Gene Regulation Biology

Transcription

1 Gene Regulation Biology Potential and Limitations of Cell Re-programming in Cancer Research Eric Blanc KCL April 13, 2010 Eric Blanc (KCL) Gene Regulation Biology April 13, / 21

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

3 Cdmb.svg 20/11/ :55 Central Dogma of Molecular Biology : Eukaryotic Model DNA Promoter Region ATG Intron Exon mrna 5 Cap TATA Transcription and mrna processing UGA Stop Codons UAA UAG 5 Un-Translated Region AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA 3 Poly A tail Protein Methionine Translation Post-Translational Modification PO 4 S S PO 4 Active Protein From Wikipedia Eric Blanc (KCL) Gene Regulation Biology April 13, / 21

4 The eukaryotic DNA organisation at different scales Eukaryotic cells pack their DNA in the nucleus (each human cell contains almost 1.8 m of DNA) The DNA is hierarchically organized, and its structure influences gene expression Davidson, Molecular Expressions, Eric Blanc (KCL) Gene Regulation Biology April 13, / 21

5 The chromatin and the nucleosome The eukaryotic DNA is organised as follows: The nucleosome contains 147 bp of DNA wrapped around 8 histone proteins (2 copies of H2A, H2B, H3 & H4) The histone proteins have N-terminal tail domains which can accommodate several modification signals (principally methylation and acetylation) Eric Blanc (KCL) Gene Regulation Biology April 13, / 21

6 The chromatin and the nucleosome The linker histone H1 connects nucleosomes to pack them tightly into the 30 nm filament, which precise structure remains elusive The chromatin filaments are very dynamic, oscillating between the unfolded (beads-on-a-string) and compact configurations Marmorstein (2001) Nat. Rev. Mol. Cell Biol Eric Blanc (KCL) Gene Regulation Biology April 13, / 21

7 The influence of chromatin on gene expression The equilibrium between folded and unfolded conformations can be shifted by varying salt concentrations While H1 shifts the equilibrium towards the folded state, the High Motility Group (HMG) proteins have the opposite effect The nucleosome is a barrier to DNA accessibility The core histone N-terminal domain mediate nucleosome-nucleosome interactions which lead to local & global condensation of chromatin The histones N-terminal domain support a combinatorial code made of post-translational modifications Typically, lysine acetylation promote gene expression Eric Blanc (KCL) Gene Regulation Biology April 13, / 21

8 Chromatin remodelling The transcription factor binds its enhancer region Upon binding, histone acetylation proteins and co-activators modify neighbouring nucleosomes histone tails The remodelling complex is recruited, which alters DNA configuration and presents additional transcription factor binding sites The complete complex is formed and transcription begins Hartl & Jones. Genetics: Analysis of Genes and Genomes, Jones & Bartlett publ. Eric Blanc (KCL) Gene Regulation Biology April 13, / 21

9 The transcription preinitiation complex Holstege et al. (1999) Cell 95(5) Eric Blanc (KCL) Gene Regulation Biology April 13, / 21

10 Transcription initiation Transcription is initiated by the recruitment of the pre-initiation complex binding to the transcription starting site The Core Promoter Elements (BRE, TATA box, INR & DPE) are required for accurate transcription initiation These elements (not always all present) bind Generic Transcription Factors (GTFs) conserved among all eukaryotes, which recruit Pol II The gene specificity is achieved by the enhancer sequence, which recruits only specific transcription factors Enhancers/Suppressors BRE TATA box INR DPE Core promoter elements Eric Blanc (KCL) Gene Regulation Biology April 13, / 21

11 Transcription initiation CpG islands as proximal promoter elements CpG islands are 0.5 to 2 kb regions rich in dinucleotide CG (otherwise rarer than other dinucleotides) Methylation of these regions suppress expression of nearby genes About such regions have been found in the human genome, i.e. 60 % of promoters These region contain binding site for transcription factor Sp1, which recruits the pre-initiation complex Enhancers/Suppressors CpG island Eric Blanc (KCL) Gene Regulation Biology April 13, / 21

12 Transcription initiation The mediator complex The mediator complex connects the specific transcription factors bound to enhancer elements to the pre-initiation complex These enhancer elements are generally upstream of the gene to be transcribed, and can be up to 100 kb away from the transcription starting site Beside enhancers, similar sequences (repressor elements) can bind transcription factors inhibiting gene expression Gene-specific TFs Mediator Pre-initiation complex Eric Blanc (KCL) Gene Regulation Biology April 13, / 21

13 Epigenetics and imprinting Histone modifications and DNA methylation patterns of CpG islands are stable regulatory signals on the DNA which can be inherited from mother to daughter cells DNA methylation patterns can cause genomic imprinting, where the gene expression levels depend on whether it has been inherited from the father or the mother The many enzymes responsible for specific modification of the histone code can generate rich patterns of modified and unmodified sites on the N-terminii of histone molecules Patterns of histone modifications at the cell level can be used as prognosis markers for clinical outcome Eric Blanc (KCL) Gene Regulation Biology April 13, / 21

14 DNA methylation Most of the CpG are methylated, except for CpG islands, for which methylation can change Methlyation (in particular of CpG islands) is associated with gene silencing, and conversely gene repression may trigger methylation Methylation patterns are stable, but can change during development: Inactivation of one X chromosome in females is achieved by extensive and almost irreversible DNA methylation during early development which results in the packaging of one copy of the X chromosome into inactive heterochromatin The paternal pronucleus is de-methylated immediately after fertilization 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 Eric Blanc (KCL) Gene Regulation Biology April 13, / 21

15 Histone modifications and the Polycomb group Polycomb and trithorax groups of proteins are repressors and activators of many genes Both achieve gene expression regulation via histone modifications The polycomb proteins group is involved in the maintenance of stem cell identity by suppressing regulators of differentiation pathways Polycomb Response Elements (PREs) are DNA motifs recruiting the Polycomb Group (PcG) (in D.melanogaster at least) Polycomb proteins were shown to repress tumor-supressor genes expression Eric Blanc (KCL) Gene Regulation Biology April 13, / 21

16 Post-transcriptional regulation Once the elongation is finished, several other steps are required before a functional 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 Eric Blanc (KCL) Gene Regulation Biology April 13, / 21

17 RNA interference Micro RNA (mirna) & small interfering RNA (sirna) Both mirna & sirna are non-coding genes regulating the activity of other mrna transcripts, usually by inhibition or degradation These gene form double stranded RNA molecules which are processed by the protein Dicer RNA-dependent gene silencing is controlled by the RNA-induced silencing complex (RISC) in the cytoplasm, to which is bound the matured mi/sirna: a single stranded RNA fragment from 20 to 25 bp The short single-stranded RNA sequences are complementary to the gene(s) which expression is controlled by the mirna/sirna When the mirna/sirna sequence is exactly complementary, the mrna is cleaved, otherwise the translation is blocked sirna mirna Eric Blanc (KCL) Gene Regulation Biology April 13, / 21

18 Post-translational modifications Large spectrum of possible modifications, mostly Attaching functional groups (phosphate, acetate, lipids, carbohydrates) to exposed side chains, and Covalent binding of disulfide bonds between cystines. Phosphorylation is a common regulatory mechanism, achieved by kinases (attach the PO 4 group) & phosphotases (remove it) Used (for example) to regulate the transport of transcription factors into the nucleus, or to tag proteins for degradation Eric Blanc (KCL) Gene Regulation Biology April 13, / 21

19 Additional regulatory mechanisms Genetic variability between individuals: Single Nucleotide Polymorphism (SNPs) and Copy Number Variation (CNV) both can affect gene expression RNA editing: nucleoside modification C to U and A to I which change the mrna sequence which is not a copy of the DNA anymore. In human, it has been demonstrated that RNA editing can be tissue specific, and it is particularly important in the brain. Gene location in the nucleus: genes in the nuclear periphery tend to be less expressed unless they sit near nucleopores. Eric Blanc (KCL) Gene Regulation Biology April 13, / 21

20 Genome comparisons Number of Proportion of Genome Size Genes Proteins coding DNA E.coli % S.cerevisiæ D.melanogaster M.musculus H.sapiens % The number of genes is the number of protein-coding genes, and the number of protein is the number of transcripts Source: ENSEMBL release 57 Eric Blanc (KCL) Gene Regulation Biology April 13, / 21

21 Some numbers For E.coli Translation rate 40 aa/sec Transcription rate 70 nt/sec Concentration 5-8 mm (protein), 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 D Eric Blanc (KCL) Gene Regulation Biology April 13, / 21