Gene Expression: Basic Mechanics of Transcription: Three critical phases 1. Initiation 2. Pause/Release 3. Elongation

What is transcription?

The process of turning DNA into RNA

What enzyme produces mRNA?

RNA pol II

What is a promoter?

A DNA region near the transcription start site where RNA pol II and general transcriptions factors assemble

What is an enhancer?

A regulatory DNA seq that increases transcription of a target gene, sometimes from a far distance

What do sequence-specific transcription factors do?

They recognize and bind to specific DNA seq in enhancers or promoters to help regulate gene expression

How can an enhancer regulate a distant promoter?

DNA looping helps bring the enhancer and promoter close together, which allows proteins on both to interact

What is the role of cohesin?

It helps organize and stabilize the loop, supporting enhancer-promoter interactions

What is the role of Mediator?

It is a coactivator which communicates with the transcription factors and connects with with RNA pol II and the general transcription machinery

What are general transcription factors (GTFs)?

They are proteins that help assemble and position RNA pol II at the promoter and initiate transcription

What is the difference between sequence-specific transcription factors and general transcription factors?

Seq specific transcription factors bind to specific DNA seq to target particular genes, while general transcription factor assemble at the promoter of the gene to help position RNA pol II for transcription

What are examples of general transcription factors?

1. TFIID

2. TFIIB

3. TFIIH

What is the overall sequence of transcription initiation?

Enhancers interact with promoters (via DNA looping) —> Sequence-specific transcription factors bind specific DNA sequences at enhancers —> Mediator connects those transcription factors with the transcription machinery —> General transcription factors assemble and position RNA Polymerase II at the promoter —> RNA Polymerase II begins transcription.

What happens after RNA polymerase II begins transcription?

RNA pol II can produce a short nascent RNA and may stop briefly near the promoter

What is promoter-proximal pausing?

A temporary pause in transcription at approx 20-60 nucleotides shortly after RNA pol I begins making RNA

Which proteins help maintain Pol II pausing?

NELF and DSIF

Why is promoter-proximal pausing important?

It allows cells to rapidly activate transcription because pol II has already initiated and is positioned near the beginning of the gene basically control when and where transcription starts

What is transcription elongation?

This is the stage when RNA polymerase II comes out of the pause and moves along DNA and produces a longer RNA molecule

What protein complex promotes the release of paused RNA polymerase II?

P-TEFb (positive transcription elongation factor b)

What kinase is part of P-TEFb?

CDK9, this adds a phosphate group to proteins through phosphorylation

Which proteins does P-TEFb phosphorylate to promote pause release?

NELF, DSIF, and the C-terminal domain of RNA pol II

What happens to NELF and DSIF during pause release?

NELF is released, while DSIF transitions towards supporting elongation.

What is chromatin?

DNA that is packaged together with proteins, including histones.

What is a nucleosome?

Basic structural unit of chromatin, made up of DNA wound around a histone

How can nucleosomes interfere with transcription?

They can cover DNA seq, preventing transcriptions factors and other regulatory factors from accessing them.

What is chromatin remodeling?

It is the process of changing nucleosome positions or structures so that transcription factors can get inside and express genes this happens from energy provided by ATP

What are three ways chromatin remodeling complexes can alter nucleosomes?

They can slide, evict, or restructure nucleosomes

How can chromatin remodeling promote transcription?

It exposes the DNA seq for transcription factors and machinery to access

What are examples of chromatin remodeling complexes?

SWI/SNF (BAF), ISWI, CHD, and INO80

Do chromatin remodelers add chemical modifications to histones?

No, primary function is to change nucleosome position or structure

What are histone modifications?

Chemical groups added or removed from histone proteins that help regulate chromatin and gene expression

What are five examples of histone modifications?

Acetylation, methylation, phosphorylation, ubiquitination, and sumoylation.

What are examples of histone modification regulators

1. Writers enzymes that add chemical modifications to histones

2. Erasers enzymes that remove chemical modifications from histone

3. Readers proteins that recognize specific histone modifications and help recruit other regulatory proteins

What are examples of histone writers?

Histone acetyltransferases (HATs), histone methyltransferases (HMTs/KMTs), and kinases.

What are examples of histone erasers?

Histone deacetylases (HDACs) and histone demethylases.

What histone modification do bromodomains (epi reader for lysine) recognize?

Histone acetylation.

What histone modification can chromodomains recognize?

Histone methylation.

How can histone acetylation promote transcription?

Weakens interactions between DNA and histones which can allow access for transcription factors and other machinery

Does histone methylation always activate transcription?

No, it can either repress or activate it depends on the specific residue on the specific histone that methyl has modified

Where is H3K27ac commonly found?

At active enhancers and promoters.

Where is H3K4me3 commonly enriched?

Near active promoters and transcription start sites.

Where is H3K79me2 commonly found?

Within transcribed gene bodies, often toward the early-to-middle region.

Where is H3K36me3 commonly enriched?

Within actively transcribed gene bodies, often increasing toward the 3′ end.

Which histone modification is strongly associated with active enhancers?

H3K27ac.

Which histone modification is strongly associated with active promoters?

H3K4me3.

Which two histone modifications are associated with transcription elongati

H3K79me2 and H3K36me3.

A transcription factor binds an enhancer thousands of base pairs away from a promoter. How can it influence transcription?

DNA looping brings the enhancer near the promoter, which allows transcription factors and coactivators to interact with transcription machinery

A transcription factor binds an enhancer thousands of base pairs away from a promoter. How can it influence transcription?

Promoter-proximal pausing, regulated by factors such as NELF and DSIF.

A cell needs to rapidly increase transcription of a stress-response gene. Why is promoter-proximal pausing useful?

Because RNA pol II has already initiated transcription and can quickly transition into productive elongation when activated

What would happen if P-TEFb activity were inhibited?

Pause release would be stopped, which would reduce elongation of genes

A nucleosome is blocking a transcription factor's binding site. What type of complex could help?

An ATP dependent chromatin remodeling complex that will evict or reposition the nucleosome

A researcher detects high H3K4me3 enrichment at a genomic region. What might this indicate?

The region is likely associated with an active promoter.

A researcher detects high H3K27ac at a distal regulatory region. What might this indicate?

The region may be an active enhancer.

A researcher observes increasing H3K36me3 enrichment toward the 3′ end of a gene. What process is this associated with?

Transcription elongation.

What is the preinitiation complex (PIC)?

Collection of RNA pol II and general transcription factors with RNA pol II and the preinitation complex

How do transcription factors recruit the transcription machinery?

They bind DNA and recruit coactivators like the mediator that help them communication with transcription machinery and assemble

A transcription factor binds an enhancer and recruits Mediator. What mechanism is occurring?

Recruitment of transcript machinery

A transcription factor recruits a HAT to a gene. What is the likely effect?

Histone acetylation increase, which weakens interaction and grants access for transcription

A transcription factor recruits an HDAC. What is the likely effect?

Histone acetylation decreases, potentially creating a chromatin environment less favorable for transcription.

RNA polymerase II has started producing RNA but remains paused near the promoter. What can help it continue?

Recruitment of P-TEFb to promote pause release.

A gene has an active transcription factor but its promoter is blocked by nucleosomes. What could help increase transcription?

Recruitment of chromatin-remodeling complexes or histone-modifying enzymes that can allow access for transcription

What would happen if a transcription factor could bind DNA but could not recruit its required coactivators?

It would be able to recognize the DNA seq that needs to be transcribed, but won’t be able to communicate that to the mediator and then it can’t recruit transcription machinery

You mutate a promoter sequence and reporter activity drops dramatically. What can you conclude?

That sequence was important for promoter activity

You mix labeled DNA with a protein extract and observe a shifted band. What does this suggest?

A protein in the extract binds the DNA fragment, forming a complex that migrates more slowly

You want to identify where transcription factor X associates with DNA throughout the genome. What technique could you use?

ChIP-Seq or CUT&RUN.

You want to compare open chromatin regions between two cell types. What technique would you use?

ATAC-Seq.

You want to determine where engaged RNA polymerases are synthesizing nascent RNA. What technique would you use?

ChRO-Seq.

What is the purpose of mutational promoter analysis?

To identify which DNA sequence with a promoter are important for regulating gene expression

What is a transcriptional reporter assay? (Technique used in mutational promoter analysis)

it is an experiment in which a promoter is attached to a reporter gene like CAT whose expression can be measured to determine promoter activity

How does mutational promoter analysis work?

Researchers attach a promoter to a reporter gene, then they mutate or delete promoter regions, and then measure how these changes affect reporter expression

What does it mean if mutating a promoter region significantly decreases reporter activity?

The mutated or deleted region likely contains a regulatory DNA element important for activating transcription

What does it mean if mutating a promoter region does not change reporter activity?

The region may not be necessary for promoter activity

Does mutational promoter analysis identify the specific transcription factor that binds a promoter?

No, it only identifies that the specific region mutated or deleted was important for transcription

What is the purpose of Electrophoretic Mobility Shift Assay?

To determine whether a protein can bind a specific DNA sequence in vitro

How does Electrophoretic Mobility Shift Assay work?

A labeled DNA fragment is mixed with a protein sample and run through a gel to determine if a DNA-protein complex forms

Why does DNA bound to a protein move more slowly through a gel?

DNA is normally small and free, when you add the protein and form a complex it slows it migration.

What is a shifted band in Electrophoretic Mobility Shift Assay?

A band representing a DNA-protein complex that migrates more slowly than free DNA.

What is free DNA in an EMSA experiment?

DNA that is not bound to a protein and typically migrates faster through the gel.

Does EMSA automatically identify which protein binds DNA?

No, if a cell extract does contain a specific binding protein more test are done to determine which one exactly contains a TF

What is a limitation of EMSA?

It measures the DNA-protein binding in a test tube, and does not prove that binding occurs in living cells

What is the purpose of Chromatin Immunoprecipitation?

To determine whether a particular protein (TF) is associated with specific DNA region in cells, basically is this the TF for that specific DNA seq

What is the purpose of ChIP-Seq?

To identify genomic regions associated with a particular protein or histone modification across the genome, basically is this region where the TF binding an enhancer or promoter for transcription

What are the four major steps of ChIP-Seq?

Crosslinking, chromatin fragmentation, immunoprecipitation, and DNA sequencing.

What happens during crosslinking in ChIP-Seq?

Proteins are chemically linked with formaldehyde to nearby DNA to preserve their interactions

Why is chromatin fragmented during ChIP?

To break the DNA into smaller pieces that can be isolated and analyzed

What is immunoprecipitation during ChIP?

An AB is selectively capture a protein of interest along with the DNA associated with it

What happens after immunoprecipitation during ChIP?

The crosslink is broken and the associated DNA is recovered and analyzed with PCR or seq

What is the difference between ChIP-PCR and ChIP-Seq?

ChIP-PCR examines selected DNA regions to find TF-DNA binding, while ChIP-Seq identifies enriched regions across the genome looking everywhere not just regions for TF-DNA binding.

What does a ChIP-Seq enrichment peak indicate?

That a genomic region is enriched for DNA associated with the protein or histone modification being studied.

What is the purpose of CUT&RUN?

It used to identify genomic regions associated with a particular protein or histone mod

How does CUT&RUN differ from ChIP-Seq?

ChIP-seq shears all cellular chromatin globally into small pieces before using an AB to immunoprecipitated the protein-DNA complex, CUT&RUN does not shear everything and directly binds the AB to the protein and uses a tethered nuclease to make a precise cut at the binding site, releasing that specific DNA fragment.

What is the first step in CUT&RUN?

An AB binds to the targer protein or histone mod in cells or nuclei

What enzyme is commonly used in CUT&RUN?

A micrococcal nuclease that is fused to protein A or protein A/G its role is to cut DNA neat the AB targeted protein

What happens to the DNA fragments generated by CUT&RUN?

They are collected and sequenced to identify the genomic regions associated with the target protein

Why can CUT&RUN have lower background than conventional ChIP-Seq?

Because the DNA cut is targeted mostly near the protein of interest rather than the whole chromatin

What is the purpose of Assay for Transposase-Accessible Chromatin using Sequencing?

To identify accessible regions of chromatin that is exposed and available for interactions with regulatory proteins across the genome

What enzyme is used in ATAC-Seq?

Tn5 transposase, It locks specifically on exposed DNA and fragments while inserting seq adapters this is called tagmentation.

What happens after Tn5 acts on accessible DNA?

The resulting DNA fragments are sequenced and mapped to the genome.

What does an ATAC-Seq enrichment peak indicate?

A region of chromatin that is relatively accessible, this does not mean the region is actively being transcribed

What is the purpose of Chromatin Run-On Sequencing?

To identify genomic regions where RNA pol are transcriptionally active by measuring nascent RNA synthesis.

What is the first step in ChRO-Seq?

Researchers isolate chromatin containing transcriptionally engaged RNA pol