Unit 3.L3-Transcription Factors & Epigenetic in Development and Stem Cells

Question 1

What experiment helped discover that differenct cell types of a multicellular organism have the same DNA blueprint?

Answer 1

A skin cell nucleus of an adult frog transplanted into an enucleated egg gives rise to an entire tadpole!

Question 2

DNA sequence (6 Billion bases) in all cells of a person is the same? So how do we achieve cell diversity and body plan?

Answer 2

Other factors determine cell differentiation to form organs and whole body: TRANSCRIPTION FACTORS

Question 3

What are the two types of differential gene expression in development?

Answer 3

• House Keeping Gene
• Tissue Specific Gene

Question 4

What is an example of a House-keeping gene? What makes it a house-keeping gene?

Answer 4

• β-actin is a House-keeping gene used by all cells all the time.
• RNA is collected from 7 human tissue cell lines (β-actin mRNA: Expression is same in 7 tissue)
• So the expression is same in all cells all the time

Question 5

What is an example of a Tissue specific gene? Where in the body is the specific gene for?

Answer 5

• Tyrosine Aminotransferase
• Highly expressed Liver tissue Specific Gene (Tyrosine aminotransferase is seen to be expressed in liver but not in the other cell types)

Question 6

What dictates cell fates in Development?

Answer 6

Active & Inactive Gene Loci

Question 7

What is an actively expressed gene in cell Type-A far more than compared to Type-B cell (in which it is not expressed)?

• Type A- Hematopoietic lineage; SYK or Spleen Tyrosine Kinase gene
• Type B- Fibroblasts

Answer 7

An actively expressed gene in cell type-A (hematopoietic lineage; SYK or Spleen Tyrosine Kinase gene) is far more DNase-I Hyper-Sensitive (DHS) than in cell type-B (fibroblasts) in which it is not expressed

Question 8

Related cells in different organs (all fibroblasts) have similar what?

Answer 8

DHS (DNase-I Hyper-Sensitive)

Question 9

Distantly related cell types to fibroblast (i.e. hematopoietic cells) have what compared to fibroblasts? But have what within themselves (hematopoietic cells)?

Answer 9

• Distantly related cell types to fibroblasts (i.e., hematopoietic cells) have different DHS signature when compared to fibroblasts
• Have similar DHS signature within themselves (hematopoietic cells)

DHS (DNase-I Hyper-Sensitive)

Question 10

• 30% of DHS in adult cells overlap with what?
• 30% of what is found in each adult cell type (~200 types)?

Answer 10

• 30% of the DHS in adult cells overlap with ES (embryonic stem) cells
• 30% of a unique DHS is found in each adult cell type (~200 types).

DHS (DNase-I Hyper-Sensitive)

Question 11

What is shown in a Dendrogram?

Answer 11

Dendrogram from all DHS maps showing relationship of gene activation in each cell type across the entire genome

DHS (DNase-I Hyper-Sensitive)

Question 12

What is the relationship between the 3 germ layers and DHS? What types of cells cluster together for each one?

Answer 12

Each Germ layer has a strong relationship

1. Mesoderm

• Fibroblast (Paraxial Mesoderm) Cluster together
• Hemocytoblasts (Lymphoid + Hematopoietic Prog.) Cluster together

2. Ectoderm

• Skin Keratinocytes/mammary epithelia Cluster together

3. Endoderm

• Airway & Esophageal Epithelia Cluster together

Question 13

What determine cell types during development?

Answer 13

Transcription Factors (TFs)

Question 14

Where does selective gene expression differentiate cell types? Where do they later differentiate and what role do they play?

Answer 14

Selective gene expression differentiate cell types in three germ layers & later in each tissue of an organ, giving structure and function to an organ & embryo.

Question 15

What is selective gene expression dictated by?

Answer 15

Tissue-specific transcription factors (TFs)

Question 16

What do TFs target and what type of expression do they have?

Answer 16

Tissue-specific transcription factors (TFs) that select target genes that have cell-lineage and stage-specific expression

Question 17

Mechanism of Cell Specification by Transcription Factors

1. How many cells types are defined by the Distinct set of TFs?
2. What turns on/off during successive generations of cells in development?
3. What does TFs activate when a cell matures and goes through different stages?
4. In subsequently cell-divisions what terminally differentiate a cell type?

Answer 17

1. Distinct set of TFs define each of the >200 cell types in humans.
2. Spatial-temporal expression of TFs turn on/off during successive generations of cells in development.
3. As cells mature and go through different stages, TFs activate a gene repertoire and change the cell type.
4. In subsequent cell-divisions, it is the combination of different TFs that terminally differentiate a cell type.

Question 18

About how many TFs in the human body is tissue specification dependent on?
What are the major families of TFs? (4)

Answer 18

• About 2500 TFs in a human body expressed in different cell types→combination→Cell & Tissue-Type
• Major Families of TFs
  1. Forkhead Box (FOX) family of TFs
  2. HOX family of TFs
  3. PAX family of TFs
  4. bHHL-family TFs

Question 19

Tissue specfic TFs bind what? And how does it affect transcription?

Answer 19

Tissue specific TFs bind DNA-elements & activate or repress transcription

Question 20

What does TFs dictate concering gene expression? What are the four parts of TFs?

Answer 20

TFs dictate cell specific gene expression for tissue & organ specification

1. Promoters
2. Enhancers
3. Silencers
4. Insulators

Question 21

Where are promoters and enhancers present on TFs? What are silencers and insulators and what are their functions?

Answer 21

1. Promoters: Present in the proximal regulatory regions
2. Enhancers (Activators): Present in the distal regulatory regions
3. Silencers: DNA elements that bind repressors and silence gene transcription (near proximal promoter)
4. Insulators: DNA boundary element that blocks the interaction between enhancers & promoters (@ either side of gene loci)

Question 22

How does DNA looping result in liver specific expression of Transthyretin Gene (TTR)?

Answer 22

DNA-looping brings Enhancers bound to liver-specific TFs (HNF1,3, 4) to the promoter & TATA-box, resulting in TTR expression only in the liver

Question 23

• How many members are part of the Forkhead Box (FOX) family?
• What is strucure of Forkhead (FKH) BOX?
• What is the function of FOX factors?
• How do FOX TFs act as?
• What part binds DNA?

Answer 23

• >40 members in FOX-family play diverse roles in development
• FKH BOX: : ~100 amino acids (wings; W1, W2 & Helix H1, H2, Helix-H3). H3 binds DNA at specific target genes (conserved domain)
• FOX factors: open chromatin & act as gateway factors to make gene loci accessible for transcription
• FOX TFs: act as transcription activators or repressors
• Helix-3 (H3) binds DNA: note W1 & W2 (Wings) that form wings

Question 24

List the different mutations that can arise for the FOX-family?

Answer 24

• FOXC1 mutations
• FOXC2 mutations
• FOXP2 mutations
• FOXP3 mutations

Question 25

# Mutation of FOX-family What developmental disorders (2) are caused by FOXC1 mutations?

Answer 25

Iris hypoplasia & Rieger syndrome

Question 26

# Mutation of FOX-family What developmental disorder is caused by FOXC2 mutations?

Answer 26

Lymphedema (**Lymphedema of the limbs**) and/or **Distichiasis syndrome** (**double rows of eyelashes**) ## Footnote Ex. Elizabeth Taylor

Question 27

# Mutation of FOX-family What developmental disorder is caused by FOXP2 mutations?

Answer 27

* Language acquisition defects * Speech defects

Question 28

# Mutation of FOX-family What developmental disorder is caused by FOXP3 mutations?

Answer 28

* IPEX Syndrome: X-linked immuno-dysregulation, poly-endocrinopathy and enteropathy (intestinal inflammation).

Question 29

What is FOXP3 a master regulator for?

Answer 29

FOXP3 is a master regulator of the **regulatory T cell lineage**

Question 30

How were the Homeobox (Hox) family genes discovered?

Answer 30

**Hox-family** of **Homeotic genes** expressing TFs were discovered in **Drosophila melanogaster**

Question 31

What does a mutation in HOM-C gene cause?

Answer 31

Antennapedia: sprout legs instead of antennae on the head

Question 32

What is Hox gene Conservation & Collinearity of expression?

Answer 32

Physical **order** & **expression** of **HOX genes along the anteroposterior axis is conserved** from flies to humans

Question 33

* What is the structure of HOX (Homeobox) family TFs? * What does it contain? * Which structure is the Recognition Helix? * What binds DNA at the TAAT or ATTA sequence? * What binds in the DNA-Major Groove? * What binds in the DNA-Minor Groove?

Answer 33

1. Is a **50 Amino Acid conserved region (180 basepairs)** 2. **Contains 3 Helices: H1, H2, H3** & an **N-terminal tail region** 3. **Helix-3 (H3)** is the **"Recognition Helix”** 4. **H3 & N-terminus** binds DNA at **TAAT** or **ATTA** sequence 5. **H3** binds in the DNA-Major Groove 6. **N-terminus** tail binds in the DNA-Minor groove

Question 34

What are the four Homeobox (HOX) mutations that can occur?

Answer 34

1. HOXA1 Mutations 2. HOXA13 & HOXD13 Mutations 3. EMX2 Homeobox gene Mutations 4. NKX-2.5 Mutations (~40 mutations)

Question 35

# HOX family Mutations What defects occur d/t HOXA1 mutation?

Answer 35

Impaired human neural development

Question 36

# HOX family Mutations What defects occur d/t HOXA13 & HOXD13 mutation? What mutation specifically happens w/ HOXD13?

Answer 36

* **Limb malformations** * Mutations in **N-terminal, non–DNA binding** part of **HoxD13 cause**→Webbing, Syndactyly, & Polydactyly.

Question 37

# HOX family Mutations What defects occur d/t EMX2 Homeobox gene mutations?

Answer 37

**Schizencephaly (type II): Unilateral Full-Thickness cortex in the ventricles**→ **Seizures, Spasticity, and Mental deficiency**

Question 38

# HOX family Mutations What defects occur d/t NKX-2.5 Mutations?

Answer 38

* **~40 mutations** * **Diverse Congential Cardiac Defects** 1. Congenital CVM (Cardiovascular Malformation) 2. **ASD: Atrial Septal Defect** 3. **AVB: Atrioventricular Block** (block in impulse from the atria→ ventricles) 4. DORV: Double Outlet Right Ventricle (PA + Aorta open in RV)] 5. TOF: Tetralogy of Fallot (VSD + Pul. Valve Stenosis + RA-hypertrophy + moved aorta) 6. **VSD: Ventricular Septal Defect** 7. TVA: Tricuspid Valve Atresia

Question 39

* What is the structure of all Paired box (PAX) family proteins? * What does PAX1 & PAX9 lack? * What is the function of PAX TFs? * What is the role of Pax6 in humans? What is it called in Drosophila? What occurs if there is a mutation? What occurs when there is ectopic expression? * What is Pax6 a master controller?

Answer 39

* All PAX proteins have a **bipartite DNA-binding motif (Paired Box; PAX)**, & most of them have a **homeodomain** * PAX1 & PAX9 **do not** have a **homeodomain** * PAX TFs **activate** or **repress transcription** of target genes * **Pax6 (human)**→ **Eye formation**; Drosophila Ortholog **eyeless** gene mutation→ **no eyes**→ **Ectopic expression of eyeless**→ **additional eyes (antenna thorax & 6 legs)** * **Pax6 is a Master controller for eye development**

Question 40

What are mutations in PAX genes that can occur?

Answer 40

1. PAX6 mutations 2. PAX3 and PAX7 Translocation and Fusion to FOXO1A 3. PAX3 Mutation

Question 41

# PAX family mutations What defects occur d/t PAX6 Mutations?

Answer 41

* Ocular malformations: **Aniridia (absence of the iris)** & **Peter anomaly (central corneal opacity)** * **PAX6 Haploinsufficiency**: Have some ocular defects. * **PAX6- null** patients have **Anophthalmia**

Question 42

# PAX family mutations What defects occur d/t PAX3 and PAX7 translocation to FOXO1A? What chimeras are formed?

Answer 42

* **Childhood Cancer: Alveolar rhabdomyosarcoma** (in the muscle) due to chromosomal translocation * Forms **PAX3:FOXO1A** or **PAX7:FOXO1A** chimeras ## Footnote Chimeras: an organism or tissue that contains at least two different sets of DNA

Question 43

# PAX family mutations What defects occur d/t PAX3 Mutations?

Answer 43

* **Waardenburg syndrome type I** (autosomal dominant disease) * **Hearing deficits, ocular defects, epicanthal fold** * **Pigmentation abnormalities** (typified by **White Forelock**)

Question 44

* What does the Basic Helix-Loop-Helix (bHLH) TFs determine? * bHLH proteins contain what two things? * bHLH TFs bind target gene promoters via what?

Answer 44

* bHLH determine **cell fate** and **differentiation** in many developing tissues. * bHLH proteins contain 1. **Basic, positively charged DNA-binding region (N-terminus)** 2. **Two Alpha-Helices separated by a loop (C-terminus)** * bHLH TFs bind target gene promoters via **E-box** DNA-sequence **(CANNTG)** ## Footnote bHLH straddles the DNA (C-terminus binds to DNA, N-terminus stays outside)

Question 45

What is the role of the bHLF TFs listed below: * bHLH-TFs (Myogenin & MYOD) * bHLH-TFs (Neurogenin + NEUROD) * bHLH-TFs (ASCL1 + NEUROD3)

Answer 45

* bHLH-TFs **(Myogenin & MYOD)**→ Differentiation to **muscles** in embryo. * bHLH-TFs **(Neurogenin + NEUROD)** → Differentiation to **neurons** in embryo * bHLH-TFs **(ASCL1 + NEUROD3)** are **proneural genes** that **convert neuroepithelium to neuroblasts**

Question 46

What is the definition of Epigenetics?

Answer 46

The change in gene expression pattern (repression or activation) that alters the phenotype **without altering the DNA sequence** of a genome

Question 47

What are the major mechanisms that causes Epigenetic regulation? (4)

Answer 47

1. Histone acetylation 2. Histone methylation 3. DNA methylation 4. miRNAs

Question 48

What are the monomers that make up a histone?

Answer 48

* H2A * H2B * H3 * H4

Question 49

How does histone monomers become a dimer?

Answer 49

Handshake interactions (evolutionarily conserved)

Question 50

What is the structure of Histone Octamers?

Answer 50

Pair of H2A, H2B, H3 + H4 subunits

Question 51

What is the structure of a Nucleosome?

Answer 51

**Histone Octamer** (Pair of H2A, H2B, H3 + H4 subunits) + **DNA (~147 bps)**

Question 52

What is the structure of a Chromatin? What are major modifications on Chromatin?

Answer 52

* **Nucleosome + Linker Histone H1= Chromatin** * Major Modifications 1. **Acetylation** 2. **Methylation**

Question 53

What are the 3 steps of the Dynamic Histone Code and what enzymes play a role in each step?

Answer 53

1. **Writing** * Acetylases * Methylases * Phosphorylases 2. **Erasing** * Deacetylases * Demethylases * Phosphatases 3. **Reading** * Bromodomain * Chromodomain * PHD finger * WD40 repeat

Question 54

What are the writer protein, eraser protein and reader protein that are involved in the Epigenetic Modifications listed below: 1. Histone Acetylation 2. Histone Methylation 3. DNA Methylation

Answer 54

1. Histone Acetylation * Writer protein: **Histone acetyltransferases (HATs)**: E1A binding protein, 300-KD (EP300) * Eraser protein: **Histone deacetylases (HDACs)**: HDAC1 * Reader protein: **Chromatin remodeling enzymes: SMARCA4** (formerly BRG1) 2. Histone Methylation * Writer protein: **Histone methylases (HMTs)**: EZH2 * Eraser protein: **Histone demethylases: JARID1C** * Reader protein: **Polycomb repressive complex: CBX2** 3. DNA Methylation (5th position on Glycine) * Writer protein: **DNA methylases: DNMT1** * Eraser protein: **Tet oncogene family members: methylcytosine dioxygenases (TET1)** * Reader protein: **MECP2**

Question 55

What are disorders of chromatin remodeling that can arise during development?

Answer 55

1. **Rett Syndrome** 2. Rubinstein-Taybi syndrome 3. Alpha-thalassemia/X-linked mental retardation syndrome 4. Many types of cancers

Question 56

What is the role of histone acetylation? Histone methylation?

Answer 56

* Histone Acetylation- **Gene Activation** * Histone Methylation-**Silencing Genes**

Question 57

What are histone tails acetylated by? What is the function of Histone Acetyl Transferases (HATs) and Histone deacetylases (HDACs)?

Answer 57

* Histone tails are acetylated by enzymes * **Histone Acetyl Transferases (HATs)**: **Add acetyl groups** (Writers) on histone tails = **Gene Activation** * **Histone deacetylases (HDACs)**, which **remove acetyl groups** (Erasers) from histone tails = **Gene Silencing**

Question 58

What are Histone Lysines on the tails modified by? What removes the modifications?

Answer 58

* Histone Lysines on the tails are modified by **methyl group** **added** by ENZYME WRITERS **histone methyltransferases (HMTs)** * These modifications are **removed** by ENZYME ERASERS: **histone demethylases (HDMs)**

Question 59

Which histone proteins acetylated/demethylated Active Chromatins?

Answer 59

**HATs** (Histone Acetyl Transferases) + **HDMs** (histone demethylases)

Question 60

Which histone proteins deacetylated/methylated Silenced Chromatins?

Answer 60

**HDACs** (Histone deacetylases) + **HMTs** (histone methyltransferases)

Question 61

* What is DNA methylation used for? * At Embryo Implantation, what is methylated? What methylates it and what is the outcome to the gene? * As Embryo develops, what is silenced by DNA methylation and why? * What happens to DNA methylation in Primordial Germ Cells (PGCs)?

Answer 61

* DNA methylation is used for the **long-term repression of genes** * **At Embryo Implantation: Cytosines** at **GC dinucleotides** are **methylated** by **DNA methyltransferases (DNMTs); most genes silenced** * **As Embryo develops: Pluripotency genes** are **silenced** by DNA Methylation→ **Cells differentiate into 3 germ layers** * In Contrast: **In Primordial Germ Cells (PGCs): DNA** Methylation is **erased** to re-express pluripotency genes

Question 62

What is Methyl-Cytosine-Binding Protein 2 (MECP2)?

Answer 62

**MECP2; a reader enzyme** is a methylation-dependent transcriptional modulator (binds to DNA on the cytosine that are methylated)

Question 63

What does a mutation in MECP2 (Methyl-Cytosine-Binding Protein 2) result in?

Answer 63

Results in abnormal expression at gene locus→leads to a **developmental disorder: Rett syndrome**

Question 64

What is Rett Syndrome? What clinical effects does it cause? Who is most affected?

Answer 64

* **Neurological developmental disorder**, which alters **brain development** * Causing a **progressive inability to use muscles** for **eye and body movements & speech** * Found **almost exclusively in girls (show frequent seizures and intellectual disability)** ## Footnote Mutation in MECP2

Question 65

What are MicroRNAs (miRNAs)? When do they act? What do they contribute to?

Answer 65

* MicroRNAs (miRNAs or miRs) are **conserved 22-nucleotide small noncoding RNAs** that act **post-transcriptionally** to **silence functional RNAs** in a cell * It contributes to **cell differentiation** and **cell-fate** without changing DNA sequence –thus is an **epigenetic event**

Question 66

What are the steps in the production of miRNA? What is the function?

Answer 66

1. **Transcription**: Long **primary-miRNA** is formed from the genome 2. **DROSHA cuts & matures** it to a **70 bp stem-loop** (pre-miRNA) in the nucleus 3. **70 bp stem-loop-miRNAs** is exported out & binds **RNase (DICER)**, which processes it to mature **22 bp miRNA duplexes** 4. **RISC-Complex** binds complementary **miRNA strand** * Function: **RISC-miRNA:**→ 1) Inhibits Translation or 2) Degrades mRNA→**NO PROTEIN** ## Footnote 3-protein interplay

Question 67

What are dieases that can be caused by altered miRNA expression?

Answer 67

* Different **developmental disorders** * **Oncomirs**→miRNA associated with cancer * **DICER1: (Germline mutations**)→**Familial Tumor Predisposition Syndrome:** * **Diseases: Pleuropulmonary blastema, Cystic nephroma, & Medulloepithelioma**

Question 68

* Totipotent (morula) stems are? * Pluripotent (inner blastula) are? * Stem cells self-renew by?

Answer 68

* **Totipotent (morula)** stem cells are→**all 3 primary germ layers** + **extra-embryonic tissues** * **Pluripotent (inner blastula)**→cell of **only 3 primary germ layers** but **NOT extra-embryonic tissues** * **Stem cells self-renew by**: **(1)Symmetric (vertical)** or **(2) Asymmetric (horizontal)** cell divisions ## Footnote Symmertric division→Replenishment Asymmetric division→ Differentiated

Question 69

What are the different types of Stem Cells? (4)

Answer 69

1. **ESCs (Embryonic Stem cells)** 2. **ASCs (Adult Stem Cells)** 3. **iPSCs (induced Pleuripitent Stem Cells)** 4. **CSCs (Cancer Stem Cells)**

Question 70

What do ESC (Embryonic Stem Cells) express? and what is there function?

Answer 70

(ESCs) express **TFs (SOX2 & OCT4)** that **repress differentiation & maintain STEMNESS** required for replenishment of stem cell pool

Question 71

How do Adult Stem cells (ASCs) or Embryonic Stem cells (ESCs) **divide symmetrically**?

Answer 71

**2 equivalent daughter stem cells** in a **vertical cell division**; the **plane of mitosis is perpendicular** to the neural ventricle surface

Question 72

How do Adult Stem cells (ASCs) or Embryonic Stem cells (ESCs) **divide asymmetrically**?

Answer 72

**Daughter stem cell + Neuroblast cell; horizontal cell division**; the **plane of mitosis is parallel to the ventricular surface** * The progenitor cell loses stem cell factors (geometric shapes) * The progenitor cell expresses new proteins→Alar and Basal Plate

Question 73

Embryonic Stem Cells (ESCs) & Induced pluripotent stem cells (iPSCs) have the capacity to do what? (4)

Answer 73

1. Self-renew to Stem Cells 2. Apoptosis/cell death 3. Become progenitor Cells 4. Differentiate into various types of Cells.

Question 74

What can Adult, differentiated somatic cells (skin fibroblast) be reprogrammed into? by expressing what?

Answer 74

Adult, differentiated somatic cells (skin fibroblasts), can be reprogrammed into **iPSCs** by **expressing master transcription factors: (SOX2 + OCT3/4 + KLF4)** ## Footnote Induced pluripotent stem cells (iPSCs)