1. BIOL 2416 Chapter 18: Regulation of Eukaryotic gene expression

2. Control of eukaryotic gene expression NO operons in eukaryotes (rare exceptions in C. elegans - but do not use polycistronic mRNA as is) Related genes are scattered throughout genome Related genes are regulated coordinately however! In eukaryotes, potential control points lie anywhere along the pathway from DNA to functional protein –in prokaryotes pretty much only see transcriptional control of operons –Transcriptional control does dominate in eukaryotes

3. Peter J. Russell, iGenetics: Copyright © Pearson Education, Inc., publishing as Benjamin Cummings. Fig. 17.1 Levels at which gene expression can be controlled in eukaryotes

4. 1.transcriptional control Core promoter DNA elements (e.g. TATA box) are required for transcription initiation (located just upstream of tx’al start site) – decides where txn starts Proximal promoter elements bind general transcription factors (proteins); required to maintain basal transcription rates – decides whether txn starts Specialized regulatory promoter elements bound by regulatory proteins specific for control of 1 or a few genes (only correct genes activated) Many of these regulatory proteins may also bind enhancers implying regulatory protein interactions:

5. transcriptional control, cont’d DNA enhancer elements can be located far upstream or downstream of the promoter: determine whether maximal txn rates occur –Bound by regulatory proteins (combinatorial gene regulation by relatively few proteins) E.g. activator proteins that can bind DNA and have a txn activation domain; activators recruit coactivator proteins (e.g. yeast mediator complex of 20 proteins) that interact with transcription factors and RNA Polymerase; causes looping of the DNA and activation/enhancement of transcription Bound regulatory proteins may also disrupt nucleosomes on TATA boxes, increasing RNA Polymerase access, tx’al rates (chromatin remodeling)

6. Peter J. Russell, iGenetics: Copyright © Pearson Education, Inc., publishing as Benjamin Cummings. Fig. 5.6 Events that may occur during the initiation of transcription catalyzed by RNA polymerase II

7. transcriptional control, cont’d Repressors are rare in eukaryotes. Can work in different ways: Sometimes, a repressor protein binds on or near an enhancer, blocking activator/enhancer binding causing silencing of the gene; enhancer is now called a silencer Or a repressor may engage in chromatin remodeling: binds and recruits a histone deacetylase complex, that causes chromatin to compact, silencing transcription:

8. transcriptional control, cont’d Chromatin remodeling: –Chromatin = (histone) proteins + DNA –Histones generally repress transcription by blocking RNA Polymerase access –Tx’ally active genes hypersensitive to DNase I (demonstrating accesibility, less coiling) –To activate a gene, can change chromatin structure around the core promoter by: Acetylating core histones to destabilize higher order chromatin structure, increasing tx’al rate, while deacetylated histones may be recognized by a silencing complex Nucleosome remodeling complexes recruited by bound activators to slide (away), transfer or restructure nucleosomes to expose promoters

9. transcriptional control, cont’d DNA methylation –May play a role in silencing transcription (good correlations, but it’s been tough to show cause and effect…) –E.g. 5 m C, usually in CG sequences –Involved in fragile X syndrome: abnormally methylated triplet repeats silence FMR-1 gene expression –Barr bodies are highly condensed and methylated –Involved in genomic imprinting - gene expression determined by maternal vs. parternal inheritance E.g seen in Prader-Willi (normal maternal alleles silenced by methylation) and Angelman syndrome (normal paternal alleles methylated/silenced); expressed alleles are disrupted/have deletions

10. DNA methylation, cont’d –Also nutritional epigenetics: vitB12, folic acid, choline and betaine dietary supplements given to pregnant mice: Causes inadvertant methylation of agouti gene cell trying to inactivate junk transposons near agouti gene (agouti codes for yellow coats)

12. transcriptional control, cont’d Steroid hormone regulation in animals –Secreted by specific cell, put into the blood stream, and affects transcription of another far-away target cell –Target cell must have steroid hormone receptor to “hear” the hormone –Hormone-receptor complexes bind target cell DNA to regulate gene expression –Steroid hormones may also affect mRNA stability and processing of mRNA precursors. Plant hormones –Poorly understood –Made by all cells, heard by all cells –Often hormone ratios are important

13. Peter J. Russell, iGenetics: Copyright © Pearson Education, Inc., publishing as Benjamin Cummings. Fig. 17.11 Model for the action of steroid hormone glucocorticoid in mammalian cells

14. Peter J. Russell, iGenetics: Copyright © Pearson Education, Inc., publishing as Benjamin Cummings. Fig. 17.9 Mechanisms of action of polypeptide hormones and steroid hormones

16. 2. RNA processing control. Determines production of different mature mRNAs resulting in different (or no) proteins By choice of polyA site (e.g. how different classes of immunoglobulins are produced, by producing different pre- mRNAs that include different exons) Or by choice of splice site: alternative splicing –plays key role in Drosophila sex determination –X:A ratio is transmitted to the sex determination genes –In response, females make shorter, functional Sxl protein causing a cascade of events that turn off genes for male differentiation, males make longer, nonfunctional Sxl protein Both mechanisms produce protein isoforms that differ structurally and functionally

17. Peter J. Russell, iGenetics: Copyright © Pearson Education, Inc., publishing as Benjamin Cummings. Fig. 17.14 Alternative polyadenylation and alternative splicing resulting in tissue- specific products of the human calcitonin gene, CALC

18. 3. mRNA transport from nucleus to cytosol Spliceosome retention model: spliceosome assembly on pre-mRNA competes with and prevents nuclear export (involves snRNPs). Normally spliceosomes are supposed to dissociate from mature mRNAs (all exons), while sticking to the excised introns; only “naked” mRNA can exit through nuclear pores mRNAs retained by spliceosomes may be degraded (never are exported to cytosol).

19. 4. Translational control Long polyA tails on mRNAs stimulate translation initiation; differential translation rates possible for different mRNAs; stored mRNAs have shorter tails polyA length may be controlled on a given mRNA: –Some mRNAs have a 3’UTR AU-rich element (“ARE”) that causes a deadenylation enzyme in the cytosol to take off a bunch of 3’ A’s, making the mRNA less translatable –Or the ARE is recognized by a polyadenylation enzyme that adds about 150 A’s to activate stored mRNA

20. 5. mRNA degradation control mRNA half lives vary from minutes to months Major control point in eukaryotic gene regulation Stability influenced by AREs and secondary structures, and effector molecules like (steroid) hormones 2 degradation pathways: –Deadenylation-dependent mechanism: deadenylation (removal of A’s from poly(A) tail) followed by decapping and rapid 5’ to 3’ degradation by exonucleases –Deadenylation-independent mechanism: direct decapping only, exposing the 5’ end to exonucleases, followed by internal cleavage and chewing up of the fragments

21. Also… NEW: RNA interference (RNAi): Transposable elements encode dsRNA. Enzymes called “Dicers” (type of Rnase III) cut these dsRNAs into 21-25 nucleotide segments called small interfering RNAs (siRNAs). The siRNAs hook up with an RNA-induced silencing complex (RISC RISC is activated with ATP and can: –hunt down and degrade homologous mRNAs in the cell (silencing expression for a given gene) –Bind target mRNA to block translation to silence the gene –Move into the nucleus to bind to its complementary DNA to recruit chromatin remodeling complex to silence the gene RNAi found in every organism studied! Very specific and simple: ideal potential tool for e.g. gene therapy

22. 6. Protein degradation control Binding of ubiquitin protein marks protein for degradation by proteases N-end rule; N-terminal amino acid determines degree of ubiquitin binding and degradation.