Chapter 17 Gene Regulation in Eukaryotes

Similarity of regulation between eukaryotes and prokaryote 1. Principles are the same: signals, activators and repressors, recruitment and allostery, cooperative binding 2. Expression of a gene can be regulated at the similar steps, and the initiation of transcription is the most pervasively regulated step.

Difference in regulation between eukaryotes and prokaryote 1. Pre-mRNA splicing adds an important step for regulation. 2. The eukaryotic transcriptional machinery is more elaborate than its bacterial counterpart. 3. Nucleosomes and their modifiers influence access to genes. 4. Many eukaryotic genes have more regulatory binding sites and are controlled by more regulatory proteins than are bacterial genes.

Enhancer: a given site binds regulator responsible for activating the gene. Alternative enhancer binds different groups of regulators and control expression of the same gene at different times and places in responsible to different signals. Activation at a distance is much more common in eukaryotes. Insulators ( 绝缘体 ) or boundary elements are regulatory sequences to ensure a linked promoter not responding to the activator binding.

Topic 1 Conserved Mechanisms of Transcriptional Regulation from Yeast to Mammals Topic 1 Conserved Mechanisms of Transcriptional Regulation from Yeast to Mammals

The basic features of gene regulation are the same in all eukaryotes, because of the similarity in their transcription and nucleosome structure. Yeast is the most amenable to both genetic and biochemical dissection, and produces much of knowledge of the action of the eukaryotic repressor and activator. The typical eukaryotic activators works in a manner similar to the simplest bacterial case. Repressors work in a variety of ways

The separate DNA binding and activating domains of Gal4 were revealed in two complementary experiments 1. Expression of the N-terminal region (DNA-binding domain) of the activator produces a protein bound to the DNA normally but did not activate transcription. 2. Fusion of the C-terminal region (activation domain) of the activator to the DNA binding domain of a bacterial repressor, LexA activates the transcription of the reporter gene. Domain swap experiment

1-2 Eukaryotic regulators use a range of DNA binding domains, but DNA recognition involves the same principles same found in bacteria Homeodomain proteins Zinc containing DNA-binding domain: zinc finger and zinc cluster Leucine zipper motif Helix-Loop-Helix proteins : basic zipper and HLH proteins

Bactrial regulatory proteins Most use the helix-turn-helix motif to bind DNA target Most bind as dimers to DNA sequence: each monomer inserts an  helix into the major groove. Eukaryotic regulatory proteins 1. Recognize the DNA using the similar principles, with some variations in detail. 2. Some form heterodimers to recognize DNA, extending the range of DNA- binding specificity.

Both of these proteins use hydrophobic amino acid residues for dimerization.

1-3 Activating regions are not well-defined structures The activating regions are grouped on the basis of amino acids content Acidic activation domains Glutamine-rich domains Proline-rich domains

Ⅱ Recruitment of Protein Complexes to Genes by Eukaryotic Activation

Eukaryotic activators also work by recruiting as in bacteria, but recruit polymerase indirectly in two ways : 2-1 Interacting with parts of the transcription machinery. 2-2 Recruiting nucleosome modifiers that alter chromatin in the vicinity of a gene.

The eukaryotic transcriptional machinery contains polymerase and numerous proteins being organized to several complexes, such as the Mediator and the TF Ⅱ D complex. Activators interact with one or more of these complexes and recruit them to the gene. Figure 17-9

Activator Bypass Experiment-Activation of transcription through direct tethering of mediator to DNA. Figure 17-10

2-2 Activators also recruit modifiers that help the transcription machinery bind at the promoter 1. Modifiers direct recruitment of the transcriptional machinery 2. Modifiers help activate a gene inaccessibly packed within chromatin

Two basic models for how these modification help activate a gene : Remodeling and certain modification can uncover DNA-binding sites that would otherwise remain inaccessible within the nucleosome. By adding acetyl groups, it creates specific binding sites on nucleosomes for proteins bearing so-called bromodomains.

Many enkaryotic activators － particularly in higher eukaryotes － work from a distance. Why? 1. Some proteins help, for example Chip protein in Drosophila. 2. The compacted chromosome structure help. DNA is wrapped in nucleosomes in eukaryotes.So sites separated by many base pairs may not be as far apart in the cell as thought. 2-2 Action at a distance: loops and insulators

Specific elements called insulators control the actions of activators, preventing the activating the non-specific genes

Transcriptional Silencing Silencing is a specializes form of repression that can spread along chromatin, switching off multiple genes without the need for each to bear binding sites for specific repressor. Insulator elements can block this spreading, so insulators protect genes from both indiscriminate activation and repression.So a gene inserted at random into the mammalian genome is often “silenced”.

Ⅲ Signal Integration and Combinatorial Control

3-1 Activators work together synergistically ( 协同的 ) to integrate signals

In eukaryotic cells, numerous signals are often required to switch a gene on. So at many genes multiple activators must work together. They do these by working synergistically: two activators working together is greater than the sum of each of them working alone. Three strategies of synergy : Two activators recruit a single complex Activators help each other binding cooperativity One activator recruit something that helps the second activator bind

3-2 Signal integration: the HO gene is controlled by two regulators; one recruits nucleosome modifiers and the other recruits mediator

3-3 Signal integration: Cooperative binding of activators at the human - interferon gene.

3-4 Combinatory control lies at the hear of the complexity and diversity of eukaryotes

3-5 Combinatory control of the mating-type genes from S. cerevisiae ( 啤酒酵母 )

The yeast S.cerevisiae exists in three forms: two haploid cells of different mating types － a and  － and the diploid formed when an a and an  cell mate and fuse. Cells of the two mating types differ because they express different sets of genes : a specific genes and  specific genes.

a cell make the regulatory protein a 1,  cell make the protein  1 and  2. A fourth regulator protein Mcm1 is also involved in regulatory the mating- type specific genes and is present in both cell types. How do these regulators work together to keep a cell in its own type?

Ⅳ Transcriptional Repressors

In eukaryotes, repressors don’t work by binding to sites that overlap the promoter and thus block binding of polymerase, but most common work by recruiting nucleosome modifiers. For example, histone deacetylases repress transcription by removing actetyl groups from the tails of histone.

Ⅴ Signal Transduction and the Control of Transcriptional Regulators

5-1 Signals are often communicated to transcriptional regulators through signal transduction pathway

Signals refers to initiating ligand (can be sugar or protein or others), or just refers to “information”. There are various ways that signals are detected by a cell and communicated to a gene. But they are often communicated to transcriptional regulators through signal transduction Pathway, in which the initiating ligand is detected by a specific cell surface receptor.

In a signal transduction pathway: initiating ligand binds to an extracellular domain of a specific cell surface receptor this binding bring an allosteric change in the intracellular domain of receptor the signal is relayed to the relevant transcriptional regulator often through a cascade of kinases.

5-2 Signals control the activities of eukaryotic transcriptional regulators in a variety of ways

Once a signal has been communicated, directly or indirectly, to a transcriptional regulator, how does it control the activity of that regulator ? In eukaryotes, transcriptional regulators are not typically controlled at the level of DNA binding. They are usually controlled in one of two basic ways : Unmasking an activating region Transport in or out of the nucleus

5-3 Activators and repressors sometimes come in pieces. For example, the DNA binding domain and activating region can be on different polypeptides. same of an activator In addition, the nature of the protein complexes forming on DNA determines whether the DNA-binding protein activates or represses nearby genes. For example, the glucocorticoid receptor (GR).

Ⅵ Gene “Silencing” by Modification of Histones and DNA

Gene “silencing” is a position effect － a gene is silenced because of where it is located, not in response to a specific environmental signal. The most common form of silencing is associated with a dense form of chromatin called heterochromatin. It is frequently associated with particular regions of the chromosome, notably the telomeres, and the centromeres.

6-1 Silencing in yeast is mediated by deacetylation ane methylation of the histones

6-1 Histone modification and the histone code hypothesis

A histone code exists ? According to this idea, different patterns of modification on histone tails can be “read” to mean different things. The “meaning” would be the result of the direct effects of these modifications on chromatin density and form. But in addition, the particular pattern of modifications at any given location would recruit specific proteins.

Transcription can also be silenced by methylation of DNA by enzymes called DNA methylases. This kind of silencing is not found in yeast but is common in mammalian cells. Methylation of DNA sequence can inhibit binding of proteins, including the transcriptional machinery, and thereby block gene expression.

Switching a gene off : A mammalian gene marked by methylation of nearby DNA sequence recognized by DNA-binding proteins recruit histone decetylases and histone methylases modify nearby chromatin This gene is completely off.

Patterns of gene expression must sometimes be inherited. These may remain for many cell generations, even if the signal that induced them is present only fleetingly. This inheritance of gene expression patterns is called epigenetic regulation. Maintenance of a phage λlysogen, can be described as an example.

Nucleosome and DNA modifications can provide the basis for epigenetic inheritance. DNA methylation is even more reliably inherited, but far more efficiently is the so-called maintenance methylases modify hemimethylated DNA － the very substrate provided by replication of fully methylated DNA.

Ⅶ Eukaryotic Gene Regulation at Steps after Transcription Initiation

In eukaryotic cells, some regulational proteins aim at elongation. At some genes there are sequence downstream of the promoter that cause pausing or stalling of the polymerase soon after initiation. At those genes, the presence or absence of certain elongation factors greatly influences the level at which the gene is expressed. Two examples : HSP70 gene and HIV

Gcn4 is a yeast transcriptional activator that regulates the expression of genes encoding enzymes that direct amino acid biosynthesis. The mRNA encoding the Gcn4 protein contains four small open reading frames (called uORFs) upstream of the coding sequence for Gcn4.

Although it is a activator, Gcn4 is itself regulated at the level of translation. In the presence of low levels of amino acids, the Gcn4 mRNA is translated (and so the biosynthetic are expressed). In the presence of high levels, it is not translated. How is this regulation achieved ?

Ⅷ RNAs in Gene Regulation

8-1 Double-standed RNA inhibits expression of genes homologous to that RNA 8-2 Short interfering RNA (siRNAs) are produced from dsRNA and direct machinery that switch off genes in various way 8-3 MicroRNA control the expression of some genes during development.

Short RNAs can direct repression of genes with homology to those short RNAs. This repression, called RNA interference (RNAi), can manifest as translational inhibition of the mRNA, destruction of the mRNA or transcriptional silencing of the promoter that directs expression of that mRNA.

The discovery that simply introducing double-strand RNA (dsRNA) into a cell can repress genes containing sequence identical to (or very similar to) that dsRNA was remarkable in 1998 when it was reported. A similar effect is seen in many other organisms in both animals and plants. How dsRNA can switch off expression of a gene ?

Dicer is an RNAse Ⅲ -like enzyme that recognizes and digests long dsRNA. The products are short double-stranded fragments. These short RNAs (or short interfering RNAs, siRNAs) inhibit expression of a homologous gene in three ways : Trigger destruction of its mRNA Inhibit translation of its mRNA Induce chromatin modifications within the promoter that silence the gene That machinery includes a complex called RISC (RNA-induced silencing complex).

RNAi silencing is extreme efficiency. Very small amounts of dsRNA are enough to induce complete shutdown of target genes. Why the effect is so strong ? It might involve an RNA-dependent RNA polymerase which is required in many cases of RNAi.

There is another class of naturally occurring RNAs, called microRNAs (miRNAs), that direct repression of genes in plants and worms. Often these miRNAs are expressed in developmentally regulated patterns.

The mechanism of RNAi may have evolved originally to protect cells from any infectious, or otherwise disruptive, element that employs a dsRNA intermediate in its replicative cycle. Now RNAi has been adapted for use as a powerful experimental technique allowing specific genes to be switched off in any of many organisms.

Summary : There are several complexities in the organization and transcription of eukaryotic genes not found in bacteria : Nucleosomes and their modification Many regulators and larger distances The elaborate transcriptional machinery Pay attention to these differences, and tell them in details by yourself.