Mod 10: Transcriptional Control Mechanisms in Eukaryotes

Yeast two-hybrid analysis (IN VIVO) technique

**involves reporter gene fusions to identify two proteins that interact with each other. This process also uses GAL gene system; GAL genes encoding enzymes for galactose metabolism are under the regulation of GAL4 protein, which binds to an upstream GAL4p regulatory sequence. GAL4 protein has two domains: DNA binding domain, and activation domain. Steps: 1) X and Y are fused to one domain of Gal4p (yeast GAL gene) that is a little upstream of a reporter gene 2) X and Y bound to their domain will interact with RNA polymerase 3) the readout is a reporter gene required for the survival of the yeast cells to grow on a particular growth medium 4) If proteins X and Y do interact then the yeast will survive on the selective growth medium and are measured by reporter gene expression

RNAi mediated by siRNA (IN VIVO):

**used to knockdown gene expression for assessment of gene function. Steps: 1) delivery of RNAi constructs to target particular mRNAs. This enables assessment of phenotypes due to reduced levels of a particular protein, which is not necessarily completely knocked out 2) Reverse transcriptase to make cDNA 3) integrate into host DNA 4) Drosha mediated processing to form hairpin 5) Export hairpin RNA to where it converts to siRNA 7) siRNA can silence mRNA now and stop transcription Knock-out strain created; tedious

Describe the steps for Steroids Hormone Signaling: Type I

1) Nuclear receptor "NR" is retained in the cytoplasm (aka cytoplasmic receptor) through its interaction with Heat shock protein 70:"Hsp70." 2) Steroid hormone easily diffuses through the membrane phospholipid bilayer due to both the hydrophobic nature of both the steroids and the fatty acid tails of the membrane. 3) Steroid binds to NR receptor, the Hsp70 protein is released 4) The NR receptor then dimerizes to expose a nuclear import signaling domain in the protein so that NR hormone complex can enter the nucleus (aka receptor hormone dimer moves to nucleus). 5) NR hormone complex functions as a transcriptional activator by binding to DNA regulatory regions called hormone response elements (HRE) --> RNA polymerase II and coactivator associate with the complex --> activates transcription.

Explain the cytokine signaling pathway: JAK-STAT

1) The transmembrane receptor binds cytokines ( e.g., interferon and interleukin), small molecules that signal cells to grow or differentiate. 2) On cytokine binding, the receptor activates JAK, which phosphorylates the receptor. 3) This, in turn, promotes binding and phosphorylation of STAT by the phosphorylated receptor. 4) Once phosphorylated, STAT dimerizes and enters the nucleus, where it binds DNA and activates the expression of genes involved in cell growth and differentiation.

Explain the Hg and Wg pathway

1) Wnt (Wg protein) binds to membrane receptor "frizzled;" 2) activation of Dsh and subsequent INactivation of Zw3 2) this causes activated/intact beta catenin that enters the nucleus where it displaces Graucho ** If there is no activation of beta catenin then it is subject to degradation and wingless target genes are transcriptionally silent due to the action of the repressor called Graucho 4) Beta catenin itself serves as a transcription coactivator for wingless target genes downstream 3) transcription of wingless target genes encodes the hedgehog signaling ligand/protein 4) hedgehog protein can signal two transmembrane proteins: patched and smoothened; hedgehog protein/ligand binds to patched directly since patched is the actual hedgehog receptor. 6) Smoothened is located by patched so it gets activated after patched. In the absence of smoothened activation, hedgehog targeted genes are repressed by cleavage by a proteolytic fragment of a protein called cubitus interruptus 7) in normal situation, intact cubitis interruptus protein translocates to the nucleus where it functions as an inducing transcription factor for which the wingless signaling ligand/protein is one of the target genes.

Dicer

1) a double-stranded RNA endonuclease in eukaryotic cells that works within the RNA-inducing silencing complex (RISC) and cleaves hairpin-shaped precursor RNAs "pre-mRNA"(~70 nucleotides long) into mature miRNAs that are 21-23 nucleotides long through the recognition of dinucleotide overhangs. Dicer also functions to cleave long double-stranded RNAs that are made in the cell or derived exogenously, and cleaves them through hydrolysis into short duplexes of 20-22 nucleotides long, such as small interfering RNAs known as siRNAs that are double stranded with 3' overhangs.

Deeper explanation of RNA silencing with Drosha and Dicer

1) endogenous primary microRNA transcripts (pri-mRNA) are produced in the nucleus via transcription 2) Drosha, endonuclease in eukaryotic cells, cleaves the endogenous primary transcript "pri-mRNA" hairpin duplex to produce precursor miRNAs(pre-mRNAs) which are about 60-70 nucleotides and double-stranded 3) pre-mRNA are loaded onto RNA-inducing silencing complex (RISC) which includes Dicer 4) Dicer, a double-stranded RNA endonuclease in eukaryotic cells, works within the RNA-inducing silencing complex "RISC" (dicer + argonaute) and cleaves hairpin-shaped precursor RNAs "pre-mRNA"(~70 nucleotides long) into mature miRNAs that are 21-23 nucleotides long (even shorter now!) through the recognition of dinucleotide overhangs. Dicer also functions to cleave long double-stranded RNAs that are made in the cell or derived exogenously, and cleaves them through hydrolysis into short duplexes of 20-22 nucleotides long, such as small interfering RNAs known as siRNAs that are double stranded with 3' overhangs. 5) After cleavage by Dicer, another endonuclease called Argonaute also cleaves 6) now RNA helicase can unwind the DNA properly into single stranded mature miRNA. Following the formation of mature mRNA, two outcomes can result: partial complementarity with cytoplasmic mRNA which results in repressed translation, or near-perfect complementarity which results in mRNA degraded.

Explain RTK: Receptor Tyrosine Kinase pathway

1) ligand (i.e insulin) binding induces auto-phosphorylation of tyrosine amino acids in the cytoplasm tails of RTK 2) cascade of phosphorylation events is set off starting with IRS-1 that nucleates the formation of a protein complex 3) phosphorylation of a series of protein kinases (Ras-Raf1- MEK-ERK) 4) activated ERK enters the nucleus and phosphorylates transcription factor Elk1 5) ELK1 stimulate expression of target genes.

Describe the process of GPCR signaling

1) ligand binds to GPCR receptor protein that is interacting with G-protein in the presence of GTP 2) Once activated, the G-protein dissociates from the receptor and activates adenylyl cyclase (converts ATP to cAMP). 3) Levels of cAMP increase 4) cAMP acts as a second messenger that carries a message received from outside the cell such (from a first messenger) to deliver it inside the cell 5) cAMP activates a cAMP-dependent protein kinase A (PKA) 6) PKA phosphorylates numerous protein targets resulting in alterations in gene expression 7) Phosphorylated PKA causes activation/phosphorylation of transcription activator CREB (cAMP responsive element binding protein) 8) CREB binds to CRE (cAMP response element) in the DNA 9) Transcription is activated through coactivator CBP (CREB binding protein).

Describe the steps for Steroids Hormone Signaling: Type II

1) nuclear receptors are bound to DNA regardless of the presence of the hormone signal Example: thyroid hormone (TR) 2) steroid hormone diffuses through both the plasma membrane and the nuclear membrane where it binds to the corepressor on the heterodimer (protein RXR and TR) bound to HRE 3) corepressor is displaced by the hormone 4) DNA bends, and complex on HRE is able to associate with Polymerase II and coactivator to activate transcription.

Immunoprecipitation (IN VITRO) technique

1) translation fusion tagging. If no Ab is known, then the cDNA of the protein of interest can be fused to a peptide sequence (epitope) for which an Ab does exist so the expressed protein of interest will have an epitope tag fused to the carboxyl terminus 2) After the translation fusion is expressed in an appropriate cell type, proteins are extracted and allowed to combine with Ab 3) the Ab-protein complex can be precipitated away(co-precipitation) from the other proteins 4) Any proteins that interact directly with tagged protein will also be precipitated (like in a second wash to that we can have different collections) 5) Gel electrophoresis will show several other bands along with the tag protein. 6) Protein can then be eluted(cut out band from gel, grind up gel and extract DNA from it) from the gel and identified using biochemical technique.

List the EUK model organisms that are critical in studying regulation side note: remember that PROK organisms were: myxococus, caelobacter, bacilus, strptomyces

1. S. cerevisiae (yeast) 2. Neurospora crassa (bread mold) 3. C.elegans (nematode/worms) 4. D. melanogaster (fruit fly) 5. A thaliana (plant) 6. M. musculis (mouse)

What are the three significances of gene regulation?

1. optimal energy utilization, 2. response to environmental and metabolic signals 3. proper development of multicellular organisms

RNA processing in the nucleus of EUK involves what three events

5' cap, splicing, 3' Poly A tail

How many different RTKs are there in humans? how many families?

60 different RTKs, 16 families

Alternative Splicing of Introns occurs in ____ % of mammalian genes

95

What is the function of committee proteins?

A complex committee of proteins interact with RNA Polymerase II and general transcription factors. Positive regulation via activators predominates, but some circumstances of negative regulation via repressors.

Drosophila Embryology (Body Plan Segment):

After just one day, the fundamental body plan for the adult has been established and cells within the embryo have already committed to differentiate into different structures of the adult. The early embryo has established its front and back and top/bottom. During later stages of pupation, cells differentiate to form the various structures of all body parts.

Chromatin remodeling coordinated by _______. Give an example of this.

Chromatin remodeling coordinated by transcription activators: Transcription activators can remodel chromatin by repositioning nucleosomes. For example, transcriptional activator called SW1/SNF binds to heterochromatin and induces chromatin remodeling through repositioning of nucleosomes. Some activators modify histones via acetylation or other chemical additions to alter affinity of histones for DNA. Chromatin remodeling exposes promoter and regulatory proteins allowing for transcriptional activation.

Explain why eukaryotic chromatin compaction levels are so significant to transcriptional regulation; and explain the role of histone modifications.

Eukaryotic chromatin compaction indicates closed conformation of binding sites which causes a ground state of gene regulation in eukaryotes to be off since promoters are inaccessible, unless there are transcription factors binding that enable transcription. The eukaryotic chromatin compaction allows for tight and highly regulated transcription. To understand histone modifications, one needs to know that histone are positively charged proteins wound by negatively-charged DNA. Histone modifications are changes (closed/open chromatin) that occur on these histones to alter their affinity for being bound by transcription factors. For example, methylation of histones causes closed conformation such that no transcription factors can bind therefore inhibiting transcription. Another example is acetylation which causes open conformation and allows transcription factor binding and transcription.

2. Neurospora crassa (bread mold)

Eukaryotic multicellular filamentous organism; growth cycle is very similar to prokaryotes streptomyces (aerial hyphae); useful for studying meiotic recombination and RNA interference

Ground State Comparisons for prok and euk: on or off? restrictive or nonrestrictive?

For PROK, most promoters are accessible to RNA polymerase, even in the absence of specific activators or presence of specifc repressors some transcription occurs, transcriptional ground state is ON (non-restrictive). In EUK, due to chromatin structure most promoters are not readily accessible to RNA polymerase so transcriptional ground state is OFF(restrictive) in the absence of regulatory proteins. Also, most euk genes are repressed as default state and require activators to induce gene expression, but negative regulation also occurs.

Post-transcriptional mechanisms: degradation and transport. They involve what mechanism

Gene expression can be controlled through modifications or degradation of encoded proteins themselves. Protein modification can activate/inactivate receptors, signal transducers, and transcription factors. Protein degradation is another way to modulate cellular response. In eukaryotes, protein degradation is regulated by covalent additions of a small 76 aa peptide called ubiquitin. Ubiquinated proteins are then degraded by passage through structures called proteomes (large-barrel like complexes of proteases which degrade the tagged proteins and then regulate the aa back into the cell).

Heterochromatin vs Euchromatin

Hetero= no = no transcription; Eu= do = transcription Causes of Euchromatin: coactivator complexes, loss of histone H1, histone modifications (acetylation, phosphorylation, methylation). Causes of Heterochromatin: histone deacetylation, dephosphorylation, demethylation, corepressor complexes. All of these control accessibility to gene promoters and regulatory regions.

Drosophila Homeotic mutants

In an attempt to understand how fly development works at a molecular level, developmental biologists have approached the study through mutagenesis to yield flies with abnormalities in larval and adult phenotypes. Typically the mutants are assigned names that are descriptive of the developmental phenotype. For example, some homeotic mutants have odd defects in development of organs/appendages. Others have antennapedia, a mutant that has legs growing where the antennas should be. Another is bithorax which has duplicate thoracic segment complete with an extra set of wings. Both antennapedia and bithorax genes encode transcription factors that have homeobox DNA binding domains. Homeobox was coined in reference to the homeotic mutant flies in which the corresponding genes were first discovered.

Restrictive ground state

In eukaryotes, this is the controlled and inactive state of the promoter and transcription machinery. Such state is enforced by highly packaged DNA and closed chromatin structure. Therefore, only regulatory proteins, such as activators/repressors create an open chromatin structure, increasing RNA polymerase affinity, and inducing transcription; otherwise the state of eukaryotes is not accessible.

How does B lymphocyte have an alternative 3' cleavage?

In the absence of Ag detection, the 3' end cleavage occurs downstream from the transmembrane domain so that IgM antibody is expressed as a surface protein receptor/ anchored in the plasma membrane because exon 4 joins M exons by the 2nd 3' end cleavage site. When Ag is detected and B lymphocyte is activated, 3' cleavage occurs upstream from the transmembrane domain coding region so that the expressed Ab protein is secreted from the cell because CstF-64 represses slicing and promotes use of 1st 3' end cleavage site. In summary, the difference is 3' cleavage downstream à expression as a surface protein receptor OR 3' cleavage upstream à protein secreted from cell.

Give an intro to RNA Silencing (2000s)

In the early 2000s, an exciting breakthrough in molecular biology occurred - a novel gene regulatory mechanism. Gene expression inhibited through action of short interfering RNA molecules (siRNAs), also called microRNAs (miRNAs). RNAi silencing can be an endogenous regulatory mechanism or introduced experimentally where the introduction of dsRNA silences genes. In brief, the steps involve dsRNA being cleaved by an endonuclease called "dicer" (widely expressed in eukaryotes), then gene silencing is achieved through the interaction of the siRNA/miRNA in the cytoplasm serving to inhibit translation or to trigger mRNA degradation.

For the cytokine signaling pathway JAK-STAT what are the two components JAK and STAT?

JAK is Janus kinase so it's a kinase. STAT is a transcription factor/activator

Life cycle of a Fruit Fly

Life cycle of a Fruit Fly: 1) Fertilization 2) Embryogenesis (early to late embryo segmentation) 3) Formation of a pupa 4) Metamorphosis 5) Hatching into a mature adult fly after 9 days

Non-restrictive ground state

Nonrestrictive indicates that there are no measures stopping or preventing transcription from occurring therefore, RNA polymerase can bind to every promoter to initiate transcription (in absence of activators/repressors). In bacteria, this is the natural state of activity for the promoter and transcriptional machinery.

Explain regulation site quantities and distances from promoter for both euk and prok.

PROK have an activator binding site, promoter, repressor binding site, and the coding regions (genes); regulation sites are controlled by one or two transcription factors with binding sites located proximally to promoter. EUK have many activator binding sites, promoter region, coding region, and then more activator binding sites. These numerous regulation sites spanning large distances can be upstream or downstream, not like in prok that they have to be near promoter.

Why does DNA have to loop/bend during regulation?

Polymerase II and transcription factors bind the promoter sequence. Gene activation requires interaction with transcript activators binding to regulatory sequences at great distances thus necessitating a looping of DNA to enable these interactions.

What is the function of steroids and what are their properties?

Steroids play diverse signaling functions in eukaryotic organisms. In humans, hormones are involved in metabolism, immune function, sexual characteristics. Steroids have a hydrophobic diffusion into cells without cell surface receptors (aka they diffuse easily). ). Instead the steroid hormone signal is transduced by binding to an intracellular hormone receptor.

T/F: Eukaryotes have more regulatory regions than prokaryotes

T

T/F: For receptor tyrosine kinase (RTK) pathway, a common outcome of growth factor activity is induction of mitosis to trigger cell proliferation

T

T/F: GPCRs receptor works for ligands that must interact with the cell surface receptor to induce activity.

T

T/F: Mutation in the hedgehog gene, this results in defective embryos resembling hedgehogs

T

T/F: RTK pathway is highly conserved

T

T/F: Wingless proteins are expressed in alternating cells, and loss of function mutation in the wingless gene results in wingless flies

T

T/F: when a G-protein is bound to GTP it is active?

T

T/F: JAK-STAT pathway is conserved in many organisms.

The JAK-STAT pathway is conserved in organisms ranging from worms to mammals, indicating its importance to cellular function. Genetic defects in this pathway are associated with immune diseases and cancer.

List the Major Signaling pathways

The following are characterized on the basis of either the signaling ligand or the receptor involved in sensing the ligand. 1. Steroid hormone receptors 2. G-protein coupled receptors (GPCRs) 3. Cytokine receptors (Jak-STAT) 4. Receptor tyrosine kinases (RTKs) 5. Wingless & Hedgehog

How many different STATS are there in mammals?

There are at least seven different STATs in mammals, each binding a different DNA sequence.

List the two types of nuclear receptor pathways (2).

Type I and Type II

In considering the seven steps of gene regulation, explain which particular steps occur only in eukaryotes.

Unlike prokaryotes that do not involve posttranscriptional processing, eukaryotes take on all the 7 steps in gene regulation. The very first step of transcription initiation is crucial because it is energy efficient, modulated by RNA polymerase binding, and guided by activator/repressors. Posttranscriptional processing (RNA processing) in eukaryotes involves transcript elongation such that the many elongation factors involved in establishing proper conditions of the transcript can be a target of regulation control. Also, modifications of mRNA termini occur: 5' capping to protect newly formed single stranded DNA that is at risk for degradation by endonucleases and 3' poly A tail which marks the end of transcription. Lastly, mRNA splicing removes introns (noncoding) regions of DNA. Subsequently, RNA stabilization involves regulation by small RNAs that interact with mRNA to silence it, leading to degradation of the mRNA or inhibition of translation when cellular needs do not require it. However, small RNAs do have various functions: control length of development, repress transposons, and destroy invading RNA viruses, and inducing heterochromatin formation. Due to eukaryotes complex multicellular structure, translation regulation involves many transcription factors ensuring that assembly at the 5' end of the mRNA in the ribosomal subunits is correct, and that the correct polypeptide is formed. The following step of protein modification is beneficial for establishing speed with which both the level of protein and activity can be altered. Transport and degradation are essential modulatating cellular response and in the termination phase of the cycle of proteins. In eukaryotes, protein degradation is regulated by covalent additions of a small 76 aa peptide called ubiquitin. Ubiquinated proteins are then degraded by passage through structures called proteomes (large-barrel like complexes of proteases which degrade the tagged proteins and then regulate the aa back into the cell).

______ and ______ are signaling genes in early development of the fruit fly model organism, Drosophila melanogaster, and other eukaryotes.. Defects are associated with dvlpt abnormality + cancer

Wingless (wg) and hedgehog (hh)

What initiates the cytokine signaling (JAK-STAT pathway)?

a cytokine

Drosha

an endonuclease in eukaryotic cells that cleaves the endogenous primary transcript "pri-mRNA" hairpin duplex to produce precursor miRNAs(pre-mRNAs) which are about 60-70 nucleotides, in length, double-stranded, and with 3' overhangs. This occurs in the nucleus prior to being exported into the cytoplasm were pre-mRNA is loaded onto RISC and undergoes many next processes.

Argonaute

an endonuclease that participates in the RISC. After cleavage by Dicer, Argonaute also cleaves so that then RNA helicase to unwind the DNA properly into single stranded mature miRNA. Following the formation of mature mRNA, two outcomes can result: partial complementarity with cytoplasmic mRNA which results in repressed translation, or near-perfect complementarity which results in mRNA degraded.

Explain Combinatorial control

an energy-conserving method of using combinations of a limited set of transcription factors, enabling activation of genes. For example, CRP is used in the metabolism of lactose/galactose and other sugars; another example is seven unique regulatory proteins (A, B, C, D, E, F, G) can bind 15 DNA sites in different patterns to control expression of 3 different genes. This would prevent the waste of energy required for making and maintaining hundreds of unique regulators that all require different committee proteins.

5. A thaliana (plant)

euk easily cultivated in the lab; amenable to recombinant DNA manipulation so it serves as a model for studying many aspects of plant molecular biology

6. M. musculis (mouse)

euk mammalian model organism; easily cultivated in lab; quick generation timel useful for genetic studies, human disease, and molecular analysis of development, immunology, and behavior

4. D. melanogaster (fruit fly)

eukaryote; early studies in 1900s; useful for studying development, behavior, and signaling pathways

on the contrary to prokaryotes, eukaryotes involve ______ , and the nucleus separates _______ from _____

eukaryotes involve post-transcriptional RNA processing (5' /3' ends and splicing of introns) nucleus separates transcription from translation thus post transcriptional regulation plays a larger role in eukaryotes than in prokaryotes

Hedgehog proteins interact with receptors on the Wg-producing cells, which ______ synthesis of Wg

increases

During early development of Drosophila, wingless and hedgehog signaling proteins are involved in a __________ reinforcing signaling loop across a field of neighboring cells. The signals generated help to shape the ultimate maturation of these cells into adult tissues.

mutually

List the strategies for Eukaryotic Gene Regulation.

o 1) In vitro: Immunoprecipitation is used for identification of interacting proteins o 2) in vivo analysis involves two options: yeast two-hybrid analysis, RNAi knockdown analysis o 3) Model eukaryotic organisms: S. cerevisiae (yeast), C.elegans (nematode/worms), N. crassa (bread mold), D. melanogaster (fruit fly), A thaliana (plant), M. musculis (mouse)

· Closing thoughts:

o Relevance of Drosophila developmental mutants o Foundation for signaling pathway research at the heart of cell-cell communication (i.e wingless, hedgehog, RTK pathways) o Significance to cancer and development of pathway-specific targeted therapies underscores importance of basic academic research

1. S. cerevisiae (yeast)

one of the simplest single-celled eukaryotes; useful for studying fundamental aspects of chromosome structure, regulation of cell division, and signaling pathways

Complexity of Signaling Pathways

pathways are a complex network of overlapping and counteracting pathways. Individual cells respond to limited set of signals depending on expressed receptors. Any single cell can express 1000s of receptors. Each cell is thereby challenged to integrate these complex sets of reactions and to behave appropriately while the complexity allows cells tremendous ability to respond to diverse signals and circumstances (diverse responsiveness). Pathways are also points of vulnerability: mutations that affect these pathways are drivers of cancer development. Most oncogenes and TS genes (cancer associated genes) encode proteins involved in signaling pathways, either as ligands, receptors, intracellular transducers, or TFs.

IS GPCR a receptor or a ligand? What does it bind?

receptor, binds G-proteins

What is the general importance of Signaling Pathways?

signaling pathways are the integration of environmental signals and gene expression. All living cells must be able to integrate extra and intracellular signals. Integration of signals occurs through signal transduction, a cascade of events involving a signal and a receptor, and many proteins that commonly results in alteration of gene expression. Alteration of gene expression involves activation of transcription factors or inactivation of repressors, resulting in transcription activation of numerous target genes (i.e metabolic enzyme, gene regulatory protein, cytoskeletal protein).

Benefits of alternative splicing

splicing patterns varies by cell type; increases the protein coding capacity of the genome; complex regulatory mechanisms control particular splicing selections

Transcriptional ground state

the involvements or state of promoter and transcriptional machinery in vivo in the absence of regulatory mechanisms in cells. There are two types: restrictive and non-restrictive ground states.

Much like prokaryotes, eukaryotes most regulated step is?

transcription initiation

In RTK signaling, is RTK a receptor or ligand?

transmembrane receptor (Receptor tyrosine kinase) in cytoplasmic domain

3. C.elegans (nematode/worms)

useful for developmental biology. Worms are transparent and composed of 959 cells that can be tracked through a microscope at every step of development starting with a fertilized egg into a complete multicellular organism with nerves, muscles, digestive tissues, and reproductive tissues. Useful for studying signaling pathways, aging. Interestingly, siRNA was first discovered in experiments involving this organism

How many distinct GPCRs with metabolic and neurological functions are there in humans?

~800

Molecular Biology 2 values

§ Essential foundation for all other life science disciplines § Reveals mechanistic details of how life works

Applications of signaling pathways

· Huge subject area of research in cell biology, developmental biology, biochemistry, molecular biology. Cell-cell communication studies (cellular and organismal level control). At a higher level, since signaling is central to cell-cell communication mechanism, it is highly relevant to hold organism studies such as in human physiology and disease. Insight in cellular and organismal cell control. Disease mechanism and drug targets: ligands, receptors and signal transducers have been the targets for development of some highly effective therapeutic drugs, such as for the treatment of cancer and inflammatory diseases.