Flashcards in gene expression Deck (65):
Gene expression is a consequence of RNA synthesis, processing, stability, and translation, as well as post-translational processing and protein stability. Examples of regulation can be found at each step.
Control of Transcription Initiation
transcription initiation is the MOST important mechanism for determining whether or not most genes are expressed, and consequently how much of which particular protein a specific cell contains.
DNA Control Elements
DNA elements that act locally. Binding of transcription factors to these elements controls expression of the gene that the element is associated with.
TATA box/Initiator sequence
a type of dna control element, generally 25-35 bps upstream of the transcription start site. It determines the site of transcription initiation and directs binding of RNA polymerase II. This is the site at which general transcription factors bind.
Promoter Proximal Elements
located within 200 bps upstream of transcription start site and are ~ 20 bps long, help to regulate transcription, and can be bound by factors in a cell type specific manner, a type of dna control element, effects the basal recruitment of transcriptional machinery
Contain multiple DNA control elements, each 8-20 bps in length, region of DNA that can be bound with proteins (activators) to activate transcription of a gene or genes. Thus, an entire enhancer can be 100-200 bps long. An enhancer can be 200-tens of kilobases upstream or downstream from the promoter or the last exon of the gene, or within an intron. Similar to promoter proximal elements, enhancers may help to regulate transcription in a cell type specific manner.
is usually a DNA sequence that contains a gene. This gene codes for a protein (or microRNA or other diffusible molecule) that will be used in the regulation of another target gene. The trans-acting gene may be on the same chromosome as the target gene, but the activity is via the intermediary protein or RNA that it encodes. Trans-acting factors can switch from repressors to activators by recruiting either HATs or HDACs (ex-thyroid hormone receptor)
cis-Regulatory Elements (CREs)
are regions of non-coding DNA which regulate the transcription of nearby genes.
what are the two basic regulators of eukaryotic gene expression?
DNA control elements (cis) and transcriptional activators/ repressors (trans)
An inherited anemia due to deficient production of b-globin protein by erythroid cells. Can occur due to different types of mutations- one of which can occur in the b-globin promoter (DNA control element), reducing the amount of b-globin mRNA and thus protein produced (is usually clinically mild).
a globin protein, which along with alpha globin (HBA), makes up the most common form of hemoglobin in adult humans, the HbA
Hemophilia B Leyden
Is an X-linked disorder that affects clotting. Affected males have 1% of normal factor IX active until puberty due to inherited mutations in a DNA control element in the promoter of the Factor IX gene (which prevents the binding of the appropriate transcriptional activators). Alternative transcriptional activators (which have an androgen receptor) can bind overlapping sites in the promoter, and at puberty it can bind at the promoter site and increase transcription such that males after puberty make ~60% the normal amount of factor IX.
Affects 1 in ~1500 males and results in mental retardation, dysmorphic facial features, and postpubertal macroorchidism. CGG repeat in the 5’ region of the FMR1 gene facilitates methylation of the cytosine residues in CpG islands and transcriptional inactivation of the FMR1 gene. Normal males have 6-50 or so CGG repeats in this region, however, affected males have an expansion of this repeat sequence (>200 copies)- leading to increased transcriptional silencing of the FMR1 gene.
Transcriptional Activators and Repressors
Proteins encoded by one gene that act on other genes to regulate their transcription. Can therefore diffuse around the nucleus and affect transcription of numerous genes. Can either activate or repress transcription. Multiple activators stimularte the transcription from both proximal and distal site. It is the combination of these protiens that lead to when, where, and how much of a protein is transcribed. the two classes inculde sepuence- specific DNA binding proteins and co-factors. Can recruit either histone acetyltransferases (HATS) or deacetylases (HDACs), eg if bound to hormone, changes conformation of TF.
Sequence-specific DNA binding proteins
bind to promoter or enhancer elements (DNA control elements) in their target genes to regulate transcription. The elements they bind to are usually 6-8 base pairs long. Usually bind DNA by inserting their a-helices into the major groove of DNA, making contacts between the amino acid side chains of the protein and the bases in the DNA . Contain two domains, DNA binding domain and activation/ repression domain, which allow you to mix and match (modularity)
DNA binding domain
The DNA binding domains (DBDs) are highly structured and evolutionarily conserved. They are folded so that they can “read” the DNA sequence and bind to their specific target DNA. Devides sequences- specific DNA binding proteins into different families, which have similar tertiary structure
helix-turn-helix structure in which three alpha helices are connected by short loop regions. Many homeodomains induce cellular differentiation by initiating the cascades of coregulated genes required to produce individual tissues and organs. Members include Hox family, Pit1, Msx, etc.
is characterized by the coordination of one or more zinc ions in order to stabilize multiple finger-like protrusions that make tandem contacts with their target molecule. Members include nuclear receptors such as estrogen receptor, androgen receptor, retinoic acid receptor
Basic leucine zipper proteins (bZIP)
contains a region that mediates sequence specific DNA binding properties and the leucine zipper that is required to hold together (dimerize) two DNA binding regions. The DNA binding region comprises a number of basic amino acids such as arginine and lysine. Members include c-fos and c-jun
Basic helix-loop-helix motif (bHLH)
is characterized by two α-helices connected by a loop. In general, transcription factors including this domain are dimeric, each with one helix containing basic amino acid residues that facilitate DNA binding. Members include MyoD, myogenin, Myf5
Craniosynostosis is characterized by the premature closure of one or more sutures in the skull and affects 1/3000 infants. One particular variant of craniosynostosis (Boston-type) occurs as a result of a mutation in the homeodomain protein MSX2. MSX2 is normally required for proper craniofacial development by affecting the transcription of a number of genes important in this process. When the DNA binding domain (or homeodomain) of this protein has a one amino acid substitution, the protein binds DNA more strongly- giving a “gain of function” or “hypermorphic allele”. This mutated hyperactive protein then affects the transcription of other genes critical for suture closure, leading to craniosynostosis.
Androgen insensitivity syndrome (AIS)-
It occurs in males who are a normal karyotype (46 X,Y), but have mutations in either the DNA binding domain or the ligand binding domain of the androgen receptor (a zinc finger DNA binding protein). This makes the patients less responsive to androgens, leading to feminization or undermasculinization of the external genitalia at birth, abnormal secondary sexual development in puberty, and infertility. Depending on the degree to which the mutation disrupts the function of the androgen receptor- varying levels of AIS can be observed (complete, partial, mild).
Waardenburg Syndrome type II
Is characterized by deafness, pigmentation anomalies of the eyes, and other pigmentation defects (hair, skin). Mutations in the microphthalmia-associated transcription factor (MITF) gene (which encodes a bHLH DNA binding protein) are observed in 15-20% of the patients. This gene encodes a transcription factor that plays a major role in the development of melanocytes.
Dimerization of sequence specific DNA binding proteins
Many sequence specific DNA binding factors bind DNA as homo or heterodimers. The Zinc finger, bZIP, and bHLH can all form heterodimers. If each monomer of the heterodimer has a different DNA binding specificity, the formation of heterodimers will increase the number of potential sequences to which that family of sequence specific transcription factors can bind. “Combinatorial Control” can both regulate expression through new combinations of activation domains and change the DNA binding site
How do transcriptional activators or repressors, once bound to DNA control elements, stimulate transcription?
1) regulate assembly of initiation complexes and rate of initiation of transcription 2) regulate changes in chromatin structure influencing the ability of general transcription factors to bind to promoters
What are the two classes of chromatic- remodelling factos?
DNA-dependent ATPases (SWI/SNF)- disrupt histone octamers and DNA and F\factors that reversibly modify histones through acetylation (HATs and HDACs)
a basic unit of DNA packaging in eukaryotes, consisting of a segment of DNA wound in sequence around eight histone protein cores. The histone core complex includes 2 molecules of H2A, H2B, H3, and H4 histones. The N-termini of histones are rich in lysine residues (basic), which can be reversibly modified by acetylation, phosphorylation, methylation, and ubiquitination. Acetylation is associated with gene control.
are electrostatic, The phosphodiester backbone of the DNA associates with positively charged amino acids (mainly lysine and arginine residues) located in the histone-fold domains. DNA is distorted as it is wrapped around the octamer
H1 does not make up the nucleosome "bead", but sits on top of the structure, keeping in place the DNA that has wrapped around the nucleosome. H1 is present in half the amount of the other four histones, which contribute two molecules to each nucleosome bead. In addition to binding to the nucleosome, the H1 protein binds to the "linker DNA" (approximately 20-80 nucleotides in length) region between nucleosomes, helping stabilize the 10nm fiber into the zig-zagged 30 nm chromatin fiber.
How does the transcription machinery gain access to its binding sites buried in chromatin?
transcription activators recruit or stimulate the use of: 1) ATP dependent chromatin remoldelers, 2) histone modifying enzymes, 3) histone chaperones to remove histones from the DNA, 4) histone eschange for more transcriptionally favorable histone variants
ATP-dependent chromatin remodelers
Use the energy of ATP hydrolysis to break histone-DNA contacts in order to move the histone octamer along the DNA, creating room for transcription factors. Is cancer related
types of histone post-translational modifications
1) covalent modification of histone proteins, 2) Modifications that indirectly regulaate chromatin structure through recruitment of chromatin- associated proteins, including histone tail modifications, 3) proteins recruited by these modifications, including TF, ATP- dependent nucleosomal remodeling enzymes
a post-translational modification (an addition to a protein after it has been made) where ubiquitin is attached to a substrate protein. The addition of ubiquitin can affect proteins in many ways: It can signal for their degradation via the proteasome, alter their cellular location, affect their activity, and promote or prevent protein interactions.
Histone acetyltransferases (HATs)
are co-activator enzymes that acetylate conserved lysine amino acids on histone proteins by transferring an acetyl group from acetyl CoA to form ε-N-acetyllysine. DNA is wrapped around histones, and, by transferring an acetyl group to the histones, genes can be turned on and off. In general, histone acetylation increases gene expression. It was first thought that HATS neutralize the positize charge of histones. But now they know that acetylation of lysines allows for the binding of specific transcription factors, and that the pattern of histone acetylation thus serves as a “code” to recruit different factors that will then affect the transcriptional state. Thus, it is more complex than originally thought. examples include CBP and p300. Many sequence specific activators recruit multiple HATs
are a class of enzymes that remove acetyl groups (O=C-CH3) from an ε-N-acetyl lysine amino acid on a histone, allowing the histones to wrap the DNA more tightly.
Histone Code Hypothesis
This model proposes that distinct patterns or combinations of modifications at conserved residues in the histone amino terminal domains are important for the recruitment of specific chromatin-associated proteins. These chromatin-associated proteins play various roles in altering local chromatin structure, with important consequences for DNA metabolic processes such as transcription, DNA repair, and recombination.
A rare genetic multisystem disorder (affects 1/125,000). Characterized by growth retardation, mental retardation, craniofacial dysmorphism, abnormally broad thumbs and great toes. Results from mutations in one copy of the CREB binding protein (CBP) gene. CBP is an essential transcriptional coactivator for many different transcription factors and is a histone acetyltransferase. It is normally recruited to many genes to activate transcription, and thus haploinsufficiency can result in widespread transcriptional changes.
occurs when a diploid organism has only a single functional copy of a gene (with the other copy inactivated by mutation) and the single functional copy does not produce enough of a gene product (typically a protein) to bring about a wild-type condition, leading to an abnormal or diseased state. It is responsible for some but not all autosomal dominant disorders.
is always heterochromatin and contains satellite DNA. Example - centromeric DNA
can change to euchromatin, depending on the cell type or developmental stage, and is enriched in LINE sequences. Example - X-inactivation
CREB binding protein (CBP)
is ubiquitously expressed and is involved in the transcriptional coactivation of many different transcription factors, mainly cAMP-response element-binding protein. CBP is an essential transcriptional coactivator for many different transcription factors and is a histone acetyltransferase. Also has intrinsic histone acetyltransferase activity and also acts as a scaffold to stabilize additional protein interactions with the transcription complex.
Chronic myelogenous leukemia (CML)
a cancer of the white blood cells. It is a form of leukemia characterized by the increased and unregulated growth of predominantly myeloid cells in the bone marrow and the accumulation of these cells in the blood. It is associated with a characteristic chromosomal translocation called the Philadelphia chromosome, leading to gain of function fusion proteins- some of which involve fusions of transcriptional regulators with HATs or HDACs, altering the activity of the regulators .
Overview of Basic Principles of Transcriptional Regulation
1. Specificity depends on binding of transcriptional activators or repressors to DNA control element 2. Regulation depends on protein-protein and protein-DNA interactions 3. The interactions affect conformation of DNA, modification of chromatin structure, formation of transcription initiation complex 4. Control is combinatorial, allows for several thousand TFs to differentially alter the expression of genes in different cell types in response to different stimuli
How are the sequence-specific DNA binding proteins regulated?
1. The conformation of the DNA-binding protein can be altered by ligand binding 2. Entry into the nucleus can be regulated 3. The amount of transcription factor in the cell can be regulated 4. DNA binding can be regulated 5. Phosphorylation of the DNA-binding protein can alter various properties including protein degradation, recruitment of co-activators, and DNA binding
Examples of sequence-specific DNA binding proteins regulation through altering ligand binding
The nuclear receptor family of Zinc finger transcription factors work by binding to steroid hormones (they have a hormone binding domain), which affects its conformation. This binding can lead to dimerization of receptor and bind to DNA, which leads to recruitment of coactivators and its translocation into the nucleus. Examples include estrogen receptor activation and glucocorticoid receptor activation
their receptors are located inside the cell within the cytoplasm of the target cell. These receptors belong to the nuclear receptor family of ligand-activated transcription factors. To bind their receptors, these hormones must first cross the cell membrane. They can do so because they are lipid-soluble. The combined hormone-receptor complex then moves across the nuclear membrane into the nucleus of the cell, where it binds to specific DNA sequences, regulating the expression of certain genes, and thereby increasing the levels of the proteins encoded by these genes.
is currently used for the treatment of both early and advanced ER+ (estrogen receptor positive) breast cancer. Some breast cancer cells require estrogen to grow. Estrogen binds to and activates the estrogen receptor in these cells. Tamoxifen is metabolized into compounds that also bind to the estrogen receptor but does not activate it or alters conformation of estrogen receptor to recruits co-reprossors (in some cell types). It is a competitive antagonism, preventing estrogen from binding to its receptor. Hence breast cancer cell growth is blocked.
is the receptor to which cortisol and other glucocorticoids bind.When the GR binds to glucocorticoids, its primary mechanism of action is the regulation of gene transcription. The unbound receptor resides in the cytosol of the cell. After the receptor is bound to glucocorticoid, the receptor-glucorticoid complex can take either of two paths. The activated GR complex up-regulates the expression of anti-inflammatory proteins in the nucleus or represses the expression of pro-inflammatory proteins in the cytosol (by preventing the translocation of other transcription factors from the cytosol into the nucleus).
is a "rapid-acting" primary transcription factors, (transcription factors that are present in cells in an inactive state and do not require new protein synthesis in order to become activated) and and is involved in cellular responses to stimuli such as stress, cytokines, free radicals, ultraviolet irradiation, oxidized LDL, and bacterial or viral antigens. It is normally held in the cytoplasm by its binding to the inhibitor of NFκB (IκB), which masks the nuclear localization signal of NFκB. In response to stimuli, IκB can be phosphorylated leading to addition of ubiquiten tag, targeting it for degradation by a proteosome. with commensal bacteria, there is a block of ubuquitination, preventing degradation. NFκB is then released and moves to the nucleus, where it turns on a number of target genes, including those involved in inflammation. Aspirin, works in part by inhibiting the phosphorylation, and thus preventing degradation of IκB and translocation of NFκB to the nucleus, thus inhibiting the transcription of genes involved in the inflammatory response. Is an example of regulating nuclear entry of sequence-specific DNA binding proteins
Nuclear factor of activated T-cells (NFAT)
a general name applied to a family of transcription factors shown to be important in immune response and proper function in the heart. High intracellular calcium (triggered by the binding of a ligand to a cell membrane receptor) activates calcineurin’s phosphatase activity, which dephosphorylates cytoplasmic NF-AT. This exposes the nuclear localization sequence, allowing NF-AT to enter the nucleus where it affects transcription of genes involved in the immune response and in heart function. ] Is an example of regulating nuclear entry of sequence-specific DNA binding proteins
is a calcuim dependent protein phosphatase, activates the T cells of the immune system and two immunosuppresants (cyclosporin and FK506) act by inhibiting calcineurin, thereby inhibiting NF-AT action. is targeted by cyclosporin, which inhibits calcineurin
is a dual function protein, regulating the coordination of cell–cell adhesion and gene transcription. In the absence of Wnt singaling, beta catenin is targeted for degradation through the ubiquitin-proteasome pathway through phosphoryation by glycogen synthase 3 (GSK3) in a complex with Axin and APC. In the presence of Wnt signaling, the Axin-APC-GSK3 complex is destabilized, preventing phosphorylation of β-catenin and leading to an increase in the cytoplasmic pool of the protein. This increase allows some of the β-catenin to move to the nucleus, where it interacts with the TCF family of transcription factors and promotes the expression of Wnt responsive genes. It is an example of regulating amount of transcription factor in the cell. This pathway is really important in proliferation of colon cells
Wnt signaling pathway
are a group of signal transduction pathways made of proteins that pass signals from outside of a cell through cell surface receptors to the inside of the cell. Plays a role in embryonic development, cell fate and proliferation. It is also associated with cancer and type II diabetes
Glycogen synthase kinase 3 (GSK3)
s a serine/threonine protein kinase that mediates the addition of phosphate molecules onto serine and threonine amino acid residues. is mediated by to Axin
Anaphase-Promoting Complex (APC)
is an E3 ubiquitin ligase that marks target cell cycle proteins for degradation by the 26S proteasome. Mutations in this gene are related to colon cancer. It is a tumor supressent gene leading to a huge build up of beta catenin. Even among those with colon cancer and without germline mutation, 80% have tumor mutation in APC
TCF family of transcription factors
a group of transcription factors which bind to DNA through a high mobility group domain. They are involved in the Wnt signaling pathway, where they recruit the coactivator beta-catenin to enhancer elements of genes they target.
is crucial in multicellular organisms, where it regulates the cell cycle and, thus, functions as a tumor suppressor, preventing cancer. It is a sequence specific DNA binding protein, which activates the transcription of genes in cell cycle assert and cell death. It is downregulated by binding to the MDM2 protein (expression is also controlled by p53, negative feedback loop) which not only masks its activation domain, but also targets it for destruction by the ubiquitin-proteasome pathway. It is an example of regulating amount of transcription factor in the cell
common in cancer of all kinds, in tumors with mutated p53, there is actualy a high level of MDM2 because p53 activates transcription of MDM2, but it also makes it dominent negative, inhibiting the wildtype protein
dominant negative mutation
A mutation whose gene product adversely affects the normal, wild-type gene product within the same cell, usually by dimerizing (combining) with it. In cases of polymeric molecules, such as collagen, dominant negative mutations are often more deleterious than mutations causing the production of no gene product (null mutations or null alleles).
an important negative regulator of the p53 tumor suppressor.
Inhibitor of DNA-binding/differentiation proteins (Id proteins)
are key regulators of development where they function to prevent premature differentiation of stem cells and are a family of proteins that heterodimerize with basic helix-loop-helix (bHLH) transcription factors to inhibit DNA binding of bHLH proteins, (It titrates out the correct co-dimer bHLH protein) by preventing DNA binding due to their lack of a basic domain. It is an example of regulating DNA binding to sequence specific DNA binding proteins
CREB (cyclic AMP response element-binding protein)
TF invloved in learning and memory. Is normaly present and bounded to its target site but is unable to activate transcription when unphosphorylated. A series of events initiated by the binding of a ligand to a guanine nucleotide binding protein coupled receptor induces the phosphorylation of the CREB protein. Once phosphorylated, the CREB protein recruits the histone acetyl transferase, CBP (CREB binding protein), which has intrinsic HAT activity and recruits RNA Pol II- leading to transcriptional activation of the gene. It is an example of how phosphorylation affects activity of trans-acting factors
mechanisms of transcription repressors
1) competitive binding with activator at same DNA binding site, 2) interaction with activation domain of bound activator from a different DNA binding site, 3) interaction with general transcription factors (eg blocking tata box
What are some other ways to control levels of gene expression?
1. Control mRNA export from the nucleus 2. Control mRNA degradation 3. Control efficiency of translation 4. miRNAs (can control mRNA degradation and translation) 5. Control protein degradation