gene regulation biology - potential and limitations of cell re … · 2010-04-15 · gene...

Gene Regulation BiologyPotential and Limitations of Cell Re-programming in Cancer Research

Eric Blanc

KCL

April 13, 2010

Eric Blanc (KCL) Gene Regulation Biology April 13, 2010 1 / 21

Outline

1 The Central Dogma of Molecular Biology

2 Expression regulation and transcription factors

3 Gene regulation at the DNA level

4 Post-transcriptional regulation


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ATG

Promoter Region

Intron Exon

AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA

UGAUAA UAG

PO4

PO4

S S

3’ Poly A tail5’ Cap

Methionine

Stop CodonsTranscription and mRNA processing

Translation

Post-Translational Modification

DNA

mRNA

Protein

5’ Un-Translated Region

TATA

Central Dogma of Molecular Biology : Eukaryotic Model

Active Protein

From Wikipedia


The eukaryotic DNA organisation at different scales

Eukaryotic cells pack their DNA in the nucleus (each human cellcontains almost 1.8 m of DNA)

The DNA is hierarchically organized, and its structure influences geneexpression

Davidson, Molecular Expressions, http://micro.magnet.fsu.edu


http://micro.magnet.fsu.edu

The chromatin and the nucleosome

The eukaryotic DNA is organised as follows:

The nucleosome contains 147bp of DNA wrapped around 8histone proteins (2 copies ofH2A, H2B, H3 & H4)

The histone proteins haveN-terminal tail domains whichcan accommodate severalmodification signals (principallymethylation and acetylation)


The chromatin and the nucleosome

The linker histone H1 connects nucleosomes to pack them tightly intothe 30 nm filament, which precise structure remains elusive

The chromatin filaments are very dynamic, oscillating between theunfolded (beads-on-a-string) and compact configurations

Marmorstein (2001) Nat. Rev. Mol. Cell Biol. 2 422-432


The influence of chromatin on gene expression

The equilibrium between folded and unfolded conformations can beshifted by varying salt concentrations

While H1 shifts the equilibrium towards the folded state, the HighMotility Group (HMG) proteins have the opposite effect

The nucleosome is a barrier to DNA accessibility

The core histone N-terminal domain mediate nucleosome-nucleosomeinteractions which lead to local & global condensation of chromatin

The histones’ N-terminal domain support a combinatorial code madeof post-translational modifications

Typically, lysine acetylation promote gene expression


Chromatin remodelling

The transcription factor bindsits enhancer region

Upon binding, histoneacetylation proteins andco-activators modifyneighbouring nucleosomes’histone tails

The remodelling complex isrecruited, which alters DNAconfiguration and presentsadditional transcription factorbinding sites

The complete complex is formedand transcription begins

Hartl & Jones. Genetics: Analysis of Genes and Genomes, Jones & Bartlett publ.


The transcription preinitiation complex

Holstege et al. (1999) Cell 95(5) 717-728


Transcription initiation

Transcription is initiated by the recruitment of the pre-initiationcomplex binding to the transcription starting siteThe Core Promoter Elements (BRE, TATA box, INR & DPE) arerequired for accurate transcription initiationThese elements (not always all present) bind Generic TranscriptionFactors (GTFs) conserved among all eukaryotes, which recruit Pol IIThe gene specificity is achieved by the enhancer sequence, whichrecruits only specific transcription factors

BRE TATA box INR DPE

Core promoter elements

Enhancers/Suppressors


Transcription initiationCpG islands as proximal promoter elements

CpG islands are 0.5 to 2 kb regions rich in dinucleotide CG (otherwiserarer than other dinucleotides)Methylation of these regions suppress expression of nearby genesAbout 29000 such regions have been found in the human genome, i.e.60 % of promotersThese region contain binding site for transcription factor Sp1, whichrecruits the pre-initiation complex

CpG islandEnhancers/Suppressors


Transcription initiationThe mediator complex

The mediator complex connects the specific transcription factorsbound to enhancer elements to the pre-initiation complexThese enhancer elements are generally upstream of the gene to betranscribed, and can be up to 100 kb away from the transcriptionstarting siteBeside enhancers, similar sequences (repressor elements) can bindtranscription factors inhibiting gene expression

Mediator Pre-initiation complexGene-specific TFs


Epigenetics and imprinting

Histone modifications and DNA methylation patterns of CpG islandsare stable regulatory signals on the DNA which can be inherited frommother to daughter cells

DNA methylation patterns can cause genomic imprinting, where thegene expression levels depend on whether it has been inherited fromthe father or the mother

The many enzymes responsible for specific modification of the histonecode can generate rich patterns of modified and unmodified sites onthe N-terminii of histone molecules

Patterns of histone modifications at the cell level can be used asprognosis markers for clinical outcome


DNA methylation

Most of the CpG are methylated, except for CpG islands, for whichmethylation can change

Methlyation (in particular of CpG islands) is associated with genesilencing, and conversely gene repression may trigger methylation

Methylation patterns are stable, but can change during development:I Inactivation of one X chromosome in females is achieved by extensive

and almost irreversible DNA methylation during early developmentwhich results in the packaging of one copy of the X chromosome intoinactive heterochromatin

I The paternal pronucleus is de-methylated immediately after fertilizationI The activation of naive T cells is an active de-methylation process of

the Interleukin-2 promoter

The exact mechanism for active de-methylation is still unknown


Histone modifications and the Polycomb group

Polycomb and trithorax groups of proteins are repressors andactivators of many genes

Both achieve gene expression regulation via histone modifications

The polycomb proteins group is involved in the maintenance of stemcell identity by suppressing regulators of differentiation pathways

Polycomb Response Elements (PREs) are DNA motifs recruiting thePolycomb Group (PcG) (in D.melanogaster at least)

Polycomb proteins were shown to repress tumor-supressor genesexpression


Post-transcriptional regulation

Once the elongation is finished, several other steps are required before afunctional protein is produced.Most of these steps can the target of some regulatory process.

Exon splicing, polyadenylation and capping

mRNA transport outside of the nucleus to the ribosomes

mRNA inhibition and decay regulation

Post-translational modifications


RNA interferenceMicro RNA (miRNA) & small interfering RNA (siRNA)

Both miRNA & siRNA are non-coding genes regulating the activity ofother mRNA transcripts, usually by inhibition or degradationThese gene form double stranded RNA molecules which are processedby the protein DicerRNA-dependent gene silencing is controlled by the RNA-inducedsilencing complex (RISC) in the cytoplasm, to which is bound thematured mi/siRNA: a single stranded RNA fragment from 20 to 25 bpThe short single-stranded RNA sequences are complementary to thegene(s) which expression is controlled by the miRNA/siRNAWhen the miRNA/siRNA sequence is exactly complementary, themRNA is cleaved, otherwise the translation is blocked

siRNA miRNA


Post-translational modifications

Large spectrum of possible modifications, mostlyI Attaching functional groups (phosphate, acetate, lipids, carbohydrates)

to exposed side chains, andI Covalent binding of disulfide bonds between cystines.

Phosphorylation is a common regulatory mechanism, achieved bykinases (attach the PO4 group) & phosphotases (remove it)

Used (for example) to regulate the transport of transcription factorsinto the nucleus, or to tag proteins for degradation


Additional regulatory mechanisms

Genetic variability between individuals: Single NucleotidePolymorphism (SNPs) and Copy Number Variation (CNV) both canaffect gene expression

RNA editing: nucleoside modification C to U and A to I which changethe mRNA sequence which is not a copy of the DNA anymore. Inhuman, it has been demonstrated that RNA editing can be tissuespecific, and it is particularly important in the brain.

Gene location in the nucleus: genes in the nuclear periphery tend tobe less expressed unless they sit near nucleopores.


Genome comparisons

Number of Proportion ofGenome Size Genes Proteins coding DNA

E.coli 4.6 · 106 4252 4252 ≥ 90 %S.cerevisiæ 12.1 · 106 6532 7547

D.melanogaster 1.3 · 108 14076 22423M.musculus 2.7 · 109 22941 82641

H.sapiens 3.1 · 109 22286 142707 2 %

The number of genes is the number of protein-coding genes, and thenumber of protein is the number of transcripts

Source: ENSEMBL release 57


Some numbersFor E.coli

Translation rate 40 aa/secTranscription rate 70 nt/sec

Concentration 5-8 mM (protein), 0.5-0.8 mM (RNA) & 0.5 nM (DNA)Volume 70 % (water), 17 % (protein) 6 % (RNA) & 1 % (DNA)Velocity 3-10 µm/s (protein), 50 µm/s (small molecule)

#(ATP)/protein 1500 (360 aa long)#(ATP)/RNA 2000 (1000 nt long)

1 glocose generates 36 to 38 ATP

Source: The CyberCell database, Sundararaj et al. (2004) Nucl. Acids Res. 32 D293-95.
