Lec2/3HumanGeneTranscription Flashcards
Why are transactivators important in development?
Expression and asymmetric distribution of transactivators to daughter cells is KEY in cell linage determination
(Also can be viewed as a circuit)
Examples of Transactivators in development
-Globin switching (Biochemical)
-Heart morphology (Morphological)
What is the path of globin switching from fetus to adult?
-Start with Epsilon globin gene cluster
-Switch to gamma with higher O2 affinity (5-6 weeks, expressed in liver)
-End with beta (+delta) with lower O2 affinity (just before birth, in bone marrow)
What is the region that allows for transcription for fetal or adult goblin genes?
Locus Control Region (LCR)
LCR: Locus Control Region
An enhancer that allows for the transactivators to express fetal or adult hemoglobin (also a regulatory element)
What happens to the cells and transactivator that express fetal hemoglobin after birth?
They will die after birth
The fetal hemoglobin system is an example of what?
Example of”synthesis” as a mechanism for regulating transactivator function
What do adult transactivator bind?
They bind the adult globin gene regulatory elements
What is the basis for hereditary persistence of fetal hemoglobin?
-Fetal cells express different transactivators in development.
-The cells expressing the transactivator would be early stage stem cells.
What does HBAC (histone deac contribute too in globin switching?
-Contributes to silencing the fetal promoters at the adult stage
-Keeps fetal globin genes from getting transcribed
How do we switch form Fetal to adult globin?
- Missing fetal globin transactivator
- HDAC keeps fetal genes condensed
What is the AA mutation in sickle cell anemia?
Glutamate —> Valine
What happens to hemoglobin because of valine?
-Causes it to become a polymer because it wants to exclude H2O due to its hydrophobicity
-these polymers cause a deformity making the sickle shape which leads to a decrease in O2 transfer (can cause pain)
Vorinostat (______ inhibitor)
- Treatment for sickle cell anemia
-HDAC
Lack of HDAC allows for
-Allow for a PIC to form a little and some fetal genes will be transcribed (still no fetal transactivator)
-Then fetal hemoglobin can insert itself into the polymers of the globin in the sickle cell and allow for better O2 transfer
Tbox article:
What are TBXs?
TBX: Transactivators (ex; TBX1 & TBX2)
Tbox article:
Mutation/Removal of TBX transactivator cause what?
Morphological changes in the heart (cardiac congenital anomaly syndromes)
What is an inducible regulation of transcription?
Signal Transduction Pathway
(leading to the activation of a transactivator and transcription)
Definition of a Signal Transduction Pathway
-An extracellular signal (eg. peptide hormone in the blood) can lead to a change in the confirmation of a RECEPTOR, and subsequently, in the way a series of cytoplasmic and nuclear proteins interact with one another.
-These changes ultimately lead to SIGNAL TRANSDUCTION thus “transducing” the extra-cellular signal to the NUCLEUS
Examples of Signal Transduction Pathways
- TNF alpha
- JAK/STAT
- Hedgehog
- RAS
5.FGF
TNF alpha Pathway
-Part of the Immune System
1. TNF alpha binds to receptor
2. Conformational change of receptor leads to a phosphorylation of IKK complex
3. IKB is bound to NFkappaB inhibiting the transactivator, IKK binds to IKB and phosphorylating IKB causing it to degrade
4. NFkB is free, translocates to nucleus binds on to a sequence of DNA and increases transcription
(IKB is a decoy)
1st step of the TNF alpha pathway
- TNF alpha binds to receptor
2nd step of the TNF alpha pathway
- Conformational change leads to a phosphorylation of IKK complex
3rd step of the TNF alpha pathway
3.IKB is bound to NFkappaB inhibiting the transactivator, IKK binds to IKB and phosphorylating IKB causing it to degrade
4th step of TNF alpha pathway
- NFkB is free, translocates to nucleus and increases transcription
JAK/STAT1 pathway
-Plays a part with interferons
1. A cytokine (IFN) will bind to the cytokine receptor and cause the two subunits to come together
2. This will cause JAK to cross phosphorylate each other and the receptor. This will attract STAT (transactivator)
3. STAT will phosphorylated, it will dimerize, hide the phosphates and translocate to the nuclease to increase transcription
1st step of the JAK/STAT1 pathway
- A cytokine (IFN) will bind to the cytokine receptor and cause the two subunits to come together
2nd step of JAK/STAT1 pathway
- This will cause JAK to cross phosphorylate each other and the receptor. This will attract STAT (transactivator)
3rd step of JAK/STAT1 pathway
- STAT will phosphorylated, it will dimerize, hide the phosphates and translocate to the nuclease to increase transcription
What Is the connection between STAT1 dimerization and position of phosphorylation sites?
Phosphates get internalized
Example of interferons that are part of the JAK/STAT pathway
-Interferon(gamma)
-Interferon (alpha)
Why is that the STAT protein phosphorylation does not interfere with the STAT protein binding to DNA?
Phosphate is in the middle of the dimer
JAK stands for
Janus kinase
STAT stands for
Signal transducer & activator of transcription
What is the disease caused by a consistently active Hedgehog pathway?
Basel cell carcinoma
(WITHOUT) Hedgehog pathway
- No hedgehog, PATCH binds to smoothened inactivating it
- Inactivated smoothened, Protease free to cleave Ci protein (transactivator)
- Cleavage of Ci protein exposes the Co-repressor binding sites
- Ci co-repessor binding site translocate to nucleus and decrease transcription (specifically proliferation genes-NO proliferation)
1st step of the (WITHOUT) Hedgehog pathway
- No hedgehog, PATCH binds to smoothened inactivating it
2nd step of the (WITHOUT) Hedgehog pathway
- Inactivated smoothened, Protease free to cleave Ci protein (transactivator)
3rd step of the (WITHOUT) Hedgehog pathway
- Cleavage of Ci protein exposes the Co-repressor binding sites
4th step of the (WITHOUT) Hedgehog pathway
- Ci co-repessor binding site translocate to nucleus and decrease transcription (specifically proliferation genes-NO proliferation)
Which of the following protein interactions is defective in basal cell carcinoma?
PATCHED to SMOOTHENED
(WITH) Hedgehog Pathway
- Hedgehog will interact with PATCHED, patched cannot inhibit smoothened
- Smoothened interacts with protease, Protease cannot cleave the Ci protein
- Ci protein is now a co-activator (rather than repressor)
- Leads to transcription of proliferation
1st step of (WITH) Hedgehog Pathway
- Hedgehog will interact with PATCHED, patched cannot inhibit smoothened
2nd step of (WITH) Hedgehog Pathway
- Smoothened interacts with protease, Protease cannot cleave the Ci protein
3rd and 4th step of (WITH) Hedgehog Pathway
- Ci protein is now a co-activator (rather than repressor)
- Leads to transcription of proliferation
Receptor Tyrosine Kinase Pathway (RAS)
- Two receptors come together and phosphorylate each other (own tyrosine kinase activity)
- Binds to lose GDP
- GTP exchange:
-Inactive State-when RAS is bound to GDP
-Active State-when RAS is bound to GTP
4.Leading to RAF, MEK, ERK which phosphorylate the transcription factors (Downstream signals)
1st step of Receptor Tyrosine Pathway
- External ligand binds to cell membrane and brings two receptors come together and phosphorylate each other (own tyrosine kinase activity)
2nd step of Receptor Tyrosine Pathway
Binds to lose GDP
3rd step of Receptor Tyrosine Pathway
GTP exchange (GDP to GTP to activate RAS prortein):
-Inactive State-when RAS is bound to GDP
-Active State-when RAS is bound to GTP
4th step of Receptor Tyrosine Pathway
Leading to RAF (MAP3K), MEK (MAP2K), ERK(MAPK) which phosphorylate the transcription factors (Downstream signals)
Fibroblast growth factor (FGF) pathway—- receptor mutation
Activating FGF mutation lead to INCREASED GROWTH of spermatocytes
How does FGF lead to increased growth of spermatocytes?
- Men age and acquire more FGF mutations
- More spermatocytes with those mutations
- More sperm have these FGF receptor mutations
FGF mutations in offspring leads to what and why does this occur?
Leads to achondroplasia (dwarfism; shortening of bones). This is because chondrocytes are in growth plates and they can divide to lengthen bone due to the the FGF receptor mutations
What are three main points of FGF
- Occurs in spermatocyte development
- Occurs in chondrocytes (cartilage)
- Signaling pathway can occur even without PIC
Peptide hormones binds to receptor inside or outside the cell? And why?
outside (because it requires an EXTRAcellular receptor)
Steroid hormones binds to receptor inside or outside
Inside—-AKA Intracellular in cytoplasm or nucleus (because steroid hormones can diffuse through lipid bilayer membrane)
Genomics based approaches to studying transcriptional regulation:
Transcriptome
What is a transcriptome?
Total collection of cytoplasmic mRNA molecules in the cell. It represents genes expressed in a given cell type
transcriptome=mRNA (just exons)
Why is the transcriptome attainable (knowable) for any human cell type?
100% of the human genome is known and is available for use in detecting mRNA’s
What is the relationship between the presence of mRNA and the translation of mRNA?
Transcriptome emphasizes the differences in the potential for protein production between cell types, BUT the presence of an mRNA does NOT automatically imply the translation of the mRNA.
The transcriptome can represent a _______ of a cell type, regardless of the functional implications of the fingerprint.
fingerprint
The transcriptome can lead to verification of what?
Whether there are protein differences between cells
RNA sequencing: A transcriptome can also be obtained by converting all of the mRNA to _______ using________ in a test tube
cDNA, reverse transcriptase (DNA SYNTHESIS)
After reverse transcriptase cDNA is amplified by
massive parallel sequencing
qualitative-can be done for each gene
quantitative: proportionality is maintained
“the more mRNA we start with, the more cDNA we get”
Which of the following genomic files depends on reverse transcriptase?
RNA seq
Which of the following steps is in the proper sequence?
RNA samples, use reverse transcriptase, you get cDNA, you bind it on the chip, you do massive parallel sequencing, you find out what genes are expressed
If we compare a normal cell to a cancer cell using aRNA seq file,that is shown in this picture, what can be determined?
If you see a large amount of transcription occurring for that specific gene compared to the norm (left), then we can conclude that the read is that gene that that is transcribing, translating, and thats the protein we are getting for proliferation (cancer)
What does the green mean in this photo?
Little/no expression
What does the red mean in this photo?
a lot of expression
What does the black mean in this photo?
no difference in expression compared to normal cell (doesn’t get affected by mutations)
What are you able to compare with clustering software on the computer?
You are able to compare gene expression between different patient/tumor samples for which certain genes are expressed to a greater or lesser degree in disease states. This is very helpful in tumor cell fingerprinting
What is the implication of the transcriptome for regulation of mRNA production?
The developmental or inducible regulation of transcription for all genes can be known.
For example, if a hormone is added to a cell, the effect of that hormone on the rates of Transcription for every single Gene can be determined
What are two examples of clinical significance to transcriptomes.
We can find the relationship of gene expression and cancer
- Prospective validation of a 21-gene expression assay in breast cancer
2 .The Peripheral Blood Transcriptome identifies the presence and extent of disease in idiopathic pulmonary fibrosis
What are the two main functions of transactivators?
Stabilize PIC and recruit HAT
What is SSDBP?
a protein that binds to a specific sequence on DNA. It’s an umbrella term
Can SSDBP/Transactivators recognize sequences when DNA is still in the nucleosome (when chromatin is closed/inactivated)?
YES
What is the purpose of recruiting chromatin remodeling complexes?
SSDBP proteins/transactivators swap histones to alter chromatin structure which requires ATP
Explain competitive DNA binding
Proteins (SSDBP) in our genome can have DNA binding domains but not transactivator domains, so they compete for the binding site by blocking activators by recruiting HDACs and will repress transcription.
SSDBP=HDAC+Bind with no transactivator domain`
What occurs if there is mutated transactivator binding site domain?
It still has its DNA binding domain, and can still bind to DNA. The transactivator domain is messed up because its mutated so transcription cannot occur.
What is the histone code?
also, know this photo
The series of histone post-translational modifications that represent recognition (binding) sites for other proteins, particularly proteins that regulate gene expression.
What is Histone Deposition?
Type of acetylation that takes histones that are coming off a ribosomes in the cytoplasm and moving them to the nucleus and having the nucleosomes form by linking them up with a chaperone
What is the order of heterochromatin silencing?
- HDAC-removes acetyl grp
- Histone methylase: methylates lysine at K9
- HP1 is recruited and can self associate, facilitating the formation
- DNMT- Binds to HP1 and adds methyl to cytosine (5-methyl cytosine). Methyl on cytosine attracts MBP.
- MBP will recruit a HDAC and will start on the next histone
- Now we have a section of chromatin that is sealed off. Can be as small as a few Nucleosomes in a reg. region or as LARGE as female x chromosome that is inactivated. (you silence a specific portion of protein)
Which of the following is consistent with no transcription?
HP1
What is Rett Syndrome?
MBP defect
What are Epigenetics?
passing information from parent to daughter cell that isn’t in the sequence. Change in information that is not caused by mutation
True or False: The methylation of the C residue interferes with W/C base pairing to G meaning that DNA cannot be created
FALSE, it does NOT interfere with WCBP and DNA can still be created
How is heterochromatin passed and how do we maintain the heterochromatin?
Heterochromatin is often passed from parent to daughter cell, and we can maintain the Heterochromatin spreading with just a methyl
How do we specifically maintain the Heterochromatin spreading with just a methyl?
DNA methyltransferases (DNMTs) are included as part of the replication machine. The symmetry of CpG makes methylating both strands in almost the same place possible.
Re-establishment of repression after DNA replication is an example of epigenetics.
What is the Encode database?
Encode database: This technology depends on antibodies specific for these post translational modifications. We can chop up the euchromatin of a cell and identify these Post translational modificaitons with the ab and we can amplifty and modify the DNA
the capacity for determining histone modifications occurring at different points in the genome are cell-type specific. Why?
This is cell type specific because the status off chromatin is very different across different cell types and transcription varies across cell type
In the example of HeLA cell what causes cervical carcinoma?
The histone post translational modifications match the immortal cell line
Random, permanent inactivation of one of the X chromosome (Transfer of shutting down the chromosome) is an example of
epigenetics
What does the transfer of shutting down the chromosome specifically mean?
Following inactivation, all daughter cells keep the same X chromosome (either paternal or maternal) inactivated throughout development.) This inactivation leads to different alleles being expressed in different cells of the adult female mammal.
What is Xist (X INACTIVATION SPECIFIC TRANSCRIPT)
scaffolding RNA with a particular affinity for histone methylases
How does Xist scaffolding work?
Xist RNA bring histone methylase, and starts heterochromatn spreading and spreads to the X chromosome inactivating it. We close the whole X chromosome, its an ex of epigentics and we can pass it from parent to daughter cells. The x chromosome that ends up being inactivated ends up transcribing the XIST RNA
(The other X chromosome wont)
What are 6 main points about epigenetics?
- Heritable change in the genome.
- Not a mutation. (SAME SEQUENCE no change in base sequence of DNA, just silenced chromatin and spread)
- Usually reflects gene silencing.
- Usually involves methylation of DNA.
- Can occur at relatively small regions in the genome, e.g., regulatory region of a single gene, or over large regions, e.g., the inactive female X-chromosome.
- The role of non-coding RNAs is less well understood for smaller regions.
Explain this picture (epigenetic reprogramming in the early embryo) in regards to methylation as a fertilized oocyte, early embryo, and developing cell
fertilized oocyte: all genes are methylated and silenced
Early embryo: After fertilization, we strip away methylation and remove cytosine, and certain genes are becoming available which is allowing them to become specific cells which will develop into a blastocyst.
Developing cells: As our cells differentiate they methylate genes (met increases) they don’t want and keeping the ones they want to become that specific cell
Imprinting
Sub-category of epigenetics where we are NOT talking about parent to daughter cells. We are talking about Parent to child
Heritable gene regulation attributable to the gene (i.e., allele) having been received from one parent or the other. (From mom or dad)
In other words, epigenetics between generations
In imprinting, if inherited from the dad does gene stay methylated or does it get demethylated.
stay methylated (stays silence)
In imprinting, if inherited from the mom does gene stay methylated or does it get demethylated.
demethylated and able to transcribe
If a dominant mutant allele is methylated, what could this prevent?
disease
What is an example of a condition that results from a defect in imprinting?
Prader-Willi Syndrome
What is Prader-Willi Syndrome?
-Chromosome 15 deletion (defect)
-Other chromosome is repressed by methylation (normal imprinting process)
-“Autism Spectrum”
Specific and nonspecific factors and DNA sequences contribute to RNA polymerase II (RNA pol II) __________ regulation in eukaryotes.
transcription
The ____________ forms by an ordered assembly of components. Most of the components assemble by contacting other proteins of the PIC, not by contacting DNA. RNA pol II elongation proceeds by __________ of the RNA pol II by TFIIH.
PIC, phosphorylation
Specific activation of transcription is due to specific DNA sequences that bind specific regulatory factors, referred to as transcriptional activators or transactivators. The transactivators bind to components of the __________, especially the TATA binding protein (TBP) associated factors (TAFs), facilitating the formation of the PIC. Transactivators can also make _____________more accessible.
TFIID, chromatin
The activation of the ____________ is regulated by various biochemical mechanisms that either prevent or allow binding of the transactivator to its _______________.
transactivator, regulatory element
Transactivators have two distinct ____________: A transactivation domain, that contacts components of the PIC, especially the TAFs; and a __________ domain that binds a specific regulatory element.
domains, DNA binding
Transactivators regulate _____________ biochemical and pattern-formation phenotypes, that is, aspects of development.
developmental
_____________signals are ________________ via intracellular activation of a transactivator, leading to the transcriptional activation of a subset of genes. There are a variety of transactivator activation mechanisms, with STAT1 and NF-kappaB representing two distinct examples.
Extracellular, transduced
Because the entire ___________ has been sequenced, __________ and developmental regulation of a transcriptome can be determined.
genome, inducible
The transcriptome is obtained by generating an __________ file. KNOW THIS
RNA sequence
The RNAseq file is generated by sequencing, mapping to the human reference genome, and counting all of the _______ synthesized from an mRNA sample.
cDNA
Because many mammalian genomes have been completely sequenced, transcriptional ____________ can be identified by evolutionary _____________ of DNA sequences.
transcriptional activators, conservation
_____________ can facilitate promoter reactivation, for example, fetal globin gene activation.
HDAC inhibitor
Sickle cell anemia is being treated by reactivation of the ____________by HDAC inhibitors. The low level expression of the fetal globin protein disrupts polymer formation caused by presence of the mutated adult globin gene.
fetal hemoglobin gene
Many human diseases are caused by defects in either ___________ or transcriptional regulatory proteins.
transactivator binding site
HPFH is caused by a mutation in the ________ regulatory elements, allowing binding of the adult globin transactivator to the fetal globin ________.
transcriptional, gene
Mutations in the FGF receptor are responsible for _______________, in turn traceable to the accumulation of FGF receptor mutations in the sperm of older men, in turn traceale to the _________ effects of these FGF receptor mutations for sperm pre-cursor cells.
achondroplasia, increase growth
Transcriptional repression can occur by repressor blockage of a ____________, by the repressor binding to that site.
transactivator binding site
HDACs can lead to the generation of methylated DNA by de-acetylating histones, which leads to histone methylation, which leads to the binding of ___________, which leads to the binding of DNMTs, which methylate cytosines.
HP1
_______ is the phenomenon of heritable changes in the genome that do not involve mutations.
epigenetics
Epigenetic mechanisms usually involve the maintenance of inaccessible _________ over many cell divisions.
chromatin
DNA ___________are present at the replication fork and are the enzymes that catalyze DNA methylation.
methyl transferase
Only _______ is methylated in mammals.
cytosine
___________bind methyl-cytosines and also bind HDACs, leading to methylation of histones.
MBPs
Certain histone_____________lead to the binding of HP-1 to the histones and the condensation of chromatin.
post translational modifications
The term___________ refers to post-translational modifications of histones which can lead to the interaction of histones with different proteins that will either repress or active transcription (KNOW this)
histone code
In the case of the inactive, mammalian female X chromosome, _________ tethers chromatin modifying proteins to the chromatin to ensure heterochromatin spreading throughout, and methylation of this chromosome.
Xist RNA
______is epigenetics over a generation rather than from parent to daughter cell. Only a very small fraction of human genes are subject to imprinting. Prader-Willi syndrome is due to a defect in the imprinting process for a particular set of genes.
Imprinting
All of the following are true regarding HDAC ‘s EXCEPT:
a. HDAC inhibitors are a treatment for sickle cell anemia to help the chances of forming the preinitiation complex for the y hemoglobin
b. An example of an HDAC inhibitors is Vemurafenib
c. HDAC’s are brought to a specific element by SSDP’s to deacetylate and close the chromatin
d. HDAC’s are recruited by methyl DNA binding protein to the next histone for heterochromatin spreading
b
Below is a visualization of Post translational modifications, this is a representation of?
a. Histone Deposition
b. Histone Code
c. Epigenetics
d. Imprinting
b
Which of the following is a correct statement regarding imprinting?
a. Imprinting is a subcategory of epigenetics and is passed down from Parent to daughter cells
b. Imprinting is seen in almost every protein coding gene
c. When inheriting a methylation, it is almost always inherited maternally
d. If a dominant allele is methylated paternally, it could help prevent disease
d
The below photo Is an example of what? And explain the gene expression for the LTB gene in MM (Multiple myeloma)
a. Clustering software, LTB has a lot of expression in MM
b. Sequencing, LTB has a lot of expression in MM
c. Clustering software, LTB has little expression in MM
d. Sequencing, LTB has little expression MM
c
Which pathway is related to interferons and involve cytokine receptors?
a. JAK/STAT
b. TNF Alpha
c. Hedgehog
d. Tyrosine kinase
a
Which of the following mutations below is matched with its respective disease
a. Hedgehog—->Cervical Carcinoma
b. HeLa—->Melanoma
c. MAPK pathway—->Basal cell Carcinoma
d. MBP—->Imprinting
e. Prader-Willi syndrome—->Rett Syndrome
f. FGF receptor—->Achondroplasia
f
If a t-box gene (TBX5) is mutated, what would be the result of this?
a. The rats would start to produce the Y globin gene which could be a useful treatment in sickle cell Anemia
b. The rats will produce heart abnormalities due to the morphological changes during development caused by the lack of the transactivator
c. The rats will have no effect due to compensation from the other allele
d. The rats will not be able to silence certain genes in their genome due to the loss of the SSDBP
b
True or False: DNMT’s are a protein in heterochromatin spreading that get passed on during replication by methylating CPG islands via the cytosine and guanine?
a. True
b. False
false
If patched is bound to smoothen in the hedgehog pathways what will be the effect?
a. The protease will cleave hedgehog, decrease transcription of pro proliferation genes
b. The protease will not be able to cleave ci therefore Ci’s co activator binding sites will be exposed, and there will be an increase in transcription of pro proliferation genes
c. The protease will be able to cleave Ci therefore Ci’s co repressors binding sites will be exposed, and there will be a decrease in transcription of pro proliferation genes
d. RAS will become active via the GTP exchange and activate the MAPK pathway
c
A transcriptome is a representation of?
a. mRNA, gene expression
b. cDNA, sequencing
c. Primary transcript, gene expression
d. Transcription
a
