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of proteins called histones. Nucleosomes resemble beads joined by short DNA linkers; the entire structure is generically called chromatin. Importantly, the winding and compaction of chromatin in any given cell varies in different genomic regions. Thus, nuclear chromatin exists in two basic forms (visualizable by standard his- tology): (1) histochemically dense and transcriptionally inactive heterochromatin and (2) histochemically dis- persed and transcriptionally active euchromatin. Because only euchromatin permits gene expression and thereby dictates cellular identity and activity, there are a host of mechanisms that tightly regulate the state of chromatin (described below). #31 в контекст | | |
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DNA methylation. High levels of DNA methylation in gene regulatory elements typically result in chroma- tin condensation and transcriptional silencing. Like histone modifications (see later), DNA methylation is tightly regulated by methyltransferases, demethylating enzymes, and methylated-DNA-binding proteins. #32 в контекст | | |
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Histone modifying factors. Nucleosomes are highly dynamic structures regulated by an array of nuclear proteins and post-translational modifications: #33 в контекст | | |
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Chromatin remodeling complexes can reposition nucleo- somes on DNA, exposing (or obscuring) gene regula- tory elements such as promoters. #34 в контекст | | |
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“Chromatin writer” complexes carry out more than 70 different covalent histone modifications generi- cally denoted as marks. These include methylation, acetylation, and phosphorylation of specific histone amino acid residues: Histone methylation of lysines and arginines is accomplished by specific writer enzymes; methylation of histone lysine residues can lead to transcriptional activation or repression, depending on which histone residue is “marked.” Histone acetylation of lysine residues (occurring through histone acetyl transferases) tends to open up chromatin and increase transcription; histone deacetylases (HDAC) reverse this process, leading to chromatin condensation. Histone phosphorylation of serine residues can variably open or condense chromatin, to increase or decrease transcription, respectively. #35 в контекст | | |
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Histone marks are reversible through the activity of “chromatin erasers.” Other proteins function as “chro- matin readers,” binding histones that bear particular marks and thereby regulating gene expression. #36 в контекст | | |
The mechanisms involved in the cell-specific epigenetic regulation of genomic organization and gene expression are undeniably complex. Despite the intricacies, learning to manipulate these processes will likely bear significant therapeutic benefits because many diseases are associated with inherited or acquired epigenetic alterations, and dysregulation of the “epigenome” has a central role in the genesis of benign and malignant neoplasms (Chapter 6). Moreover—unlike genetic changes—epigenetic alterations (e.g., histone acetylation and DNA methylation) are readily reversible and are therefore amenable to intervention; indeed, HDAC inhibitors and DNA methylation inhibitors are already being used in the treatment of various forms of cancer. #37 в контекст | | |
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Another mechanism of gene regulation depends on the functions of noncoding RNAs. As the name implies, these are encoded by genes that are transcribed but not trans- lated. Although many distinct families of noncoding RNAs exist, only two examples are discussed here: small RNA molecules called microRNAs and long noncoding RNAs >200 nucleotides in length. #39 в контекст | | |
• Micro-RNAs (miRNAs) are relatively short RNAs (22 nucleotides on average) that function primarily to modulate the translation of target mRNAs into their corresponding proteins. Posttranscriptional silencing of gene expression by miRNA is a fundamental and evolutionarily conserved mechanism of gene regula- tion present in all eukaryotes (plants and animals). Even bacteria have a primitive version of the same general machinery that they use to protect themselves against foreign DNA (e.g., from phages and viruses). #40 в контекст | | |
• The human genome contains almost 6000 miRNA genes, only 3.5-fold less than the number of protein-coding genes. Moreover, individual miRNAs appear to regu- late multiple protein-coding genes, allowing each miRNA to coregulate entire programs of gene expres- sion. Transcription of miRNA genes produces a primary transcript (pri-miRNA) that is processed into progres- sively smaller segments, including trimming by the enzyme Dicer. This generates mature single-stranded miRNAs of 21 to 30 nucleotides that associate with a multiprotein aggregate called RNA-induced silencing complex (RISC; Fig. 1.3). Subsequent base pairing between the miRNA strand and its target mRNA directs the RISC to either induce mRNA cleavage or to repress its translation. In this way, the target mRNA is posttran- scriptionally silenced. #41 в контекст | | |
Taking advantage of the same pathway, small interfering RNAs (siRNAs) are short RNA sequences that can be intro- duced into cells. These serve as substrates for Dicer and interact with the RISC complex in a manner analogous to endogenous miRNAs. Synthetic siRNAs that can target specific mRNA species are therefore powerful laboratory tools to study gene function (so-called knockdown technol- ogy); they also are promising as therapeutic agents to silence pathogenic genes, e.g., oncogenes involved in neo- plastic transformation. #42 в контекст | | |
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Long noncoding RNA (lncRNA). The human genome also contains a very large number of lncRNAs—at least 30,000, with the total number potentially exceeding coding mRNAs by 10- to 20-fold. lncRNAs modulate gene expression in many ways (Fig. 1.4); for example, they can bind to regions of chromatin, restricting RNA polymerase access to coding genes within the region. The best-known example of a repressive function involves XIST, which is transcribed from the X chromo- some and plays an essential role in physiologic X chro- mosome inactivation. XIST itself escapes X inactivation, but forms a repressive “cloak” on the X chromosome from which it is transcribed, resulting in gene silencing. Conversely, it has been appreciated that many enhanc- ers are sites of lncRNA synthesis, with the lncRNAs expanding transcription from gene promoters through a variety of mechanisms (Fig. 1.4). Ongoing studies are exploring the role of lncRNAs in diseases like athero- sclerosis and cancer. #44 в контекст | | |
Fig. 1.3 Generation of microRNAs (miRNA) and their mode of action in regulating gene function. miRNA genes are transcribed to produce a primary miRNA (pri-miRNA), which is processed within the nucleus to form pre- miRNA composed of a single RNA strand with secondary hairpin loop structures that form stretches of double-stranded RNA. After this pre- miRNA is exported out of the nucleus via specific transporter proteins, the cytoplasmic enzyme Dicer trims the pre-miRNA to generate mature double- stranded miRNAs of 21 to 30 nucleotides. The miRNA subsequently unwinds, and the resulting single strands are incorporated into the multiprotein RISC. Base pairing between the single-stranded miRNA and its target mRNA directs RISC to either cleave the mRNA target or to repress its translation. In either case, the target mRNA gene is silenced posttranscriptionally. #45 в контекст | | |
Fig. 1.4 Roles of long noncoding RNAs (lncRNAs). (A) Long noncoding #46 в контекст | | |
RNAs (lncRNAs) can facilitate transcription factor binding and thus promote gene activation. (B) Conversely, lncRNAs can preemptively bind transcription factors and thus prevent gene transcription. (C) Histone and DNA modifica- tion by acetylases or methylases (or deacetylases and demethylases) may be directed by the binding of lncRNAs. (D) In other instances lncRNAs may act as scaffolding to stabilize secondary or tertiary structures and/or multisub- unit complexes that influence general chromatin architecture or gene activity. (Adapted from Wang KC, Chang HY: Molecular mechanisms of long noncoding RNAs, Mol Cell 43:904, 2011.) #47 в контекст | | |
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Exciting new developments that permit exquisitely specific #49 в контекст | | |
genome editing stand to usher in an era of molecular revo- lution. These advances come from a wholly unexpected source: the discovery of clustered regularly interspaced short palindromic repeats (CRISPRs) and Cas (or CRISPR- associated genes). These are linked genetic elements that endow prokaryotes with a form of acquired immunity to phages and plasmids. Bacteria use this system to sample the DNA of infecting agents, incorporating it into the host genome as CRISPRs. CRISPRs are transcribed and pro- cessed into an RNA sequence that binds and directs the nuclease Cas9 to a sequences (e.g., a phage), leading to its cleavage and the destruction of the phage. Gene editing repurposes this process by using artificial guide RNAs (gRNAs) that bind Cas9 and are complementary to a DNA sequence of interest. Once directed to the target sequence #50 в контекст | | |
by the gRNA, Cas9 induces double-strand DNA breaks. #51 в контекст | | |
Repair of the resulting highly specific cleavage sites can lead to somewhat random disruptive mutations in the targeted sequences (through nonhomologous end joining [NHEJ]), or the precise introduction of new sequences of interest (by homologous recombination). Both the gRNAs and the Cas9 enzyme can be delivered to cells with a single easy-to-build plasmid (Fig. 1.5). However, the real beauty of the system (and the excitement about its genetic engi- neering potential) comes from its impressive flexibility and specificity, which is substantially better than other previ- ous editing systems. Applications include inserting specific mutations into the genomes of cells to model cancers and other diseases, and rapidly generating transgenic animals from edited embryonic stem cells. On the flip side, it now is feasible to selectively “correct” mutations that cause hered- itable disease, or—perhaps more worrisome—to just elimi- nate less “desirable” traits. Predictably, the technology has inspired a vigorous debate regarding its application. #52 в контекст | | |
Fig. 1.5 Gene editing with clustered regularly interspersed short palin- #53 в контекст | | |
dromic repeats (CRISPRs)/Cas9. In bacteria, DNA sequences consisting of CRISPRs are transcribed into guide RNAs (gRNAs) with a constant region and a variable sequence of about 20 bases. The constant regions of gRNAs bind to Cas9, permitting the variable regions to form heteroduplexes with homologous host cell DNA sequences. The Cas9 nuclease then cleaves the bound DNA, producing a double-stranded DNA break. To perform gene editing, gRNAs are designed with variable regions that are homologous to a target DNA sequence of interest. Coexpression of the gRNA and Cas9 in cells leads to efficient cleavage of the target sequence. In the absence of homologous DNA, the broken DNA is repaired by non homologous end joining (NHEJ), an error-prone method that often introduces disruptive insertions or deletions (indels). By contrast, in the presence of a homologous “donor” DNA spanning the region targeted by CRISPR/Cas9, cells instead may use homologous DNA recombination (HDR) to repair the DNA break. HDR is less efficient than NHEJ, but has the capacity to introduce precise changes in DNA sequence. Potential applications of CRISPR/Cas9 coupled with HDR include the repair of inherited genetic defects and the creation of pathogenic mutations. #54 в контекст | | |
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The viability and normal activity of cells depend on a variety of fundamental housekeeping functions that all dif- ferentiated cells must perform. #56 в контекст | | |
Many normal housekeeping functions are compart- mentalized within membrane-bound intracellular organ- elles (Fig. 1.6). By isolating certain cellular functions within distinct compartments, potentially injurious degradative enzymes or reactive metabolites can be concentrated or stored at high concentrations in specific organelles without risking damage to other cellular constituents. Moreover, compartmentalization allows for the creation of unique intracellular environments (e.g., low pH or high calcium) that are optimal for certain enzymes or metabolic pathways. #57 в контекст | | |
New proteins destined for the plasma membrane or secretion are synthesized in the rough endoplasmic reticulum (RER) and physically assembled in the Golgi apparatus; pro- teins intended for the cytosol are synthesized on free ribo- somes. Smooth endoplasmic reticulum (SER) may be abundant in certain cell types such as gonads and liver where it serves as the site of steroid hormone and lipoprotein syn- thesis, as well as the modification of hydrophobic com- pounds such as drugs into water-soluble molecules for export. #58 в контекст | | |
Cells catabolize the wide variety of molecules that they endocytose, as well as their own repertoire of proteins and organelles—all of which are constantly being degraded and renewed. Breakdown of these constituents takes place at three different sites, ultimately serving different functions. #59 в контекст | | |
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Proteasomes are “disposal” complexes that degrade denatured or otherwise “tagged” cytosolic proteins and release short peptides. In some cases the peptides so generated are presented in the context of class I major histocompatibility molecules to help drive the adaptive immune response (Chapter 5). In other cases, protea- somal degradation of regulatory proteins or transcrip- tion factors can trigger or shut down cellular signaling pathways. #61 в контекст | | |
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Lysosomes are intracellular organelles that contain enzymes that digest a wide range of macromolecules, including proteins, polysaccharides, lipids, and nucleic acids. They are the organelle in which phagocytosed microbes and damaged or unwanted cellular organelles are degraded and eliminated. #63 в контекст | | |
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Peroxisomes are specialized cell organelles that contain catalase, peroxidase and other oxidative enzymes. They play a specialized role in the breakdown of very long chain fatty acids, generating hydrogen peroxide in the process. #65 в контекст | | |
The contents and position of cellular organelles also are subject to regulation. Endosomal vesicles shuttle inter- nalized material to the appropriate intracellular sites or direct newly synthesized materials to the cell surface or targeted organelle. Movement of both organelles and pro- teins within the cell and of the cell in its environment is orchestrated by the cytoskeleton. These structural proteins also regulate cellular shape and intracellular organiza- tion, requisites for maintaining cell polarity. This is par- ticularly critical in epithelia, in which the top of the cell (apical) and the bottom and side of the cell (basolateral) are often exposed to different environments and have distinct functions. #66 в контекст | | |
Most of the adenosine triphosphate (ATP) that powers cells is made through oxidative phosphorylation in the mitochondria. However, mitochondria also serve as an important source of metabolic intermediates that are needed for anabolic metabolism. They also are sites of syn- thesis of certain macromolecules (e.g., heme), and contain important sensors of cell damage that can initiate and regu- late the process of apoptotic cell death. #67 в контекст | | |
Cell growth and maintenance require a constant supply of both energy and the building blocks that are needed for synthesis of macromolecules. In growing and dividing cells, all of these organelles have to be replicated (organel- lar biogenesis) and correctly apportioned in daughter cells following mitosis. Moreover, because the macromolecules and organelles have finite life spans (mitochondria, e.g., last only about 10 days), mechanisms also must exist that allow for the recognition and degradation of “worn out” cellular components. The final catabolism occurs in lysosomes. #68 в контекст | | |
With this as a primer, we now move on to discuss cellular components and their function in greater detail. #69 в контекст | | |
Plasma Membrane: Protection and Nutrient Acquisition #70 в контекст | | |
Plasma membranes (and all other organellar membranes) are more than just static lipid sheaths. Rather, they are fluid bilayers of amphipathic phospholipids with hydro- philic head groups that face the aqueous environment and hydrophobic lipid tails that interact with each other to form a barrier to passive diffusion of large or charged molecules (Fig. 1.7A). The bilayer is composed of a hetero- geneous collection of different phospholipids, which are distributed asymmetrically—for example, certain mem- brane lipids preferentially associate with extracellular or cytosolic faces. Asymmetric partitioning of phospholipids is important in several cellular processes: #71 в контекст | | |
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Phosphatidylinositol on the inner membrane leaflet can be phosphorylated, serving as an electrostatic scaffold for intracellular proteins; alternatively, polyphosphoinosit- ides can be hydrolyzed by phospholipase C to generate intracellular second signals such as diacylglycerol and inositol trisphosphate. #73 в контекст | | |
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Phosphatidylserine is normally restricted to the inner face where it confers a negative charge and is involved in electrostatic interactions with proteins; however, when it flips to the extracellular face, which happens in cells undergoing apoptosis (programmed cell death), it becomes an “eat me” signal for phagocytes. In the special case of platelets, it serves as a cofactor in the clotting of blood. #75 в контекст | | |
Glycolipids and sphingomyelin are preferentially expressed on the extracellular face; glycolipids (and particularly gangliosides, with complex sugar linkages and terminal sialic acids that confer negative charges) are important in cell–cell and cell–matrix interactions, including inflam- matory cell recruitment and sperm–egg interactions. #76 в контекст | | |
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Certain membrane components associate laterally with each other in the bilayer, leading to distinct domains called lipid rafts. Because inserted membrane proteins have differ- ent intrinsic solubilities in various lipid domains, they tend to accumulate in certain regions of the membrane (e.g., rafts) and to become depleted from others. Such nonran- dom distributions of lipids and membrane proteins impact cell–cell and cell–matrix interactions, as well as intracel- lular signaling and the generation of specialized membrane regions involved in secretory or endocytic pathways. #78 в контекст | | |
