Chapter 12: Regulation of Gene Expression in Eukaryotes

what are insulators? barrier insulators? enhancer-blocking insulators?

*Insulators* are DNA segments that function as a boundary between two genes. Insulators protect a gene from the regulatory effects of a neighboring gene. *Barrier insulators* prevent changes in chromatin structure by stopping the spread of heterochromatin. (protecting genes to it gets expressed) *Enhancer-blocking insulators* prevent neighboring enhancers from activating the gene. (shutting down gene expression "protecting from gene expression")

what are the effects of activators and repressors on transcription initiation?

- activator proteins bind to enhance stimulating transcription (by recruiting RNA Poly II and GTF's) - repressor proteins bind to silencers so its RNA poly and GTF's cannot bind to the promoter, therefore, transcription is inhibited.

what can ATP-dependent remodeling complexes (eq, SWI-SNF) do?

- change nucleosome position - evict histones (exposing more DNA) - add histone variants

the differeneces between bacterial and eukaryotic gene regulation include what four diffferences?

1. Bacterial genes are transcribed by the same RNA polymerase (vs. *three* different RNA polymerases). 2. Bacterial mRNA is translated "as is" (vs. *mRNA processing step* 3' adenlyation, 5' capping, sliping reactions). 3. Bacterial RNA polymerase is smaller and less complex (vs. RNA polymerase II that synthesizes RNA and carries out cotranscriptional mRNA processing). 4. Bacterial gene expression is generally "on," whereas most eukaryotic genes are "off" at any one time.

what are the proteins that control eukaryotic genes?

1. RNA Pol II and associated GTFs bind to the promoter. 2. One or more *activators* may stimulate the ability of RNA Pol II to initiate transcription. 3. One or more *repressor* may inhibit the ability of RNA Pol II to initiate transcription. 4. The function of activators and repressors may be *modulated* in a variety of ways, including the binding of (i) small effector molecules, (ii) protein-protein interactions, and (iii) covalent modifications. 5. Regulatory proteins may alter the composition or arrangements of *nucleosomes* in the vicinity of a promoter, thereby affecting transcription. 6. *DNA methylation* may inhibit transcription, either by preventing the binding of an activator protein or by recruiting proteins that cause the chromatin to become more compact. Note: A cell can change the expression of its genes in response to external signals (eg, hormones) that may affect the function of these proteins.

what are the effects of activators and repressors on TFIID and mediator. 1. transcriptional activation via TFIID 2. transcriptional repression via TFIID 3. transcriptional activation via mediator 4. transcriptional repression via mediator

1. The activator/coactivator complex recruits TFIID to the core promoter and/or activates its function. Transcription will be activated. 2. The repressor protein inhibits the binding of TFIID to the core promoter or inhibits its function. Transcription is repressed. 3. The activator protein interacts with mediator. This enables RNA polymerase to form a preinitiation complex that can proceed to the elongation phase of transcription. 4. The repressor protein interacts with mediator so that transcription is repressed.

RTFs can be modulated by?

1. binding of an effector molecule (eg, hormone). 2. protein-protein interaction (eg, dimerization). 3. covalent modification (eg, phosphorylation). RTFs can bind to DNA as homodimers or heterodimers. homodimers: same two RTFs interacting hetrodimers: two different RTFs interacting

Gal4, like other activators, can recruit the transcriptional machinery to the promoter via:

1. binding to TBP of TFIID. 2. interacting with the mediator complex. when Gal4 binds to UAS

what are the four major mechanisms that alter chromatic structure?

1. changing nucleosome position by *chromatid remodeling* 2. removing histones, known as *histone eviction* 3. replacing the common histones with *varient histones* 4. adding or removing functional groups of histones, resulting in *histone modification*

what are the two types of epigenetic inheritance?

1. genomic imprinting 2. dosage compensation

inactivation occurs in three phases. what are the three phases?

1. initiation: Occurs during embryonic development. The number of X-inactivation centers (Xics) is counted and one of the X chromosomes remains active and the other is targeted for inactivation. 2. spreading: Occurs during embryonic development. It begins at the Xic and progresses toward both ends until the entire chromosome is inactivated. The Xist gene encodes an RNA that coats the X chromosome and recruits proteins that promote its compaction into a Barr body. 3. maintenance: Occurs from embryonic development through adult life. The inactivated X chromosome is maintained as such during subsequent cell divisions Having two active X chromosomes is a lethal condition for human female embryos.

CREB is an activator (RTF) modulated by?

1. phophoralation (covalent modification) 2. protein-protein interaction (dimerization) CREB is a dimer already present in the nucleus but it cannot make target seq until it is phosporalated. CRE: cAMP response elements CREB: cAMP response elements-binding proteins

CpG methylation inhibits transcription by?

1. preventing activators from binding. 2. causing methyl-CpG-binding proteins (MECPs) to bind, which recruit HDACs, resulting in DNA compaction. HDACs: remove aceyl gr. off histone which makes in further compacted.

what are the DNA sequences that control eukaryotic genes?

1. promoter (TATA Box) 2. Regulatory elements (enhancers and silencers) 3. promoter-proximal elements (near the promoter) (GC-rich box and CCAAT box)

GCR is an activator (RTF) modulated by?

1. protein-protein interaction (dimerization) 2. binding of small effector molecules (hormone glucocorticoid) NLS: nuclear localization seq - seq of amino acids that target that protein to go into the nucleus GRE: GC response elements

RTFs (activators and repressors) contain one or more of the following domains that do what?

1. recognize a regulatory element (*DNA-binding domain*) 2. interact with other RTFs (*transactivation domain*) 3. interact with RNA Pol II and/or GTFs (*activation domain*) 4. influence chromatin condensation 5. act as a sensor of physiological conditions within the cell

what is genomic imprinting?

A DNA segment is marked, and that marked is retained throughout the individual's lifetime. Paternal and maternal alleles can be distinguished based on the presence or absence of a mark. This results in *monoallyic expression*: only 1 of 2 alleles is expressed; the other is silenced.

what are nucleosomes?

A nucleosome is composed of DNA wrapped around a histone core called the octamer (octamer: two each of H2A, H2B, H3, and H4).

If a deletion of AS or PWS gene occrus what is developed?

AS develops if a *deletion* in chromosome 15 is inherited from the *mother* PWS develops if a *deletion* in chromosome 15 is inherited from the *father*

Imprinting plays a role in the inheritance of certain human genetic diseases. what are these diseases?

Angelman syndrome (AS) - thin, hyperactive, seizures, mental deficiencies Prader-Willi syndrome (PWS) - obese, reduced motor function, mental deficiencies AS gene: *maternal* copy is active in the brain; *paternal* is silent PWS gene: *paternal* copy is active in the brain; *maternal* is silent Both genes are located next to each other on chromosome 15. to have normal brain function you have to have both expressed

dosage compensation in mice

Black and white mice may be heterozygous females: XBXb (B = black; b = white) at the beginning both are expressed but over time one is inactive resulting in patches of black and white fur.

dosage compensation in cats

Calico cats are heterozygous females: XOXo (O = orange; o = black)

in genomic imprinting DNA seq. are marked by what?

DNA segments are marked by methylation of cytosine bases (via DNMTs) in the *imprinting control region (ICR)*. ICR acts as an enhancer-blocking insulator when bound by *CTC-binding factor (CTCF)*, and thus prevents Igf2 transcription. marked by DNMTs

what is chromatin?

Eukaryotic DNA is packaged in the form of chromatin. *Chromatin* is the substance of chromosomes, composed of DNA and chromosomal proteins. you can have euchromatin and heterochromatin

what are histones? what do they include?

Histones are small, basic (+) proteins composed of ~20-30% *lysine* and *arginine* residues that bind to DNA in the *minor groove.* Histones include H1, H2A, H2B, H3, and H4.

If a CpG island is unmethylated is it active or inactive? if a CpG island is methylated is the gene active or inactive?

If unmethylated, the gene is active (eg, housekeeping genes). If methylated, the gene is inactive. Thus, *DNA methylation typically decreases gene expression.*

The mechanism of X inactivation is known as? what happens?

Lyon hypothesis. When the Xist gene is expressed, the Xist RNA coats the X chromosome, leading to its inactivation. When the Tsix gene is expressed, Tsix RNA inhibits transcription of the Xist gene, thereby preventing X inactivation. *Xist* is expressed in inactive X chromosomes. *Tsix* is expressed in active X chromosomes. Xic: X-inactivation center Xist: X-inactive specific transcript

diagram of everything necessary to get transcription going

NFR: nuclear free regions A simplified model for the transcriptional activation of a eukaryotic gene

eukaryotic transcription usually on or off?

OFF TATA box is usally hidden due to DNA being compact. therefore transcription is off until activator proteins bind to enhancer regions, mediator proteins, GTF's, and poly II

bacterial transcription usually on or off?

ON activator protein is bond RNA pol and transcribes until repressor protein binds therefore bacterial is repressible

what is the additional step added in eukaryotic gene regulation?

RNA processing an additional step when compared to prokaryotes who only have 1. transcription 2. translation 3. post-transitional modification

what is the yeast GAL system and what is Gal4?

Several genes in the yeast genome encode enzymes that convert galactose into a form of glucose that can be metabolized. These genes are transcribed only when glucose is absent and galactose is present. *Gal4* is a RTF activator protein that regulates the expression of the enzyme genes by binding to enhancer sequences known as *upstream activation sequences (UASs).* it works as a dimer

what happens in acetylation? what reverses it?

The acetylation of *lysine* neutralizes *its positive charge*, thereby loosening the interaction between histones and DNA. histone acetyltransferases (HATs) vs. histone deacetylases (HDACs) -adding actyl gr. you loosen DNA -remove acetyl gr. you tighten DNA

what are reporter genes?

The control of gene expression by the binding of an RTF to a regulatory element can be measured by reporter genes. In reporter gene constructs, the reporter gene (eg, lacZ, GFP) is linked to the regulatory element. *The DNA-binding* and *activation domains* can then be investigated. *link reporter gene to regulator element* which we can then measure the ability of RTF to bind to that element

what is dosage compensation?

The expression of genes on the *X* chromosomes is similar in both sexes. XX expression = XY expression In female mammals, the inactivated X chromosomes can be seen as a highly condensed structures, called a *Barr body*, in the nucleus.

what happens in methylation? what reverses it?

The methylation of *lysine and arginine* *does not alter their positive charge*. However, the number of methyl groups creates different binding sites for other proteins to activate or repress gene expression. histone methyltransferases (HMTs) vs. histone demethylases (HDMs)

what happens in phosphorylation?

The phosphorylation of *serine* adds a *negative* charge, thereby loosening the interaction between histones and DNA. bc DNA is negative

True or False? Promoter-proximal elements are necessary for efficient transcription?

True. if you mutate or take away some of the NT in the GC-rich box you can see *transcription levels decrease* this is the same with CCAAT and TATA boxs. therefore one can see how important these elements are in producing normal transcription

GTFs are necessary for what type of transcription? what do RTFs control

basal *regulatory transcription factors* (RTFs; activators and repressors) control the *rate* of transcription of target genes. RTFs recognize and bind to specific DNA sequences called *regulatory* or *response elements* (enhancers and silencers). RTF amino acid residues and DNA nucleotides interact in the *major groove* via H-bonds, ionic bonds, and hydrophobic interactions.

Histone modifications can be inherited, a form of ___________ inheritance?

epigenetic During DNA replication, nucleosomes are disassembled in the parental strands and reassembled in the daughter molecules. The old histones are randomly distributed to the daughter molecules and guide the modification of new histones.

DNA methylation can be inherited, a form of _________ inheritance?

epigentic DNA methylation can also be inherited, and is also a form of epigenetic inheritance. In DNA methylation, the Cs of *CpG islands* are methylated via *DNA methyltransferases (DNMTs).* CpG islands occur near many gene promoters, and are ~1000-2000 bp in length.

what is the difference between euchromatin and heterochromatin?

euchromatin = less condense; usually transcriptionally active heterochromatin = more condense; usually transcriptionally inactive - two types: always heterochromatin: constitutive hetero can convert to euchromatin: facultative hetero

Igf2 is an example of what?

genomic imprinting. Example: Igf2 expression in mice Only the paternal Igf2 allele is expressed; the maternal Igf2 allele is silent. Igf2 is normal. Igf2- is a loss-of-function allele.

what is the advantage of heterodimerization?

increase DNA recognition sites

what is epigenetic inheritance?

is a modification of a gene or chromosome or DNA segment that alters gene expression during gametogenesis or early embryogenesis. Epigenetic inheritance affects the *phenotype* but not the *genotype* of an individual during its lifetime.

Histone modifications are _____________ modifications, and include:

post-translational modifications 1. acetylation (lys) 2. methylation (lys and arg) 3. phosphorylation (ser) The pattern of histone modifications is known as the *histone code*. Different patterns of modification result in different levels of chromatin compaction to hide or expose genes for expression. tails on histones contain amino acids that can modified giving different levels of transcription

Although different types of cells of a multicellular organism contain the same DNA, they make different sets of proteins (eg, hepatocyte vs. neuron). What makes these cells different? (what is different in the two cells)

regulatory transcription factors different RTF's transcribe different proteins resulting in different cells

True or False? The effects of enhancer location and orientation can also be investigated using reporter gene constructs.

true. promoter has to stay in the same region but enhancer can move around. 1. just promoter 2. neither 3. just enhancer d,e,f,g have the same level of transcription from these we can determin enhancer region, what orrentation it is in, and how far away it is located.