Gene expression and genetics

Differentiated cells

perform specific functions
Different patterns of protein expression / distribution / activity

Requires different patterns of gene expression

Different cell-types express the same proteins

Common structural proteins and enzymes etc:

e.g. histones, RNA polymerases, β-actin, tubulin, glycolytic enzymes etc

Encoded by ‘housekeeping genes’

Neurons = same genome = Skeletal muscle

neurons = Specialised proteins

E.g. neurofilaments, microtubule, associated protein-2 (MAP2)

skeletal muscles = Specialised proteins

E.g. myosin heavy chain

Gene expression

Most cells express 10,000 - 15,000 genes

“Housekeeping” proteins required by all cells
“Specialised” proteins not essential for cell viability - but are for specific functions in the organism

Not all genes are expressed at an equal rate:abundant: ~1000 – 10 000 mRNAs / cell
scarce: <10 - 100 mRNAs / cell = Majority

Genes can be either:

1. constitutively expressed (always on)

2. conditionally expressed (inducible)

Transcriptional control

Hypoxia inducible factor called F1 is primed within the cell so it is expressed all the time but it's downregulated.

Is there as a sensing mechanism for low oxygen levels.

If the cell experiences low oxygen, then that protein is suddenly stabilised/the processed involved in breaking down that protein very rapidly are halted = rapid controlling

The mRNA population is regulated by :

1. Which genes are transcribed (e.g. active vs. repressed)

2. The rate at which they are transcribed • The transcriptional start site used*

mRNA processing control

The protein coding sequence is interrupted

INTervening sequence = INTron

EXpressed sequence = EXon

~ 3 % of genes don’t have introns

Splicing

removing non-coding sequence (introns)

Highly controlled, same time as transcription = co-transcriptional process

Capping

addition of 5’methyl cap

Polyadenylation

addition of 3’ poly(A) tail

RNA polymerase has complexes of proteins around it that allow polyadenylation to occur - also involved in splicing

Pol II acts as a transcription “factory”

Pol II associates with factors forming a complex to perform:

- capping

- splicing

- poly(A)n

Co-transcriptional process

• ‘Spliceosome’ complex

Introns and exons

Gene organisation can vary considerably

Mean exons / gene = 8.8 (80% <200bp long)

< 5% of genes contain a single exon
Intron size varies (10 - 100,000 bp)
24 - 28% of the human genome = introns

Only 1% of the human genome = exons

Why have introns?

Exons often encode discrete protein functional domains

Important in molecular evolution “exon shuffling”

Allows for alternative splicing
i.e. different proteins from same gene

Role of mRNA splicing

Integral role in generating the cellular repertoire of mRNA transcripts

Virtually all multi-intronic genes are subject to alternative mRNA splicing

The splicesome – mRNA Splicing machinery

Small nuclear ribonucleoprotein complexes (snRNPs) - U1, U2, U4, U5 & U6

RNA binding proteins (RNBPs) recognise splicingenhancers and repressors – mRNA regions that differentially regulate splicing. e.g. hnRNPs, SRSFs

Affected by rate of RNA pol II elongation and the epigenetic environment of the gene locus

Splicing site choice is governed by transcriptional environment of the gene / cellular context

snRNPs – Small nuclear ribonucleoprotein complexes

U1 & U2 snRNPs recognise the 5’ donor & branch pointnucleotide to initiate mRNA splicing

Splicing involves snRNPs

U1 and U2 snRNPs 5’ donor & branch point nucleotidenucleotide to initiate mRNA splicing

hnRNPs (heterogeneous nuclear ribonucleoproteins) bind to cap, exons and poly(A)

Process of snRNP splicing involves sequential assembly and activation of the splicesome

The tri-snRNP U4/U6.U5 joins the U1/U2 complex
Removal of U1 and U4 activates the spliceosome
The 5’ end is spliced first followed by 3’end
spliced mRNA product is released from the post-splicing (P) complex

Regulation of mRNA splicing

Serine and arginine-rich splicing factors (SRSFs) – bind exonic splicing enhancers (ESE) promote splicing by U2 auxiliary factor (U2AF), or the U1 snRNP

Heterogeneous nuclear ribonucleoproteins (hnRNP) – bind exonic splicing silencers (ESS) and can inhibit this process

Alternative splicing has functional implications for the cell

Cell-type specific expression (e.g. IgM in B cells) = Different proteins from the same gene

Alternative splicing I

1. Alternative selection of promoters (e.g., myosin primary transcript)

2. Alternative selection of cleavage/polyadenylation sites (e.g., tropomyosin transcript)

3. Different exons = different protein domains - altered function

Alternative splicing II

1. Intron retaining mode (e.g., transposase primary transcript)

2. Exon cassette mode (e.g., troponin primary transcript)

3. Additional exons = larger proteins – additional / altered function

Alternative splicing III

Tissue specific expression
= different proteins from the same gene

Gene expression / product is controlled at multiple levels

Transcription is the predominant level of control of protein production

What is a gene?

Genes are transcriptional units composed of:

1. Structural information coding for a protein

2. Regulatory sequences giving instructions for expression of the gene

5’ regulatory sequences

5’ regulatory sequence controls transcription initiation = Gene promoter
Core promoter ~ 50 bp includes TSS

RNA polymerases

3 distinct RNA polymerases functional specialization transcribe different non-overlapping subsets of genes

Pol I – rRNA (5.8S, 18S, 28S)
Pol II – mRNA (+ ncRNAs)
Pol III – tRNA, rRNA (5S), nc/RNAs/miRNAs, etc

RNA Pol II transcription initiation

RNA Pol II is a 12-subunit enzyme

Requires an array of additional proteins/protein complexes to recognise the start of the gene (TFIID) & initiate transcription

Pol II transcription – multiple layers of regulation

 

1. RNA is present with a complex as well as other factors

2. TFIID is the complex that comes in and does the recognition at the start site in the core promoter region - It will come in and it will dock

3. This will cause other complexes that are associated with it to be assembled with all the machinery that’s involved in making the transcript and regulating it

4. E.g. C-terminal domain (CTD) is involved in binding, becoming phosphorylated and binding factors that are involved in splicing

5. So all the spliceosome proteins end up being associated with the proteins that are involved in capping and polyadenylation are latching on, making a big complex

6. There are other proximal promoters or enhancers further upstream or downstream

7. In case of enhancers, there are other gene specific regulators that were all involved in enhancing the activity of the complex and getting genes to be activated to a high degree

8. There are regions where factors combine and switch off the genes - silencers are present

9. All dock onto the TFIID

Transcription initiation I

RNA Pol II cannot recognise transcription start sites • most cells (probably) express 20,000+ genes – Chromatin represses ‘adventitious’ initiation
Core promoters contain a ‘consensus sequence motif’ which allows the binding factors to initiate transcription – e.g. highly-conserved TATA box (~25 - 30bp upstream of TSS)

TBP (TATA binding protein) recognises & binds the TATA box

Transcription initiation II

General transcription factors (‘basal machinery’) recruit RNA polymerase II to start site

TFIID is a multi-protein complex comprising: - TATA Binding Protein (TBP)
TAFs (TBP-associated factors)

TBP distorts the promoter DNA

Saddle’ shaped protein
- Introduces almost 90o bend.
- Role in promoter recognition?

- Binds in combination with other proteins = TFIID

TBP or TBP-related proteins in association with TAFs are also responsible for the activation of TATA-less genes

TFIID + TFIIA and TFIIB

TFIID forms a horseshoe shaped structure around DNA

- TBP at top of cavity
- TFIIA and TFIIB alter shape of cavity

- Subsequent binding of other TAF components

- RNA polymerase ‘holoenzyme’

(including non-phosphorylated C-terminal domain)

Pol II C-terminal domain (CTD)

Chromatin remodellers

RNA polymerase II holoenzyme

Histone
modifying enzymes

Pol II holoenzyme
enables transcription through chromatin

Transcription initiation IIII

TFIIH contains -

• DNA-dependent helicase activity (unwinds DNA template)

• Protein kinase activity (phosphorylates CTD)

• Releases Pol II from other general transcription factors

  TFIID remains to promote re-initiation

Transcription initiation

Initiation complex assembly is via stepwise binding of the general transcription factors

= “Basal” level of transcription
In vitro transcription with only ‘basal factors’ are very inefficient.

Transcription + gene specific transcription factors = ‘Activated’ transcription.

Enhancer sequences

DNA sequence elements that are specifically associated with gene promotors

Or

Can be a considerable distance away from the promotor (>500Kbp) – both 5’ and 3’

Bind DNA sequence-specific transcription factors

Act as switches

Transcriptional activators

are modular

DNA binding domain

Activation domain interacts with basal transcription complex (typically TAFs)

DNA binding Basic A.A (lysine, arginine)

CREB protein ‘leucine zipper’ motif

genomics

the study of the body’s genes, their functions and their influence on the growth, development and working of the body

mutations in cancer

Most are somatic (occur de novo in the precursor cells of the tumour)

But inheriting mutations can also predispose to cancer (germline)

Importance of genetics and genomics

Effects of inherited mutation are generally:
Small where the genetic variant is common (frequent)

and
Large in the case of very rare mutations

Importance of genetics and genomics

In many other clinical problems- e.g.

1. Type 2 diabetes,

2. Cardiovascular disease

3. Psychiatric disorders

Again, the effects of mutation are generally small where the genetic variant is common (frequent) and large in the case of very rare mutations

Genetic factors in type 2 diabetes

The genetic architecture of type 2 diabetes.Fuchsberger et al, 2018, Nature 536;41-47

• There is compelling evidence that the individual risk of type 2 diabetes (T2D) is strongly influenced by genetic factors

• Over the past decade, successive waves of T2D genome wide association studies—with ever larger samples— have delivered more than 80 robust association signals

• However, in these studies, the alleles associated with T2D were predominantly common (minor allele frequency (MAF) >5%), with individually modest impacts on T2D risk

Genetic factors in coronary heart disease

• Myocardial infarction (MI), a leading cause of death around the world, displays a complex pattern of inheritance

• When MI occurs early in life, genetic inheritance is a major component to risk. Previously, rare mutations in low-density lipoprotein (LDL) genes have been shown to contribute to MI risk in individual families

• Whereas common variants at more than 45 loci have been associated with MI risk in the population

Genetics and Genomics of psychiatric disease

There are confirmed associations between genetic variants that increase the risk of
•schizophrenia (SCZ),
•autism spectrum disorder (ASD),

•major depression, •Bipolar disorder (BPD),

and in some cases the underlying gene(s) have been identified

Mutations and their effects

Mutations in DNA can cause loss or gain of gene function and may involve:

1. a single gene (Mendelian inheritance)

2. a chromosomal segment (or whole chromosome) and so affect thousands of genes

3. several genes acting with environmental influences (multifactorial inheritance)

Single Gene Disorders

Mutations in single genes (often causing loss of function)

• Autosomal dominance

• Autosomal recessive

• • X-linked

classification of genetic disorders

Single Gene Disorders - Mutations in single genes (often causing loss of function)

Multifactorial diseases - Variants in genes causing alteration of function

Chromosome disorders - Chromosomal imbalance causes alteration in gene dosage

Mitochondrial disorders - Generally affect organ systems with high energy requirement.

Somatic mutations - Cause cancer. Inactivation of both alleles (two “hits”) of a gene involved in growth

genotype

pair of alleles at a locus

If alleles in population are ‘A’ and ‘a’ then the combinations of

genotypes possible are:

AA, Aa, aA and aa

that give the phenotype

Autosomal dominance

What we call a dominant allele (mutation) will determine a phenotype when only one copy is present in the genome of the individual.

usually only the wild type and heterozygous mutant phenotypes are encountered

The homozygotes are usually very severe phenotypes – lethal - and not seen

examples of Autosomal dominant disorders

Achondroplasia – dwarfism (mutation of FGFR3 gene)

Marfan Syndrome – connective tissue disorder- patients very tall (mutation of FBN1 gene)

Neurofibromatosis

Features of autosomal dominant inheritance

1. Affected individuals in every generation

2. Male and female equally likely to be affected

3. Inherited from one OR other affected parent

But can be a new mutation e.g. neurofibromatosis, achondroplasia

Autosomal recessive disorders

A recessive allele has no effect on the organism’s phenotype if only one copy of that allele is present in the genome

You could be carrying a mutation in a gene and be totally unaware of this - this is the case for carriers of recessive diseases

How to tell difference between a dominant and recessive mutant allele

In the heterozygote with 1 normal and 1 mutant allele - Aa,

Is the residual amount of normal gene product sufficient to perform its function?

• If yes, then the mutant allele and its associated disorder is recessive

• If answer is no then the mutant allele and its disorder is dominant

Origin of recessive mutations

Most random mutations will either be neutral or inactivating

Default state of mutations is therefore recessive

In the homozygous state there will be no function and consequences will be seen in clinical state

In the heterozygous state there will be no effect of the allele on the organism

Autosomal recessive

Most mutations are recessive to wild type
Recessive behaviour of most mutations arises from physiology of gene action.

Diploidy may have evolved because it protects against recessive mutations

How does dominance of a mutation occur?

Dominant disorders outnumber recessive disorders

A dominant allele (mutation) will determine a phenotype when only one copy is present in the genome

What aspects of a mutant allele’s function cause it to affect the phenotype in the presence of a wild type allele?

In the heterozygote (Aa) with one normal and one mutant allele is the residual amount of gene product sufficient to perform its function?

If answer is no then the mutant allele and its disorder is dominant

Molecular basis of genetic dominance

Reduced gene dosage (haploinsufficiency)

Increased gene dosage
Altered expression of mRNA
Increased protein activity

Dominant negative effects

Altered structural proteins

Gain of new function

Remember- dominant when in the heterozygote the level of normal product (one allele) is sufficient to perform function.

Reduced gene dosage (haploinsufficiency)

Where expression from one normal allele is not sufficient for function. Threshold dosage effects

Increased gene dosage

Due to presence of 3 copies of mutant gene- very rare- but example of Trisomy 21( several 10s of genes)
Duplication of PMP2 may be principal cause of Charcot Marie-Tooth Type 1
Gene amplification (especially in some tumours)

Altered expression of mRNA

Due to loss of control of regulation of mRNA expression. Hereditary persistence of fetal haemoglobin (HPFH)

Increased or constitutive protein activity

Increased half life or loss of normal inhibitory regulation Constitutive (Ras gene mutation in cancer)

Dominant negative effects

In the heterozygous state these mutants antagonise the activity of the remaining normal allele.
They are often multimeric proteins (eg tetramer of p53)

Altered structural proteins

A mixture of normal and abnormal structural alterations will disrupt the whole structure. Eg fibrillin in Marfans, myosin heavy chains in hypertrophic cardiomyopathy.

Gain of new protein function

Best examples are chromosome translocations giving rise to new genes/protein- fusion proteins (C-Abl-BCR in chronic myeloid leukaemia

Characteristics of X-linked inheritance

1. Incidence of the trait is much higher in males than females

2. The gene responsible for the condition is transmitted from an affected man through all his daughters

3. The gene is never transmitted from father to son

4. Sons always inherit their X chromosome from their mother

Multifactorial inheritance

are caused by a combination of small inherited variations in genes, often acting together with environmental factors

Heart disease, diabetes, and most cancers are examples of such disorders

Behaviors are also multifactorial, involving multiple genes that are affected by a variety of other factors

Genetic contribution to behavioral disorders such as alcoholism, obesity, mental illness and Alzheimer's disease

Chromosome disorders

are caused by an excess or deficiency
of the genes that are located on chromosomes, or by structural changes Within chromosomes

Down syndrome, for example, is caused by an
extra copy of chromosome 21 (trisomy 21), although no Individual gene on the chromosome is abnormal

Prader-Willi syndrome, on the other hand, is caused by the absence or non-expression of a group of genes on chromosome 15

Chronic myeloid leukemia, CML) may be caused by a Chromosomal translocation, in which portions of two Chromosomes (9 and 22) are exchanged. No chromosomal material is gained or lost, but a new, abnormal gene is formed that leads to formation of cancer

Mitochondrial inheritance

those leading to a primary defect in oxidative phosphorylation- main source of cellular ATP

Mitochondrial diseases are marked by the variation in their clinical presentation

Can present in childhood (nuclear genes) or in any decade of adulthood (mainly mt- genes).

Can be severe and progressive

The mitochondrial genome as a hotspot for pathogenic mutation

Mitochondrial DNA is small ~15.4kb circular and there are numerous copies (~1000) in each cell

mt DNA is inherited from the maternal oocyte
which has many more mitochondria than somatic cells have

Approximately 100,000 copies of mtDNA are inherited in the fertilized oocyte

Frequency of mitochondrial disorders is high

How is the mutation expressed in all the mt genomes?

•In the inherited mitochondria (up to 10exp5) 99.9% are identical (homoplasmy)

•If a new mutation arises in the mt population and spreads there will be two populations (heteroplasmy)

•As a result mitochondrial disorders are characterized by by reduced penetrance, variable expression and pleiotropy

How are new mitochondrial mutations fixed

mtDNA bottleneck

The number of mt DNA molecules within each oocyte is reduced before being greatly amplified in the mature oocyte. This restriction and subsequent amplification is called the mitochondrial genetic bottleneck

Only a small number of mtDNA molecules are selected for further amplification- explains the variability in % of mutant mt DNA. Molecules seen in offspring of mothers carrying a mt DNA mutation

When is a DNA sequence change pathogenic?

consider deletions, nonsense mutations, frameshifts to be true pathogenic mutations

Not every sequence variation is necessarily pathogenic (polymorphisms). Any sequence change seen in more than 1% of individuals may be a polymorphism

If the ‘polymorphism’ is rare in the population and is found in an A- T patient how can we know whether it is pathogenic

Some clues can be gained by considering the actual sequence change

A missense mutation is more likely to pathogenic if it:

i. Affects a functionally important part of the protein

ii. If the amino acid is a conserved over evolution, it is more likely to be important

iii. If amino acid substitutions are non-conservative (polar for non polar, acidic for basic) they are more likely to be pathogenic

This is an important problem – finding out how severe the effects are of a missense mutation – affects clinical severity

Most missense mutations will result in a mutant protein with no activity

But some missense mutations will give a protein with low level activity - that will affect patient

Loss of function mutations

Total loss of function results from total absence of the protein.

When a disorder results from total loss of function of a protein, any mutation resulting in inactivation of the protein will give the same severe clinical result.

total loss of function may occur even when some protein is expressed

ATM kinase activity – we can measure this

Has to be induced by DNA damage (protein level is not induced, just activity)

Commonly use ionizing radiation or a radiomimetic drug to activate

Immunoprecipitate the ATM protein using an ATM Ab and use it to phosphorylate a protein target (p53) in vitro

OR

Use phosphospecific antibodies to detect the phosphorylated target

Possible consequences of mutant ATM protein

Some may have some retained function:

- Could allow some degree of normal response to radiation

- Could affect level of radiosensitivity - measurable
- Gain of function?

May result in altered interactions with other proteins – cancer?

Dominant negative effects

A non-functional mutant polypeptide can interfere with a normal protein from the normal allele giving a dominant negative effect

Conclusions from molecular pathology – indications for prognosis?

No protein expression at all – Associated with classical disease, Outlook for patient associated with classical prognosis

Some protein expression - May be associated with a less severe picture, Difficult to suggest to a patient that this will mean a better outlook, even if this is suggested by the presence of residual protein function – matter of confidence, Need a lot of data

Is there is quantitative relationship between protein function and improved clinical outlook?

What about other interacting genes in the family

Modifying genes

The same mutant allele can have different phenotypic effects on different genetic backgrounds.

This depends on particular alleles at other gene loci

e.g. Less severe features of Beta-thalassemia in homozyotes who also inherit an alpha-thalassemia mutation
- which acts as a modifier

Locus heterogeneity

Same disease can be caused by mutation in a different gene.

Proteins interact with other proteins, form complexes or are part of a biochemical pathway.

Nonsense mediated mRNA decay

Destabilisation of mRNA

It is not possible to obtain any sequence data from the cDNA because the RNA transcript is unstable and is not produced.

Therefore no cDNA can be synthesized

Chromosomal non-disjunction

Results from a chromosome pair failing to separate

Non-disjunction of sex chromosomes

sex chromosome abnormalities

individuals can appear normal

Compare with Down Syndrome patients who have an additional chromosome 21- the smallest chromosome - severe learning difficulties

X chromosome inactivation

In early embryonic development one X chromosome in normal XX female is inactivated.

In females the second X chromosome remains active.
In females the progeny of each cell have the same X chromosome active.

Gives a mosaic effect with respect to the two X chromosomes.

Where there is more than two X chromosomes (Eg triple X female), rule is all X chromosomes except one is inactivated.

Aneuploidy

1. Loss of one autosome - monosomy (monosomy for a single autosome is lethal)

2. Gain of one chromosome - trisomy E.g. Down syndrome.

Deletions

Deletion of a small segment of a chromosome. Leads to monosomy for that segment
Eg. Prader-Willi syndrome, chromosome 15;
Cri-du-chat syndrome, chromosome 5.

Chromosome translocations

Breakage of two chromosomes and exchange between them.

Can occur in the germline.
Can occur in somatic cells and there they are important in the pathogenesis of cancer.

An apparently normal chromosome complement may be pathogenic if they have the wrong parental origin.

• Some genes are imprinted with their parental origin and are expressed differentially according to their origin (maternal or paternal)

• E.g. a hydatidiform mole is an abnormal conceptus, 46XX, where all the chromosomes are of paternal origin

• Ovarian teratoma - disorganised embryonic tissue, 46XX, exclusively of maternal origin.