Control of Eukaryotic Genes “Epigenetics” CHAPTER 19 Control of Eukaryotic Genes “Epigenetics” 2007-2008

The BIG Questions… How are genes turned on & off in eukaryotes? How do cells with the same genes differentiate to perform completely different, specialized functions?

REVIEW Evolution of gene regulation Prokaryotes single-celled evolved to grow & divide rapidly must respond quickly to changes in external environment exploit transient resources Gene regulation = (?) Operons turn genes on & off rapidly flexibility & reversibility adjust levels of enzymes for synthesis & digestion prokaryotes use operons to regulate gene transcription, however eukaryotes do not. since transcription & translation are fairly simultaneous there is little opportunity to regulate gene expression after transcription, so control of genes in prokaryotes really has to be done by turning transcription on or off.

Evolution of gene regulation Eukaryotes Multicellular = only expresses a fraction of its genes evolved to maintain constant internal conditions even with changing conditions (?) Homeostasis must REGULATE the body as a whole rather than serve the needs of individual cells Also need to regulate: (?) growth & development long term processes (?) specialization turn on & off large number of genes Specialization each cell of a multicellular eukaryote expresses only a small fraction of its genes Development different genes needed at different points in life cycle of an organism afterwards need to be turned off permanently Continually responding to organism’s needs homeostasis cells of multicellular organisms must continually turn certain genes on & off in response to signals from their external & internal environment

Points of control The control of gene expression (?)can occur at any step in the pathway from gene to functional protein 1. packing/unpacking DNA 2. transcription 3. mRNA processing 4. mRNA transport 5. translation 6. protein processing 7. protein degradation

Structural Organization Chromatin is packed into chromosomes =ordered into higher structural levels compared to prokaryotes Figure 19.1

1. DNA packing as gene control Unfolded chromatin has the appearance of beads on a string Each “bead” is a (?) nucleosome Made up of (?) histones 2 nm 10 nm DNA double helix Histone tails His- tones Linker DNA (“string”) Nucleosome (“bead”) Histone H1 (a) Nucleosomes (10-nm fiber) Figure 19.2 a

2. Where does transcription occur on chromosome Degree of packing of DNA regulates transcription If tightly wrapped around histones = (?) no transcription = (?) genes turned off heterochromatin darker DNA (H) = tightly packed euchromatin lighter DNA (E) = loosely packed H E

Higher Levels of DNA Packing The next level of packing Forms the 30-nm chromatin fiber Nucleosome 30 nm (b) 30-nm fiber Figure 19.2 b

(c) Looped domains (300-nm fiber) The 30-nm fiber, in turn Forms looped domains, making up a 300-nm fiber Protein scaffold 300 nm (c) Looped domains (300-nm fiber) Loops Scaffold Figure 19.2 c

(d) Metaphase chromosome In a mitotic chromosome The looped domains coil and fold = metaphase chromosome 700 nm 1,400 nm (d) Metaphase chromosome Figure 19.2 d

What regulates Chromatin Structure ? Acetylation of histones (?) unwinds DNA (?) attachment of acetyl groups (–COCH3) (?) enables transcription (?) genes turned on transcription factors have easier access to genes Figure 19.4 b (b) Acetylation of histone tails promotes loose chromatin structure that permits transcription Unacetylated histones Acetylated histones

DNA methylation Methylation of DNA (?) blocks transcription factors (?) attachment of methyl groups (–CH3) to cytosine  (?) genes turned off nearly permanent inactivation of genes ex. inactivated mammalian X chromosome = Barr body

Epigenetic Inheritence =Terminology for gene expression ** DNA sequence NOT changed, just the expression of the gene (on or off) Can Chromatin modifications be passed offspring? (sometimes – poorly understood) **** In a new embryo, all tags are removed except for “imprinted” tags for getting development started

Examples of “epigenetics” Morphogenesis and specialization

Examples of “epigenetics” FTO gene & obesity Cytogenetic Location: 16q12.2 Molecular Location on chromosome 16: base pairs 53,703,962 to 54,114,466

Examples of “epigenetics” Cancer

Examples of “epigenetics” Twin Studies…”epigenetic drift” http://learn.genetics.utah.edu/content/epigenetics/twins/

III. PROCESS CONTROLS (?) transcription controls seem to be the most “important” factor in gene expression

A. The Roles of Transcription Factors To initiate transcription (?) RNA polymerase requires transcription factors (proteins) to bind to the (?) promotor region

B. Role of Enhancers enhancer DNA sequence (?) upstream from promotor Activator protein – “enhance” (high level) or transcription

Model for Enhancer action Enhancer DNA sequences (?) distant control sequences Activator proteins (?) bind to enhancer sequence & stimulates transcription Repressor proteins bind to enhancer sequence & (?)block gene transcription Much of molecular biology research is trying to understand this: the regulation of transcription. Silencer proteins are, in essence, blocking the positive effect of activator proteins, preventing high level of transcription.

Transcription complex Activator Proteins • regulatory proteins bind to DNA at distant enhancer sites • increase the rate of transcription Enhancer Sites regulatory sites on DNA distant from gene Enhancer Activator Activator Activator Coactivator B F E RNA polymerase II A TFIID H Coding region T A T A Core promoter and initiation complex Initiation Complex at Promoter Site binding site of RNA polymerase

C. Significance of protein-mediated bending Activators BEND TOWARD transcription factors stimulating transcription (influence chromatin structure) Distal control element Activators Enhancer Promoter Gene TATA box General transcription factors DNA-bending protein Group of Mediator proteins RNA Polymerase II RNA synthesis Transcription Initiation complex Chromatin changes RNA processing mRNA degradation Translation Protein processing and degradation A DNA-bending protein brings the bound activators closer to the promoter. Other transcription factors, mediator proteins, and RNA polymerase are nearby. 2 Activator proteins bind to distal control elements grouped as an enhancer in the DNA. This enhancer has three binding sites. 1 The activators bind to certain general transcription factors and mediator proteins, helping them form an active transcription initiation complex on the promoter. 3 Figure 19.6

2. Post-transcriptional controls = RNA modification * Gene expression can be blocked or stimulated during RNA modification

A. Role of RNA Processing (?) exons joined after introns cut out Chromatin changes Transcription RNA processing mRNA degradation Translation Protein processing and degradation Exons DNA Primary RNA transcript RNA splicing or Figure 19.8

B. Role of mRNA Degradation The life span of mRNA molecules in the cytoplasm (hours to weeks) Degradation of the leader (5’cap) and trailer regions (poly-A tail) by enzymes Prokaryotes vs. Eukaryotes lifespan

(?) Regulatory proteins – attach to 5’ end of mRNA 3. Translation Control (?) Regulatory proteins – attach to 5’ end of mRNA Prevent attachment of ribosome Block translation

B. Protein Processing After translation (?)protein processing/modification are controlled by cellular events (Endo. System) = folding, cleaving, adding sugar groups, targeting for transport

Protein degradation (Figure 19.10) Ubiquitin tagging Proteasomes Chromatin changes Transcription RNA processing mRNA degradation Translation Protein processing and degradation Ubiquitin Protein to be degraded Ubiquinated protein Proteasome and ubiquitin to be recycled Protein fragments (peptides) Protein entering a proteasome Enzymatic components of the proteasome cut the protein into small peptides, which can be further degraded by other enzymes in the cytosol. 3 The ubiquitin-tagged protein is recognized by a proteasome, which unfolds the protein and sequesters it within a central cavity. 2 Multiple ubiquitin mol- ecules are attached to a protein by enzymes in the cytosol. 1 Figure 19.10

Ubiquitin 1980s | 2004 “Death tag” Nobel Prize 2004 mark unwanted proteins with a label 76 amino acid polypeptide = ubiquitin labeled proteins are broken down rapidly in "waste disposers“ = proteasomes Since the molecule was subsequently found in numerous different tissues and organisms – but not in bacteria – it was given the name ubiquitin (from Latin ubique, "everywhere") Nobel Prize 2004 Aaron Ciechanover Israel Avram Hershko Israel Irwin Rose UC Riverside

Proteasome (?) Protein-degrading “machine” cell’s waste disposer breaks down any proteins into 7-9 amino acid fragments cellular recycling A human cell contains about 30,000 proteasomes: these barrel-formed structures can break down practically all proteins to 7-9-amino-acid-long peptides. The active surface of the proteasome is within the barrel where it is shielded from the rest of the cell. The only way in to the active surface is via the "lock", which recognises polyubiquitinated proteins, denatures them with ATP energy and admits them to the barrel for disassembly once the ubiquitin label has been removed. The peptides formed are released from the other end of the proteasome. Thus the proteasome itself cannot choose proteins; it is chiefly the E3 enzyme that does this by ubiquitin-labelling the right protein for breakdown play Nobel animation

RNA interference Small interfering RNAs (siRNA) short segments of RNA (21-28 bases) bind to mRNA create sections of double-stranded mRNA “death” tag for mRNA triggers degradation of mRNA causes (?) gene “silencing” post-transcriptional control (?) turns off gene = no protein produced Nobel Prize 2006 UMASS!!! siRNA

Action of siRNA siRNA mRNA degraded functionally turns gene off Hot…Hot new topic in biology Action of siRNA dicer enzyme mRNA for translation siRNA double-stranded miRNA + siRNA breakdown enzyme (RISC) mRNA degraded functionally turns gene off

Now that the complete sequence of the human genome is available 98.5% does not code for proteins, rRNAs, or tRNAs Exons (regions of genes coding for protein, rRNA, tRNA) (1.5%) Repetitive DNA that includes transposable elements and related sequences (44%) Introns and regulatory (24%) Unique noncoding DNA (15%) DNA unrelated to (about 15%) Alu elements (10%) Simple sequence DNA (3%) Large-segment duplications (5-6%) Figure 19.14

Another post-transcriptional control…. RNAi RNA interference by microRNAs (miRNAs) Can lead to degradation of an mRNA or block its translation The micro- RNA (miRNA) precursor folds back on itself, held together by hydrogen bonds. 1 2 An enzyme called Dicer moves along the double- stranded RNA, cutting it into shorter segments. One strand of each short double- stranded RNA is degraded; the other strand (miRNA) then associates with a complex of proteins. 3 The bound miRNA can base-pair with any target mRNA that contains the complementary sequence. 4 The miRNA-protein complex prevents gene expression either by degrading the target mRNA or by blocking its translation. 5 5 Chromatin changes Transcription RNA processing mRNA degradation Translation Protein processing and degradation Degradation of mRNA OR Blockage of translation Target mRNA miRNA Protein complex Dicer Hydrogen bond Figure 19.9