Eukaryotic gene expression

Eukaryotic gene expression • Bacterial genes have a ground state that permits transcription • Without CAP site or operator, the sigma subunit will locate a gene • Eukaryotic genes require complex systems to turn them on • Chromatin structure must be relaxed in order for RNA polymerase to gain access to DNA sequence information • Eukaryotic genes are positively regulated. They are not transcribed in the absence of active mechanisms. • The regulatory components and systems are more complex than bacteria • Transcription is removed from translation • There are no systems equivalent to attenuation in eukaryotes

The evidence for alteration of chromosomal structure during transcription • DNase I cleaves chromatin at the linker junctions between nucleosomes • When run on an agarose gel, the DNA resulting from cleavage forms ladders reflecting discrete units increasing in size by 200 nucleotides • This means that nucleosomes cover the bulk of DNA and protect from Dnase digestion • But this procedure reveals the structure of DNA, and not any specific genes

DNAse digestion of heat shock genes • When genes are identified within these ladders, they are found in two forms • Genes that are not transcribed are also found to form ladders in response to DNAse I • Genes that are transcribed are fragmented into smaller pieces • The nucleosomes are gone in the upstream regions of genes undergoing transcription • A heat shock gene was digested over time following heat shock and the upstream region identified with a specific probe • Following heat shock, the control regions of the gene become hypersensitive to digestion

Where there are no nucleosomes • The nucleosome free sites are not throughout the entire gene, but in certain places called hypersensitive sites • Hypersensitive sites correspond to regions of DNA that bind transcription factors • Thus hypersensitive sites are found upstream of the coding region of genes • They also may be found wherever transcription factors bind • For eukaryotic genes, that is not always just 5’ to the transcriptional start

Other alterations to transcriptionally active DNA • Loss of histone H1 • This is the histone that exists between the DNA/histone octomer coils • Loss of methyl group from 5 methyl cytosine in CpG islands • Transcriptionally silent DNA tends to have more 5 methyl cytosine than active DNA

A clinical example - thalassemia • The shutdown of g globin is due to methylation of the upstream region of the genes before and after birth • In the human disease thalassemia, b and d globin chains are lost due to mutation of the globin genes. • One therapy involves administration of 5 azacytidine • Incorporation of this nucleotide results in a loss of methylation • This results in the activation of fetal globin genes which assume some of the oxygen carrying capacity of the mutant globin

Histone acetylation • Histones have two functional domains • One for binding other histones and wrapping DNA around the nucleosome core • The other is a modification site for control of histone assembly • Multiple lysine residues are presented to the exterior of the histone • Histones are acetylated prior to import into the nucleus following their synthesis on ribosomes • They are actively assembled on DNA by an enzymatic mechanism Acetylation sites Of Histone H4

Acetylation near transcriptionally active genes • A nuclear histone acetylase further acts on histones H3 and H4 • Increasing acetylation decreases the affinity of the histone octomer for DNA • This makes the DNA more available for binding interactions with other proteins • Histones are moved out of the way by an ATP driven process involving a multiprotein complex • Repressors may stimulate deacetylation • Activators may stimulate acetylation

Eukaryotic promoters • Why are they subject to positive regulation? • The genes within are sequestered because of chromatin structure • The size of the genome favors non-specific binding of regulatory proteins at random • In a diploid genome of 6 billion nucleotide pairs, a short sequence capable of binding regulatory proteins would occur many times by chance • So regulatory systems demand that multi-protein complexes form before a gene is transcribed • It is more efficient to negatively regulate the entire genome with a single mechanism (chromatin structure) and then specifically turn on the set of genes needed by the cell than to specifically negatively regulate every gene of a eukaryote • That would mean tens of thousands of repressors for each cell type

Promoters and Enhancers • Promoters include, for example, TATAA boxes, GC boxes and CAAT boxes that are responsible for positioning RNA polymerase II at the beginning of a gene • Polymerase II has no affinity for the TATAA box on its own. • Assembly of a transcriptional complex depends on the sequence around the 5’ end of the gene • Enhancers are sequences that are distant from the promoter but positively affect its function • They may be pointed in either orientation

Three classes of transcription factors • Basal (general) transcription factors • These interact directly with RNA polymerase II or with each other in building a complex around the promoter • They also recognize the promoter sequences • The TATA box is highly conserved • The TATA binding protein + transcription factors for polymerase II (TF II) assemble and provide the minimal assembly for transcription • But transcription still requires a positive signal • This complex marks the spot where RNA polymerase is to bind and begin transcription

Enhancer binding proteins • Also known as DNA binding transactivators • These bind enhancers that are far away from the promoter • They recognize the specific enhancer sequence • Some enhancer binding proteins work on a large number of genes, permitting coordinate control of transcription • Others are specific to a single gene • They then loop inward toward the promoter so that the enhancer binding protein can interact with the basal transcription factors at the promoter site • Protein-protein interactions are mediated through motifs such as the leucine zipper and the helix loop helix

Coactivator proteins • These bind RNA polymerase II complexes and enhancer binding proteins and mediate the signaling between them • RNA polymerase II may carry the coactivator proteins with it as it transcribes • Coactivators are necessary for transcription

The process of transcriptional activation • Remodeling chromatin • May involve • Demethylation of 5 methyl C • Acetylation of histones • Binding of basal transcription factors • Transactivator binding enhances the remodeling of chromatin and facilitates opening up chromatin structure • This helps other enhancer binding proteins to interact with exposed DNA sequence • Transactivators interact with coactivators and help RNA polymerase position itself on the transcription complex at the TATA box

Induction and repression • Inducibility and especially repression is not as common a phenomenon in eukaryotic cells • Especially higher eukaryotic cells • The larger the organism, the more stable the environment a cell experiences • So it needn’t respond to radical changes in the environment • However some transcriptional regulation is still necessary • Transactivators can serve the function of inducers or repressors • A repressor generally inhibits the function of an inducer by some mechanism • Competitive binding • To DNA • To basal transcription factors • Directly binding the activator

Induction and repression • Binding to a small molecule can result in an increase or decrease in the ability of a transactivator to work • Steroid hormone receptor becomes a functional DNA binding transactivator in response to binding its ligand • Binding to a ligand displaces HSP90 (a heat shock protein) and permits translocation to the nucleus and subsequent DNA binding • In the absence of ligand, it interferes with transcription and thus becomes a repressor AD: activator domain DBD: DNA binding domain LBD: ligand binding domain

A specific example • The GAL genes of yeast • These are a set of individual genes under coordinate control • Eukaryotes don’t have operons • Each gene has a promoter and set of enhancers (called UAS) • Turning on one of the GAL genes means activating a set of enhancer binding proteins and coactivators that turn on all of the other GAL genes • The products of the genes are needed for importation of galactose and its metabolism

The GAL genes can be repressed • The logic is the same as with bacteria • When glucose is present, there is no necessity to make galactose importation and metabolizing enzymes, so the genes are shut down • This repression overrides induction

Induction • Gal4p is a transactivator that induces transcription at a GAL locus by interacting with the coactivator assembly at promoter • In the absence of galactose, Gal4p is sequestered by another regulatory protein Gal80 • Gal4p is displaced from Gal80 by Gal3p when Gal3p binds galactose • Thus in contrast to bacterial inducers, the ligand binding and the DNA binding proteins are not the same

Minimum structure of enhancer binding proteins • Each must • Bind its target DNA • Bind the promoter complex and/or activating proteins • The ability of a protein to perform each function is due to functional domains in the protein • The domains permit interaction with other proteins and specific recognition of DNA sequence • In addition to DNA and protein interaction domains, there are 3 common types of activator domains • Acidic – Gal 4p • Glutamine rich – SP1 • Proline rich - CFT1

GAL 4p • This has a domain resembling a zinc finger • Instead of two cys and two his coordinating a Zn , it has 6 cys residues • It is a homodimer, bound by interactions of two coiled coils • The two zinc fingers interact with a palindromic sequence • The protein is controlled by another domain that is rich in aspartic and glutamic acid residues • This was identified by constructing mutants of the Gal4p gene that substituted other amino acids in this domain • The mutants lost function

SP1 and CTF1 • SP1 binds the GC box • GC boxes are located close to the TATA sequence • SP1 is a very common enhancer binding protein • Many genes lack a GC box • There are 3 Zn fingers for DNA binding • Two glutamine rich activator domains • CTF1 binds the CAAT box • The DNA binding domain is unique and is neither helix turn helix or a zinc finger • The activation domain is proline rich

Domain swapping • Since the domains for DNA binding and activation are distinct, their domains may be separated on the level of DNA • By taking a domain for DNA binding and adding it to a domain for activation, a new protein may be engineered • This binds the DNA sequence specified by one gene, and responds to the signals of another • Such experiments permit the manufacture of proteins with unique control abilities • Although not therapeutically useful right now, they are important experimental tools in defining the way that genes respond to external signals.

Regulated gene expression • Gene expression in multicellular organisms is often controlled by intercellular signaling • Some genes are directly responsive to environmental stimulus however • UV induction of DNA repair enzymes • Stress response (heat shock) genes • Signaling takes two forms • Hormones may be bound by • Membrane bound receptors • Diffusible receptors

Diffusible receptors • Diffusible receptors act by directly binding a hormone and then moving into the nucleus • Hormone binding induces a conformational change that permits the receptor to act as a transcriptional activator • Diffusible molecules can be transactivators • “trans” means something that acts on a gene that originates from another site. In this fashion they resemble the activators of bacteria • However hormones are made by one cell in order to command a transcriptional response in another cell • Bacterial effectors are nutrients or their metabolites or analogs

Steroid hormones • These are • endocrine hormones • hydrophobic molecules that are synthesized using cholesterol as a precursor • made by certain cell types and secreted in response to biochemical, developmental or neurological signals • carried by the blood from their cell of origin to target cells either dissolved or by a protein carrier • Many are too hydrophobic to dissolve directly in blood • They enter a cell by dissolving in the plasma membrane and diffusing to their receptor

The steroid hormone receptor • This is a DNA binding protein with a hormone binding domain at the carboxyterminal end of the protein • There are several related types • Each receptor has a specific complement of transcription factors it must interact with which vary from one receptor to another and one cell type to another • They are all related in structure • The DNA binding domain contains two Zn fingers and is in the middle of the protein • The domain that interacts with transcription factors is amino terminal and varies in structure • The hormone binding region is highly variable in structure • Each must recognize and bind its cognate ligand

MUTATIONS • Loss of responsiveness to a hormone can be caused by changes in any of the three domains • Hormone-ligand complexes may serve either positive or negative regulatory functions • Mutations prevent transcriptional activation or repression in response to hormone binding • Mutation in the androgen binding domain of the androgen receptor creates androgen unresponsiveness • Mutation in the DNA binding or transcription activation domains would mean the protein could bind androgen, but nothing would happen • This results in developmental abnormalities such as XY females • To the left are four XY siblings suffering from androgen insensitivity

The cis elements • Cis refers to sequence involved in gene expression • Trans elements interact with cis elements but arise from other genes • The glucocorticoid responsive element (GRE) and estrogen responsive element (ERE) share sequence homology • The cis elements that are important in hormone responsiveness are the binding sites for the hormone-receptor complexes • Hormone responsive elements: HRE • These are direct repeats that interact with the Zn finger domains • The consensus sequences for these receptors are very similar • This reflects the similarity in the Zn finger domains among the various receptors

Receptor – DNA binding • Following binding of a hormone, the receptor diffuses to the nucleus and binds the HRE • The receptor is a dimer • Each subunit of the dimer binds to one of the two repeat elements • The strength of binding is determined by the variation of the HRE away from the consensus sequence • The stronger the binding between the receptor and HRE, the longer the receptor will remain bound and the longer transcription will be activated • Binding is an all or none event • If bound, activation due to the receptor is full

Phosphorylation of transcription factors • Transcription factors are subject to phosphorylation on serine and threonine residues • This is the result of second messenger activation of serine-threonine kinases or ras activation • In abnormal, though common, conditions, such as mutations or viral infections, gene expression is deregulated and genes are inappropriately expressed because of deregulated phosphorylation mechanisms • This is because second messengers activate a complex cascade of enzymatic steps that can be perturbed at many different points • Here the transcription factor Elk-1 is activated through phosphorylation

Repression • This occurs at the transcriptional and translational level • Genes are usually turned off as a default at the transcriptional level • But this does not mean the mRNA is gone • It could have been stabilized through sequestration • Translational regulation permits rapid responsiveness • The primary transcript of a gene may take several minutes to synthesize because of its size • It also must be spliced and transported to the ribosomes • A sequestered transcript that is released in response to a signal is faster

Translational repression affects more than just ribosomal proteins • The distribution of mRNA within some cells creates a distribution of protein inside a cell • This results in intracellular protein gradients that are important in development

Regulatory mechanisms of translational initiation • Inhibition of initiation factors through phosphorylation • eIF phosphorylation inhibits its function and can be reversed through dephosphorylation • Inhibition of initiation factors by binding to specific factors • Interference with eIF4E and eIF-4G activity by 4E-BP’s. • Inhibition of specific mRNA by binding of inhibitory proteins to sequences in the 3’untranslated region

Phosphorylation of eIF-2 inhibits its activity • Maturation of red blood cells involves a stage in which reticulocytes translate mRNA left behind after the loss of the nucleus • Reticulocytes regulate the amount of globin synthesized by phosphorylating eIF-2 • When there is heme deficiency globin synthesis is wasteful since hemoglobin cannot be synthesized • Low heme activates HCI which phosphorylates eIF-2 • Phosphorylated eIF2 binds eIF2 binding protein and is unavailable for translational initiation

eIF4E inhibition • eIF4E is necessary to bind the 5’ CAP in order to from an initiation complex for translation • Normally it binds eIF4G • Maskin binds eIF4E (preventing it from binding eIF4G) when it is bound to an mRNA through interaction with CEPB

Developmental control of gene expression • The study of fruit fly development resulted in the discovery of a number of genes involved in human disease • Although fruit fly development is greatly different in the processes leading to the final form, the activation of genes and the structure of gene products and their participation in the formation of patterns and structures have parallels in human gene regulation and the structure of human regulatory gene products

Fly development is controlled by gene expression • The conceptually difficult part of this is to understand how a single cell can create multiple, morphologically different structures starting from a seemingly symmetrical, undifferentiated state merely by dividing. • It is easier to think of the process in parts and then add up the whole than to see a cell turn into a fly and attempt to understand the entire process at once

Three gene families are responsible for early development • Maternal genes • Made by the female and exist within the egg at the time of fertilization • Responsible for establishing the polarity of the early embryo • Zygotically acting genes • Segmentation genes • These establish a repeating pattern of body segments • Homeotic genes • These establish the identity of the segments

Polarity • This is a distinction in structure established between two poles. • The distinction needn’t be great, only a morphological difference is enough to create polarity • Without polarity, further structures would have no way of organizing themselves • Segments would be repeating structures that are all the same • Establishing polarity is thus the earliest developmental event • Polarity is actually established by the assymetry of the egg • This yields pole cells on one end of the zygote

Segmentation • This is obvious in the formation of the fly abdomen • Repeating abdominal segments are very similar in appearance • However this patterning extends from end to end of the fly • The patterns are given different identities by homeotic genes • Thus the head and abdomen begin as segments similar to abdominal segments • But polarity makes them different, and therefore the genes that are expressed within each segment differs • Segments are created and further divided into smaller segments • Gap genes create the largest segments • Pair rule and segment polarity genes subdivide the largest segments

Homeotic genes • These give rise to the dramatic mutants of Drosophila • Once segments are established with the proper polarity, homeotic genes create unique structures • Mutation of a particular homeotic gene results in the formation of a structure that is due to the action of another homeotic gene • The homeotic genes are controlled by their position within a gradient of polarity • So if one segment was destined to give rise to antennae, but lacked the homeotic gene due to mutation, it would create the next most available structure

Antennaepedia • Antennaepedia represents the mutation of a gene that would create an antennae • Antennapedia is a transcription factor that coordinately controls expression of genes, that when expressed result in an antennae • In its absence, the next most similar structure is a leg • The normal leg is also formed in the next segment • The gene encoding the protein controlling leg development is expressed at lower concentrations in the head segment than antennapedia gene, but in the absence of antennapedia, it is the most highly expressed protein capable of activating genes that result in a structure

Key genes are expressed early • Maternal genes establish polarity due to formation of gradients • Front to back and top to bottom gradients establish anterior posterior and dorsal ventral gradients • When cells are formed in the blastoderm, they form within an environment in which the concentration of transcription factors will vary along one axis • This varies the type and numbers of genes that are expressed within any cell • To the left is bicoid RNA (upper) and bicoid protein (lower) in the early embryo • The RNA gradient is present in the egg and establishes the protein gradient

A few examples of developmental genes • Bicoid is a maternal gene that controls expression of segmentation genes • It is a transcription factor that activates segmentation genes • And a translational repressor • It appears in the anterior of an egg, and its concentration falls of towards the posterior • The gradient is maintained during formation of the larvae • Experiments with bcd mutants • (a) A failure of bicoid to be expressed means a fly develops with two posteriors rather than an anterior and a posterior • (b)injecting cytoplasm from a normal embryo rescues the embryo (makes an anterior) • ( c) injecting bicoid mRNA also rescues

What bicoid does • It represses translation of caudal in the anterior of the fly larvae • Caudal is a transcription factor found uniformly throughout the larvae, and it creates the posterior end • And activates expression of hunchback • Hunchback is a transcription factor that creates the anterior end • The bicoid gradient means it has these effects only in the anterior end • Without bicoid, caudal is not repressed in the anterior end and hunchback is not transcribed

Nanos • This is a translational repressor that is found at highest concentrations in the posteror of a fly larvae • It acts in concert with the uniformly distributed pumilio gene product to translationally repress hunchback • This results in establishment of high caudal gene product and inactivated hunchback mRNA, meaning the posteriorizing effect of caudal dominates

Some Gap genes • Gap genes create a gross form of segmentation in the early embryo • They are overlayed onto the pair-rule gene expression to create complex transcriptional signals • These are the expression patterns of the hb-z, Kr and kni genes

fushi tarazu and eve • These are two segmentation genes known as pair rule genes that split a segment in two • Ftz establishes the “pair rule” • Two segments form out of one • Without it the fly forms 7 rather than 14 segments • Ftz (blue) is expressed in each segment, in the anterior half of the segment • This expression pattern is again the result of the action of an anterior posterior gradient, but now within each segment • Eve (brown) for even-skipped are expressed in the posterior half of each segment • Both ftz and eve are homeodomain transcription factors that control expression of genes expressed in the segments

Eukaryotic gene expression