Regulation of Eukaryotic Gene Expression 2015

Key concepts in Expression of Eukaryotic Genomes EACH CELL IN YOUR BODY CONTAINS ALL OF THE SAME DNA ; what distinguishes one cell from another (i.e. eye cell vs. heart cell) is which genes are expressed. Eukaryotic cells typically only express a small number of specific genes. Default expression is usually off; Complex regulation and control in timing and degree of expression is required to turn genes on.

Figure 19.7 Opportunities for the control of gene expression in eukaryotic cells TRANSCRIPTION IS MOST HEAVILY REGULATED STEP; HOWEVER REGULATION CAN OCCUR AT ANY POINT OF PROTEIN SYNTHESIS

Fig. 18-UN4 Genes in highly compacted chromatin are generally not transcribed. Chromatin modification DNA methylation generally reduces transcription. Histone acetylation seems to loosen chromatin structure, enhancing transcription. Chromatin modification Transcription RNA processing Translation mRNA degradation Protein processing and degradation mRNA degradation Each mRNA has a characteristic life span, determined in part by sequences in the 5 and 3 UTRs. Protein processing and degradation by proteasomes are subject to regulation. Protein processing and degradation Initiation of translation can be controlled via regulation of initiation factors. Translation ormRNA Primary RNA transcript Alternative RNA splicing: RNA processing Coordinate regulation: Enhancer for liver-specific genes Enhancer for lens-specific genes Bending of the DNA enables activators to contact proteins at the promoter, initiating transcription. Transcription Regulation of transcription initiation: DNA control elements bind specific transcription factors.

Link to eukaryotic gene regulation Link to eukaryotic gene regulation

Euchromatin (Contains active genes, unique sequences) vs Heterochromatin

Active vs Inactive Genes

Acetylation / DeAcetylation of Histones

Fig Histone tails DNA double helix (a) Histone tails protrude outward from a nucleosome Acetylated histones Amino acids available for chemical modification (b) Acetylation of histone tails promotes loose chromatin structure that permits transcription Unacetylated histones Recall DNA backbone is - charged; histone protein surface is + charged Recall HIGH methylation in DNA ↓ acetylation of histones Link to Video Link to chromatin structure gene expression animation

DNA Packing is Key Component of Regulation of Transcription ACTIVE GENESINACTIVE GENES DNA BINDING TO HISTONE LOOSE PACKING AND WEAK BONDING TO HISTONE COMPACT PACKING AND TIGHT BINDING TO HISTONE DNA METHYLATION ? NO YES HISTONE ACETYLATION? YESNO DNA bases Modified by Adding CH 3 group Histones modified by acetyl groups

Epigenetic Inheritance Epigenetics – which genes are expressed based on modifications of DNA/histones Patterns of DNA methylation can change based on environment Patterns of DNA methylation can be passed to offspring Differences in DNA methylation patterns can cause cancer even when no mutations are present.

Twin studies are often used to determine the role of genetics on health. A woman developed breast cancer at age 60. Her identical twin did not. When the sequences of genes known to be linked with breast cancer were compared in the twins, no differences were detected. Propose a hypothesis to account for the presence of cancer in one twin but not the other and an experiment to test your hypothesis. Answer: It was observed that DNA methylation patterns differed on the relevant genes in the two twins, presumably the result of different environmental exposures. Link to chromatin structure gene expression animation CASE STUDY IN EPIGENETICS

Review Questions Chromatin What is epigenetics? What is epigenetics? Ans: Describes which genes are expressed. Ans: Describes which genes are expressed. Describe the how the packing of DNA, level of methylation of bases, and level of histone acetylation impact gene expression. Describe the how the packing of DNA, level of methylation of bases, and level of histone acetylation impact gene expression.  Ans: ACTIVELY TRANSCRIBED GENES - Loosely packed DNA; loose binding between DNA and histone proteins - Loosely packed DNA; loose binding between DNA and histone proteins - DNA has LOW level of METHYLATION - DNA has LOW level of METHYLATION - Histones have HIGH level of ACETYLATION - Histones have HIGH level of ACETYLATION

TRANSCRIPTION FACTOR PROTEINS AND ACTIVATOR PROTEINS ARE KEY TO REGULATING TRANSCRIPTION Eukaryotic Gene Expressions requires the binding of transcription factors to the promoter region and activator proteins (specific transcription factors) to enhancer regions (Note: Campbell’s calls activator proteins specific transcription factors; enhancers is a general term for a set of binding sites for activator proteins Different Enhancer Regions bind different Activator Proteins ) Activator Proteins and Transcription Factors can be produced or activated as a result of different chemical signals originating from both inside and outside of cell.

Link to enhancer animations Link to enhancer animations DNA Interactive Transcription animation DNA Interactive Transcription animation

Fig Enhancer TATA box Promoter Activators DNA Gene Distal control element

Fig Enhancer TATA box Promoter Activators DNA Gene Distal control element Group of mediator proteins DNA-bending protein General transcription factors

Fig Enhancer TATA box Promoter Activators DNA Gene Distal control element Group of mediator proteins DNA-bending protein General transcription factors RNA polymerase II RNA polymerase II Transcription initiation complex RNA synthesis

Fig Control elements Enhancer Available activators Albumin gene (b) Lens cell Crystallin gene expressed Available activators LENS CELL NUCLEUS LIVER CELL NUCLEUS Crystallin gene Promoter (a) Liver cell Crystallin gene not expressed Albumin gene expressed Albumin gene not expressed Different enhancers means genes respond to different activator proteins

Case Study: Transformation of Cells One of the most dramatic discoveries in biology in recent years has been the demonstration that differentiated (specialized) of one type (e.g. skin cells) can be converted to other types of differentiated cells (e.g. blood cells). Describe the DNA differences present in the different types of cells. Answer: None. The differences are not in the gene sequences but rather which genes are being expressed. The approach used by researchers was to search for proteins that were expressed in skin cells but not in blood cells. Different combinations of the proteins were systematically added to colonies of skin cells until a combination was found that caused the cells to transform. Speculate on the identify of the proteins that caused the transformation. Answer: The proteins that caused the transformation where all identified as transcription factors (activator proteins), that cause different genes to be expressed in the cells.

Case Study: The presence or absence of black spots on the wings of a species of fruit fly has been traced to the expression of a single gene. The DNA sequences of two flies, one with spots and one without were compared. The DNA sequences within the genes were found to be absolutely identical. A mutation (difference in DNA sequence) was detected in a region that is 500 bp upstream from the startpoint of the gene. Black spots on wing Why does one have spots and the other does not? How could you test your hypothesis experimentally? No spots on wing

Answer: One fly expresses the gene while the other fly does not because an enhancer mutation. The two flies differ in the sequence of DNA upstream from the gene which is a noncoding segment of DNA. The mutated DNA is an enhancer site for binding of activator proteins. When a “corrected” enhancer region was added to the DNA of developing non-spotted flies they became spotted. Note: Promoters are 25 to 200 bp in length and overlap the start of the gene. A mutation 500 bp upstream is much too far away to be a promoter mutation. Another plausible answer is that the mutation is in a gene that codes for a transcription factor that regulates expression of the spot gene.

Link to signal transduction animation Link to signal transduction animation Link to regulated transcription Link to regulated transcription Signal Cascade blood clot Cold spring Signal Cascade blood clot Cold springSignal Cascade blood clot Cold springSignal Cascade blood clot Cold spring

Figure Nuclear response to a signal: the activation of a specific gene by a growth factor

Fig Hormone (estradiol) Hormone-receptor complex Plasma membrane Estradiol (estrogen) receptor Activation of a gene caused by a hormone

Fig Hormone (estradiol) Hormone-receptor complex Plasma membrane Estradiol (estrogen) receptor DNA Vitellogenin mRNA for vitellogenin Activation of a gene caused by a hormone

Fig Enhancer (distal control elements) Proximal control elements Poly-A signal sequence Termination region Downstream Promoter Upstream DNA Exon Intron REGULATION OF EUKARYOTIC GENE EXPRESSION BY USING ALTERNATE SPLICING PATTERNS OF pre -mRNA

Fig Enhancer (distal control elements) Proximal control elements Poly-A signal sequence Termination region Downstream Promoter Upstream DNA Exon Intron Cleaved 3 end of primary transcript Primary RNA transcript Poly-A signal Transcription 5 Exon Intron ROLE OF RNA PROCESSING IN REGULATING EUKARYOTIC GENE EXPRESSION

Fig Enhancer (distal control elements) Proximal control elements Poly-A signal sequence Termination region Downstream Promoter Upstream DNA Exon Intron Exon Intron Cleaved 3 end of primary transcript Primary RNA transcript Poly-A signal Transcription 5 RNA processing Intron RNA Coding segment mRNA 5 Cap 5 UTR Start codon Stop codon 3 UTR Poly-A tail 3 ROLE OF RNA PROCESSING IN REGULATION EUKARYOTIC GENE EXPRESSION UTR regions = Untranslated Region - binding site for micro interfering RNA or regulatory proteins that impact stability of mRNA

Fig or RNA splicing mRNA Primary RNA transcript Troponin T gene Exons DNA ALTERNATIVE SPLICING DIFFERENT SPLICING PATTERNS PRODUCES DIFFERENT mRNA

Regulation of Translation Life time of mRNA is important in determining amount of protein produced. Link to translation regulation animation Link to translation regulation animationLink to translation regulation animation Rate of Translation is determined in part by the binding of translation initiation protein factors Recent Discoveries suggest that Micro -Interfering RNA (miRNA) that is complementary to the mRNA can also suppress translation

Fig miRNA- protein complex (a) Primary miRNA transcript Translation blocked Hydrogen bond (b) Generation and function of miRNAs Hairpin miRNA Dicer 3 mRNA degraded 5 REGULATION OF TRANSLATION BY INTERFERENCE OF mRNA Link to miRNA video

Figure Degradation of a protein by a proteasome Rate at which protein is degraded in the cell is also a critical mechanism for regulating gene expression (amount of protein product present in the cell)

Figure 19.7 Opportunities for the control of gene expression in eukaryotic cells TRANSCRIPTION IS MOST HEAVILY REGULATED STEP; HOWEVER REGULATION CAN OCCUR AT ANY POINT OF PROTEIN SYNTHESIS