1. Eukaryotic Gene Regulation

2. Introduction Difference between eukaryotic and prokaryotic DNA Regulation at chromosome level Regulation at the transcription level Post transcriptional regulation – Alternative Splicing – microRNA… Post-translation modification

3. Prokaryotic DNA V Eukaryotic DNA Prokarytoic is circular, eukaryotic is straight. Eukarytoic DNA is in the form of chromatin [prokaryotic is not]; essentially Eukaryotic is surrounded by a histone envelope. The promoter region of a prokaryotic gene [coding region] is immediately “upstream” of the gene. In eukaryotic DNA there are more that one promoter region which can be a large distance from the gene Prokaryotic genes are in the form of operons[polycistronic]; e.g. lac operon, while eukaryotic normally are associated with one gene and are regulated by silencers and enhancers. These enhancers can be controlled by more that one regulatory element [trans] The coding region of Eukaryotic DNA consists of exons [ regions that are translated] interjected with introns [regions that are not translated]. Prokaryotic coding regions do not have introns;

4. Overview of Eukaryotic gene regulation

5. level 1: Regulation at the chromatin level Histones are proteins that surround and “protect” DNA and form chromatin While the histones conceal the DsDNA so no RNA/DNA polymerase can bind to it. Chromatin modification can be considered to be the first step of gene regulation: – Prerequisite for some gene(s) transcription – Simultaneous with others [dna exposed and then transcribed] Forms the basis of the field of epi-genetics: modification of the phenotype with any change to the genotype or DNA sequences.

6. level 2: Expression Ctrl at the transcription level Much more complex than prokaryotic consists of: Promoter: like prokaryotics is the region where RNA polymerase binds. [ refer to p region in the lac operon] – There different promoter “regulatory” sites : e.g. core (basal promoter), distal (upstream )promoter. Enhancers: regions that increase transcription levels Silencers: regions that decrease the level of transcription Both enhancers and silencers can be thousands of bp away form the transcription site

7. level 2: Expression Ctrl at the transcription level Ref [1] p 321 The Core promoters regions : Just upstream of where RNA polymerase binds and transcription starts [transcription start site] initiating low level transcription; Contains TATA and/or CAAT boxes and/or CG rich

8. level 2: Expression Ctrl at the transcription level Enhances: – DNA sequences that can be located at some distance on either side of the gene or within it – Required to achieve maximum level of expression – There position is not fixed and they seem to be generic to an extent (an enhancer need not be gene specific ([1] p 322) – They can also be inside the gene they regulate; Ig heavy chain enhancer. – Can enhance more than one gene; e.g. β and ε globins in chickens (ref [1] p. 322) – Time and tissue specific (play a part in organism development.

9. level 2: Expression Ctrl at the transcription level Silencers : – Cis-acting transcription regulatory element – Acts upon the gene to repress the level of transcription that was initiated by the corresponding promoter. – Are tissue specific and temporoal-specific – E.g. found in gene that produces a hormone involved in thyroid production/stimulation. This hormone is only produced in pituitary cells. Expression only occurs in these cells because of a silencer that binds a cellular factor which repress transcription. However, in cells that are required to produce the hormone the effect of the silencer is itself neutralised by an enhancer located 1.2 kb upstream of the promoter of the gene and is only “activated” in the cells [thyrotrophs] that must produce this hormone

10. Level 3: Post transcription regulation Alternative splicing: The coding region [gene] of Eukaryotic DNA consists of regions called exons interjected with introns. Prior to translation these introns must be “cut out” spliced from the pre mRNA [ mRNA] to produce mature mRNA However, for many genes the introns can be spliced in more a number of ways or produces alternative spliced mature mRNA strands. Only mature mRNA strands are translated into amino acid strands. The consequence of this process [Alternative Splicing] is that one DNA coding region can produce many mature mRNA strands and so many proteins. With some genes being able to produce ~38,000 different mature mRNA strands [splices]. The are a number of splicing process and it is regulated like DNA transcription

11. Illustration of Alternative splicing 40-60% of genes have alternative splicing forms. Frequencies of splices can vary 1 - thousands. – Encoding proteins at nodes highly connected interaction networks e.g. neural tissue Adapted from [3]

12. Types of Alternative splicing A more comprehensive description A.S. can be found at ref [5 and 6]

13. Alternative splicing: the effects Alternative splicing can lead to: 1.use of a different site for translation initiation (alternative initiation); alternative promoter/exon. 2.a different translation termination site by the addition/removal of a stop codon in the coding sequence (alternative termination). Note a poly A tail is a sequence of adenine (A) RNA molecules added to the end (3’ end) of the mature mRNA. In addition a “CAP” complex [modified G RNA molecule] is added to the 5’ start of the mature mRNA; both play a part in protecting the mRNA from degradation while it is being transported to the ribosomes.. 3.Alternative splicing can also change the internal region because of an in- frame insertion or deletion.

14. AS regulation[2] Normal regulation Cis-acting splicing disorder Indirect Trans-acting splicing disorder

15. Examples of abnormal A.S. regulation [2] Cis-acting disorder: – Found in neurological disease such as spinal muscular atrophy Indirect transacting-acting disorder: – Found in Prader Willi syndrome; ocd, autism Direct trans-acting disorder. – Cases of epilepsy and mental retardation.

16. Level 5: Regulation via RNA degradation Small fragments of RNA strands called Micro RNA (miRNA) (22 nucleotides in length). Can regulated gene expression in a number of ways: – Degrade the target (mature) mRNA – Prevent the early stages on translation by ribosome “drop off” – Affect chromatin Remodelling by causing histones to bind more tightly to the DNA and so prevent pre- translation expression. This process can hace a significant affect as it can “knock out” large segments of the DNA (100 to 1000s of genes An aside: It seems that the origins of the process was protecting cells from viral infection.

17. Level 6: Translation/post translational Modification The protein levels and activities can also be controlled via: – Regulation of the protein stability and modification – An example of such a protein is p53 a transcription factor for a number of important cell cycle genes. – Normally its low but in a damaged cell levels increase and helps express these important genes – When cells are damaged P53 Protein modification results in its activation and furthermore the modification decrease its rate of degradition. – However P53 is controlled by a negative feedback loop so eventually the levels go back to their original low levels ([1] p 327) Insulin is an example of a molecule that undergoes post- translational modification. When it is translated it is in an inactive form

18. Exam question A bacterial genome is different from a animal/plant genome discuss how the effect of such differences on the the regulation of gene expression in Eukaryotic cells animal cells to be more complex. “Alternative splicing is a critical reason as to why the genome of humans is much smaller than would be expected”. Discuss how the alternative splicing causes more proteins are produced than the number of genes present in the genome of Eukaryotic cells.

19. References Klug Essentials of Genetics: chapter 15 7 th Edition 2.Licatalosi, D.D. and Darnell, R.B. 2006. Splicing Regulation in Neurologic Disease. Neuron 52, 93-101

20. References [3] http://www.ncbi.nlm.nih.gov/Class/MLACourse/ Modules/MolBioReview/alternative_splicing.html http://www.ncbi.nlm.nih.gov/Class/MLACourse/ Modules/MolBioReview/alternative_splicing.html [4] Modrek, B. and Lee, C. 2002. A genomic view of alternative splicing. Nature genetics 30, 13-19. [5] http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1 370565/ http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1 370565/ [6] IntronsIntron Retention RetentionIntron Retention