Flashcards in Gene Transcription Deck (19)
How did he start the lecture
He started off with saying that we all arise from a single cell. In this lecture we talked about gene promoters (which are also known as core promoters or proximal promoters), transcription enhancers and transcription silencers.
He then showed us a slide where we can see that the promoter regions is actually adjacent to the gene where transcription begins, it defines where the transcription will begin.
Relationship between DNA and gene expression
Gene expression programs determine the morphology and function of each cell
DNA sequence information specifies when, where and how much transcription occurs of each gene.
Which polymerase is involved in gene transcription? What other kinds of pol's are there?
RNA pol 2
There are 2 other RNA pols that transcribes tRNA and rRNA that are in the nucleus and there is another RNA pol that is found in the mitochondria.
Our discussion was solely about RNA pol 2 since it is the one involved in all regulatory process of gene transcription
DNA silencers and enhancers
Now we talk about the DNA enhancers and silencers that occur in DNA. These are separate from the region where the promoter are found. They have specific conserved nucleotide sequences, which we saw are the actual binding sites for proteins that regulate transcription.
These regulatory elements can be near, far, upstream or downstream. They can even be inside introns. Each regulatory element binds with different transcriptional proteins also known as transcription factors. Transcription factors alter the activity of associated promoters.
3 year old boy with a heart murmur case study
We then talked about a case study where a 3 year old was observed with symptoms of breathlessness and had a below average weight and height. He have had several respiratory tract infections in the past. Upon physical examination it was observed that he had a heart murmur and upon further testing he was found to have an atrial septal defect (which is a hole between the left and right atrial wall).
Lineage determining factor: a transcription factor that, if present in a progenitor cell, alters the gene transcriptional program such that the cell commits to a specific lineage.
He then explained to us on one slide that there are lineage determining factors for cardiomyocytes and one of them is GATA4 which if defected can cause atrial septal defect.
We looked closely at GATA4. All transcription factors have domain architecture. They have DNA binding domains, Transcriptional activation domains and dimerization/protein interaction domains.
These mutations help us define functional domains as well as the mechanisms by which these domains achieve gene activation.
He then talked about mutations in GATA4 that leads to atrial septal defect. There are protein interaction domains that have mutations on them so the proteins do not interact right and can lead to atrial septal defect. GATA4 binds with Zinc Fingers binding domains (which are one of the types of DNA binding domains).
The hallmark of all these details is that even though the mutation is outside the DNA binding domain, somehow the mutation affects the protein binding and leads to a hole in the atria of our patient.
NOTE: It is important to go back and look into this case in more detail to know about all of the working mechanisms.
How does the TF bind to DNA
It is important to know how does transcription factors recognize DNA sequences that are to be transcribed. First of all it is important to know that DNA is packed with histones so transcription depends upon whether that part of the DNA is accessible or not. Then the transcription factors can know which regions of the DNA are to be transcribed by Protein-DNA specificity. This is accomplished in 2 ways: the first is that these transcription factors can read the base sequences in the DNA, they can read the bases in the major groove and minor groove which are determined by hydrogen bonding, hydrophobic interactions and Van Der Waals forces (this is called touching the edges of the nucleotide bases). The second mechanism is that these transcription factors can read the shape of the helix that is also determined by the underlying sequence of the DNA
Homodimerization: transcription factor binds itself to create two-protein complex
Heterodimer: transcription factor binds a different protein to create a two-protein complex
Hetero- or homo-multimer: rather than two proteins involved in the complex, it’s 3 or more proteins.
He then talked about how a mutation at the end of one of the zinc finger motif leads to less extent of DNA binding with the transcription factors but also that since this mutation is towards the end of the zinc finger motif the next transcription factor will also bind to the DNA to a less extent.
All of these mutations alter the ability of transcription factors to facilitate transcription of DNA.
What are the 3 ways that transcription factors regulate promoter activity (or transcription)
There are 3 ways by which transcription factors activate transcription from a promoter:
1. b/r proteins that alter the chromatin structure
2. b/r general transcription factors
3. b/r kinase that phophorylates RNA pol 2
Model of transcriptional activation
1. GATA4 and TBX5 recruits HAT and CRC. HAT and CRC peels the DNA off of the nucleosomes
2. Transcriptional activation domain binds and recruits proteins that alter the chromatin structure (basically this makes the promoter accessible to protein binding). There is more detail here, histone acetylase (HAT) and chromatin remodeling complex (CRC) modifies the histones, allowing the gene to be accessible.
3. This results in binding and recruitment of general transcription factors, this results in the formation of pre-initiation complex)
4. DNA helicase also comes in now and opens up the DNA. I'm guessing single stranded proteins called RPA holds the other strand of the DNA away from the other to maintain the open helix structure.
5. Binding and recruitment of kinase (Ptef-B) takes place that phosphorylates RNA pol2 which then transcribes the gene. It is important to remember the name of the kinase which is Ptef-B.
Now we talk about Nucleosome acetylation. We know the action of mechanism of HAT that it adds a carboxylic group to the N atom of the R group of the amino acid lysine, neutralizing the positive charge by adding the carboxylic group to it, forming an amide.
Histone deacetylase works the opposite way (HDAC).
He then said on the slide that transcriptions factors bound to silencers can recruit HDACs to repress transcription (to stop transcription, it is important to know that it is the transcription factors (the same factors we talked about before) that bind to silencers to stop transcription.
He showed us a picture of one of the CRC, CRCs are huge, we can see a tiny strand of DNA around a big globular protein.
Straight up facts about CRCs
Straight up facts about CRCs: they have several families with multiple members, these are multiple protein complexes all of which contain ATPase units, subunits are domain that can read histone modifications (called bromodomain), these peels off DNA from nucleosomes (it had a comment that said slides or removes core octamer making DNA accessible), energy is used from ATP in the process.
Introduction of so-called “Yamanaka” factors (Oct4, c-myc, Nanog, Sox9) into fiblroblasts result in production of induced pluripotent stem cells
Then there was a slide that introduced us to Yamanaka factors which are transcription factors when introduced in a terminally differentiated cell can cause the cell to become induced pluripotent stem cell which hypothetically can be used to make an entire another organism asexually.
• Transcription factors (TFs) specify tissue lineage by regulating gene expression programs
• “Master regulator” TFs can re-program cell lineage by altering gene transcription (not all of the transcription factors can do that)
• Defects in TFs can result in failure of these programs and defects in cell/tissue function
• Mutations (sometimes leading to amino acid substitutions) can disrupt TF function by disabling specific biochemical activities
Summary Slide 2
Transcriptional Activator Proteins facilitate events that must occur for productive transcription.
Activator proteins can, via their transcriptional activation domains:
1. Contact general transcription factors to assist in their recruitment/stabilize binding to promoter
2. Recruit HAT enzymes and chromatin remodeling complexes which create open chromatin environment at promoter and transcriptional start site
3. Recruit kinase Ptef-B to phosphorylate RNAPII so it can move beyond initiation and enter productive elongation (promoter clearance).
Hypothetical case of a bad doctor
He was trying to ask here with this case presentation that how does the cells know how to respond in different situation (like the macrophages and the fibroblast) as obviously their response before and after the injury are very different. The answer to this is because of PAMPs (Pathogen associated molecular patterns) that are present on the bacteria's cell membrane and these bind to the receptors in macrophages (and other cells) that leads to a signalling cascade.
The protein I kappa B is phosphorylated and degraded, this releases NF-kappa B. NF kappa B then goes to the nucleus and activates transcription of pro-inflammatory genes
He then gave us an outline of what events happen as bacteria gets inside our body:
1. Detection of bacteria
2. Activation of signaling pathway
3. Change in transcription factor location (goes into the nucleus)
4. Transcription of genes that encode for the inflammatory proteins
5. Killing of bacteria and alerting the rest of the immune system
We dive into the details of what NF-kappa-B does. It recruits HAT and CRC, causes the opening of the DNA, makes promoter accessible, this leads to the binding and recruitment of the transcription factors, this stimulates the kinase Ptef-B to phosphorylate RNA pol 2 which then starts transcription.
• Signaling pathways often terminate with activation/repression of transcription factor function
• Change gene transcription in response to external signals: infectious agents, hormones, toxins, temperature, oxygen levels…
• The same basic mechanisms to control gene transcription are used by essentially all transcription factors
We then talked about Crohn's disease which is involved with excessive prodution of inflammatory proteins.
Know the symptoms and other details about Crohn's disease:
• Idiopathic, chronic inflammatory process that can affect any part of the gastrointestinal tract
• Chronic inflammation from T-cell activation leading to tissue injury is implicated in the pathogenesis of Crohn disease.
• Inflammatory cytokines (IL-12, TNF-α) stimulate the inflammatory response
• Recruited inflammatory cells release nonspecific inflammatory substances which result in direct injury to the intestine.
Why did the physicians decide to use methylprednisolone for the treatment of Crohn's disease?
The answer to this is that it is a glucocorticoid steroid that help with controlling the inflammation by developing and anti inflammatory response.
It is important to know the mechanism of action of these type of drugs.
Glucocorticoid steroids work by activating the transcription of proteins that counteract the inflammatory proteins and they also initiate trans repression of pro inflammatory proteins.
The GR is usually in the cytoplasm bound to the heat shock protein in their natural inactive form. When glucocorticoid is administered in a patient, they go right through the cell membrane, bind with GR, GR dissociated from the heat shock protein and then GR goes on to bind with GRE (present on the DNA) (glucocorticoid response element) which then initiates transcription of anti inflammotory proteins.
The GR can also bind to NF kappa B, causing the repression of NF kappa B genes, inhibiting the production of pro inflammatory proteins.
GR can also cause silencing of anti inflammatory proteins. It is important to know that the same receptor can cause silencing and enhancing of genes.
GRE: glucocorticoid response element, specific DNA sequence to which the glucocorticoid receptor (GR) binds
All steroid receptors (e.g. estrogen, progesterone, androgens, glucocorticoids, mineralocorticoids) as well as some vitamin and non-steroid hormone receptors (e.g. vitamin D receptor, thyroid hormone receptor) belong to a super-family known as the nuclear hormone receptors. These are ligand activated transcription factors. Some are resident in the cytoplasm and, once bound with ligand, typically dimerize and enter the nucleus. There, they bind specific DNA sequences and regulate gene transcription. Other nuclear hormone receptors are already bound to DNA, and the ligand changes how they affect transcription (e.g. without ligand they repress transcription, with ligand they activate transcription).
How is typically a single gene regulated
One of the principle that we need to know is that the same gene is regulated by many regualtors, the outcome is that many factors can be taken into account to regulate a gene, multiple signals come into the cell and then the cell makes its decision on how to transcribe a gene based on the regulatory mechanism.
• Transcription factor function is altered directly and indirectly by drugs
– The response to a given drug will depend (partially or mostly) on
changes in gene transcription
• Most (all) genes are under the control of multiple regulatory elements: integration of signals