ch 12 (lectures 24-25)

Question 1

What are the five levels at which gene expression can be regulated, from DNA to protein activity?

Answer 1

• Transcriptional Regulation– Controls how much RNA is made from DNA
• mRNA Stability– Determines how quickly mRNA is degraded
• Translational Regulation– Controls how much protein is made from mRNA
• Protein Stability– Determines how long a protein lasts before it’s degraded
• Post-Translational Modifications (PTMs)– Regulate protein activity, localization, and interactions

Question 2

How does transcriptional regulation differ between bacteria and eukaryotes?

Answer 2

Bacteria:
RNA polymerase can bind promoters easily.
➕ Activators increase transcription
➖ Repressors decrease transcription

Eukaryotes:
RNA polymerase cannot bind promoters on its own.
* Requires multiple regulatory mechanisms to enable efficient binding and transcription.

Question 3

What roles do promoter-proximal elements and enhancers play in eukaryotic transcription regulation?

Answer 3

Promoter-proximal elements:
* DNA sequences just upstream of the promoter
* Bind general transcription factors
* Necessary for efficient transcription

Enhancers:
* Can be far from the gene
* Bind specific transcription factors
* Control subset of genes/tissues

Mutating promoter-proximal elements significantly reduces transcription.

Question 4

What domains are commonly found in transcription factors (TFs)?

Answer 4

Key domains in transcription factors:
* DNA-binding domain: Binds specific DNA sequences
* Protein-protein interaction domain: Interacts with other TFs or RNA polymerase
* Enzymatic activity domain: Modifies histones (e.g., acetylation, methylation)
* Sensor domain: Responds to cellular/physiological signals

Question 5

What makes budding yeast (S. cerevisiae) a useful model system in molecular biology?

Answer 5

Key features of budding yeast:
* Single-celled eukaryotic microorganism
* Can divide mitotically as both haploid and diploid
* Diploid cells can undergo meiosis to produce haploid spores

Question 6

How is the Gal pathway regulated in yeast?

Answer 6

In presence of galactose:
* Expression of galactose-metabolizing genes increases ~1000x
* UAS (Upstream Activation Sequence)= enhancer that binds Gal4

Gal4 is a modular transcription factor with:
* DNA-binding domain → targets UAS (enhancer)
* Activation domain → interacts with RNA Pol-associated proteins

Domains can function independently, but both are required together for transcriptional activation

Question 7

What is the purpose of a reporter gene in transcriptional studies?

Answer 7

A reporter gene is used to monitor transcriptional activity

Linked to a regulatory sequence (like UAS-Gal4 system)

Transcriptional activity is visualized via an easily detectable phenotype

Example:
🔹 LacZ (from bacteria) can be expressed in yeast
🔹 LacZ breaks X-gal → blue color appears
🔹 Blue = gene is ON, No color = gene is OFF

Question 8

What experiment shows that Gal4 is a modular transcription factor?

Answer 8

Modular= Has separable functional domains
Reporter gene: LacZ (turns colonies blue with X-gal if ON)
Evidence:

Wild-type Gal4:
* Has both DNA binding + activation domains
* 🟦 LacZ is ON → Blue colonies

Separated domains:
* DNA binding and activation domains expressed separately
* ⚪ LacZ is OFF → White colonies

LexA-Gal4 hybrid:
* LexA (DNA binding domain) fused to Gal4 activation domain
* 🟦 LacZ is ON → Blue colonies
* Shows functionality retained when domains recombined

Question 9

How is Gal4 regulated by Gal80 and galactose? What role does the mediator complex play in transcription?

Answer 9

Gal80:
* Binds Gal4’s activation domain
* Prevents transcription in absence of galactose
* Corepressor (does not bind DNA)

Galactose present:
* Galactose binds to Gal3
* Gal3 interacts with Gal80 → removes Gal80 from Gal4
* Gal4 now free to activate transcription
* Gal3 is a coactivator

Transcription Machinery Recruitment:
Gal4 recruits:
* Basal transcription factors
* Mediator complex

Mediator complex:
* Coactivator
* Bridges activators like Gal4 to RNA polymerase II
* Does NOT bind DNA
* Essential for transcription at most eukaryotic promoters

Question 10

What are the different yeast cell types, and how is gene expression regulated in each?

Answer 10

Yeast Forms:
🧬 Haploid a
* a cells
* Express a genes
* Mate with α cells
* Secrete a-factor pheromone

🧬 Haploid α
* α cells
* Express α genes
* Mate with a cells
* Secrete α-factor pheromone

🧬 Diploid (a/α)
* Suppress a genes and α genes
* Express haploid-specific genes
* Cannot mate

Question 11

How is gene expression regulated in a cells in budding yeast?

Answer 11

MCM1: A general activator of a and α genes
* Binds only a gene promoters in a cells

a1: Expressed in a cells
* Has no function alone in this context

Question 12

How is gene expression regulated in α cells in budding yeast?

Answer 12

MCM1: General activator

α1 & α2: Both are expressed in α cells
* α1: Recruits MCM1 to α gene promoters → activates α genes
* α2: Blocks MCM1 from activating a genes

Question 13

How is gene expression regulated in diploid cells in budding yeast?

Answer 13

MCM1: Activator

a1 + α2: Both are expressed
* α2: Blocks MCM1 activation of a genes
* a1 + α2 complex: Represses Haploid-specific genes and α1 gene

Question 14

How do combinations of MCM1, a1, α1, and α2 regulate gene expression in yeast?

Answer 14

Protein Roles:
* MCM1– General activator
* a1– Repressor, only when paired with α2
* α1– Activator
* α2– Repressor

The same proteins can have different effects depending on what other proteins are present.
* Different combinations → different transcriptional outcomes.
* Gene regulation is context-dependent— the same protein can activate, repress, or do nothing depending on its binding partners.

Question 15

What is the basic structure of chromatin in eukaryotic cells?

Answer 15

Chromatin = DNA + histone proteins
Basic unit: Nucleosome

~150 bp of DNA wrapped around a histone octamer

Histone octamer: 2 copies each of H2A, H2B, H3, H4

Chromatin compacts DNA to fit into the nucleus and regulates gene expression

Question 16

What is chromatin remodeling and how does it affect transcription?

Answer 16

Chromatin remodeling= repositioning or removal of histones to expose DNA
* Makes regulatory sequences (e.g., promoter, TATA box) accessible to transcription factors & RNA polymerase
* Required for stable binding of RNA polymerase to many eukaryotic promoters
Promoters (like those with a TATA box) are often hidden by nucleosomes- remodeling exposes these sites to initiate transcription
SWI/SNF complex: chromatin remodeling complex that moves or ejects nucleosomes to expose promoter regions

Question 17

What are histone tail modifications, and how do they affect gene expression?

Answer 17

Histone modifications occur mainly on N-terminal tails of histone proteins.
* These tails protrude from the nucleosome and are accessible for enzyme activity.

Modifications are post-translational and include:
* Acetylation
* Methylation
* Phosphorylation
* Ubiquitination

Each modification is specific to certain amino acids (e.g., lysine, serine).
* Modifications are reversible.
Together, they form the “histone code” (~150 known positions/mods).

This code helps regulate chromatin structure and gene expression:
* Acetylation of H3K9 → open chromatin → active transcription
* Methylation of H3K27 → compact chromatin → gene silencing

Question 18

How does acetylation of histone tails affect chromatin and gene expression?

Answer 18

Acetylation is a histone modification that generally activates gene expression.
* Targets lysine residues on histone tails.

Neutralizes positive charge on lysine:
* Reduces interaction between histones and negatively charged DNA.
* Results in looser, more open chromatin (euchromatin).
* Facilitates access of transcription machinery.
* Can create binding sites for chromatin-associated proteins.

HATs (Histone Acetyl Transferases):
* Add acetyl groups to histones.

HDACs (Histone Deacetylases):
* Remove acetyl groups, often leading to gene repression.

Question 19

How is GAL1 transcription negatively regulated through histone deacetylation?

Answer 19

GAL1 is negatively regulated by the Mig1-Tup1 complex.
* Tup1 acts as an HDAC (histone deacetylase)- removes acetyl groups from histones.
* –> Leads to chromatin condensation and gene repression.
* Mig1 localizes to the nucleus when glucose is high, recruiting Tup1.

In high glucose, this repression overrides Gal4 activation.

In presence of galactose:
* Gal80 is removed from Gal4.
* Gal4 activates GAL1 transcription.

Question 20

What is histone methylation and how does it affect gene expression?

Answer 20

Histone methylation is a post-translational modification on lysine or arginine residues of histone tails.
* It can occur in mono-, di-, or tri-methylated forms on each amino acid.

Unlike acetylation, methylation does not change histone charge.
* Instead, it creates binding sites for specific proteins
* These proteins can activate or repress transcription, depending on the site and degree of methylation.

Examples:
* H3K4me (methylation on histone H3 lysine 4) → Activation of gene expression; enriched at transcription start sites.
* H3K9me and H3K27me → Repression of gene expression.

Question 21

Can methylation and acetylation occur on the same lysine residue of a histone?

Answer 21

Yes, both methylation and acetylation can target the same lysine residue.
* However, a single lysine on a histone can only be modified by one of them at a time—not both simultaneously.

This creates a form of regulatory competition, influencing whether the chromatin is in an active or repressed state.

Question 22

What is epigenetic inheritance and what mechanisms are involved?

Answer 22

Epigenetic inheritance is the passing of chromatin state from one generation to the next, without changing the DNA sequence.

Mechanisms include:
* DNA methylation
* Histone positions on DNA
* Histone modifications (e.g., acetylation, methylation)
* Histone variants (core histones replaced by similar proteins with different functions)

Question 23

How is chromatin state inherited during cell division?

Answer 23

During DNA replication, both the DNA sequence and chromatin structure are faithfully passed on.

• Old histones from existing nucleosomes are randomly distributed to daughter DNA strands.
• New histones are added to fill in the gaps.

This random distribution preserves the “memory” of histone modifications, helping maintain epigenetic states across cell generations.

Question 24

What is DNA methylation and how is it inherited during cell division?

Answer 24

DNA methylation= addition of methyl groups to DNA, usually at cytosine in CG dinucleotides.

In mammals, 70–80% of CG sites are methylated.

After DNA replication, the DNA is hemimethylated (only one strand is methylated).

Maintenance methyltransferases recognize hemimethylated DNA and methylate the new strand.

DNA methylation is more stable than histone modifications and is often linked to long-term gene silencing.

Question 25

What are enhanceosomes and how do they help activate transcription?

Answer 25

Enhanceosome = a large complex of regulatory proteins bound to an enhancer that **synergistically** activates transcription (greater than additive effect). Formed by binding of multiple transcription factors to tightly spaced sites within an enhancer. Act over **long distances** (>50 kb from promoter). Precise spacing between binding sites is critical for synergy. Enhanceosomes recruit both **transcriptional machinery** and **chromatin remodelers** Function in **β-interferon** activation: * Viral infection induces β-interferon. * Enhanceosome forms ~100 kb upstream. * GCN5 **acetylates** histones at the enhancer. * Acetylated histones recruit chromatin remodelers. * Histones are moved, **exposing the promoter**. * RNA polymerase II binds → **transcription begins**.

Question 26

What is an enhancer-blocking insulator and how does it work?

Answer 26

**Insulator**: A **cis-acting** DNA element that restricts the spread of chromatin state to a **specific region** of the genome. **Enhancer-blocking insulator**: A type of insulator that prevents an enhancer from activating a promoter when placed between them. Likely mechanism: * DNA-binding proteins attach to the insulator. * These proteins form complexes that fold DNA and promote long-range DNA interactions, physically separating the enhancer from the promoter.

Question 27

How does spreading heterochromatin silence genes?

Answer 27

**Chromatin** = DNA + associated proteins. **Heterochromatin** = Condensed chromatin; transcriptionally **inactive**. **Euchromatin** = Open chromatin; transcriptionally **active**. Spreading heterochromatin: Heterochromatin can extend into neighboring regions, silencing genes by making DNA **inaccessible** to transcription machinery. Constitutive heterochromatin: Regions **always** heterochromatic, like: * Centromeres * Telomeres * Clusters of repetitive DNA

Question 28

What is Position Effect Variegation (PEV), and what causes it?

Answer 28

**Position Effect Variegation (PEV):** A phenomenon where gene expression **varies between cells**, depending on the gene’s chromosomal **position**, even though the cells are from the **same tissue**. * Occurs when a gene is relocated **near heterochromatin**, leading to **random spreading** of repressive chromatin into the gene in some cells but not others. * The white gene in Drosophila: * -- When moved near heterochromatin, it gets silenced in some ommatidia, causing a **mottled eye color**. Key Proteins: HP1 (Heterochromatin Protein 1): Binds to H3K9-methylated histones and is required for maintaining repressed chromatin. Genetic Screens:

Question 29

What is a barrier insulator, and what is its function?

Answer 29

**Barrier Insulator**: A DNA element that functions to **block the spread** of heterochromatin into neighboring euchromatic (active) regions. * Maintains gene expression by **preventing silencing** from spreading repressive chromatin into active gene regions. * Acts as a **boundary** between heterochromatin and euchromatin.

Question 30

What is genomic imprinting and how does it differ between maternal and paternal imprinting?

Answer 30

**Genomic Imprinting**: A form of epigenetic gene regulation where gene expression depends on the parent of origin. * One allele is **silenced** (repressed) based on whether it came from the mother or the father. **Paternal Imprinting**: * The allele from the father is repressed. * **Maternal Expression** **Maternal Imprinting**: * The allele from the mother is repressed. * **Paternal Expression** Result: Only **one parental copy** of the gene is **active**, while the other is **silenced**.

Question 31

What are the steps required for genomic imprinting and how is the imprint established?

Answer 31

**Erasure** of Existing Imprints: * In primordial germ cells, previous imprinting marks on both chromosomes are erased. **Re-establishment** of New Imprints: * Imprints are re-established according to the sex of the individual: * Sperm and oocytes acquire different imprinting patterns. **Mechanism** of Imprinting: * Imprints are mainly imposed through **DNA methylation**. * Methylation prevents DNA-binding proteins from interacting with DNA, leading to **gene silencing**.

Question 32

How does genomic imprinting regulate Igf2 and H19 expression using insulators?

Answer 32

Paternal allele: * ICR is methylated → **CTCF cannot bind** * Enhancer activates Igf2 → **Igf2 expressed**, **H19 repressed** Maternal allele: * ICR is unmethylated → **CTCF binds the ICR** * CTCF blocks enhancer from accessing Igf2 * Enhancer activates H19 → **H19 expressed, Igf2 repressed** Key concepts: * Enhancer can only activate **one gene** (Igf2 or H19) at a time * CTCF binds **only unmethylated** ICR, acting as an insulator * Methylation status of ICR determines which gene is **activated**

Question 33

How can genomic imprinting result in unusual inheritance patterns in heterozygous individuals?

Answer 33

Imprinting can cause a **null** (loss-of-function) phenotype even in heterozygous individuals, depending on parental origin of the allele. A recessive allele can **appear dominant or recessive** depending on which parent it was inherited from and which allele is imprinted (silenced).

Question 34

What is X-chromosome inactivation and how is it regulated?

Answer 34

**X-chromosome inactivation** is the process where one of the two X chromosomes in female cells becomes **transcriptionally inactive** to **balance gene dosage** with XY males. * Which X chromosome is inactivated is **random**, but once selected, that same X **remains inactive** in all descendant (progenitor) cells.

Question 35

What is the role of Xist in X-inactivation?

Answer 35

Xist (**X-inactive specific transcript**) is a noncoding RNA (ncRNA) * It is transcribed from the X-inactivation center of the inactive X chromosome. Xist RNA coats the chromosome it’s transcribed from. * Coating by Xist leads to the formation of **silent chromatin** on that X. Xist acts as a **“loading dock”** for chromatin-modifying proteins that induce gene silencing. * The other X chromosome (active X) does **not express Xist**.