Unit 3.L3-Transcription Factors & Epigenetic in Development and Stem Cells Flashcards
What experiment helped discover that differenct cell types of a multicellular organism have the same DNA blueprint?
A skin cell nucleus of an adult frog transplanted into an enucleated egg gives rise to an entire tadpole!
DNA sequence (6 Billion bases) in all cells of a person is the same? So how do we achieve cell diversity and body plan?
Other factors determine cell differentiation to form organs and whole body: TRANSCRIPTION FACTORS
What are the two types of differential gene expression in development?
- House Keeping Gene
- Tissue Specific Gene
What is an example of a House-keeping gene? What makes it a house-keeping gene?
- 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
What is an example of a Tissue specific gene? Where in the body is the specific gene for?
- Tyrosine Aminotransferase
- Highly expressed Liver tissue Specific Gene (Tyrosine aminotransferase is seen to be expressed in liver but not in the other cell types)
What dictates cell fates in Development?
Active & Inactive Gene Loci
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
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
Related cells in different organs (all fibroblasts) have similar what?
DHS (DNase-I Hyper-Sensitive)
Distantly related cell types to fibroblast (i.e. hematopoietic cells) have what compared to fibroblasts? But have what within themselves (hematopoietic cells)?
- 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)
- 30% of DHS in adult cells overlap with what?
- 30% of what is found in each adult cell type (~200 types)?
- 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)
What is shown in a Dendrogram?
Dendrogram from all DHS maps showing relationship of gene activation in each cell type across the entire genome
DHS (DNase-I Hyper-Sensitive)
What is the relationship between the 3 germ layers and DHS? What types of cells cluster together for each one?
Each Germ layer has a strong relationship
- Mesoderm
- Fibroblast (Paraxial Mesoderm) Cluster together
- Hemocytoblasts (Lymphoid + Hematopoietic Prog.) Cluster together
- Ectoderm
- Skin Keratinocytes/mammary epithelia Cluster together
- Endoderm
- Airway & Esophageal Epithelia Cluster together
What determine cell types during development?
Transcription Factors (TFs)
Where does selective gene expression differentiate cell types? Where do they later differentiate and what role do they play?
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.
What is selective gene expression dictated by?
Tissue-specific transcription factors (TFs)
What do TFs target and what type of expression do they have?
Tissue-specific transcription factors (TFs) that select target genes that have cell-lineage and stage-specific expression
Mechanism of Cell Specification by Transcription Factors
- How many cells types are defined by the Distinct set of TFs?
- What turns on/off during successive generations of cells in development?
- What does TFs activate when a cell matures and goes through different stages?
- In subsequently cell-divisions what terminally differentiate a cell type?
- Distinct set of TFs define each of the >200 cell types in humans.
- Spatial-temporal expression of TFs turn on/off during successive generations of cells in development.
- As cells mature and go through different stages, TFs activate a gene repertoire and change the cell type.
- In subsequent cell-divisions, it is the combination of different TFs that terminally differentiate a cell type.
About how many TFs in the human body is tissue specification dependent on?
What are the major families of TFs? (4)
- About 2500 TFs in a human body expressed in different cell types→combination→Cell & Tissue-Type
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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
Tissue specfic TFs bind what? And how does it affect transcription?
Tissue specific TFs bind DNA-elements & activate or repress transcription
What does TFs dictate concering gene expression? What are the four parts of TFs?
TFs dictate cell specific gene expression for tissue & organ specification
- Promoters
- Enhancers
- Silencers
- Insulators
Where are promoters and enhancers present on TFs? What are silencers and insulators and what are their functions?
- Promoters: Present in the proximal regulatory regions
- Enhancers (Activators): Present in the distal regulatory regions
- Silencers: DNA elements that bind repressors and silence gene transcription (near proximal promoter)
- Insulators: DNA boundary element that blocks the interaction between enhancers & promoters (@ either side of gene loci)
How does DNA looping result in liver specific expression of Transthyretin Gene (TTR)?
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
- 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?
- >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
List the different mutations that can arise for the FOX-family?
- FOXC1 mutations
- FOXC2 mutations
- FOXP2 mutations
- FOXP3 mutations
Mutation of FOX-family
What developmental disorders (2) are caused by FOXC1 mutations?
Iris hypoplasia & Rieger syndrome
Mutation of FOX-family
What developmental disorder is caused by FOXC2 mutations?
Lymphedema (Lymphedema of the limbs) and/or Distichiasis syndrome (double rows of eyelashes)
Ex. Elizabeth Taylor
Mutation of FOX-family
What developmental disorder is caused by FOXP2 mutations?
- Language acquisition defects
- Speech defects
Mutation of FOX-family
What developmental disorder is caused by FOXP3 mutations?
- IPEX Syndrome: X-linked immuno-dysregulation, poly-endocrinopathy and enteropathy (intestinal inflammation).
What is FOXP3 a master regulator for?
FOXP3 is a master regulator of the regulatory T cell lineage
How were the Homeobox (Hox) family genes discovered?
Hox-family of Homeotic genes expressing TFs were discovered in Drosophila melanogaster
What does a mutation in HOM-C gene cause?
Antennapedia: sprout legs instead of antennae on the head
What is Hox gene Conservation & Collinearity of expression?
Physical order & expression of HOX genes along the anteroposterior axis is conserved from flies to humans
- 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?
- Is a 50 Amino Acid conserved region (180 basepairs)
- Contains 3 Helices: H1, H2, H3 & an N-terminal tail region
- Helix-3 (H3) is the “Recognition Helix”
- H3 & N-terminus binds DNA at TAAT or ATTA sequence
- H3 binds in the DNA-Major Groove
- N-terminus tail binds in the DNA-Minor groove
What are the four Homeobox (HOX) mutations that can occur?
- HOXA1 Mutations
- HOXA13 & HOXD13 Mutations
- EMX2 Homeobox gene Mutations
- NKX-2.5 Mutations (~40 mutations)
HOX family Mutations
What defects occur d/t HOXA1 mutation?
Impaired human neural development
HOX family Mutations
What defects occur d/t HOXA13 & HOXD13 mutation? What mutation specifically happens w/ HOXD13?
- Limb malformations
- Mutations in N-terminal, non–DNA binding part of HoxD13 cause→Webbing, Syndactyly, & Polydactyly.
HOX family Mutations
What defects occur d/t EMX2 Homeobox gene mutations?
Schizencephaly (type II): Unilateral Full-Thickness cortex in the ventricles→ Seizures, Spasticity, and Mental deficiency
HOX family Mutations
What defects occur d/t NKX-2.5 Mutations?
- ~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
- 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?
- 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
What are mutations in PAX genes that can occur?
- PAX6 mutations
- PAX3 and PAX7 Translocation and Fusion to FOXO1A
- PAX3 Mutation
PAX family mutations
What defects occur d/t PAX6 Mutations?
- Ocular malformations: Aniridia (absence of the iris) & Peter anomaly (central corneal opacity)
- PAX6 Haploinsufficiency: Have some ocular defects.
- PAX6- null patients have Anophthalmia
PAX family mutations
What defects occur d/t PAX3 and PAX7 translocation to FOXO1A? What chimeras are formed?
-
Childhood Cancer:
Alveolar rhabdomyosarcoma (in the muscle) due to chromosomal translocation - Forms PAX3:FOXO1A or PAX7:FOXO1A chimeras
Chimeras: an organism or tissue that contains at least two different sets of DNA
PAX family mutations
What defects occur d/t PAX3 Mutations?
- Waardenburg syndrome type I (autosomal dominant disease)
- Hearing deficits, ocular defects, epicanthal fold
- Pigmentation abnormalities (typified by White Forelock)
- What does the Basic Helix-Loop-Helix (bHLH) TFs determine?
- bHLH proteins contain what two things?
- bHLH TFs bind target gene promoters via what?
- 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)
bHLH straddles the DNA (C-terminus binds to DNA, N-terminus stays outside)
What is the role of the bHLF TFs listed below:
- bHLH-TFs (Myogenin & MYOD)
- bHLH-TFs (Neurogenin + NEUROD)
- bHLH-TFs (ASCL1 + NEUROD3)
- 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
What is the definition of Epigenetics?
The change in gene expression pattern (repression or activation) that alters the phenotype without altering the DNA sequence of a genome
What are the major mechanisms that causes Epigenetic regulation? (4)
- Histone acetylation
- Histone methylation
- DNA methylation
- miRNAs
What are the monomers that make up a histone?
- H2A
- H2B
- H3
- H4
How does histone monomers become a dimer?
Handshake interactions (evolutionarily conserved)
What is the structure of Histone Octamers?
Pair of H2A, H2B, H3 + H4 subunits
What is the structure of a Nucleosome?
Histone Octamer (Pair of H2A, H2B, H3 + H4 subunits) + DNA (~147 bps)
What is the structure of a Chromatin?
What are major modifications on Chromatin?
- Nucleosome + Linker Histone H1= Chromatin
- Major Modifications
1. Acetylation
2. Methylation
What are the 3 steps of the Dynamic Histone Code and what enzymes play a role in each step?
- Writing
- Acetylases
- Methylases
- Phosphorylases
- Erasing
- Deacetylases
- Demethylases
- Phosphatases
- Reading
- Bromodomain
- Chromodomain
- PHD finger
- WD40 repeat
What are the writer protein, eraser protein and reader protein that are involved in the Epigenetic Modifications listed below:
- Histone Acetylation
- Histone Methylation
- DNA Methylation
- 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)
- Histone Methylation
- Writer protein: Histone methylases (HMTs): EZH2
- Eraser protein: Histone demethylases: JARID1C
- Reader protein: Polycomb repressive complex: CBX2
- DNA Methylation (5th position on Glycine)
- Writer protein: DNA methylases: DNMT1
- Eraser protein: Tet oncogene family members: methylcytosine dioxygenases (TET1)
- Reader protein: MECP2
What are disorders of chromatin remodeling that can arise during development?
- Rett Syndrome
- Rubinstein-Taybi syndrome
- Alpha-thalassemia/X-linked mental retardation syndrome
- Many types of cancers
What is the role of histone acetylation? Histone methylation?
- Histone Acetylation- Gene Activation
- Histone Methylation-Silencing Genes
What are histone tails acetylated by? What is the function of Histone Acetyl Transferases (HATs) and Histone deacetylases (HDACs)?
- 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
What are Histone Lysines on the tails modified by? What removes the modifications?
- 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)
Which histone proteins acetylated/demethylated Active Chromatins?
HATs (Histone Acetyl Transferases) + HDMs (histone demethylases)
Which histone proteins deacetylated/methylated Silenced Chromatins?
HDACs (Histone deacetylases) + HMTs (histone methyltransferases)
- 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)?
- 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
What is Methyl-Cytosine-Binding Protein 2 (MECP2)?
MECP2; a reader enzyme is a methylation-dependent transcriptional modulator (binds to DNA on the cytosine that are methylated)
What does a mutation in MECP2 (Methyl-Cytosine-Binding Protein 2) result in?
Results in abnormal expression at gene locus→leads to a developmental disorder: Rett syndrome
What is Rett Syndrome? What clinical effects does it cause? Who is most affected?
- 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)
Mutation in MECP2
What are MicroRNAs (miRNAs)? When do they act? What do they contribute to?
- 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
What are the steps in the production of miRNA? What is the function?
- Transcription: Long primary-miRNA is formed from the genome
- DROSHA cuts & matures it to a 70 bp stem-loop (pre-miRNA) in the nucleus
- 70 bp stem-loop-miRNAs is exported out & binds RNase (DICER), which processes it to mature 22 bp miRNA duplexes
- RISC-Complex binds complementary miRNA strand
- Function: RISC-miRNA:→ 1) Inhibits Translation or 2) Degrades mRNA→NO PROTEIN
3-protein interplay
What are dieases that can be caused by altered miRNA expression?
- Different developmental disorders
- Oncomirs→miRNA associated with cancer
- DICER1: (Germline mutations)→Familial Tumor Predisposition Syndrome:
- Diseases: Pleuropulmonary blastema, Cystic nephroma, & Medulloepithelioma
- Totipotent (morula) stems are?
- Pluripotent (inner blastula) are?
- Stem cells self-renew by?
- 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
Symmertric division→Replenishment
Asymmetric division→ Differentiated
What are the different types of Stem Cells? (4)
- ESCs (Embryonic Stem cells)
- ASCs (Adult Stem Cells)
- iPSCs (induced Pleuripitent Stem Cells)
- CSCs (Cancer Stem Cells)
What do ESC (Embryonic Stem Cells) express? and what is there function?
(ESCs) express TFs (SOX2 & OCT4) that repress differentiation & maintain STEMNESS
required for replenishment of stem cell pool
How do Adult Stem cells (ASCs) or Embryonic Stem cells (ESCs) divide symmetrically?
2 equivalent daughter stem cells in a vertical cell division; the plane of mitosis is perpendicular to the neural ventricle surface
How do Adult Stem cells (ASCs) or Embryonic Stem cells (ESCs) divide asymmetrically?
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
Embryonic Stem Cells (ESCs) & Induced pluripotent stem cells (iPSCs) have the capacity to do what? (4)
- Self-renew to Stem Cells
- Apoptosis/cell death
- Become progenitor Cells
- Differentiate into various types of Cells.
What can Adult, differentiated somatic cells (skin fibroblast) be reprogrammed into? by expressing what?
Adult, differentiated somatic cells (skin fibroblasts), can be reprogrammed into iPSCs by expressing master transcription factors: (SOX2 + OCT3/4 + KLF4)
Induced pluripotent stem cells (iPSCs)
