Chapter 4- Genes and development

1. Chapter 4- Genes and development _______________- expression of traits ________________-transmission of traits Embryology Embryology Embryology 1920s Hostility Genetics Genetics 1970s Genetics 1870s- What controls inheritance- ___________ or _______________?

2. Huge conceptual problems(Early 1900’s) • Can identical chromosomes really result in distinct cell types?- defined as _______________________________ • Do genes ___________ embryogenesis? • How can ___________________ affect sex in reptiles?

3. 1. Can identical chromosomes really result in distinct cell types?- defined as genome equivalence Evidence for • _____________________observed (transformation of one differentiated cell type to another, e.g. iris cells become lens cells (1950s) • Amphibians can be cloned from intestinal _________ cells (1960s) -nuclear transfer techniques developed • Sheep cloned from ________ tissue • (1997)- “Dolly”, then cows and mice (1998) Fig. 4.8

4. Differential gene expression Three tenets • The DNA of all ______________ cells are identical in a given __________ • All ____________ genes can be expressed • Only a ____________ of genes are expressed in a given _______

5. Techniques to know to understand differential gene expression • ___________- Detect DNA • _____________- Detect RNA • ___________ Detect protein • ____________________ Detect DNA or RNA • __________________ to amplify copies of DNA

6. 1. Northern blot 3. _______ (blot) onto nylon membrane 1. ______ RNA 2. ______ on gel 4. _______ membrane with radiolabled RNA/DNA A developmental Northernblot Fig. 4-14

7. 2. Southern blot Similar to __________ blot, except 1. Must chop up DNA to smaller pieces (restriction enzymes or general shearing with strong acid) Note- ______________________ are proteins that recognize specific double-stranded DNA sequences and cleave the DNA 3. Western • Load isolated protein onto a ____. • _________ onto nylon membrane • _______ with _________ to specific proteins (these ________ • can be conjugated to fluorescent molecule or an enzyme for • detection)

8. 4. ____________________- shows exactly where protein is expressed in organism • Mount thin slices of _______onto slide. • Incubate with _________ probe Fig. 4.15 Whole-mount in situ hybridization is also possible a. Add Dioxygenin- labeled DNA b. Add antibodies (alkaline phosphatase linked) to Dioxygenin c. Add alkaline phosphatase substrate Fig. 4.16

9. 5. Polymerase Chain Reaction (PCR) One copy Fig. 4.17 many copies

10. Genome-wide analysis- yeast

11. ______________ organisms- a great way to study gene function • Getting the DNA (a gene) into a cell- • ______________ • __________________ (mix DNA with cells) • Retrovirus _____________ (infect a cell) • P element in Drosophila- a transposable element that allows a gene to be inserted into specific positions in the fly genome

12. The _______________ mouse 1. Isolate _________________ cells from trophoblast, expand and transfect 2. Introduce modified ES cells into new _____________ 3. Place modified trophoblast into uterus, then cross the ___________ normal mice 4. Cross two _________________ to generate a homozygote Fig. 4.19

13. Gene targeting (______________) 3. Generate heterozygote, then ______________ mice 1. Isolate _________________ cells from trophoblast, expand and _________ with __________________ gene 2. _________and expand ES cells with integrated transgene

14. Other techniques • _______________ to inhibit function- antisense RNA produced binds to mRNA and cause degradation • __________________________- short RNA fragments result in degradation of specific mRNA Example- antisense Kruppel affects fly development Fig. 4.23

15. From fertilized _______ ES cell lines established – ___ lines available Human ES cell transplantation therapy- ethics ES cells from developing embryo coaxed into _______ cell types Fig. 4.22

16. Chapter 5- Differential Gene Expression DNA wrapped around histones

17. Overview of eukaryotic transcription/translation Chromosome- 1.5 x 108 base pairs containing about 3000 genes UAA UAA mRNA Also see figs. 5.2 and 5.3 0.4% of a chromosome, containing 10 genes 1. Exon F B C D E TAA 2. Intron ATG DNA 5’ 3’ 3’ 5’ +1 AATAAA Transcription 3. _____________ AUG Pre-mRNA Promoter 4._________ RNA Processing Cap PolyA 5. ______ 6. ____________ E A B C D F 7mG AAAAAA….. AUG Translation 7. ________________ Protein COO- NH2 Know this terminology

18. Chromatin and transcription A. Levels of organization • DNA must be compact- each mammalian • cell has _______________ of DNA a._______________- 700 nm b. ______________- 200 nm c. ______________-30 nm d. ________________ -10 nm- histones + DNA

20. Ac Ac Ac Ac Histone tails H4 H4 H3 H3 H2A H2A H2B H2B Ac Ac Ac Ac Peptide-CH-CH2-CH2-CH2-CH2-NH3 Peptide-CH-CH2-CH2-CH2-CH2-NH-C-CH3 O Acetylation of lysine • Chromatin and transcription (Chapter 13) (continued) • B. ________________ • Large amount of ________ residues, positive charge • can interact with DNA • five types of histones • ________________________ • H1 in _______ regions (8-14 bp) • H2A, H2B, H3 and H4 form _________ around ________ of DNA • _______________ of histone tails ________________ as gene activity increases

21. How do histones get acetylated? By ________________________________) -Three histone acetyltransferases • Two type of HATs • ________- in nucleus, involved in gene regulation e.g. p55 and Gcn5 • ________ - in cytoplasm, acetylates newly made histones • Note- CBP (a coactivator) and TAFII250 (a TFIID subunit)and • P/CAF also display HAT activity

22. How do histones get acetylated? By Histone deacetylases (HDACs) - Model for histone ______________________ in transcriptional repression G. Histone deacetylation ____________________ bind to _________________________ and to histone deacetylase to repress transcription Examples of co-repressors - Sin3A and Sin3B (mammals) - NcoR/SMRT (mammals)

23. Transcription inititation • Regulatory elements- RNA Pol II • 1. __________________ • TATA box- initiate __________________, fixed position • ________________ at -70 • _____________ at -110 • _______________ promoters instead of TATA • “________________” genes (constitutively active in most cells) • some “___________ “genes (tissue specific expression) • 2. _____________- orientation /position independent • affects level of transcription, not determine _______ status • contains DNA sequences which _______ transcription factors • A major determinant of ____________ gene transcription

24. Activating transcription RNA Polymerase ________________ now binds and initiates transcription _______ binds TATA box via ______, followed by ordered binding of other factors Fig. 5.4

25. Function of __________ • 1. interaction with core ___________ • TAFs extend area of protection to +35 bp • Question- Which other TAFs interact with DNA? TFIID = TBP + several TAFs 2. Enable TBP to bind _________________ promoters. 3. Interact with upstream _____________ 4. Chromatin remodeling- Example: TAF250 is histone acetyl transferase (HAT)

26. How do we achieve differential transcription?

27. Transcription activators in eukaryotes • ___________________ (DNA binding proteins, transacting factors) • 1. ________________ proteins • 2. bind to specific DNA sequences on promoter or enhancer • 3. modify transcription of gene by altering _________________ • loading Three domains (domain- a cluster of amino acids that carry out specific functions) • 1. _______________ domains- • Zinc-containing (e.g. zinc finger) • homeodomain- 60 AA • b-barrels • b-ZIP and bHLH motifs 3. ________________________ domain e.g, Leucine zipper • 2. _______________________ domains • acidic domains • Glutamine-rich domians • Proline-rich domains Trans-acting factor NH3 COOH Protein-protein interaction domain Activation domain (30-100 amino acids) DNA binding domain

28. An example of a developmental transcription factor • MITF is a transcription factor that activates pigment genes CBP __________________ domain ___________________ interaction domain ___________________ domain DNA Fig. 5.8

29. Muscle-spec. promoter B-Gal Eye-specific promoter GFP How do we determine where a given enhancer/promoter is active? Answer- Fuse enhancer/promoter to _______________ (B-gal) or __________________ (GFP) gene, then introduce the fusion gene into the organism Fig. 5.7

30. Big themes in regulation HNF4 C/EBP HNF1a HNF3 Liver-specific genes 1. Hepatic gene regulation occurs primarily at the level of ___________________________ 2. Tissue-specific expression is due to __________________ of transcription factors HNF3a

31. Transcription factors in early liver development Anterior-posterior axis HNF3 Primitive streak Hepatic Determination HNF3 Endoderm differentiation HNF3, HNF4 Onset of liver gene expression Foregut endoderm migration into mesenchyme HNF1, c-jun Amplification of liver gene expression Organ formation C/EBP Liver

32. Other forms of gene regulation • What activates expression of the activators? • The Pax6 gene has 4 distinct ______________, each utilized in four distinct_________ to drive Pax6 transcription Pancreas Lens Neural tube Retina Fig. 5.15 2. ____________- sequences that block ________________ Albumin gene promoter Albumin expression silenced by inhibitor until birth Fig. 5.16 L1 promoter is silenced in all tissues except neuronal due to silencer element NRSE Fig. 5.17 Silencer elements are rare! Delete NRSE sequences

33. Other forms of gene regulation 3. _________________- Human b-globin gene cluster • Five erythroid-specific genes • Arranged in _____________________ • LCR is upstream cluster of 5 (actually 7) _______ • Each HS site binds numerous _______

34. Proposed LCR functions • Open ______________ • prevent variegated ______________________ • Affects timing of _________________ • Keeps promoters ___________-free • Change subcellular localization of b locus • LCR transcription affects rest of locus expression • Recruit ________

35. Other forms of gene regulation 4. ___________-a major method of transcriptional regulation in vertebrates Model- Methylation represents a biochemical specialization of large genomes that participate in allele-specific expression, whereas differentiation does not depend on covalent modification. Globin gene cluster ________ Fig. 5.20

36. 4. Methylation-continued Interesting methylation facts • 3% of Cs are methylated in mammals, • 90% of these at CG • As methylation increases, transcription decreases • GC-rich regions are preferentially found in 5’ regions • mice lacking methyl transferase die during embryogenesis • Model- methylation groups interfere with factor • binding on DNA • Importance of methylation question due to lack of • methylation in Drosophila • CG sequence occurs at only 10% of expected frequency • 70-80% of these are methylated • patterns reset during gamete formation • methylation status of a panel of tissue-specific genes could not • be correlated with expression in tissues of fetal and newborn • mice • Methylation deficient mice- observe biallelic expression of imprinted genes.

37. Differential expression of two _______alleles Only occurs in ________(placental, nonmarsupial) mammals Not in other ___________ Of 20-some identified genes Many involved in _____________ Igf2, IgF2r, H19, Grb1 ________________ Prader-Willi syndrome PS) Angelman syndromes (AS) Peg1/Mest 5. Genomic _______________

38. A potential mechanism of genomic imprinting female male • A single enhancer drives either the Igf2 or the H19 gene, but ______. • ____________ binding prevents enhancer from acting on Igf2 gene. • CTCF cannot bind if region is __________; hence Igf2 is expressed.

39. 6. X chromosome ___________- A. Introduction- • ____________ first described in females in 1949 • _____ syndrome (45,X) are Barr body negative; ________syndrome (47, XXY) are Barr body positive • ___hypothesis- one of the two X chromosomes in female is inactivated; all but one is inactivated if multiple X chromosomes - referred to as “________________________”

40. Other forms of gene regulation X chromosome inactivation 7. Dosage compensation • Introduction- • X-chromosome inactivation occurs at _______ of embyrogenesis • Inactivation process is _______ • Inactivation state __________ throughout life • A few genes remain active in the inactive X chromosome, including XIST at Xq13

41. Dosage compensation comparisons 1X 1X 2X 2X 2-fold ________ in males 2-fold ________ in females Stably inactivate ___ X chromosome

42. 13 p 12 11 12 13 14 q 21 24 mouse autosome X-inactivation- observations • Xist is necessary and sufficient for X inactivation (using 450kb YAC) • insert Xist transgene on autosome results in inactivated autosome

43. Inactivated X chromosome Xist RNA

44. Xist Gene Random inactivation Blocking factors Preferential inactivation Prevent inactivation delete X-inactivation Mechanism in mammals • If mutate Xist promoter- preferential X inactivation on chromosome with mutation • possibly due to failure to compete with blocking factor • Delete Xist exons 1-5- mutant chromosome chosen but not inactivated

45. Other forms of gene regulation 8. Differential RNA ____________ a. RNA selection-only certain RNAs are exported to _________ b. Differential RNA ____________- Different spliced forms of a RNA

46. Other forms of gene regulation 9. RNA ____________ a. mRNA longevity- minutes to _____ Lin-14 RNA Lin-4 RNA • b. Selective_______ of translation- • e.g. the C. Elegans lin-4 RNA binds lin-14 mRNA to ________ translation Lin-14 mRNA Untranslated region Fig. 5.32