A number of cell surface receptors mediate extracellular information to the nucleus…

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2. A number of cell surface receptors mediate extracellular information to the nucleus…

3. EPO binds to its cell surface receptor The cell surface EPO receptor is associated with JAK, but its kinase activity is very low in unbound conditions. Ligand binding causes conformational changes that lead to homodimerisation and weakly active JAK can phosphorylate the other receptor associated JAK kinase. Stimulated kinase activity then activates the second receptor-associated JAK, both of which phosphorylate tyrosine (Y) residues in the intracellular domain of the EPO receptor. Y-phosphorylation is recognised by the SH2 domains of STAT transcription factor which is in turn Y-phosphorylated by JAK. Y-phosphorylation of STAT promotes homodimerisation and exposes its NLS.

4. GTP-binding activates Ras and induces dissociation from the RTK complex Active Ras dissociates from the RTK/Sos/GRB complex and will contact downstream effectors to transduce the cellular signal.

5. A conserved kinase cascade is activated resulting in specific transcriptional changes Activated Raf will initiate a kinase cascade the ultimately leads to phosphorylation of specific transcription factors Raf is an oncogenic kinase. Its N-terminal domain negatively regulates its C-terminal kinase activity

6. Chromatin Modifications

7. But how can the transcription complex assemble on DNA when it is all wound up? • DNA is not naked in cells. • It is associated with Histone proteins (H1-H4) that bind it strongly and wind it up into higher-order structures called nucleosomes • The nucleosomes are necessary to package the extraordinary lengths of genomic DNA into a cell’s nucleus in the form of chromosomes taken from Nature,2003.

8. DNA gets “unwound” by modification of the chromatin • Modification of the chromatin results in significant changes in gene expression/transcription. • Unwinding the chromatin in a given region facilitates the expression of the genes within that domain. • This may be carried out by large complexes that include histone modifying enzymes, chromatin remodelling complexes and components of the RNA Polymerase II protein complex.

9. How did was this all worked out? The power of yeast genetics and biochemistry • Three genetic loci on chromosome III genetically control the mating type of Saccharomyces cerevisiae. • The HMLaand HMRaloci must be silenced otherwise the cells will be diploid a/a and cannot mate. • Transcriptional repression depends on silencer sequences. This silencer works outside the context of mating and can even block expression of tRNA genes (RNA Pol III). • Genetic experiments indicate that histones affect repression, while regions around telomeres behave similarly. Silent Mating Type Loci

10. Genetic screens identified factors required for repression of the silent mating type loci • RAP1- binds to DNA in the region of the silencer - also binds to repetitive sequence in telomeres • SIR1 (Silent Information Regulator)cooperates with RAP1 andis important for binding the silencer region in the silent mating type loci • SIR2, 3, 4 -binds to hypoacetylated histone tails (H3, H4) and recruits further SIR2 -forms large complexes with telomeric DNA

11. SIR3 co-localises with telomeric DNA

12. Transcriptional repressors may act through histone deacetylation complexes (HDACs) • Positive charge of N-terminal histone tail interacts electrostatically with the DNA phosphate groups. • Transcriptional pre-initiation complex cannot form on TATA boxes within regions of condensed chromatin (hypoacetylated). • Acetylation neutralizes the electrostatic interaction and permits complex formation. • Rpd3p is required for repressing some genes in yeast. It has substantial homology to a histone deacetylase and demonstrates deacetylase activity. • Specific targeting requires Ume6p (which binds URS) and finally Sin3p. Regions around the URS are hypoacetylated in wt and hyperacetylated in rpd3 and sin3 mutants Co-repressor

13. …while activators often recruit Histone Acetyl Transferases (HATs) • Some transcriptional activators can overcome the repressed chromatin state by inducing acetylation of histone tails through associated proteins • ie… Gcn4p and Gcn5p CBP, p300 Co-activators

14. The Histone Code leaves it mark… Specific modifications on tails of H3 and H4 induce changes in chromatin structure typical of EUCHROMATIN vs HETEROCHROMATIN…….but be careful not to generalise!!! Methylation on H3 K4->ACTIVE Methylation on H3 K9->INACTIVE

15. Heterochromatin vs Euchromatin • Euchromatin is delicate and thread-like. • It is abundant in actively transcribing cells. • It may represent DNA that is unwound to provide a transcriptional template.

16. Heterochromatin vs Euchromatin • Heterochromatin is a condensed form of chromatin that localises at the nuclear envelope often near the nuclear pores • Heterochromatin is considered transcriptionally inactive • Transcriptionally inactive regions of the genome are maintained in a “heterochromatinized” state. Their transcription could be detrimental to the cell/organism.

17. One X chromosome must be inactivated: Dosage Compensation • Females must inactivate one X chromosome early during embryonic development. Inactive X chromosomes are referred to as Barr Bodies • Once inactivated the targeted X-chromosome will remain inactive in all cells of the organism. • The patched nature of the fur of calico cats indicate regions where the inactivation of the X chromosome was altered, therefore providing coat colour information from two different X chromosomes. XX female XXX female (n-1) rule From Gilbert, Developmental Biology. 2002 Anti-acetylated H4 (green) From Gilbert, Developmental Biology. 2002

18. XIST is a seXIST gene product… Xist RNA • The XIST locus encodes long non-coding RNA • It binds to discrete regions of the X chromosome and spreads along the X to eventually X-tinguish gene expression. • Although many mechanisms have been proposed it is still not clear how this RNA functions and what is involved in its activation or specificity.

19. Epigenetic marks have to be propagated • EPIGENETIC TRAITS - transmitted independently of the DNA sequence itself • Inactive X (Xist,histone methylation and heterochromatin spreading) • Imprints (DNA methylation) • Developmental restrictions (legs vs antennae…Polycomb) -DNA marks (methylation) are propagated and recognized by mSin3 -Histone marks (ie..H3 K9 methylation) can nucleate other proteins such as HP1 and/or histone methyltransferases to repress gene activity across an entire genetic region. These marks can be (often are) heritable following cell divisions. -To ensure that every cell daughter acquires the appropriate fate following division epigenetic markers must be faithfully inherited.

20. Epigenetic marks have to be propagated Spreading Histone methyltransferase • EPIGENETIC TRAITS - transmitted independently of the DNA sequence itself” • Inactive X • Imprints • Developmental restrictions (legs vs antennae) • Histone marks can nucleate complexes to repress gene activity. These marks can be heritable. • To ensure that every cell daughter acquires the appropriate fate following division epigenetic markers must be faithfully inherited. Propagation Histone methyltransferase taken from Nature, 2003.

21. Other non-coding RNAs nucleate chromosomal silencing • Centromeric regions are associated with the kinetocore-a structure that is very important for accurate cell division. Chromatin within the centromeres must be silenced. • In S. pombe small RNAs are required for this silencing mechanism. • dsRNA nucleates a complex that involves a RNA-dependent RNA polymerase, Swi6p, and Clr4p, which is involved in directing the methylation of lysine 9 on Histone H3. • A similar mechanism functions in vertebrates to silence centromeric regions. taken from Science, 2002.