Genetics: From Genes to Genomes Powerpoint to accompany Genetics: From Genes to Genomes Third Edition Hartwell ● Hood ● Goldberg ● Reynolds ● Silver ● Veres Chapter 18 Prepared by Malcolm Schug University of North Carolina Greensboro Copyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display

Gene Regulation in Eukaryotes Copyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display

Outline of Chapter 18 How we use genetics to study gene regulation Using mutations to identify cis-acting elements and trans-acting proteins How genes are regulated at the initiation of transcription Three polymerases recognize three classes of promoters. Trans-acting proteins control class II promoters. Chromatin structure affects gene expression. Signal transduction systems DNA methylation regulates gene expression. How genes are regulated after transcription RNA splicing RNA stability mRNA editing Translation Posttranslational modification A comprehensive example of sex determination in Drosophila Copyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display

Regulatory elements that map near a gene are cis-acting DNA sequences. cis-acting elements Promoter – very close to gene’s initiation site Enhancer Can lie far way from gene Can be reversed Augment or repress basel levels of transcription Figure 18.1 a Fig. 18.1 a Copyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display

Reporter constructs are a tool for studying gene regulation. Sequence of DNA containing gene’s postulated regulatory region, but not coding region Coding region replaced with easily identifiable product such as b-galactosidase or green fluorescent protein Reporter constructs can help identify promoters and enhancers by using invitro mutatgenesis to systematically alter the presumptive regulatory region. Copyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display

Regulatory elements that map far from a gene are trans-acting DNA sequences. Genes that encode proteins that interact directly or indirectly with target genes cis-acting elements Known genetically as transcription factors Identified by: Mapping Biochemical studies to identify proteins that bind invitro to cis-acting elements Figure 18.1 b Fig. 18.1 b Copyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display

In eukaryotes three RNA polymerases transcribe different sets of genes. RNA polymerase I transcribes rRNA. rRNAs are made of tandem repeats on one or more chromosomes. RNA polymerase I transcribes one primary transcript which is broken down into 28s and 5.8s by processing. Figure 18.2 a Fig. 18.2 a Copyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display

RNA polymerase III transcribes tRNAs and other small RNAs (5s rRNA, snRNAs). Figure 18.2 b Fig. 18.2 b Copyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display

RNA polymerase II recognizes cis-acting regulatory regions composed of one promoter and one or more enhancers. Promoter contains initiation site and TATA box. Enhancers are distant from target gene. Sometimes called upstream activation sites Figure 18.2 c Fig. 18.2 c Copyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display

RNA polymerase II transcribes all protein coding genes. Figure 18.2 c RNA polymerase II transcribes all protein coding genes. Primary transcripts are processed by splicing, a poly A tail is added to the 3’ end, and a 5’ GTP cap is added. Fig. 18.2 c Copyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display

Large enhancer region of Drosophila string gene Fourteenth cell cycle of the fruit fly embryo A variety of enhancer regions ensure that string is turned on at the right time in each mitotic domain and tissue type. Figure 18.3 Copyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Fig. 18.3

Trans-acting proteins control transcription from class II promoters. Basal factors bind to the promoter. TBP – TATA box binding protein TAF – TBP associated factors RNA polymerase II binds to basal factors. Figure 18.4 a Fig. 18.4 a Copyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display

Activator proteins Also called transcription factors Bind to enhancer DNA in specific ways Interact with other proteins to activate and increase transcription as much as 100-fold above basal levels Two structural domains mediate these functions: DNA-binding domain Transcription-activator domain Copyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display

Transcriptional activators bind to specific enhancers at specific times to increase transcriptional levels. Figure 18.5 a Copyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Fig. 18.5 a

Examples of common transcription factors Zinc-finger proteins and helix-loop-helix proteins bind to the DNA binding domains of enhancer elements. Figure 18.5 b Copyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Fig. 18.5 b

Some proteins affect transcription without binding to DNA. Coactivator – binds to and affects activator protein which binds to DNA Enhancerosome – multimeric complex of proteins Activators Coactivators Repressors Corepressors Copyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display

Localization of activator domains using recombinant DNA constructs Fusion constructs from three parts of gene encoding an activator protein Reporter gene can only be transcribed if activator domain is present in the fusion construct. Part B contains activation domain, but not part A or C. Figure 18.6 Copyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Fig. 18.6

Most eukaryotic activators must form dimers to function. Eukaryotic transcription factor protein structure Homomers – multimeric proteins composed of identical subunits Heteromers – multimeric proteins composed of nonidentical subunits Figure 18.7 a Fig. 18.7 a Copyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display

Leucine zipper – a common activator protein with dimerization domains Figure 18.7 b Fig. 18.7 b Copyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display

Repressors diminish transcriptional activity Figure 18.8 a.b Fig. 18.8 Copyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display

Repressors Reduces transcriptional activation but does not affect basal level of transcription Activator-repressor competition Quenching (corepressors) Some repressors stop basal level of transcription. Binding directly to promoter Bind to DNA sequences farther from promoter, contact basal factor complex at promoter by bending DNA causing a loop where RNA polymerase can not access the promoter Copyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display

Transcription factors may act as activators or repressors or have no affect. Action of transcription factor depends on: Cell type Gene it is regulating Copyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display

Specificity of transcription factor can be altered by other molecules in the cell. yeast a2 repressor – determines mating type Haploid – a2 factor silences the set of “a” genes Diploid – a2 factor dimerizes with a1 factor and silences haploid-specific genes Figure 18.9 Fig. 18.9 Copyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display

Myc-Max system is a regulatory mechanism for switching between activation and repression. Figure 18.10 Myc polypeptide has an activation domain. Max polypeptide does not have an activation domain. Fig. 18.10a Copyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display

Myc-Max system is a regulatory mechanism for switching between activation and repression. As soon as a cell expresses the myc gene, the Myc-Myc homodimers convert to Myc-Max heterodymers that bind to the enhancers. Induction of genes required for cell proliferation Figure 18.10 Fig. 18.10b Copyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display

Gene expression results only when the Max polypeptide is made in the cell. max gene Figure 17 Figure 18.10 b Copyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Fig. 18.10 c

Gene activation occurs when both Myc and Max are made in the cell. Figure 18.10 Copyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Fig. 18.10 d

The locus control region is a cis-acting regulatory sequence that operates sequentially. Human b-globin gene cluster contains five genes that can all be regulated by a distant LCR (locus control region). Figure 18.12 Fig. 18.12 a Copyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display

Proof that cis-acting factor such as LCR is needed for activation of b-globin gene Figure 18.12 b Fig. 18.12 b Copyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display

One mechanism of activation that brings LCR into contact with distant globin genes may be DNA looping. Figure 18.12 c Fig. 18.12 c Copyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display

Other mechanisms of gene regulation Chromatin structure Slows transcription Hypercondensation stops transcription. Genomic imprinting Silences transcription selectively if inherited from one parent Some genes are regulated after transcription. RNA splicing can regulate expression. RNA stability controls amount of gene product. mRNA editing can affect biological properties of protein. Noncoding sequences in mRNA can modulate translation. Protein modification after translation can control gene function. Copyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display

Normal chromatin structure slows transcription /figure 18.13 a,b Copyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Fig. 18.13 a-b

Remodeling of chromatin mediates the activation of transcription. Figure 18.13 c,d Copyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Fig. 18.13 c-d

Hypercondensation over chromatin domains causes transcriptional silencing. Figure 18.14 Fig. 18.14 a Copyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display

In mammals hypercondensation is often associated with methylation. Figure 18.14 It is possible to determine the methylation state of DNA using restriction enzymes that recognize the same sequence, but are differentially sensitive to methylation. Fig. 18.14 b Copyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display

Genomic imprinting results from chromosomal events that selectively silence genes inherited from one parent. 1980s, invitro fertilization experiments demonstrated pronuclei from two females could not produce viable embryos. Fig. 18.14 a Copyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display

Figure 18.15 a Experiments with transmission of Ig f 2 deletion showed mice inheriting deletion from male were small. Mice inheriting deletion from female were normal. Fig. 18.15 a Copyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display

Figure 18.15 d H19 promoter is methylated during spermatogenesis and thus the H19 promoter is not available to the enhancer and is not expressed. Fig. 18.15 d Copyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display

Figure 18.15 c Epigenetic effect – whatever silences the maternal or paternal gene is not encoded in the DNA. The factor is outside the gene, but is heritable. Methylation can be maintained across generations by methylases that recognize methyl groups on one strand and respond by methylating the opposite strand. Fig. 18.15 c Copyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display

RNA splicing helps regulate gene expression. Figure 18.16 Fig. 18.16 a Copyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display

Figure 18.16 b Fig. 18.16 b Copyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display

Micro-RNAs mediate RNA interference. 2000-2005 – micro-RNAs identified and characterized RNA interference – trans-acting single stranded micro-RNAs that regulate eukaryotic gene expression ¼ from introns of protein coding transcripts ¾ from products of primary transcripts devoid of ORFs Number of miRNAs may exceed the number of protein coding genes. Copyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display

Micro-RNAs mediate RNA interference. miRNAs distinguished by 60-120 ribonucleotide-long segments of reverse sequence complimentarity – will snap back spontaneously into hairpin stem-loop structures. Involved in posttranscriptional regulation and trans acting transcription factors Provides a method for development of powerful new RNA-based therapies for treatment of diseases. Copyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display

Examples of primary transcripts from micro-RNA-containing genes Figure 18.17 Fig. 18.17 Copyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display

Micro-RNA processing and modes of action Immediately after transcription, pri-miRNAs are recognized by Drosha which crops out pre-miRNA stem loops from larger RNA. Pre-miRNAs undergo active transport from nucleus to cytoplasm where they are recognized by Dicer. Dicer reduces the pre-miRNA into a short-lived miRNA:miRNA duplex which is released and picked up by RISC. Figure 18.18 Fig. 18.18a Copyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display

Two modes of RNA interference If miRNA and its target mRNA contain perfectly complementary sequences, miRISC cleaves the mRNA. RNase rapidly degrades cleavage product. If miRNA and its target mRNA have only partial complementarity, cleavage does not occur. miRISC remains bound to its target and represses its movement across ribosomes. Figure 18.8b Copyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display

Protein modifications after translation provide a final level of control over gene function. Ubiquitination targets proteins for degradation. Ubiquitin – small, highly conserved protein Covalently attaches to other proteins Ubiquitinized proteins are marked for degradation by proteosomes Figure 18.19 Copyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Fig. 18.19 a

Sex specific traits in Drosophila Sex determination in Drosophila A comprehensive example of gene regulation Sex specific traits in Drosophila Figure 18.20 Fig. 18.20 Copyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display

Table 18.2 Copyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display

Table 18.3 Copyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display

The X:A ratio regulates expression of the Sex lethal (sxl) gene. Key factors of sex determination: Helix-loop-helix proteins encoded by genes on the autosomes Denominator elements Helix-loop-helix proteins encoded by genes on the X chromosome Numerator elements – monitor the X:A ratio through formation of homodimers or heterodimers sisterless-A and sisterless-B Copyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display

Figure 18.21 Fig. 18.21 Copyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display

Hypothesis to explain why flies with more numerator homodimers transcribe Sxl early in development Numerator subunit homodimers may function as transcription factors that turn on Sxl. Females Some numerator subunits remain unbound by denominator elements. Free numerator elements act as transcription factors at Pe promoter early in development. Males Carry half as many X-encoded numerator subunits All numerator proteins are bound by abundant denominator elements. Pe promoter is not turned on. The Sxl protein expressed early in development in females regulates its own later expression through RNA splicing. Sxl protein produced early in development catalyzes the synthesis of more of itself through RNA splicing of the PL transcript. No Sxl transcript in early development results in an unproductive transcript in later development from the PL promoter with a stop codon near the beginning of the transcript. Copyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display

Effects of Sxl mutations Recessive Sxl mutations making gene nonfunctional Females – lethal Absence of Sxl allows expression of dosage compensation genes on X chromosome. Increase transcription of X-linked genes is lethal. Males No Sxl expression No affect on phenotype Dominant Sxl mutations that allow expression even in XY embryos Females No affect because they normally produce the protein Repression of genes used in dosage compensation No hypertranscription of X-linked genes and do not have enough X-linked gene product to survive Copyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display

Sxl triggers a cascade of splicing. Sxl influences splicing of RNAs in other genes. e.g., transformer (tra) Presence of Sxl produces functional protein. Absence of Sxl results in nonfunctional protein. Figure 18.22 a Fig. 18.22 a Copyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display

Cascade of splicing continues e.g., doublesex (dsx) Tra protein synthesized in females along with Tra2 protein (produced in males and females) influences splicing of dsx. Females - Produces female specific Dsx-F protein Males – No Tra protein and splicing of Dsx produces Dsx-M protein Figure 18.22 b Fig. 18.22 b Copyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display

Dsx-F and Dsx-M are transcription factors that determine somatic sexual characteristics. Figure 18.23 Alternative forms of Dsx bind to YP1 enhancer, but have opposite effects of expression on YP1 gene. Dsx-F is a transcriptional activator. Dsx-M is a transcriptional repressor. Copyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Fig. 18.23

Tra and Tra-2 proteins also help regulate the expression of Fruitless. Figure 18.24 Primary fru mRNA transcript made in both sexes Presence of tra protein in females causes alternative splicing encoding fru-F. Absence of tra protein in males produces fru-M. Fig. 18.24 Copyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display