Chapter 17 Gene Regulation in Eukaryotes

Eukaryotic cells have two most significant complexities in transcriptional regulation comparing to prokaryotic cells. Nucleosomes and their modifiers: Nucleosomes present a problem not faced in bacteria, but their modification also offers new opportunities for regulation. More regulators and more extensive regulatory sequences: The more extensive signal integration is found in muticellular eukaryotes.

CONSERVED MECHANISMS OF TRANSCRIPTIONAL REGULATION FROM YEAST TO MAMMALS Activators have separate DNA binding and activating functions. Eukaryotic activators have separate DNA binding and activating regions, very often on separate domains. Gal4 activates GAL1 gene 103 fold.

Domain swap experiments reveal that the DNA binding domain serves merely to tether the activating region to the promoter In some cases, the DNA binding domain and the activating region are carried on two separate proteins. Ex) Oct1 + VP16

Eukaryotic regulators use a range of DNA-binding domains, but DNA recognition involves the same principles as found in bacteria Eukaryotic DNA binding domains can bind as monomers, homodimers, and heterodimers using an a helix inserted into the major groove. Heterodimers extend the range of DNA binding specificities. homeodomain, zinc finger, leucine zipper, bHLH

basic zipper, basic HLH

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Activating regions are not well-defined structures Activating regions do not always have well-defined structures. Interaction with their target may induce defined structures. It is believed that activating regions consist of small units, each of which has a weak activating capacity on its own. Acidic activating regions have the characteristic pattern of acidic and hydrophobic residues: Gal4, VP16 Q-rich activating region: SP1 P-rich activating region: CTF1 Acidic activating regions are strong and universally work in any eukaryotic organism.

RECRUITMENT OF PROTEIN COMPLEXES TO GENES BY EUKARYOTIC ACTIVATORS Activators recruit the transcriptional machinery to the gene Eukaryotic activators recruit polymerase indirectly or recruit other factor needed after polymerase has bound. Activators interact with parts of the transcription machinery, nucleosome modifiers, and factors needed for initiation and elongation. ex) TFIID, Mediator.

Chromatin immunoprecipitation (ChIP) The recruitment of the transcription machinery is the major activation mechanism although allostery may work together with recruitment.

Activators also recruit nucleosome modifiers that help the transcription machinery bind at the promoter or initiate transcription Nucleosome modifiers come in two types: histone modifiers (HAT and HDAC) and chromatin remodeling factors (SWI/SNF, etc). They uncover DNA-binding sites that would otherwise remain inaccessible. Addition of acetyl groups creates specific binding sites on nucleosomes for bromodomain proteins.

Specific requirements for transcription are different from gene to gene. Gal4 appears to contact three components: Mediator, TFIID, and SAGA (Spt-Ada-Gcn5-acetyltransferase). SAGA contains TAFs and is needed in place of TFIID at some promoters. The ability of an acidic activator such as Gal4 to work at genes with different requirements can be explained by its ability to interact with multiple targets.

Activators recruit an additional factor needed for efficient initiation or elongation at some promoters The fly HSP70 gene is controlled by the GAGA-binding factor and HSF. The GAGA factor recruits the transcription machinery but the initiated polymerase stalls 25 bp downstream from the promoter. In response to heat shock, HSF binds at the promoter and recruits P-TEF (positive transcription elongation factor). P-TEF phosphorylates CTD of RNAPII and allow the stalled polymerase to proceed. --- rapid response to heat shock HIV promoter is controlled by P-TEF. The polymerase stalls soon after transcription initiation. TAT binds a specific RNA sequence near the start of the HIV RNA and recruits P-TEF. It is not clear whether P-TEF is needed at most genes. P-TEFb = CDK9 + CyclinT

Action at a Distance: Loops and Insulators. Many eukaryotic activators work from a distance. Enhancers are found hundreds of kilobases upstream or downstream of genes they control. In these cases, bringing the promoter and the activator into close proximity is lost. How to help communication between distantly bound proteins: By binding to multiple sites between the enhancer and the promoter, Chip in fly may facilitate formation of multiple miniloops in the intervening DNA. Chromatin may form special structures that actively bring enhancers and promoters closer together.

Insulators control the actions of activators by blocking communication between enhancers and promoters. They also inhibit gene silencing by blocking the spread of chromatin modifications.

Appropriate regulation of some groups of genes requires locus control regions The different globin genes are expressed at different stages of development. LCR is found upstream of the whole cluster of globin genes.

LCR is made up of multiple sequence elements LCR is made up of multiple sequence elements. LCR is in close proximity to each promoter as that promoter is activated.

SIGNAL INTEGRATION AND COMBINATORIAL CONTROL Activators work together synergistically to integrate signals In eukaryotic cells, signal integration is used extensively. Numerous signals are required to switch a gene on. Multiple activators work together synergistically. Synergy can result from multiple activators recruiting a single component of the transcription machinery, multiple activators recruiting different components, or multiple activators helping each other bind to their sites.

Synergy can result from multiple activators help each other bind to their sites by cooperativity.

Signal Integration: the HO gene is controlled by two regulators; one recruits nucleosome modifiers and the other recruits mediator The HO gene in yeast is expressed only in mother cells and only at a certain point in cell cycle. SWI5 (active only in mother cell) can bind to its site but cannot activate the HO gene. It recruits HAT and SWI/SNF, which are needed for the binding of SBF (active only at the correct stage in cell cycle).

Signal Integration: Cooperative binding of activators at the human β-Interferon gene The human β-Interferon gene is activated upon viral infection. NFkB, IRF and Jun/ATF bind coopertively to sites in the enhancer forming an enhanceosome. HMGA1 helps the assembly of the enhanseosome. They recruit CBP.

Essentially every base of DNA within the enhancer is involved in activator binding. The enhancer is highly conserved, even more than the coding sequence itself. DNA-binding domain only

Combinatorial control lies at the heart of the complexity and diversity of eukaryotes There is extensive combinatorial control in eukaryotes. Multipe activators or multiple copies of a single activator work synergistically. Different activators can work together in so many combinations because each can use any of an array of targets.

Combinatorial control lies at the heart of the complexity and diversity of eukaryotes Heterodimers of DNA-binding proteins offer another level of combinatorial control.

Another example of combinatorial control: liver-specific expression of transthyretin

Combinatorial control of the mating-type genes from S. cerevisiae

TRANSCRIPTIONAL REPRESSORS Histone modifications (deacetylase, methyltransferase, and silencing) are the most common repression in eukaryotes.

Histone modifications affect chromatin condensation and provide protein binding sites.

In the presence of glucose, GAL genes are switched off by Mig1 In the presence of glucose, GAL genes are switched off by Mig1. Mig1 recruits a repressing complex containing Tup1. Tup1 recruits histone deacetylases and directly interacts with the transcription machinery to inhibit transcription initiation.

SIGNAL TRANSDUCTION AND THE CONTROL OF TRANSCRIPTIONAL REGULATORS Signals are often communicated to transcriptional regulators through signal transduction pathways A given gene is expressed often depending on environmental signals. Signals often control transcriptional regulators. JAK-STAT pathway

Ras-MAPK pathway

Signals control the activities of eukaryotic transcriptional regulators in a variety of ways In eukaryotes, transcriptional regulators are not typically controlled at the level of DNA binding. Instead usually controlled by the following two ways. 1. unmasking an activating region: repression by Gal80 and repression by Rb

2. transport into and out of the nucleus: NFkB-IkB and Dorsal-Cactus

Activators and repressors sometimes come in pieces 1.The DNA-binding domain and activating region can be on separate polypeptides. 2. Nature of the complex on DNA determine whether the DNA-binding protein activates or represses target genes. Ex) E2F-Rb 3. The nature and arrangement determines the activation or repression. Ex) GR. Without its ligand, GR is complexed with Hsp90. Upon ligand binding, GR moves to the nucleus. GREs come in two types. For activation, it recruits the coactivator CBP. For repression, it binds a histone deacetylase.

GENE “SILENCING” BY MODIFICATION OF HISTONES AND DNA Transcriptional silencing is a position effect: a gene is silenced because of where it is located. Silencing can spread over large stretches of DNA. The most common form of silencing is associated with heterochromatin found in the telomeres and the centromeres. About 50% of the genome is estimated to be in some form of heterochromatin. Modification of nucleosomes and methylation of DNA can silence transcription.

Silencing in yeast is mediated by deacetylation and methylation of histones Mutations have been isolated in which silencing is relieved. The products of SIR (silent information regulator) genes form a complex that associates with silent chromatin. Sir2 is a histone deacetylase. A specific DNA-binding protein (Rap1) recruits the silencing complex, which triggers local deacetylation of histone tails. The deacetylated histones are directly recognized by the silencing complex, spreading deacetylation along the chromatin.

A specific DNA-binding protein and RNA molecules define where the silencing complex forms. The insulator elements and other kinds of histone modifications (e.g., methylation of histone H3) block spreading of silencing. In higher eukaryotes and S. pombe, silencing is associated with chromatin containing histones that are not only deacetylated , but methylated as well (e.g., Lys-9 in the H3 tail).

In Drsophila, HP1 recognizes methylated histones and condenses chromatin When a gene is moved into a region of heterochromatin, variegation is seen sometimes, in which that gene switches between the silenced and expressed state at random. Su(Var)3-9, a suppressor of variegation, is a H3 lysine-9 methyltransferase and methylated histones recruit HP1, which participates in the compaction of the heterochromatin.

Chromatin condensation by Polycomb proteins: epigenetic control Polycomb repressive complex 1 and 2: repression by methylation of H3 K27 Trithorax proteins: activation by methylation of H3 K4

DNA methylation is associated with silenced genes in mammalian cells DNA methylation is often seen in regions that are also heterochromatic. Methylated sequences are often recognized by methylated DNA-binding proteins (such as MeCP2), which recruit histone deacetylases and histone methylases.

Two alleles from the father and mother are expressed at comparable levels in most cases. But in a few cases, one copy of a gene is expressed while the other is silent. The maternal copy of H19 is expressed, whereas the other copy is switched off; the paternal copy of Igf2 is on and the maternal copy is off.--- imprinting An insulator called the imprinting control region (ICR) and its methylation state regulate H19 and Igf2 genes.

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EPIGENETIC GENE REGULATION The inheritance of gene expression patterns, in the absence of both mutation and the initiating signal, is called epigenetic regulation. Some states of gene expression are inherited through cell division even when the initiating signal is no longer present Once established, the lysogenic state of lambda is maintained stably by autoregulation until an inducing signal is received.

Another mechanism of epigenetic regulation is provided by DNA methylation. DNA methylation is reliably inherited through cell division by maintenance methylases. DNA methylation may be the primary marker of regions of the genome that are silenced. Nucleosome modification could in principle provide another basis for epigenetic inheritance. Methylated histones are distributed equally between the two daughter duplexes. Methylated nucleosomes recruit proteins bearing chromodomains, including the histone methylase itself.