1. Regulation of Gene Expression in Eukaryotes
Chapter 12

2. Activation of Genes in a Chromatin Environment
Enhanceosomes help recruit the transcriptional machinery
As you have seen so far… transcription of eukaryote genes has to be turned on and off during the lifetime of an organism. To understand how eukaryotes regulate genes during their lifetime, it is necessary to see how chromatin changes during transcriptional activation
The β-interferon enhanceosome. In this case, the transcription factors recruit a co-activator (CBP), which binds both to the transcription factors and to RNA polymerase II, initiating transcription.
DNA binding proteins (enhancers) bind to cis acting sequences can have a synergistic effect on transcription
DNA binding proteins bind to cis acting sequences (enhancers) can have a synergistic effect on transcription

3. The β-interferon enhanceosome acts to move nucleosomes by recruiting the SWI-SNF complex.
β-interferon encodes the antiviral protein interferon, and it is normally switched off except in the case of viral infection.
The key to activation of this gene is the assembly of transcription factors into an enhanceosome about 100bp upstream of the stat site.

4. Enhancer-blocking insulators
Enhancer-blocking insulators prevent enhancer activation (promiscuous activation) by enhancers
Regulatory elements such as enhancers can act over tens of thousands of base pairs which could interfere with the regulation of nearby genes: Enhancer-blocking insulators prevent gene activation when placed between an enhancer and a promoter.

5. Model for how enhancer-blocking insulators might work
One proposal is that enhancer-blocking insulators create new loops that physically separate a promoter from its enhancer.
Eukaryotic enhancers can act at great distances to modulate the activity of the transcriptional apparatus. Enhancers contain binding sites for many transcription factors, which bind and interact cooperatively. These interactions result in a variety of responses, including the recruitment of additional co-activators and the remodeling of chromatin.

6. Which of the following statements best describes the “ground state” for expression of genes in eukaryotic cells?
Gene expression is “on” unless specifically inhibited by the binding of repressor proteins.
Gene expression is “off” unless specifically turned on by the binding of specific regulator proteins.
Gene expression is constitutively euchromatin.
Gene expression is constitutively “off” in heterochromatin but is constitutively “on” in euchromatin.

7. Long-Term Inactivation of Genes in a Chromatin Environment
Mating-type switching and gene silencing
Reminder: most genes are “off” and inactive for the life of an organism
Why do organisms have genes that are always inactive?
How do organisms keep genes in an inactive state for their entire lifetime?

8. Heterochromatin and euchromatin compared
Highly condensed chromatin
Stay condensed throughout cell cycle
Telomeres
Around centromeres
Composed of repetitive sequences
Closed structure
Euchromatin
Less condensed
Condense during mitosis but return to less condensed from after mitosis
Composed of mostly genes
Open structure to facilitate gene expression
The chromatin of eukaryotes is not uniform. Highly condensed heterochromatic regions have fewer genes and lower recombination frequencies than do the less-condensed euchromatic regions.

9. Position-effect variegation in Drosophila reveals genomic neighborhoods
When genes are relocated to regions that are heterochromatic can cause genes to be silenced
This phenomenon has been called position-effect variegation (PEV)
Chromatin structure therefore is able to regulate gene expression by determining if a DNA sequence will be active or silenced
Long before we understood mating type switching in yeast, geneticist discovered an interesting effect in drosophila. Active genes that are relocated to genomic neighborhoods that are heterochromatic may be silenced if the heterochromatin spreads to the genes.
Chromosomal rearrangement produces position-effect variegation. Chromosomal inversion places the wild-type white allele close to heterochromatin. The spread of heterochromatin silences the allele. Eye facets are white instead of the wild-type red wherever the allele has been silenced.

10. Active genes that are relocated to genomic neighborhoods that are heterochromatic may be silenced if the heterochromatin spreads to the genes.
Chromosomal rearrangement produces position-effect variegation. Chromosomal inversion places the wild-type white allele close to heterochromatin. The spread of heterochromatin silences the allele. Eye facets are white instead of the wild-type red wherever the allele has been silenced.

11. Genetic analysis of PEV reveals proteins necessary for heterochromatin formation
Some gene products enhance or suppress the spread of heterochromatin
HP1 binds to H3K9me
Heterochromatin protein 1
Repression
Directs histone deacetylation
AND DNA methylation
GCN5 – histone
acetyltransferase activity
Activates genes
HP1 – heterochromatin protein 1

12. Heterochromatin may spread farther in some cells than in others
4 genetically identical diploid cells
Heterochromatin spreads enough to knock out a gene in some chromosomes but not others.
Heterochromatin and euchromatin are represented by orange and green sphere
The spread of heterochromatin into adjacent euchromatin is variable. In four genetically identical diploid cells, heterochromatin spreads enough to knock out a gene in some chromosomes but not others. Heterochromatin and euchromatin are represented by orange and green spheres, respectively.
So how do we stop the spread of heterochromatin?

13. Barrier insulators stop the spread of heterochromatin
Histone acetyltransferase
In this model, barrier insulators recruit enzymatic activities such as histone acetyltransferase (HAT) that promote euchromatin formation. The letter “M” stands for methylation, and the letter “A” for acetylation.
The isolation of critical proteins necessary for the formation of heterochromatin, including HP-1 and HMTase, was made possible by the isolation of mutant strains of Drosophila that suppressed or enhanced PEV
Barrier insulators prevent the spreading of heterochromatin by creating a local environment that is not favorble to heterochromatin formation.
IN this example, the barrier insulator binds to HAT which hyperacetylated histons around it to protect the euchromatin

14. Gender-Specific Silencing of Genes and Whole Chromosomes
Genomic imprinting explains some unusual patterns of inheritance
Genomic imprinting – phenomenon in which a gene inherited from one of the parents is not expressed even though both copies are functional.
Imprinted genes are methylated and inactivated in the formation of male or female gametes
Maternal imprinting – Paternal inheritance, mother’s gene is silenced – Igf2
Paternal imprinting – Maternal inheritance, father’s gene is silenced – H19
IGF2-one of three protein hormones that share structural similarity to insulin
It is believed to be a major fetal growth factor in contrast to Insulin-like growth factor 1, which is a major growth factor in adults
H19 is a gene for a long noncoding RNA, found in humans and elsewhere. H19 has a role in the negative regulation (or limiting) of body weight and cell proliferation.[1] This gene also has a role in the formation of some cancers and in the regulation of gene expression

15. Genomic imprinting requires insulators
ICR-Imprinting control region
CCCTC-binding factor
Genomic imprinting in the mouse. The imprinting control region (ICR) is unmethylated in female gametes and can bind a CTCF dimer, forming an insulator that blocks enhancer activation of Igf2. Methylation (M) of the ICR in male germ cells prevents CTCF binding, but it also prevents the binding of other proteins to the H19 promoter.
CCCTC-binding factor

16. Parental Imprinting Can greatly affect pedigree analysis
A mutation in the allele inherited from the other parent will appear to be dominant
However, the allele is expressed because only one of the two homologs is active for this gene

17. Unusual inheritance of imprinted genes
A mutation (represented by an orange star) in gene A will have no effect if inherited from the male. Abbreviations: M, methylation; ICR, imprinting control region

18. Many steps required for imprinting
As these primordial germ cells become fully formed gametes, imprinted gene receive the sex-specific mark that will determine whether the gene will be active or silent after fertilization.
How Igf2 and H19 are differentially imprinted in males and females.
As these primordial germ cells become fully formed gametes, imprinted gene receive the sex-specific mark that will determine whether the gene will be active or silent after fertilization.

19. But what about Dolly and other cloned mammals?
Why is cloning successful?
As we just learned, a mutation in one imprinted gene can be lethal or lead to serious disease
Truth is, we don’t know….BUT…we do know that cloning is extremely hard in all species tested
One clone can take 1000’s of attempt
One theory is viable organisms need the epigenetic mechanisms of gene regulation in eukaryotes

20. Cloning video Epigenetic https://www.youtube.com/watch?v=kp1bZEUgqVI
Introduction to cloning
Possible species to benefit to cloning

21. Silencing an entire chromosome: X-chromosome inactivation
Females have two X chromosomes but only express one.
The X chromosome has about 1000 genes
Y chromosome has very few that contribute to “maleness” but NO vital genes reside only on the Y chromosome (possibly hair in ears)
Extra chromosome is dealt with via dosage compensation – makes the amount of gene products from two copies equivalent to the single dose
In mammals, the 2nd X chromosome (Xi; Barr body) is inactivated, highly condensed, heterochromatic structure

22. Silencing an entire chromosome: X-chromosome inactivation
A model for X-chromosome inactivation
For most diploid organisms, both alleles of a gene are expressed independently. Genomic imprinting and X inactivation are examples of only a single allele being available for expression. In these cases, epigenetic mechanisms silence a single chromosomal locus or one copy of an entire chromosome, respectively.

23. Post-Transcriptional Gene Repression by miRNAs
Xist is one example of rapidly growing class of functional RNAs.
Functional RNAs do not encode proteins
They direct proteins or protein complexes to places in the cell where services are needed
Xist directs proteins associated with heterochromatin formation to one of the two X chromosomes

24. X inactivation Video https://www.youtube.com/watch?v=n330FzHpI90
Calico cats are almost always female
Exception is an XXY combination is a genetic rarity that occasionally shows up in cats (people, too).
And if both X chromosomes carry the calico blueprint, you're looking at one rare cat: a male calico.

25. miRNA video

26. Which of the following is likely a functional domain of a regulatory protein that modulates the transcription of specific genes in a eukaryotic cell?
a) a DNA-binding domain b) a domain that allows the regulatory protein to interact with CBP or a mediator complex c) a domain that influences chromatin condensation d) All of the above.

27. Which of the following is likely to be associated with chromatin remodeling?
a) the movement or “repositioning” of nucleosomes b) selective post-translational modification of nucleosome proteins c) assembly of a protein complex known as the enhanceosome d) All of the above.

28. Analogous to the genetic code, the particular combination of biochemical modifications to histone tails that dictates a particular chromatin state is dubbed the:
a) enhancer code. b) upstream activation code. c) reporter code. d) histone code.

29. Which of the following is the primary functional consequence of methylation of nitrogenous bases in DNA?
a) It changes the DNA sequence, ultimately changing the codons in the mRNA transcript of a protein-coding gene. b) Chromatin remodeling occurs, typically resulting in transcriptional activation of nearby genes. c) The DNA is chemically “marked” by the added methyl group(s), altering its functional state (i.e., whether genes are inactivated or activated). d) The modified DNA is selectively degraded, as DNA methylation always indicates DNA damage.

30. A gene that is normally transcriptionally active can be “silenced” if it is relocated (through experimental manipulation) to certain regions of the chromosome (e.g., near the centromere, which is “heterochromatin-rich”). This phenomenon is an example of
a) position effect. b) epigenetic silencing. c) post-transcriptional gene silencing. d) both a and b

31. A mutant yeast strain can convert galactose to UDP-galactose, but is unable to carry out the next step of galactose metabolism because it cannot make the Gal10 enzyme. All of the following are possible explanations for this phenotype EXCEPT
a) a mutation in the protein-coding region of the GAL10 gene. b) a deletion of the UAS of the GAL10 gene. c) a deletion affecting the Gal4 protein-binding site upstream of the GAL10 gene. d) a mutation affecting the GAL4 gene that makes its activation domain nonfunctional.

32. A student is growing yeast cells in galactose-rich media
A student is growing yeast cells in galactose-rich media. Which of the following statements is likely true of these cells?
a) Gal3 will be bound to the activation domain of Gal4. b) Gal80 will be bound to the activation domain of Gal4. c) Gal3 will bind to Gal80, and no protein will be bound to the activation domain of Gal4. d) The GAL1 gene will not be transcribed in these cells.

33. Which of the following epigenetic changes could shut down transcription of a particular gene?
a) hyperacetylation of histones associated with that gene b) hypoacetylation of histones associated with that gene c) methylation of C residues near the promoter of that gene d) both b and c

34. Which of the following is NOT an example of gender-specific epigenetic silencing?
a) X-inactivation in female mammals b) maternal imprinting c) paternal imprinting d) position effect variegation

35. The control of yeast mating type: combinatorial interactions
In multicellular organisms, distinct cell types differ in expression of hundreds of genes
Gene regulation is a highly coordinated event
Cell type regulation: Mating type of yeast.
a - haploid
α - haploid
a/α - diploid
If you remember from Thursday… Explore two yeast gene regulatory systems the first on was the galactose system and the second one is going to be the yeast mating types
And as a reminder… i have already mentioned that in multicellular organisms there are distinct cell types that differ in expression of hundred of genes and gen regulation is a highly coordinated event

36. Yeast mating types
α and a can not be distinguish phenotypically but they can be differentiated by other characteristics
α cell mates only with an a cell and vice versa
α secrete an oligopeptide pheromone (sex hormone) called α factor that arrests a cell cycle (and vice versa)
Cell arrest of both participants is necessary for successful mating
α/a cell does not mate, is larger than the haploids and it does not respond to mating hormones

37. Combinations of regulatory proteins control cell types
Genetic analysis of mutants defective in mating has shown that cell type is controlled by a single genetic locus, the mating type locus MAT
MCM1 – a protein not encoded by MAT locus plays a key role in regulating cell type
In diploid yeast, all genes involved in cell mating are shut down….as well as the genes encoded in haploid specific genes
Control of cell-type-specific gene expression in yeast. The three cell types of S. cerevisiae are determined by the regulatory proteins a1, α1, and α2, which regulate different subsets of target genes. The MCM1 protein acts in all three cell types and interacts with α1 and α2.
Cell type specific patterns of gene expression are governed by combinations of interacting transcription factors
Cell type specific patterns of gene expression are governed by combinations of interacting transcription factors

38. Possible models for the repression of translation by miRNA
In chapter 8 i introduced you to miRNA and siRNA….here we will focus on miRNA

39. Review 10 6 12 6 7 1 6 1 1 2 3 8 5 9 4 13