNA (cDNA). Primers complementary to a portion of the mRNA are allowed to anneal. For unknown sequences, as in the synthesis of cDNA libraries, a primer complementary to the poly (A) tail is used, i.e., poly (dT). Reverse transcriptase added along with all four deoxynucleotides (dNTPs) will transcribe mRNA in the 3′ to 5′ direction to make copy DNA. The mRNA template is removed by RNases, and double-stranded cDNA is made using DNA polymerase. In primer extension analysis, the 5′ end of mRNA (the cap site) is identified by annealing primers of a known sequence near the 5′ end of mRNA.

(Reprinted from Physiology of the gastrointestinal tract. 4th ed. 2006.)

In the age of whole genome analysis, the characterization of gene function has lagged behind the generation of transcript mapping. In other words, the biochemical assays such as DNase-seq, ATAC-seq (assay for transposase- accessible chromatin), ChIP-seq, and 3C (chromatin conformation capture) genome-based methods do not provide an assessment of function. This has led to the development of high-throughput methods to identify changes in gene transcription levels (both coding and noncoding). These include RNA-seq and STARR-seq (self-transcribing active regulatory region sequencing. In addition, CRISPR/Cas9 methods of activating or silencing gene in situ have permitted the development of functional readouts for enhancer modification within its endogenous environment.

We now know that these additional DNA sequences might encode noncoding RNA that regulates gene expression in addition to the well-described enhancer sequences. Specific DNA elements called insulator elements mark the boundary of genes. These elements, originally identified on the globin gene, bind an 11-zinc finger transcription factor called CTCF, which is capable of blocking histone acetylation spreading between adjacent genes. More recently, it is now understood that gene expression occurs in insulated neighborhoods generated by chromosomal loops formed by the binding site for CTCF and the cohesion complex. Thus, enhancer or repressor sequences that are kilobases away from the transcriptional start site (TSS) can brought closer to the genes that they regulate by forming gene-enhancer/repressor “neighborhoods” called topologically associated domains (TADs). It has recently been shown that CTCF-binding site mutations that prevent the formation of TADs can cause disease.

Given the requirement for larger and larger pieces of DNA to recapitulate native expression in transgenic mouse models, techniques have been developed to clone and manipulate large pieces of DNA (over 50 kilobases), for example, yeast artificial chromosomes (YACs) and bacterial artificial chromosomes (BACs). Recombineering is a powerful technique performed in bacteria that permits the introduction of foreign DNA or point mutations into these large plasmids that are eventually introduced into transgenic mice, but has been superceded by a powerful new technology called CRISPR-Cas9.

CRISPR/Cas represents the latest and to date the most powerful breakthrough in our ability to modify or manipulate the genome with precision. The term CRISPR stands for clustered regularly interspaced short palindromic repeats and Cas is the abbreviation for CRISPR-associated protein. Cas9 is a nuclease that uses guide RNA to direct the enzyme to the specific DNA sequence to be modified by forming Watson-Crick base pairing. Thus, the technique is a simple, RNA-guided method by which bacteria and Archaea defend themselves from the DNA of invading bacteriophages (adaptive immune mechanism). In short, the technology originates from studying the bacterial immune system and consists of two parts: a DNA-binding domain that recognizes the sequence to be modified and an effector domain that mediates double-strand DNA breakage. These two steps activate the host cell’s sequence-specific endonucleases to repair the break by nonhomologous recombination resulting in modification of the targeted sequence. The specificity of the technology lies in the ability to program the guide RNA. Prior to CRISPR/Cas, zinc-finger nucleases (ZFNs) and transcription activator-like effector nucleases (TALENs) were the primary methods used to execute programmable genome editing.

1.2.1

DNA Methylation

DNA methylation is a postsynthesis modification that normal DNA undergoes after each replication. This modification is catalyzed by DNA methyltransferases (DNMTs) and occurs on the C-5 position of cytosine residues within CpG dinucleotides located primarily in the promoter of a gene. There are three major DNMTs (DMNT1, 3A, 3B). Each DNMT plays a distinct and critical role in cells. Murine knockouts of DNMT1 and DNMT3b exhibit embryonic lethality. The DNMT3a homozygous mouse appeared normal at birth but died by 4 weeks of age. In humans, mutations of DNMT3b are linked to ICF syndrome ( I mmunodeficiency, C entromere instability, F acial anomalies). DNMTI functions as the “maintenance” methyltransferase since it functions during cell division to methylate the newly synthesized DNA strand as dictated by the hemi-methylated complementary strand. DNMT3a plays a central role in the methylation of neural specific genes.

Sixty percent of human genes contain a CpG island. While methylation can also occur in other parts of the gene, CpG dinucleotides tend to be underrepresented in the genome and when they are found appear in clusters ranging from 0.5 to several kilobases with GC content greater than 55%. About 15% of CpG dinucleotides cluster in short DNA segments known as CpG islands. The remaining 85% of the islands are spread throughout the genome in repetitive hypermethylated segments that are transcriptionally silent. Methylation of “CpG islands” is a late evolutionary development and functions to maintain genome stability by repressing transposons and repetitive DNA elements.

DNA methylation is an important event in many processes, including transcriptional repression, X chromosome inactivation and genomic imprinting. CpG islands locate in the promoter region of genes about 60% of the time and are normally hypomethylated particularly in the germ cells. Collectively, these CpG clusters or islands cover only about 0.7% of the entire genome, which is still equivalent to several million nucleotides. Hypermethylation at CpG islands induces transcriptional silencing that in turn is stably inherited. Thus as cells differentiate, a significant percentage of these CpG islands become methylated in a tissue specific manner. Typically these would be genes involved in cell renewal. As observed with HDACs and deacetylation, the methylation status of cancers might seem contradictory. Yet, aberrant de novo hypermethylation of CpG islands is a hallmark of some human cancers and occurs early during carcinogenesis. Tumor suppressor genes are locally hypermethylated by some cancers to silence their expression; whereas, oncogenes might be hypomethylated.

The DNA of tumor cells is globally hypomethylated, a process that is linked to nutritional status, for example, B ₁₂ or cobalamin absorption. Cobalamin is required for the synthesis of S -adenosylmethionine, the primary methyl donor in the cell. In this way, reduced cobalamin absorption as sometimes observed in Crohn’s or pernicious anemia would provide an environment favorable to cancer. Niacin required to form NAD, which is necessary for ADP-ribosylation of histones, also affects chromatin structure.

The most precise approach to assessing DNA methylcytosines is through bisulfite sequencing. Treating DNA with sodium bisulfite converts unmethylated cytosines to uracil that when subjected to conventional DNA sequencing are read as thymines. Methylated cytosines are still read as cytosines. Although bisulfite sequencing is not as easy to scale up as a genome-wide analysis by methylation-sensitive restriction enzyme (MSRE) analysis, sequencing is the most accurate way to determine the methylated sites in DNA or the methylome.

Genomic imprinting occurs in gametogenesis and is necessary for development. One of the X chromosomes in females is not expressed due to the heavy methylation of the inactive X chromosome. The epigenetic phenomenon whereby expression of a gene depends on whether it is inherited from the mother or the father is called imprinting and is due to differential methylation of specific cytosine bases on the maternal versus the paternal genes. Recent genome-wide analysis of genomic imprinting in the mouse identified 1300 loci that exhibit parental bias in the expression of specific mRNA transcripts. The gene loci identified control neural systems associated with feeding and behavior. In addition, the authors in a separate article showed preferential selection of the X chromosome inherited from the mother as opposed to the one from the father in glutamatergic neurons of the female cortex. The interleukin-18 gene was identified as an important locus controlling sex-specific preferences.

1.2.2

Histone Modifications

The basic repeating unit of chromatin is the nucleosome. Each nucleosome is composed of 147 bps of DNA wrapped twice around a histone protein octamer consisting of two molecules of each of the four core histones (H2A, H2B, H3, and H4). The linker histone H1 sits alone between each core nucleosome facilitating further compaction. Each histone contains a structured globular domain with a histone-fold motif important for nucleosome assembly, and a highly charged unstructured amino-terminal tail of 25–40 residues, which protrudes from the body of the nucleosome to latch onto the phosphate backbone. The amino-termini are the major sites for histone modifications. Histones can be modified by acetylation, methylation, phosphorylation, ADP-ribosylation, ubiquitination, and sumoylation ( Table 1.1 ). The mixture of these covalent modifications create a “code” on the surface of the histone molecule that is subsequently recognized by a class of chromatin-binding proteins, for example, bromo- and chromodomain-containing proteins that mediate chromatin compaction, transcription, and DNA repair. Acetylation, methylation, ubiquitination, and sumoylation occur on the lysine residues while methylation also occurs on arginine residues. Phosphorylation occurs on serines and threonines, ADP-ribosylation on glutamic acids. Most of these modifications, particularly acetylation, alters the charge distribution on the amino-terminus and alters nucleosome structure, which can in turn regulate chromatin structure. Some covalent modifications act as molecular switches, enabling or disabling subsequent covalent modifications, which explains the functional complexity of epigenetic modifications. Each modification correlates with a specific physical status of chromatin. The next several sections will highlight the most common histone modifications.

1.2.3

Chromatin-Binding Proteins

The remaining histone methyltransferase families also recognize methyl groups on regulatory proteins other than histones and therefore are discussed here. The second group of SET domain methyltransferase proteins is related to the Drosophila protein Enhancer of Zeste, with the prototypical mammalian protein named Ezh2. Ezh2 is part of a complex of proteins called the Polycomb group (PcG). The two variations of these complexes have been designated Polycomb repression complexes 1 (PRC1) and 2 (PRC2). Ezh2 belongs to the PRC2 complex that also includes Eed, Suz12; whereas, PRC1 includes the Ring finger proteins (Ring1a,b, Rnf, Hpc, Edr, and Bmi1). Conditional deletion of Eed in the intestinal crypt resulted in crypt degeneration. Therefore, the PRC2 complex is required for normal stem cell maintenance. Ezh2 has recently garnered significant attention due to its overexpression and therefore oncogeneic function in several epithelial cancers including prostate and breast, compared to its tumor suppressor function in some hematopoietic cancers. Consistent with its oncogenic role in epithelial cancers, Ezh2 is also overexpressed in colon, gastric, and liver cancer. A genome-wide analysis of prostate cancers recently revealed an androgen- dependent fusion protein called TMPRSS2-ERG that in Chip-Seq analysis was found to transcriptionally target Ezh2. Bmi1 has received increased attention because it is an important marker of normal and cancerous hematopoeitic stem cells. Bmi1 is also associated with the + 4 reserve stem cell in the intestinal crypt zone. In addition, the PRC1 complex contains proteins that have E3 ubiquitin ligase activity. The Polycomb group of proteins with their SET domains not only participates in histone lysine methylation, but both PRC1 and 2 complexes are also important in recognizing the methylated protein imprint.

The human homolog of the Drosophila Trithorax (Trx) protein is the mixed leukemia gene (MLL1). There are four human MLL homologs. MLL1 has been shown to be a specific methyltransferase of H3 at K4. It in turn forms protein-protein interactions with coactivators, for example, CBP and corepressor chromatin remodelers, for example, SWI/SNF. Other Trithorax homologs, for example, Ash1, Trx, form complexes with different coregulatory complexes. Collectively, the Trithorax group (TrG) of proteins can either activate or repress transcription depending on the coregulator with which it associates. Nevertheless, the TrG proteins characteristically oppose the activity of the Polycomb group (PcG). The tumor suppressor protein menin (positionally cloned gene product of the MEN type 1 locus) interacts with MLL1 and normally induces the cyclin-dependent inhibitor p27 ^Kip.

RIZ (retinoblastoma protein-interacting zinc finger protein), SMYD3, and MDS-EVI1 form a fourth family of SET-domain proteins because they have two isoforms that exhibit opposing functions. The isoform containing the SET domain has tumor suppressor function while the isoform missing the SET domain is cancer promoting. This “ying-yang” theory put forth by Huang is especially true for RIZ and MDS-EVI1 in which the cancer by an unclear mechanism disturbs the normal ratio between the two isoforms. The SMYD3 protein contains another DNA-binding domain called MYND in addition to a SET domain and is overexpressed in colorectal and hepatocellular carcinomas.

Crosstalk between DNA methylation and the histone modifications exists. These interactions were revealed by the observation that HDAC1 forms a complex with DNMT1 and 5-methyl-cytosine-binding protein (MBP) on a methylated promoter to silence gene expression. Similar crosstalk occurs between HDACs, Suv39, and HP1; HDACs, PRC2, and PRC1; HATs, MLL1, and BRM. The enzymes that epigenetically modify the genome are categorized in terms of those that “erase” chromatin marks; add chromatin marks (“writers”) or “read” chromatin marks ( Table 1.2 ).

1.2.4

Epigenetics and Development

The epigenetic control of gene expression is a fundamental feature of mammalian development, as indicated by developmental arrest or abnormalities in methylation or acetylation-deficient mutants. X-chromosome inactivation is an example of sequence-identical alleles being stably maintained in different functional states. In humans, X-linked inactivation serves to normalize the level of expression of X-linked genes in females (XX) and males (XY). Mutations in genes that affect global epigenetic profiles can give rise to human diseases. For example, the Fragile X syndrome results when a CGG repeat in the FMR1 (fragile X mental retardation gene 1) 5′ regulatory region expands and becomes methylated de novo, causing the gene to be silenced and creating a visible “fragile” site on the X chromosome under certain conditions. On a more global level, mutations in the DNMT3b (which regulates DNA methylation) gene lead to ICF syndrome and CBP (with acetyltransferases activity) mutations cause RSTS (Rubinstein-Taybi syndrome). Discovered in 2004, lysine demethylases (LSDs) appear to play an essential role in stem cell pluripotency versus lineage specification.

1.2.5

Epigenetics and Cancer

Epigenetic changes play an important role in tumorigenesis. The major epigenetic changes that take place during cancer development are generally the aberrant DNA methylation of tumor suppressor genes and histones. Genomic methylation patterns are frequently altered in tumor cells, with global hypomethylation accompanying region-specific hypermethylation events. When hypermethylation events occur within the promoter of a tumor suppressor gene, this can silence expression of the associated gene and provide the cell with a growth advantage in a manner similar to deletions or mutations. Although cancer cells are hypomethylated in the genome compared to normal tissues, many tumor-suppressor genes are silenced in tumor cells due to hypermethylation. This aberrant methylation event occurs early in tumor development and increases progressively, eventually leading to the malignant phenotype. For example, a high percentage of patients with sporadic colorectal cancers with a microsatellite instability phenotype show methylation and silencing of the gene-encoding MLH1 (MutL protein homolog 1). Other methylated tumor suppressors loci include CDKN2A (p16 ^INK ) , p14ARF , Rb , E-cadherin , and BRCA1 . Deregulation of genomic imprinting can also play a role in cancer development, as exemplified by loss of IGF2 gene imprinting in Wilms’ tumor.

Chromatin remodeling also plays an important role during tumorigenesis. Loss or misdirection of HATs has been linked to embryonic aberrations in mice and to human cancers. Misdirection of HAT activities as a result of chromosomal translocations is associated with multiple human leukemias. In acute promyelocytic leukemia, the oncogenic fusion protein PML-RARα (promyelocytic leukemia-retinoic acid receptor-α) recruits an HDAC to repress genes essential for the differentiation of hematopoietic cells. Similarly, in acute myeloid leukemia, AML1-ETO fusions recruit the repressive N-CoR-Sin3-HDAC1 complex that in turn inhibits normal myeloid development.

More recently, noncoding RNAs transcribed from intervening (intronic) sequences have been linked to epigenetic changes cell cycle regulation, immune surveillance and cancer. These large intervening noncoding RNAs (originally called lincRNAs, currently called lncRNAs) in some instances redirect the repressive polycomb repressor complex 2 (PRC2) to genes that promote cell renewal. In particular, the lncRNA called HOTAIR is overexpressed in breast cancer and redirects the PRC2 complex, which methylates H3K27, an epigenetic change that tends to condense chromatin. Perhaps not surprising, many epigenetic marks target the developmentally relevant homeobox class of transcription factors (Hox genes), which in turn are master regulators of embryonic development and stem cell pluripotency that when altered can lead to disease.

The fact that many human diseases, including cancer, have an epigenetic etiology has encouraged the development of a new therapeutic option called “epigenetic therapy”. Many agents have been discovered that alter methylation patterns on DNA or the modification of histones, and several of these agents are currently being tested in clinical trials.

1.3.1

DNA Elements

RNA polymerase II (Pol II) and its accessory factors bind to a DNA sequence called the promoter located upstream of protein-coding sequences to direct RNA transcription. Without the promoter, the genetic sequences that encode the information to make a functional peptide product will not be transcribed. Other 5′ flanking sequences or DNA elements that participate in transcription are sequence-specific binding sites for proteins that regulate the fidelity, rate, and timing of Pol II binding, formation of the preinitiation complex, and initiation of transcript elongation under basal and regulated conditions. These sequences are defined as cis-acting elements because they are a part of the same (cis) gene. DNA elements are categorized according to their ability to regulate transcription as a function of their distance and orientation from the promoter. Sequences that are contained within the first 30–100 bp of the promoter are considered promoter-dependent cis-acting elements. If they are positive-acting elements and increase the rate of transcription, they are considered activating DNA elements , whereas if they are negative-acting DNA elements and decrease or repress the rate of transcription, they are considered repressor or silencer elements.

The RNA core promoter consists of two types—focused and dispersed. Focused promoters contain either one or a tight cluster of start sites over a few bp; whereas, a dispersed promoter contains several start sites over about 100 bp and are typically found at CpG dinucleotide sites. Critical promoter elements include TATA elements, which lie upstream of the transcription start site, the initiator sequence (Inr) that spans the start site, upstream regulatory elements that bind either transcriptional activators or repressors and finally downstream poly(dA-dT) elements. The TATA element or “TATA box” is an element whose DNA sequence is TATA or variants thereof. This sequence resides at a fixed distance 25–30 bp upstream from the transcriptional start site in many Pol II promoters, and its location relative to the start site is position- and distance dependent. However, many genes do not have TATA sequences. These “TATA-less promoters” still remain dependent on the TATA-binding protein (TBP) to assemble at the promoter to form the preinitiation complex (PIC) but the recruitment of TBP is not rate limiting.

Initiator elements (Inr) although initially identified at the “TATA-less promoters” have subsequently been found at both TATA-containing and TATA-less promoters. Their role appears to be in directing the accuracy of Pol II initiation. These Inr elements reside within the first 60 bp of the transcriptional start site, directly overlap the start site itself, but do not have a clearly defined consensus sequence. Many of the genes-encoding gastrointestinal peptides (e.g., gastrin, somatostatin, cholecystokinin, glucagons, and secretin) contain TATA elements ; however, the gene-encoding the growth factor, transforming growth factor alpha (TGFα), does not.

1.4.1

Structural Methods

Once functional regulatory DNA elements have been identified, assays that assess DNA-protein interactions are performed. Indeed, in circumstances where a long sequence (> 50 bp) must be analyzed, it is simpler to identify DNA-protein interactions first and then determine if these DNA elements are involved in transcriptional regulation. DNase I footprinting assays are used to identify DNA-binding elements that interact with crude or purified nuclear proteins by protecting them from chemical or enzymatic cleavage. Such assays are particularly well suited for studying cooperative interactions among proteins bound to adjacent DNA elements. The technique can be carried out in vivo or in vitro. However, in vivo footprinting has been superceded by chromatin immunoprecipitation (ChIP) assays described below. Electrophoretic gel mobility shift assays (EMSA, gel shift, gel delay, or band shift assays) permit a more detailed analysis of (a) the type of protein complexes that bind to individual DNA elements and (b) the specificity of the protein interaction with specific bp ( Fig. 1.8 ). This assay is also rapid and easier to perform than footprinting assays. Methylation interference assays extend the power of the gel shift assay by identifying specific nucleotide contacts that are required for DNA binding. DNA affinity precipitation (DAPA) is a DNA-protein interaction assay that uses a biotinylated DNA-binding site to identify the proteins that are recruited to an element. The assay uses the DNA element to isolate the protein factors along with immunoblots to identify the proteins that form both the protein-DNA and protein-protein interactions. Southwestern blot analysis takes advantage of specific DNA elements that are used to detect nuclear proteins separated on a denaturing gel and transferred to nitrocellulose or produced by a phage expression library.

Electrophoretic mobility shift assay (EMSAs, gel shift). A DNA element ~30–100 bp in length is labeled and then incubated with crude nuclear extract or purified protein. A band on the autoradiogram is detected if the radiolabeled probe is retarded and does not migrate to the bottom of the gel. The specificity of binding is determined by competing with unlabeled DNA sequences. Competitor 1 is related to the probe sequence, whereas Competitor 2 is unrelated to the probe sequence.

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