Biol/Chem 473 Schulze lecture 5: Eukaryotic gene regulation: Early Drosophila development

Maternal genes Required before the embryo starts transcribing its own genes Mother expresses these genes during oogenesis (mum packs a lunch…) Localization of maternal gene products is the first step in the cascade…

Gap genes The gap genes are expressed by the zygote (activated in the syncytial blastoderm) Their expression patterns are regulated by the maternal gene products, and they regulate each other They divide the embryo into broad, unique domains containing different combinations of transcription factors

Pair-rule genes Pair-rule genes are expressed just before cellularization in patterns of 7 stripes (half the number of segments in a wild type larva) Some pair-rule genes respond to patterns already laid down by the maternal and gap genes, while others are regulated by other pair-rule genes Pair rule gene expression is very dynamic! (pretty pictures)

Segment polarity genes Segment polarity genes are expressed at the onset of gastrulation They are expressed in 14 segment bands, refining the patterns of all the previous genes in the hierarchy. Maternal, gap and pair- rule gene expression begins to fade away

Stripe boundaries are precisely defined

Control regions upstream of pair-rule genes are complex

ASSIGNED PAPER: Transcriptional regulation of a pair-rule stripe in Drosophila Small, S. et al., Genes & Development (1991) 5:

Main players Even-skipped (eve) is a homeo-box containing transcription factor. Hunchback (hb) has multiple Zn fingers. Bicoid (bic) is a homeo-box containing transcription factor. Kruppel (Kr) contains a Zn finger. Giant (gt) contains a Leucine zipper.

Wild type expression patterns of putative regulators of eve stripe 2 even-skipped protein is red hunchback protein is green overlap of expression is yellow

even-skipped protein is red Kruppel protein is green overlap of expression is yellow Wild type expression patterns of putative regulators of eve stripe 2

even-skipped protein is green giant protein is red Overlap of expression is yellow Wild type expression patterns of putative regulators of eve stripe 2

High levels of gt High levels of Kr

Wild type expression patterns of putative regulators of eve stripe 2 What predictions arise from this picture? High levels of gt High levels of Kr

eve-lacZ gene fusion This is a transgene: gets injected into the embryo (“P transformed embryos”), integrates into the genome and is expressed stably along with all the other genes. BUT: this transgene will express lacZ in an even-skipped-stripe 2 dependent manner! OUCH! Posterior end of embryo (where germ line will form)

eve-lacZ gene fusion This is a transgene: gets injected into the embryo (“P transformed embryos”), integrates into the genome and is expressed stably along with all the other genes. BUT: this transgene will express lacZ in an even-skipped-stripe 2 dependent manner!

Questions to ask about in vivo expression patterns How do we know the order in which patterning genes act in development? How were potential trans-acting regulators of stripe 2 identified? What would happen to the domain of expression of eve stripe 2 in giant or Kruppel mutant embryos? Do these in vivo expression patterns indicate direct interaction between Kr and gt and the eve stripe 2 regulatory region?

Are these interactions direct? Control lanes Sequencing lanes (Ye Olde Fashionde Maxim Gilbert) Footprints represent regions in the eve upstream regulatory region that are protected by the bicoid protein. What does this imply?

Questions to ask about DNA footprinting analysis Do the sites show differing affinity for their respective trans-acting factors? Do the sites for different trans-acting factors overlap? What could be the explanation for differential affinity?

Regulator binding sites in the eve stripe 2 regulatory region eve skipped promoter What does the tight linkage of these sites suggest? (Hint: compare bcd and Kr binding site sequences)

Regulator binding sites in the eve stripe 2 regulatory region eve skipped promoter Bicoid binding sites are solid circles hunchback binding sites are shaded circles Kruppel binding sites are hatched boxes giant binding sites open boxes

Using reporter genes to dissect regulatory sequences

CAT in this paper Hsp70 TATA in this paper bcd OR hb OR Kr OR gt protein coding sequence ACT5 promoter Schneider cells (cultured fly cells) in this paper

CAT assays

Recipe for CAT assay CAT = chloramphenicol acetyltransferase Perform assay in cell system (transform/transfect cells etc) Extract proteins Mix with “hot” (radioactively labeled) chloramphenicol Add substrate acetyl coenzyme A If CAT has been produced by your extract, it will transfer acetyl groups from Acetyl coenzyme A to chloramphenicol Thin layer chromatography to separate out the products Autoradiography to measure amount of acetylated, radioactive chloramphenicol (which is proportional to how much CAT was made which is a reflection of how well the CAT reporter was activated).

Organic Chickenwire figure

CAT assay results: Table 2

Questions to ask about CAT assays What is Kr 9 ? What does this experiment control for? How does figure 6 support the implication of this control?

Discussion How strict is this developmental hierarchy in Drosophila development? (Example?) What contributes to sharpening the borders of gene expression? (Example?) How do stripe initiation elements function autonomously of each other?

Control regions upstream of pair-rule genes are complex General principle: regulatory regions of eukaryotic genes are complex; the promoter of any given gene has to integrate a large amount of combinatorial inputs that will define its activity depending on the context of the cell in which the gene resides. (Are bicoid levels high or low? What about Kruppel? Etc. )

Exam stats: Prody Average= 40/60

Exam stats: Schulze Mean = 25/40 Median = 26/40