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Unformatted text preview: Bio 106 Fall 2009 Professor Owen 11/1/2009 Action of Enhancers Gene Expression and Development Without an enhancer, the gene is transcribed a a low basal level. Addition of the enhancer stimulates transcription. The enhancer is active when upstream or downstream, close or far away!
Cooper 7.21 Genes
TATA-binding protein (TBP TBP) or activators or repressors 5’ enhancer RNA pol II Transcription Factors for RNA Polymerase II (TF TF) TF TATA TBP start site of transcription gene 3’ Gene Regulation Occurs at a Distance (Eucaryotes) ECB3 8-10 DNA contains not only the blueprint but also coordination information -- how much, when, where in 3in 3-D space. Review: Nucleosomes Regulatory Proteins ECB3 8-12 combinatorial control ECB3 8-11 Combinations of specific regulatory proteins work together to control the proper timing and place of gene transcription. Cells-23: Regulation of Gene Expression 1 Bio 106 Fall 2009 Professor Owen 11/1/2009 Enhancers Gene Activation Occurs at a Distance Cooper 7.23 An example: the immunoglobulin protein enhancer example: the immunoglobulin protein enhancer spans about 200 bases and contains nine binding regions. Enhancers: have roles in development; respond to hormones and growth factors. Regulatory proteins bind to the enhancer bi th site and “tickles” the promoter. They are like the volume control on an MP3 player. Compared to the basal level, it can be activated ↑ or repressed ↓ by activator or repressor proteins.
ECB2 8-13 Enhancers Enhancers – Two Types
• Transcriptional Activators
– Stimulate transcription – Bind DNA and RNA polymerase complex (mediator) Drosophila melanogaster A Developmental Model • Transcriptional Repressors
– Inhibit or slow transcription – Bind to DNA – can block binding of activator or inhibit transcription Who has worked with Drosophila (fruit Drosophila flies)? Drosophila Embryo Organization A. I have B. I have NOT C. What’s a Drosophila? Drosophila? D. What’s a fruit fly? Cells-23: Regulation of Gene Expression 2 Bio 106 Fall 2009 Professor Owen 11/1/2009 Drosophila – From Larva to Adult
Mouthparts Antennae, eyes Legs Wings As every cell in the fruit fly contains identical DNA, what makes the body form different parts? Genitalia The divisions in the larva maintain expression patterns into the adult A. migration of different mRNAs B. migration of different proteins diff C. selective expression of different genes D. maternal expression of different hormones Regulatory Proteins Enhancer proteins interact with ______ to increase or decrease ______ Many enhancer proteins contribute to gene expression – different combinations can turn on the same gene at different times/places/amounts. ECB3 8-12 A. DNA polymerase; DNA replication B. TATA box; peptide bond formation box; peptide bond formation C. TFs & TBP; translation D. TFs, TBP and RNA polymerase; transcription E. ribosomes; translation Drosophila Larva
A1 T3 T2 T1 A2 A5 A6 A7 A11 A8 A9 A10 It’s All About eve … eve A3 A4 Ventral Even skipped (“eve”; red) and odd-skipped (green) expression patterns.
This picture is at cleavage cycle 14. Anterior Posterior Cells-23: Regulation of Gene Expression 3 Bio 106 Fall 2009 Professor Owen 11/1/2009 Drosophila – Segmentation
Parasegments Nonuniform Nonuniform Distribution of Four Gene Regulatory Proteins
Each stripe of eve is defined by a combination of maternal eve factors, transcription activators, and repressors, These are patterned in the egg either prior to fertilization or prior to cellularization. cellularization. 14 12 1213 34 5 6 7 8 9 10 11 Expression of eve (even-skipped) and ftz evenftz (fushi tarazu; “odd-skipped”) genes. odd- ECB2 8-17 Distribution of bicoid mRNA and Protein bicoid
The mRNA for maternal genes such as bicoid can be unequally bicoid distributed. Here it is localized in the anterior end (note dark staining). Th bi The bicoid protein distribution is pro di similar. Thus, the translation of the mRNA and diffusion of the protein produces an anterioanterioposterior gradient in the embryo.
Wolpert (1998) Fig. 5.5 Drosophila and the French Flag
All cells have potential to be red, white or blue Protein (morphogen) gradient gradient Thresholds Their fate depends upon their position along the protein gradient eve eve
The gene even-skipped (eve) is expressed in 7 bands or evenstripes corresponding to 7 of Drosophila's 14 segments Drosophila's (skipping the even-numbered ones). even- Important Genes in Drosophila Development
1. Maternal genes bicoid 2. Gap genes Krüppel, giant, and giant and hunchback hunchback 3. Pair-rule genes Paireveneven-skipped (eve) and fushi tarazu (ftz) 4. Polarity of segment genes Each stripe of eve is defined by a combination of eve maternal factors, transcription activators, repressors and specific enhancers. Cells-23: Regulation of Gene Expression 4 Bio 106 Fall 2009 Professor Owen 11/1/2009 Specification of eve in Stripe 2 eve
bicoid and hunchback hunchback proteins activate eve eve in a broad domain, while the anterior the anterior and posterior borders are formed through repression by giant and Krüppel proteins.
Wolpert (1998) Fig. 5.22 Enhancers can be through of as: A. DNA B. a volume control volume control C. a stop sign D. a megaphone E. RNA polymerase eve
Each stripe has a stripe-specific enhancer upstream of the structural gene. Enhancers
For example, in the case of eve stripe 2, a minimal eve stripe 2 enhancer has been identified between -1.5 and -1.0 kb upstream from the eve start site. eve ECB3 8-10 Reporter Gene
Can you design an experiment to determine if the fragment of DNA you have is the upstream regulatory region of eve stripe 2? eve
Hint: you can use a reporter gene system.
Regulatory gene eve Regulatory Genes
The regulatory region for stripe 2 was removed and inserted upstream of the lacZ th gene. When reintroduced into the Drosophila genome Drosophila embryo, the resulting embryo expressed -galactosidase precisely in the position of the 2nd of the seven eve stripes eve (blue band). antibody stain
to Eve protein ECB3 8-15 A regulatory region can be inserted ahead (upstream) of the E. coli lacZ E. gene, which encodes for the enzyme -galactosidase. The enzyme gives a blue product with the substrate X-gal Xonly if it is expressed (it reports on expression). Cells-23: Regulation of Gene Expression 5 Bio 106 Fall 2009 Professor Owen 11/1/2009 eve Regulatory Genes
The stripe pattern of eve transcription is governed by eve four regional specific transcriptional activators and repressors. Drosophila – Segmentation
Expression of eve and ftz is regulated by different combinations and concentrations of TFs in each parasegment Promoters are very long 500 bp blocks Each responds to different set of TFs (or TF concentrations) 1
bicoid giant 2 3 4
-1550 krüppel eve Induction
-1070 eve promoter Combinatorial control ECB3 8-16 hunchback Repression Bicoid Bicoid Regulates eve Stripe 2 eve
Mutations in the Bicoid binding sites of eve eve stripe stripe 2 prevent Bicoid from binding to the site. This mutation causes a decrease in eve stripe eve 2 expression. Bicoid is an activator of eve eve stripe 2. 2. Stanojevic, Small and Levine. Science 1991. Giant Regulates eve eve
Mutations in the Giant binding sites that prevent Giant from binding cause an expansion of eve an expansion of eve stripe stripe 2. Giant is a repressor of eve eve stripe 2. 2. If If a mutation in the gene for Giant caused eve stripe 2 to shrink instead of grow, what would one call Giant? A. B. C. D. E. an enhancer a repressor a TBP an RNA pol Stupid Cells-23: Regulation of Gene Expression 6 Bio 106 Fall 2009 Professor Owen 11/1/2009 Bicoid, Giant, Krüppel Krü
Enhancers should really be called … A. B. C. D.
Bicoid activates eve stripe 2 protein expression stripe Giant represses eve stripe 2 protein expression Krü Krüppel represses eve stripe 2 protein expression
ECB2 8-17 repressors light switches activators/repressors activators Cells-23: Regulation of Gene Expression 7 ...
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