1. Developmental Genetics  Overview of gene structure  Mechanisms of gene regulation  Drosophila: Genetics model for Developmental Biology

2. What is a gene? CDS Regulatory Promoter Core Promoter -10 -35 +1 3 UTR AAAAAA AUG UAA

3. Developmental Genetics  Differentiation of cells requires regulation of specific genes.  Timing of Gene Regulation can determine when a tissue develops or when cells undergo Apoptosis.  How can we regulate gene expression?

4. Chromatin Remodeling

5. Histone Code: Modification of Histone tails.  Acetylation, methylation, and phosphorylation.  Charged residues in tail.

6. Histone Methylation  Methylation of residues on H3 tail  Methylation of H3K4(meH3K4) + high acetylation of H3/H4 tails)= active transcription.  meH3K9+low acetylation of H3/H4= highly repressed  meH3K9, meH3K27, meH4K20 =highly repressed. 

7. Example: Hox genes  Hox genes are responsible for determining the fate of segments along the anterior-posterior axis.  Trimethylation of H3K27 represses Hox genes.  Demethylase for me3H3K27 is recruited in differentiated cells to open up the promoters for Hox genes.

8. Epigenetic Memory  Trithorax- keeps genes active.  Counteract Polycombs  Modify nucleosomes  Keep H3K4 trimethylated  Polycomb- keeps genes inactivated.  H3K27 methylase  Bind meH3 tails

9. Methylation of DNA  Once gene is turned on and needs to stay on  CpG-usually associated with repression.   -globin promoter.  Methylation status of promoter can change during development.  Ex. Embryonic hemoglobin (  -globin)  fetal hemoglobin (  -globin)

10. Methylation and Transcriptional repression  Can block transcription factor binding  Recruit Histone methylases and acetylases  Ex. MeCP2  Dnmt3 and Dnmt1 result in the heritability of methylated DNA regions in progeny cells.  Methylation can inactivate entire chromosomes.

11. Transcriptional regulation  cis factors  trans factors

12. Promoters/enhancers  Core promoter  RNA Pol II binds to this region.  Directly upstream of transcription start site.  Regulatory Promoter  Region upstream of core promoter  Contains enhancers/regulatory binding sites  Highly modular.

13. Promoter fusions  Identifying new enhancers  Reporter genes:  -gal and GFP

14. Enhancer Modulation  Multiple transcription factors can bind to a single Promoter region.  Pax6 enhancer region- differential transcription factor binding for different tissue types. Pax6 1 Core promoter 123 4

15. Pay it forward  Pax6 is a transcriptional regulator.  Pax6 works with Sox2 and L -Maf to regulate the crystallin gene.  Pax6 also regulates Pancreas related genes  Insulin, glucagon, and somatostatin  Pax6 regulates itself!

16. Transcription Factors  Core Promoter  TFIID/TBP stabilized by TFIIA  TFIIH binds, RNA Pol II with TFIIE/TFIIF binds subsequently.  TFIIH phosphroylates RNA Pol II CTD.  Regulatory Promoter  Wide array of factors bind to enhancer regions. (Pax6 regulation).

17. Transcription Factor Domains.  DNA binding domain  Transactivating Domain  Protein-Protein Interaction domain. MITF Transcription Factor- Ear development And Pigment production

18. “Pioneer” transcription factors.  Pioneer transcription factors can penetrate repressed chromatin.  FoxA1  Pax7  Pbx

19. Gene Silencing  Neural restrictive silencer element (NRSE) found represses neural genes in non-neural cells. NRSF binds to this element.  L1 is critical for brain development.  Transgene reporter with NRSE= neural tissue expression only  Transgene reporter without NRSE= all tissues.

20. Regulation of mRNA  mRNA must undergo splicing for protein to be made.  Regulation of mRNA  Determination of which transcripts get translated  Splicing combinations to make new protein.

21. Splicing: different gene products from one gene.  Bcl-x transcript.  Bcl-x L and Bcl-x S  Tropomysin  Leads to multiple proteins that differ in function and location.  Splicing enhancers-found in specific tissues.  Splicing can silence as well- splice excludes an exon from mRNA.

22. Translational Regulation  Stored oocyte mRNAs- selective inhibition of translation.  Genes turned on quickly post-fertilization.  Relies on the recruitment of proteins involved in making a polyA tail.  Drosophila Bicoid gene product.

23. miRNA  miRNA ancient form of regulation.  Protection against viral pathogens  Quick regulation and turnover of mRNA.

24. Drosophila Development: Genetics at work!

25. Development of Drosophila

26. Anterior-Posterior Polarity  Starts during oogenesis  Gurken mRNA transferred to oocyte.  Signal sent back to oocyte post Gurken/Torpedo interaction.  Par-1 mediated organization of microtubules to posterior side of oocyte. Minus end (posterior) and plus end (anterior).

27. Anterior-Posterior Polarity

28. Dorsal-Ventral Patterning

29. Dorsal Gradient