1. Bio 127 - Section I Introduction to Developmental Biology Developmental Genetics Gilbert 9e – Chapter 2

2. This chapter is all about making different cells from equivalent DNA sequence.... 1. Every somatic cell has the complete genome 2. A small percentage of the genome is expressed by an individual cell type and a portion of what is expressed is unique to that cell type 3. Cell differentiation during development is the process by which cells select the DNA that makes them unique 4. Unused genes are not lost and the cell retains the potential to use them in the future

3. All non-sex cells have the same DNA – all of it – and are capable of reusing the parts they don’t use daily! Genomic Equivalency of all somatic cells was demonstrated by the cloning of Dolly from adult mammary epithelium. “somatic nuclear transfer” or reproductive cloning

4. Figure 2.2 The kitten “CC” sheep cats g. pigs rabbits rats mice dogs horses cows

5. Differential Gene Expression A small percentage of the genome is expressed by any given cell and a portion of what is expressed is unique to that cell

6. So, where are the points of differential control? 1. Differential gene transcription to RNA 2. Selective RNA processing in the nucleus 3. Selective mRNA translation in the cytosol 4. Differential peptide modifications

7. The steps in gene expression 12341234

8. Unlike the prokaryotes, our DNA is complexed 50:50 with protein in a very highly regulated structure called chromatin Control of transcription by histone modification

9. Heterochromatin is too tight packed to transcribe easily Euchromatin is accessible

10. 3 Stages of Transcription 1. Initiation 2. Elongation 3. Termination Acetylation promotes InitiationMethylation can go either way (lysine amino acid residues) People are figuring out the “histone code”

11. Differential Gene Transcription: Epigenetic Memory OK. So a cell differentiates to become a blood vessel smooth muscle cell...... How come all of its mitotic descendents don’t have to go through differentiation? Trithorax proteins bind to open nucleosomes and keep them open. Polycomb proteins methylate nucleosomes and then bind to them to keep them tight. These effects can then be directly passed through mitotic cell division to the offspring.

12. What mRNA sequence makes up the start codon? the stop codon? What amino acid do they produce? Anatomy of a Gene Control of Gene Transcription at the Promoter

13. Nucleotide Sequence Nomenclature Exon means sequence that exits the nucleus Intron means sequence that stays inside the nucleus Standard Formatting This shows the ‘sense’ strand, the DNA that matches the RNA. It is NOT the DNA strand that is used to template the RNA!

14. Differential Gene Transcription: Gilbert Terminology Promoter – Binding sites for TF II family transcription factors – Site of RNA Pol II recruitment, stabilization, activation – Made up TATAbox and CpG islands Enhancer – True transcriptional determinant – Binding sites for tissue-specific transcription factors – Recruit histone acetyltransferases to unwind DNA – Stabilize Transcription Initiation Complex

15. Differential Gene Transcription: The eukaryotic transcription pre-initiation complex The Transcription Initiation Complex forms on every gene that gets expressed. Its presence there is really determined by the tissue specific transcription factors that bind to enhancer cis-elements. The TF II proteins are commonly called General Transcription Factors.

16. TF-II proteins and RNA polymerase can only bind promoters positively identified by tissue specific transcription factors

17. Tissue-Specific Transcription Factors

18. How do Transcription Factors Function? 1.The primary feature is DNA binding domain sequence homology. - Small changes in sequence in this domain can significantly alter DNA binding site sequence. 2.The trans-acting domain. - Recruits acetyl- and/or methyl-transferases to loosen nucleosome - Stabilizes the TF-II pre-initiation complex 3. The protein-protein interaction domain - Dimerization, combinatorial functions, rate control

19. Three-dimensional model of the homodimeric transcription factor MITF (one protein in red, the other in blue) binding to a promoter element in DNA (white)

20. Differential Gene Transcription: Enhancer Modularity Enhancer sequence is the same in all cells A. Most enhancers have many tissue-specific TF binding sites – The combination of tissue-specific TFs present determines the rate of transcription in that cell type If A,B,C present than high transcription rate If A,B,Z present than low transcription rate If A,Y, Z present than zero transcription rate B. Some genes also have multiple enhancers – The combination of tissue-TFs present determines the presence of transcription in different cell types If A,B,C present than high transcription rate If X,Y,Z present than zero transcription rate

21. Example A. Modular Enhancers Pax-6, Sox2 and L-Maf are all required for crystallin expression Pax-6, Pbx1 and Pdx1 are all required for somatostatin expression

22. Example B. Multiple Enhancers in a Gene The pax-6 gene has four enhancers and is expressed exclusively in those four tissue types.

23. Pax-6 has a positive feedback loop A really important idea in cell differentiation is that there must be a molecular mechanism that keeps a cell differentiated. – The pax-6 gene has a Pax-6 site in its enhancer – When it is present the transcription rate is maximal – This mechanism is repeated in several cell types

24. Differential Gene Transcription: Coordinated Expression Put what you know about cells, enhancers and TFs together.... – What do you think the enhancers of genes for SkM actin and SkM myosin are like? – How about for SkM actin and SmM actin?

25. How Does a Cell Change Its Transcription: Silencers Zinc-finger NRSF binds to the NRSE and stops transcription

26. “Pioneer” Transcription Factors MyoD and E12 displace the inhibitor as long as Pbx gets there first.

27. DNA methylation can block transcription by preventing transcription factors from binding to the enhancer region

28. DNA Methylation of Globin Genes in Development

29. Transmitting DNA methylation to daughter cells

30. Modifying nucleosomes through methylated DNA DNA methylation can lead indirectly to histone methylation through recruitment of transferase activity.

31. Differential Gene Transcription: X-inactivation and Genomic Imprinting X-chromosome inactivation – Promoter methylation in one X-chromosome – Random between maternal and paternal – Achieves X-linked Dosage Compensation Genomic Imprinting – Re-distribution of methylation during gametogenesis – At least 80 genes in mammals – Only mom’s or only dad’s allele is expressed

32. Differential RNA Processing Two major ways differential RNA processing can effect development a. Selection and release of different sets of nRNA to the cytosol b. Splicing different mRNAs from the same nRNA using different exons

33. Differential RNA Processing

34. Selection and release of different sets of nRNA to the cytosol – More genes are transcribed in the nucleus than than are allowed to be mRNA in the cytosol – The unused nRNAs are degraded or used for non- coding RNA species in the nucleus

35. Cell-Specific RNA Processing Splicing different mRNAs from the same nRNA using different exons – Alternative splicing occurs in ~92% of human genes – “Splice sites” are formed from consensus sequences found at the 5’ and 3’ ends of introns – Different splicosome proteins made in different cells recognize different consensus sequences – The result is families of related proteins from the same gene in different cell types

36. Figure 2.26 Some examples of alternative RNA splicing (Part 1)

37. Figure 2.26 Some examples of alternative RNA splicing (Part 2)

38. Figure 2.27 Alternative RNA splicing to form a family of rat α-tropomyosin proteins

39. The Dscam gene of Drosophila can produce 38,016 different proteins by alternative nRNA splicing The proteome in most eukaryotes dwarfs the genome in complexity!

40. Dscam protein is specifically required to keep dendrites from the same neuron from adhering to each other Dscam complexity is essential to the establishment of the neural net by excluding self-synapses from forming

41. Splicing Enhancers and Recognition Factors - These work much like transcription enhancers and factors - Enhancers are RNA sequences that bind protein factors to promote or silence spliceosome activity at splice site - Many of these RNA sequences and trans-factors are cell type- specific, eg. muscle cells have specific sequences around all of their splice sites, thus make muscle-specific variants

42. Muscle hypertrophy through mispliced RNA Splice site mutations can be very deleterious, rarely can be advantageous

43. Control of Gene Expression at the Level of Translation A.Differential mRNA longevity can determine total protein expression B.Selective inhibition of mRNA translation can determine time of expression C.Selective destruction of active mRNA can determine cessation of expression D.Selective placement of mRNA can determine localization of expression E.Selective alteration of peptides post-translationally can determine cell-specific expression

44. Differential mRNA Longevity - The longer the poly-A tail, the longer mRNA life - Sequence of 3’-UTR determines length of tail - External regulation: hormones, growth factors - Internal regulation: stabilizing proteins

45. Figure 2.31 Degradation of casein mRNA in the presence and absence of prolactin

46. Selective Inhibition of mRNA Translation - Dormant mRNA in oocyte awaits fertilization - Proteins that control the cleavage cell cycle - Proteins to control cell differentiation - Ion signals that follow sperm entry set them off

49. Translational regulation in oocytes

50. Protein binding in Drosophila oocytes Bicoid in flies acts like maskin - but only on the caudal gene!

51. Selective destruction if active mRNA: MicroRNAs - Naturally occurring antisense RNA that stop expression of the mRNA they hybridize with - Built-in system to actively shut down expression that you wanted earlier - Can be found as different gene, in introns of same gene, even out in the “junk DNA” - miRNA are transcribed, processed in nucleus, transported into cytosol and further processed there

52. Current model for the formation and use of microRNAs

53. Figure 2.34 Hypothetical model of the regulation of lin- 14 mRNA translation by lin-4 RNAs

54. miRNA complex can block translation in several ways

55. Identified Pathways which use miRNAs - Larval development in C. elegans - Cell fate decision in B and T lymphocytes - Removal of maternal mRNAs after its needed - Ventricle differentiation in the heart development - Muscle cell division rate in response to myostatin - Can even affect the methylation pattern in histones which can alter the entire gene expression pattern

56. The lymphoid precursor can generate B or T cells

57. Cytoplasmic Localization - Most mRNAs are placed in specific locations in the cell prior to being translated - 3 major mechanisms of localization 1. diffusion and local anchoring through local protein trapping 2. localized protection – degraded everywhere but target 3. active transport along the cytoskeleton (the big one) - Often secured once there in actin cytoskeleton - Critical in brain, in the neuronal dendrite

58. Figure 2.38 Localization of mRNAs

59. Example: Stored mRNAs in Brain Cells - mRNA actively transported to dendrite on microtubules - Proteins that are critical in initial building of the synaptic connections - The same process must be active for us to learn as the connections must be remade -

60. Figure 2.39 A brain-specific RNA in a cultured mammalian neuron

61. Post-Translational Regulation of Gene Expression Let’s remember some basics.... - primary protein structure - secondary protein structure - tertiary protein structure - quaternary protein structure