3002 week 1 - 6

RNA pol 1

makes rRNA

RNA pol 2

makes mRNA, eRNA, ncRNA

RNA pol 3

makes tRNA

Chromatin

DNA + Histone Octamer

two forms of chromatin

• heterchromatin (inactive, condensed)

• euchromatin (active, less condensed)

3 steps involved in transcription

1. initiation

2. elongation

3. termination

enhancer

regulatory region which may be proximal or distal to promotor, TF bind her and increases transcription

chromatin loop

enables contact between promotor and enhancers (Loops bring regulatory DNA (enhancers) close to target genes.)

TSS

transcription start site

transcription process

1. Binding of TF, activities machinery on accessible gene region

2. Transcription progresses through defined cycle involves mediator and integrator complexes (help load RNA pol, initiate transcription and coordinate early rna processing) 

3. Initiation = rna polymerase produces short RNA, caused by mediator complex

4. Paused step = RNA pol 2 waits for activation by integrator complex, this complex gives rise to phosphorylation of rna pol 2 (activation) which starts elongation of RNA

5. During elongation - there is also splicing (removal of introns (non coding))

6. Finally - once entire gene is transcribed, there is step of termination, and addition of 3’ poly A tail

regulated in time, it is not linear, may pause for various times

trancription burst

high burst of transcriptional activity, usually followed by silence period where no RNA production

transcription burst classification

classified by size of burst and frequency

• can have high frequency with low burst size

understanding bursts can explain gene expression heterogeneity for a same cell population.

4 regulatory layers of transcription

1. epigenetics

2. transcription factors

3. 3D organisation (chromatin archetecture)

4. temporaral dynamics (time dependent)

transcription factor

• core unit of transcription

• speficic class of protein (1600 in human genome)

• read the genome, TF binds DNA and can bend it?

• DNA binding domain (of TF) recognises specific part of DNA that correlates to the organisation in the nucleotide (the nucleotide sequence which the tf recognises = the tf binding site)

TF classes

• eg, nuclear receptors → have ligand binding domain which is essential for TF activity

• binding of ligand changes protein configuration, allowing protein protein interaction which can enable basal transcriptional machinery allowing transcription

• TF is a molecular switch, turn on and off basal transcriptional machinery

other molecular functions of TFs

1. Open compacted chromatin (pioneers) - ability to bind to nucleosomal DNA in heterochromatin, through specific mechanisms they can decondense the chromatin to provide easier access for other tf’s

2. Maintain open chromatin (settlers) - when initiation of chromatin remodelling has occurs, these settlers bind in the region to maintain the new structure

3. Transcriptional activation/repression (migrants) - enter and exit the open region, they can modify transcription rate of RNA pol 2

pioneers

TFs that open heterochromatin for transcription

settlers

TFs that maintain open chromatin structure, when initiation of chromatin remodelling has occurs, these settlers bind in the region to maintain the new structure

migrants

TFs that activate or repress transcription rate by entering and exiting open region. modify transcription rate of RNA pol 2

Function of activator in transcription

Binds to DNA-binding domain that contains DNA binding motif and help general TF and RNApol assembles

Mode of action of DNA-binding domain (DBD)

Recognises and binds specific DNA sequences in promoters, enhancers, or response elements to target the correct gene

Mode of action of Ligand-binding domain (LBD)

Binds a ligand (e.g., hormone) and undergoes conformational changes that enable the transcription factor to recruit co-activators or release co-repressors; indirectly regulates the basal transcription machinery

lack of TF binding motif

no binding / affinity of the TF to remain in teh area

PPI

protein protein interactions (drives transcription)

TF’s can recruit

enhancers and silencers to alter transcription rate

a combo of regulatory elements come together with PPI to drive basal transcription machinery

gene regulatory regions

enhancers + promoters

Mode of action of Protein-protein interaction (activation/repression) domain

Interacts with co-activators, co-repressors, Mediator, or general transcription factors to stimulate or repress transcription

TFs work alongside epigenetic mechanisms such as

• Histone modification

• DNA methylation

• Non coding

These factors help establish stable gene expression state

TF activity is critical for

embryonic stem cell self renewal and lineages differentiation

3 types of embryonic stem cells

• totipotent

• pluripotent

• multipotent

KEY TF for Pluripotency

OCT4

SOX2

NANOG

maintain pluripotency of stem cell by repressing differentiation cues and activating stem cell specific genes. these keep embryonic stem cells in an undifferentiated state.

SOX2 + OCT4 + NANOG

• At physiological level - SOX2 and OCT4 activate NANOG expression, and they also control each other expression level

• when SOX2 increases and too elevated, then negative feedback loop reduces SOX2 and OCT4 and therefore NANOG

• increased OCT4, represses own transcription and NANOG

totipotent stem cells

• Can generates all cell types. Including extra-embryonic tissue

pluripotent stem cells

 Can generate all cell types, Derivative of 3 germ layers

multipotent

•  Can generate multiple related cell types

NANOG

maintains stem cell pluripotency

OCT4, SOX2

actively repress differentiation program to remain pluripotent

Totipotent stem cells (Stage, location, potency)

Stage: 2-4 (2 cell - morula stage)

Location: zygote & early blastomeres

Potency: can generate all cell types including extra-embryonic tissue

Pluripotent stem cells (Stage, location, potency)

Stage: ~7 (late blastocyst)

Location: Inner cell mass os blastocyst

Potency: can generate all cell types derivative of 3 germ layers (endoderm, mesoderm, ectoderm)

Three germ layers

1. Endoderm
2. Mesoderm
3. Ectoderm

Multipotent stem cells (Stage, location, potency)

Stage: >9

Location: Specific germ layer tissues

Potency: can generate multiple cell types within one lineage

Transcription factors that regulate stem cell fate (Pluripotency maintenance)

OCT4, SOX2, NANOG

What is epigenetics?

The inheritance of altered characteristics by mechanisms that do not involve changes to the DNA sequence

• may result in enganced gene activation or gene repression

• not permanent changes

• added or removed by specific enymes, allowing flexibility in gene expression

Role of epigenetics

Cell growth, differentiation, autoimmune disease, cancer

Three mechanisms of epigenetics

1. DNA methylation
2. Histone modification
3. Non-coding (regulatory) RNA

what enzyme used to establish DNA methylation

DNA methyltransferase (DNMT)

passive DNA demethylation

loss of DNA methylation due to failure to maintain methylation during DNA replication

• absent / inhibted DNMT

methylated cytosine is diluted from genome due to an absence of sufficient methylation maintenance enzymes

active DNA demethylation

direct removal of 5-mC from DNA independent of DNA replication

• occurs in sequential manner by modifying cytosine bases. TET enzyme mediates

where does DNA methylation mainly occur in somatic cells

CpG sites (C-G rich)

what is DNA methylation

process where methyl groups are added to DNA by methytransferases

• a major epigenetic mechanism, it affects gene activity

• increased methylation in gene promotors reduces gene expression? reduced methylation can activate genes

two classes of methylators

1. maintenance (add methyl to newly made DNA at location opposite to mehtyl groups of old strand)

2. de novo (change in pattern of methylation by adding new methyl groups

DNMT1

involves in maintenance methylation

DNMT3A/B

involved in de novo methylation

durng methylation SAM

donates the methyl, and in turn becomes SAH

DNA demythlation

• done by demethylases

• two methods - active or passive

CpG islands

DNA regions with large numbers of CpG sites. mainly present in upstream regulatory regions in front of genes.

• over 60% of genes have promotors embedded in CpG islands

• CpG methylation supresses transcription by directly inhibiting binding of TF to DNA, or inhibiting the chromatin modifying enzymes

CpG sites

• where DNA methylation occurs (on the C)

• A space where a C is immediately followed by a G nucleotide in the 5’ to 3’ direction. p = phosphate

• (the backbone)

what enzymes regulate histone modification

histone methyltransferase (HMT) and histone demethylases (HDMT)

function of histone methylation

Either activate or repress gene transcription, depending on the amino acid residue modified and the extent of methylation

• transfer f methyl group from SAM to residues of histone proteins

• regulated by HMTs and HDMT

histone modification

• Changes the chromatin structure

• 8 different types: methylation, acetylation, ubiquitination, phosphorylation, SUMOylation, GlcNAcylation, carbonylation, and ADP-ribosylation

What are SAM and SAH in the context of methylation?

SAM transfer a methyl group to cytosine bases in DNA and become SAH

what enzymes regulate histone acetylation

histone acetyltransferases (HATs) and histone deacetylases (HDACs)

what is histone acetylation

promotes gene activation by masking +ve charge of amino acidic side chains, decreases chromatin condensation

• addition of acetyl group from acetyl CoA

whilst histone deacetylation silences gene expression - increases chromatin condensation

acetylated histones

increased gene activation

• acetyl added by HAT (acetyl CoA becomes CoA

What are non-coding RNAs (ncRNAs)?

A cluster of RNAs that do not encode functional proteins

• a large part of the genome is transcribed into non coding RNA, not all RNA bases regulation is epigenetic

Function of microRNA (miRNA)

Bind complementary mRNA to degrade mRNA or inhibit translation (post translational regulation)

• small non coding RNA, about 22 nucleotides

• indirect epigenetic effects by targeting epigenetic modulators such as DNMTs and HDACs, altering DNA methylation and histone modification patterns

• miRNAs can be regulated by epigenetic changes themselves, creating reciprocal contro

• deregulation can contribute to cancer progession

How does miRNA create indirect epigenetic effects?

By targeting epigenetic modulators such as DNMTs and HDACs, altering DNA methylation and histone modification patterns.

Size of microRNA

about 22 nucleotides

Function of long non-coding RNA (lncRNA)

• Recruit epigenetic regulators such as DNMTs and HMTs to specific genomic loci

• Act as scaffolds or guides for chromatin-modifying complexes to control chromatin structure

• role = chromatine structural changes

size of lncRNA

• more than 200 bp

Four roles of epigenetics in health and disease

1. Parental imprinting (one allele silenced by methylation of DNA and histone)
2. X chromosome inactivation
3. Genome integrity (Silence/stabilise mobile/defect elements)
4. Development and differentiation

parental imprinting

certain genes are marked based on which parent they came from, 1 is silenced, 1 is expressed

• One allele is silenced by methylation of the DNA and histones

• Errors are linked to diseases such as Prader-Willi Syndrome

Genome integrity

• nearly half of genome has mobile elements, most defective, uncontrolled replication or movement of active ones can cause big damage to genome. 

environment + epigenetics

• Environmental factors (eg. diet, stress, toxins) can trigger epigenetic changes

• Untangling the effects of genetics, epigenetics and the environment is challenging especially for complex diseases such as obesity or type 2 diabetes

• Epigenetics can generate differences between identical twins

  • Eg. In identical twins where only one suffered from systemic lupus erythematosus: the affected twin had lower levels of methylation on ~50 of 800 genes analysed

BRCA1

• a tumor suppressor gene involved in DNA repair via homologous recombination

• implicated in breast and ovarian cancers

What happens with BRCA1 silencing?

Mimics loss-of-function mutation where CpG islands in BRCA1 promoter become heavily methylated, forming condensed heterochromatin around the region

• loss of BRCA1 function increases risk of breast + ovarian cancers

• BRCA1 methylation status can guide personalized therapy and predict response to PARP inhibitors and platinum drugs

3 therapeutic targets for epigenetic modificaiton

1. DNMT inhibitors
2. HDAC inhibitors
3. Combination therapy potential

DNMT inhibitor (Mechanism, examples, effect, clinical use)

Mechanism: block DNMT, reduce CpG island hypermethylation

Examples: Azacitidine, decitabine

Effect: reactivate silenced tumour suppressor genes (eg, BRCA1, MLH1)

Clinical use: hematologic malignancies, under investigationsolid tumours

HDAC inhibitor (Mechanism, examples, effect, clinical use)

Mechanism: prevent removal of acetyl groups from histones so chromatin becomes more open

Examples: Vorinostat, Romidepsin

Effect: Restore transcriptional activity of epigenetically silenced genes

Clinical use: certain lymphomas, trial for breast and ovarian cancers

combination therapy (DNMT + HDAC inhibitors)

show synergistic effects in reactivating silenced genes.

• May enhance sensitivity to chemotherapy and targeted agents (e.g., PARP inhibitors).

a potential for the future?

what is splicing

• removal of non-coding introns from pre-mRNA

• joining of the remaing exons to form mature coding mRNA

before 1970s

we thought all DNA was coding, that everything in the DNA appeared in RNA

• 1977 → study of mRNA found mature mRNA did no contain complete RNA template, large portions missing

what is RNA splicing

RNA splicing is the process that removes noncoding introns from eukaryotic pre-mRNA and joins the coding exons to form mature mRNA

• >95% of human genes contain introns that undergo splicing

splicing method

1. Genes on DNA are transcribed into precursor messenger RNA (pre-mRNA) - consists of introns and exons

2. Splicing removes noncoding tracts (introns) and coding segments (exons) are spliced together to form mature mRNA

3. Mature mRNA comprises a continuous sequence that is translated into a polypeptide

where does RNA splicing occur

inside the nucleus, is done by spliceosome

how does RNA splicing occur

RNA splicing occurs in the nucleus through two sequential transesterification reactions catalysed by the spliceosome.

spliceosome

large RNA-protein complex comprised of 5 small nuclear ribonucleoproteins (snRNPs, “snurps”) and dozens of non-snRNP proteins (really complex)

snRNPs

small nuclear ribonucleoproteins (snRNPs)

• bind conserved sequences on the intron to initiate splicing including: 5’ GU splice site, branch point adenosine (A), polypyrimidine tracts (10-20 nucleotide), and 3’ AG splice site

when does splicing occur

• co transcriptionally (more common)

• post transcriptionally

what makes up the sliceosome

- 5 snRNPs

- many non-snRNPs

splicing - step by step

1. Recognition: U1 snRNP binds to intron conserved sequence (e.g. 5'' GU splice site)

2. Prespliceosome: U2 base-pairs to branch point adenosine making it bulge out

3. Tri-snRNP joins (U4/U5/U6): snRNPs rearrange and U1&4 are released and spliceosome is catalytically active

4. 1st transesterification: branchpoint adenosine attacks 5' splice site and lariat intron forms (hoop)

5. 2nd transesterification: exon 1 attacks 3' splice site, releasing lariat and joining exons
   Spliceosome disassembles and is recycled

alternative splicing

there is more than one way to splice a pre-mRNA, to produce various mature mRNAs

A single pre-mRNA can be spliced in multiple ways to produce distinct mRNA isoforms and protein variants from one gene. 

increased protein diversity, and effects mRNA stability and output

Functional consequences of alternative splicing

• Allows single gene to produce many isoforms

• mRNA transcript variants can differ in coding sequence, regulatory elements, or untranslated regions

• Variants can influence mRNA stability, localisation, and translation output to modulate cellular function

• Expands protein isoform diversity - enables same gene to perform different functions in different contexts

• Cell-type and tissue-specific expression…distinct identities

• Context-dependent and time-depending splicing of genes

Splicing of BRCA1 Codes for Breast cancer type 1 susceptibility protein

• Chr 17

• 41 transcripts (different splicing events) 

• Canonical splice = most common variant

Splicing in disease

• Normally highly accurate at the nucleotide level to maintain coding sequence integrity

• Pathological mis-splicing can arise through genetic mutations or altered RNA-binding protein levels/availability to cause:

• Exon skipping

• Intron retention

• Cryptic splice site usage (use of hidden splice site within exon/intron)

Pathological mis-splicing is a major contributor

 to disease (e.g., BRCA1 in breast cancer, MLH1/MSH2 in colorectal cancer, SMN1 in spinal muscular atrophy)

Therapeutic targeting of splicing

Correcting splicing defects can restore normal gene expression

Strategies:

• Small molecules that are chemical inhibitors of RNA splicing. Limitation – Cannot discriminate between normal and pathogenic splicing

Oligonucleotide-based therapeutic approaches

• Antisense oligonucleotides

• Isoform-specific RNA interference

what percentage of genome is coding

1%

• 99% is non coding - including regions which control gene acitvity

regulatory regions of DNA

determine when and where genes are turned on and off

• provide sites for TFs to bind to which will either activate or repress gene transcription