Gene Regulation in Eukaryotes

Outline of Chapter 17 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

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 17.1 a Fig. 17.1 a

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 β-galactosidase (Lac Z) or green fluorescent protein (GFP) Reporter constructs can help identify promoters and enhancers by using in vitro mutagenesis to systematically alter the presumptive regulatory region

Regulatory elements that map far from a gene are trans-acting DNA sequences because they encode transcription factors 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 in vitro to cis-acting elements Figure 17.1 b Fig. 17.1 b

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, 5.8S, and 18S by processing Figure 17.2 a Fig. 17.2 a

RNA polymerase III transcribes tRNAs and other small RNAs (5S rRNA, snRNAs) Figure 17.2 b Fig. 17.2 b

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 17.2 c Fig. 17.2 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 Figure 17.2 c

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 17.3 Fig. 17.3

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 17.4 a Fig. 17.4 a

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

Transcriptional activators bind to specific enhancers at specific times to increase transcriptional levels Figure 17.5 a Fig. 17.5 a

Examples of common transcription factors zinc-finger proteins and helix-loop-helix proteins bind to the DNA binding domains of enhancer elements Figure 17.5 b Fig. 17.5 b

Some proteins affect transcription with out binding to DNA Coactivator – binds to and affects activator protein which binds to DNA Enhancerosome – multimeric complex of proteins Activators Coactivators Repressors Corepressors

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 17.6 Fig. 17.6

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 Fig. 17.7 a Figure 17.7 a

Leucine zipper – a common activator protein with dimerization domains Figure 17.7 b Fig. 17.7 b

Repressors diminish transcriptional activity Figure 17.8 a.b Fig. 17.8

Repressors Reduction of transcriptional activation but do 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

Transcription factors may act as activators or repressors or have no affect Action of transcription factor depends on Cell type Gene it is regulating

Specificity of transcription factor can be altered by other molecules in 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 17.9 Fig. 17.9

Myc polypeptide has an activation domain Myc-Max system is a regulatory mechanism for switching between activation and repression Figure 17.10 Myc polypeptide has an activation domain Max polypeptide does not have an activation domain Fig. 17.10

Myc-Max system is a regulatory mechanism for switching between activation and repression As soon as a cell expresses the myc gene, the Max-Max homodimers convert to Myc-Max heterodimers that bind to the enhancers Induction of genes required for cell proliferation Figure 17.10 Fig. 17.10

Gene repression results only when the Max polypeptide is made in the cell max gene Fig. 17.10 b Figure 17 Figure 17.10 b

Gene activation occurs when both Myc and Max are made in cell Figure 17.10 Fig. 17.10

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 17.12 Fig. 17.12 a

Proof that cis-acting factor such as LCR is needed for activation of b-globin gene Figure 17.12 b Fig. 17.12 b

One mechanism of activation that brings LCR into contact with distant globin genes may be DNA looping Figure 17.12 c Fig. 17.12 c

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

Normal chromatin structure slows transcription /figure 17.13 a,b Fig. 17.13

Remodeling of chromatin mediates the activation of transcription Figure 17.13 c,d Fig. 17.13

Hypercondensation over chromatin domains causes transcriptional silencing. This is achieved by the methylation of cytosine residues Figure 17.14 Fig. 17.14

In mammals hypercondensation is often associated with methylation Figure 17.14 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. 17.14

Genomic imprinting results from chromosomal events that selectively silence genes inherited from one parent 1980s, in vitro fertilization experiments demonstrated pronuclei from two females could not produce a viable embryos

Figure 17.15 a Experiments with transmission of Ig f 2 deletion showed mice inheriting deletion from male were small. Mice inheriting deletion from female were normal. Figure 15.15 a

Figure 17.15 d H19 promoter is methylated during spermatogenesis and thus the H19 promoter is not available to the enhancer and is not expressed

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 Figure 17.15 c Fig. 15.15 c

RNA splicing helps regulate gene expression Figure 17.16 Fig. 17.16

Figure 17.16 b Fig. 17.16 b

Cellular enzymes slowly shorten the poly-A tail. mRNA then degrades. RNA stability provides a mechanism for controlling the amount of gene product Cellular enzymes slowly shorten the poly-A tail. mRNA then degrades. Length of poly-A tails of mRNAs affects the speed at which mRNAs are degraded after they leave the nucleus. Histone transcripts receive no poly-A tail mRNA quickly degrades after S phase of cell cycle

Specialized example of regulation through RNA stability Figure 17.17 Note also the untranslated sequences that help modulate their translation Fig. 17.17

mRNA editing can regulate the function of protein products – e. g mRNA editing can regulate the function of protein products – e.g., AMPA receptor gene in mammals Figure 17.18 Fig. 17.18

Protein modifications after translation provide a final level of control over gene function Ubiquitination targets proteins for degredation Ubiquitin – small, highly conserved protein. Covalently attaches to other proteins Ubiquitinized proteins are marked for degredation by proteosomes Figure 17.19 Fig. 17.19 a

Sex specific traits in Drosophila Sex determination in Drosophila A comprehensive example of gene regulation Figure 17.20 Sex specific traits in Drosophila Fig. 17.20

Table 17.2

Table 17.3

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

Figure 17.21 Fig. 17.21

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 a unproductive transcript in later development from the PL promoter with a stop codon near the beginning of the transcript

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

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 17.22 a Fig. 17.22

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 17.22 b Fig. 17.22

Dsx-F and Dsx-M are transcription factors that determine somatic sexual characteristics Figure 17.23 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 Fig. 17.23

Tra and Tra-2 proteins also help regulate the expression of Fruitless Figure 17.24 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. 17.24