Genetics Chapter 12

paternal imprinting

The expression of a gene only when inherited from the mother, because the allele of the gene inherited from the father is inactive due to methylation in the course of gamete formation. -a mouse H19 allele is expressed only if it is inherited from the mother; H19 is an example of paternal imprinting because the paternal copy is inactive.

enhanceosome

The macromolecular assembly responsible for interaction between enhancer elements and the promoter regions of genes. -One characteristic of enhancers is that they can activate transcription when they are located at great distances from the promoter (>50 kb), either upstream or downstream from a gene or even in an intron. -Eukaryotic enhancers can act at great distances to modulate the activity of the transcriptional apparatus. Enhancers contain binding sites for many transcription factors, which bind and interact cooperatively. These interactions result in a variety of responses, including the recruitment of additional co-activators and the remodeling of chromatin.

corepressor

a repressor that facilitates gene repression but is not itself a DNA-binding repressor

reporter gene

A gene whose phenotypic expression is easy to monitor; used to study tissue-specific promoter and enhancer activities in transgenes.

co-activator

A special class of eukaryotic regulatory complex that serves as a bridge to bring together regulatory proteins and RNA polymerase II.

Hypoacetylation

An underabundance of acetyl groups on certain amino acids of the histone tails. Transcriptionally inactive chromatin is usually hypoacetylated. -inactive genes are underacetylated

maternal imprinting

The expression of a gene only when inherited from the father, because the copy of the gene inherited from the mother is inactive due to methylation in the course of gamete formation. -an Igf2 allele is expressed in a mouse only if it is inherited from the mouse's father—an example of maternal imprinting because the copy of the gene derived from the mother is inactive.

GAL system

-In yeast cells growing in media lacking galactose, the GAL genes are largely silent. But, in the presence of galactose (and the absence of glucose), the GAL genes are induced. -The key regulator of GAL gene expression is the Gal4 protein, a sequence-specific DNA-binding protein. -In the presence of galactose, the expression levels of the GAL1, GAL2, GAL7, and GAL10 genes are 1000-fold or more higher than in its absence. -the normal function of the Gal80 protein is to somehow inhibit GAL gene expression. While, Gal3 normally promotes expression of the GAL genes. -Extensive biochemical analyses have revealed that the Gal80 protein binds to the Gal4 protein with high affinity and directly inhibits Gal4 activity. Specifically, Gal80 binds to a region within one of the Gal4 activation domains, blocking its ability to promote the transcription of target genes. The Gal80 protein is expressed continuously, so it is always acting to repress transcription of the GAL structural genes unless stopped. The role of the Gal3 protein is to release the GAL structural genes from their repression by Gal80 when galactose is present. -Gal3 is thus both a sensor and inducer. When Gal3 binds galactose and ATP, it undergoes an allosteric change that promotes binding to Gal80, which in turn causes Gal80 to release Gal4, which is then able to interact with other transcription factors and RNA pol II to activate transcription of its target genes. Thus, Gal3, Gal80, and Gal4 are all part of a switch whose state is determined by the presence or absence of galactose

methylation control

-Methylation acts as a block to the binding of proteins needed for transcription. Only the unmethylated (female) ICR can bind a regulatory protein called CTCF. When bound, CTCF acts as an enhancer-blocking insulator that prevents enhancer activation of Igf2 transcription. However, the enhancer in females can still activate H19 transcription. In males, CTCF cannot bind to the ICR and the enhancer can activate Igf2 transcription (recall that enhancers can act at great distances). The enhancer cannot activate H19, however, because the methylated region extends into the H19 promoter. The methylated promoter cannot bind proteins needed for the transcription of H19. -CTCF-binding site is methylated only in chromosomes derived from the male parent. The methylation of the CTCF-binding site prevents CTCF binding in males and permits the enhancer to activate Igf2.

X chromosome inactivation

-This dosage imbalance is corrected by a process called dosage compensation, which makes the amount of most gene products from the two copies of the X chromosome in females equivalent to the single dose of the X chromosome in males. In mammals, dosages are made equivalent by randomly inactivating one of the two X chromosomes in each cell at an early stage in development. -The inactivated chromosome, called a Barr body, can be seen in the nucleus as a darkly staining, highly condensed, heterochromatic structure. -X-chromosome inactivation is an example of epigenetic inheritance. First, most of the genes on the inactivated X chromosome (called Xi) are silenced, and the chromosome has epigenetic marks associated with heterochromatin, including H3K9me, hypoacetylation of histones, and hypermethylation of its DNA. Second, most but not all of the genes on the inactivated chromosome remain inactive in all descendants of these cells, yet the DNA sequence itself is unchanged. -The mechanism that converts a fully functional X chromosome into heterochromatin. -For most diploid organisms, both alleles of a gene are expressed independently. Genomic imprinting and X inactivation are examples of only a single allele being available for expression. In these cases, epigenetic mechanisms silence a single chromosomal locus or one copy of an entire chromosome, respectively.

regulatory proteins possess one or more of the following functional domains:

1. A domain that recognizes a DNA regulatory sequence (the protein's DNA-binding site). 2. A domain that interacts with one or more proteins of the transcriptional apparatus (RNA polymerase or a protein associated with RNA polymerase) 3. A domain that interacts with proteins bound to nearby regulatory sequences on DNA such that they can act cooperatively to regulate transcription 4. A domain that influences chromatin condensation either directly or indirectly 5. A domain that acts as a sensor of physiological conditions within the cell

Eukaryotic transcriptional regulation

1. In bacteria, all genes are transcribed into RNA by the same RNA polymerase, whereas three RNA polymerases function in eukaryotes. RNA polymerase II, which transcribes DNA into mRNA. 2. RNA transcripts are extensively processed during transcription in eukaryotes; the 5′ and 3′ ends are modified and introns are spliced out. 3. RNA polymerase II is much larger and more complex than its bacterial counterpart. One reason it is more complex is that RNA polymerase II must synthesize RNA and coordinate the special processing events unique to eukaryotes. -ensure that the expression of most genes in the genome is off at any one time while activating a subset of genes and generate thousands of patterns of gene expression.

epigenetic marks

A heritable alteration, such as DNA methylation or a histone modification, that leaves the DNA sequence unchanged. -Chromatin structure is inherited from cell generation to cell generation because mechanisms exist to replicate the associated epigenetic marks along with the DNA. In this way, the information inherent in the histone modifications and the existing DNA methylation patterns serve to reconstitute the local chromatin structure that existed before DNA synthesis and mitosis. In contrast, histone variants can be used to rapidly change chromatin in a replication-independent pathway.

Euchromatin

A less-condensed chromosomal region, thought to contain most of the normally functioning genes.

activation domain

A part of a transcription factor required for the activation of target-gene transcription; it may bind to components of the transcriptional machinery or may recruit proteins that modify chromatin structure or both. -Eukaryotic activators recruit RNA polymerase II to gene promoters through two major mechanisms. First, activators can interact with subunits of the protein complexes having roles in transcription initiation and then recruit them to the promoter. Second, activators can recruit proteins that modify chromatin structure, allowing RNA polymerase II and other proteins access to the DNA. Many activators, including Gal4, have both activities. We'll examine the recruitment of parts of the transcriptional initiation complex first. -One way that Gal4 works to activate gene expression is by binding to TBP at a site in its activation domain. Through this binding interaction, it recruits the TFIID complex and, in turn, RNA polymerase II to the promoter.

genomic imprinting

A phenomenon in which a gene inherited from one of the parents is not expressed, even though both gene copies are functional. Imprinted genes are methylated and inactivated in the formation of male or female gametes. -The consequence of parental imprinting is that imprinted genes are expressed as if there were only one copy of the gene present in the cell even though there are two. Importantly, no changes are observed in the DNA sequences of imprinted genes; that is, the identical gene can be active or inactive in the progeny, depending on whether it was inherited from mom or dad. This then represents an epigenetic phenomenon. -during the development of gametes, methyl groups are added to the DNA in the regulatory regions of imprinted genes in one sex only. We saw earlier that DNA of genes that are shut down for an entire lifetime are usually highly methylated. However, it is important to note that DNA methylation is one of several epigenetic marks associated with the long-term inactivation of genes.

mediator complex

A protein complex that acts as an adaptor that interacts with transcription factors bound to regulatory sites and with general initiation factors for RNA polymerase II-mediated transcription. -A second way that Gal4 works to activate gene expression is by interacting with the mediator complex, a large multiprotein complex that, in turn, directly interacts with RNA polymerase II to recruit it to gene promoters. The mediator complex is an example of a co-activator, a term applied to a protein or protein complex that facilitates gene activation by a transcription factor but that itself is neither part of the transcriptional machinery nor a DNA-binding protein. -Eukaryotic transcriptional activators often work by recruiting parts of the transcriptional machinery to gene promoters. -In yeast and in multicellular eukaryotes, cell-type-specific patterns of gene expression are governed by combinations of interacting transcription factors.

Hyperacetylation

An overabundance of acetyl groups attached to certain amino acids of the histone tails. Transcriptionally active chromatin is usually hyperacetylated. -histones associated with the nucleosomes of active genes are rich in acetyl groups -The addition of acetyl groups to histone residues neutralizes the positive charge of lysine residues and reduces the interaction of the histone tails with the negatively charged DNA backbone. This results in more open chromatin as the electrostatic interactions between adjacent nucleosomes and between nucleosomes and adjacent DNA are reduced. -histone acetylation, in conjunction with other histone modifications, influences the binding of regulatory proteins to the DNA. A bound regulatory protein may take part in one of several functions that either directly or indirectly increase the frequency of transcription initiation.

histone modification

Covalent alteration of one or more amino acid residues of the histone protein. Modifications include acetylation, phosphorylation, and methylation. -The nucleosome core contains eight histones, two subunits of each of the four histones: histones 2A, 2B, 3, and 4 (called H2A, H2B, H3, and H4) organized as two dimers of H2A and H2B and a tetramer of H3 and H4. Surrounding the nucleosome core is a linker histone, H1, which can compact the nucleosomes into higher-order structures that further condense the DNA. -Chromatin can be dynamic; nucleosomes are not necessarily in fixed positions on the chromosome. Chromatin remodeling changes nucleosome density or position and is an integral part of eukaryotic gene regulation. -Gal4 also binds to the SWI-SNF chromatin-remodeling complex and recruits it to activated promoters. Yeast strains containing a defective SWI-SNF complex show a reduced level of Gal4 activity. Why might an activator use multiple activation mechanisms? 1. target promoters may become less accessible at certain stages of the cell cycle or in certain cell types (in multicellular eukaryotes). 2. many transcription factors act in combinations to control gene expression synergistically.

Heterochromatin

Densely staining condensed chromosomal regions, believed to be for the most part genetically inert.

epigenetic inheritance

Heritable modifications in gene function not due to changes in the base sequence of the DNA of the organism. Examples of epigenetic inheritance are paramutation, X-chromosome inactivation, and parental imprinting. -The eukaryotic replisome performs all the functions of the prokaryotic replisome; in addition, it must disassemble and reassemble the protein-DNA complexes called nucleosomes.

enhancer-blocking insulator

Regulatory elements positioned between a promoter and an enhancer. Their presence prevents the promoter from being activated by the enhancer. -A regulatory element, such as an enhancer, that can act over tens of thousands of base pairs could interfere with the regulation of nearby genes. -When positioned between an enhancer and a promoter, enhancer-blocking insulators prevent the enhancer from activating transcription at that promoter. Such insulators have no effect on the activation of other promoters that are not separated from their enhancers by the insulator.

histone variants

Uncommon histones that can replace the consensus histones (H2A, H2B, H3, or H4), thereby altering chromatin structure and/or histone code. -Histones are known to be the most conserved proteins in nature; that is, histones are almost identical in all eukaryotic organisms from yeast to plants to animals. -Unlike the common (also called consensus) histones that are added during DNA replication, eukaryotes also have other histones, called histone variants, that can replace the consensus histones that have already been assembled into nucleosomes.

barrier insulators

a DNA element that prevents the spread of heterochromatin by serving as a binding site for proteins that maintain euchromatic chromatin modifications such as histone acetylation.

upstream activation sequence (UAS)

a DNA sequence of yeast located 5' of the gene promoter; a transcription factor binds to the UAS to positively regulate gene expression -Because the Gal4-activated enhancers are located upstream (5′) of the genes they regulate, they are also called upstream activation sequences (UAS). -The binding of sequence-specific DNA-binding proteins to regions outside the promoters of target genes is a common feature of eukaryotic transcriptional regulation. -Many eukaryotic transcriptional regulatory proteins are modular proteins, having separable domains for DNA binding, activation or repression, and interaction with other proteins. -The ability of Gal4, as well as other eukaryotic regulators, to function in a variety of eukaryotes indicates that eukaryotes generally have the transcriptional regulatory machinery and mechanisms in common.

enhancer

a cis-acting regulatory sequence that can elevate levels of transcription from an adjacent promoter. many tissue-specific enhancers can determine spatial patterns of gene expression in higher eukaryotes. enhancers can act on promoters over many tens of kilobases of DNA and can be 5' or 3' of the promoters that they regulate.

post-translational modification

an alteration of amino acid residues that occurs after the protein has been translated -Post-translational modification of histones is associated with the activation and repression of gene expression. While acetylation of histones acts directly to reduce chromatin density and activate gene expression, histone methylation of specific amino acids creates binding sites for proteins that activate or repress gene expression.

chromatin remodeling

changes in nucleosome position along DNA -Compared with eukaryotic DNA, bacterial DNA is relatively "naked," making it readily accessible to RNA polymerase. In contrast, eukaryotic chromosomes are packaged into chromatin, which is composed of DNA and proteins (mostly histones). The basic unit of chromatin is the nucleosome, which contains ~150 bp of DNA wrapped 1.7 times around a core of histone proteins. -prokaryotic genes are generally accessible and "on" unless repressed, eukaryotic genes are inaccessible and "off" unless activated. Therefore, the modification of chromatin structure is a distinctive feature of many eukaryotic processes. -three major mechanisms to alter chromatin structure: 1. moving nucleosomes along the DNA, also called chromatin remodeling. 2. histone modification in the nucleosome core. 3. replacing the common histones in a nucleosome with histone variants.

constitutive heterochromatin

chromosomal regions of permanently condensed chromatin usually around the telomeres and centromeres -The chromatin of eukaryotes is not uniform. Highly condensed heterochromatic regions have fewer genes and lower recombination frequencies than do the less-condensed euchromatic regions.

transcriptional gene silencing

occurs when a gene cannot be transcribed because it is located in heterochromatin -Gene silencing is a very different process from gene repression: silencing is a position effect that depends on the neighborhood in which genetic information is located.

histone code

refers to the pattern of modification of the histone tails that may carry information required for correct chromatin assembly

histone tails

the end of a histone protein protruding from the core nucleosome and subjected to post-translational modification.

Histone deacetylases (HDACs)

the enzymatic activity that removes an acetyl group from a histone tail, which promotes the repression of gene transcription. -Unlike acetylation, the addition of methyl groups can either activate or repress gene expression. -methylation of specific lysine residues, which does not affect charge, creates binding sites for other proteins that either activate or repress gene expression depending on the residues modified.

promoter-proximal elements

the series of transcription-factor binding sites located near the core promoter. -Generally speaking, promoters and promoter-proximal elements are bound by transcription factors that affect the expression of many genes. Enhancers are bound by transcription factors that control the regulation of smaller subsets of genes. Often, an enhancer will act in only one or a few cell types in a multicellular eukaryote. Much of the strategy of eukaryotic transcriptional control hinges on how specific transcription factors control the access of general transcription factors and RNA polymerase II. -The promoters, promoter-proximal elements, and enhancers are all targets for binding by different trans-acting DNA-binding proteins. -Transcription requires the binding of general transcription factors to additional promoter-proximal elements that are commonly found within 100 bp of the transcription initiation site of many (but not all) genes. One of these elements is the CCAAT (pronounced "cat") box, and often another is a GC-rich segment farther upstream. The general transcription factors that bind to the promoter-proximal elements are expressed in most cells, and so they are available to initiate transcription at any time.

position-effect variegation (PEV)

variegation caused by the inactivation of a gene in some cells through its abnormal juxtaposition with heterochromatin -chromosomal neighborhoods exist that can silence genes that are experimentally "relocated" to adjacent regions of the chromosome. -First, that the expression of a gene can be repressed by virtue of its position in the chromosome rather than by a mutation in its DNA sequence. Second, that epigenetic silencing can be inherited from one cell generation to the next. -Findings from subsequent studies in Drosophila and yeast demonstrated that many active genes are silenced in this mosaic fashion when they are relocated to neighborhoods (near centromeres or telomeres) that are heterochromatic. Thus, the ability of heterochromatin to spread into euchromatin and silence genes is a feature common to many organisms. -It provides powerful evidence that chromatin structure is able to regulate the expression of genes—in this case, determining whether genes with identical DNA sequence will be active or silenced. -Active genes that are relocated to genomic neighborhoods that are heterochromatic may be silenced if the heterochromatin spreads to the genes. -The isolation of critical proteins necessary for the formation of heterochromatin, including HP-1 and HMTase, was made possible by the isolation of mutant strains of Drosophila that suppressed or enhanced PEV