1. Chapter 21 Regulation of Transcription

2. 21.1 Introduction 21.2 Response elements identify genes under common regulation 21.3 There are many types of DNA-binding domains 21.4 A zinc finger motif is a DNA-binding domain 21.5 Steroid receptors are transcription factors 21.6 Steroid receptors have zinc fingers 21.7 Binding to the response element is activated by ligand-binding 21.8 Steroid receptors recognize response elements by a combinatorial code 21.9 Homeodomains bind related targets in DNA 21.10 Helix-loop-helix proteins interact by combinatorial association 21.11 Leucine zippers are involved in dimer formation 21.12 Transcription initiation requires changes in chromatin structure 21.13 Chromatin remodeling is an active process 21.14 Activation of transcription requires changes in nucleosome organization at the promoter 21.15 Histone acetylation and deacetylation control chromatin activity 21.16 Polycomb and trithorax are antagonistic repressors and activators 21.17 An LCR may control a domain 21.18 Insulators block enhancer actions 21.19 Insulators can vary in strength 21.20 A domain has several types of elements 21.21 Gene expression is associated with demethylation 21.22 CpG islands are regulatory targets

3. Activation of gene structure Initiation of transcription Processing the transcript Transport to cytoplasm Translation of mRNA 21.1 Introduction

4. Table 21.1 Incucible transcription factors bind to response elements that identify groups of promoters or enhancers subject to coordinate control. 21.2 Response elements identify genes under common regulation Regulatory Agent ModuleConsensusFactor Heat shock HSECNNGAANNTCCNNGHSTF GlucocorticoidGRETGGTACAAATGTTCTReceptor Phorbol ester TRETGACTCAAP1 SerumSRECCATATTAGGSRF

5. Figure 21.1 The regulatory region of a human metallothionein gene contains regulator elements in both its promoter and enhancer. The promoter has elements for metal induction; an enhancer has an element for response to glucocorticoid. Promoter elements are shown above the map, and proteins that bind them are indicated below. 21.2 Response elements identify genes under common regulation

6. Figure 21.2 The activity of a regulatory transcription factor may be controlled by synthesis of protein, covalent modification of protein, ligand binding, or binding of inhibitors that sequester the protein or affect its ability to bind to DNA. 21.3 There are many types of DNA-binding domains

7. Figure 28.19 Oncogenes that code for transcription factors have mutations that inactivate transcription (v-erbA and possibly v- rel) or that activate transcription (v-jun and v-fos). 21.3 There are many types of DNA- binding domains

8. Figure 21.3 Transcription factor SP1 has a series of three zinc fingers, each with a characteristic pattern of cysteine and histidine residues that constitute the zinc- binding site. 21.4 A zinc finger motif is a DNA-binding domain

9. Figure 21.4 Zinc fingers may form  -helices that insert into the major groove, associated with  -sheets on the other side. 21.4 A zinc finger motif is a DNA-binding domain

10. Figure 21.5 The first finger of a steroid receptor controls specificity of DNA-binding (positions shown in red); the second finger controls specificity of dimerization (positions shown in blue). The expanded view of the first finger shows that discrimination between GRE and ERE target sequences rests on two amino acids at the base. 21.4 A zinc finger motif is a DNA-binding domain

11. Receptor is a transmembrane protein, located in the plasma membrane, that binds a ligand in a domain on the extracellular side, and as a result has a change in activity of the cytoplasmic domain. (The same term is sometimes used also for the steroid receptors, which are transcription factors that are activated by binding ligands that are steroids or other small molecules.) 21.5 Steroid receptors have several independent domains

12. Figure 21.6 Several types of hydrophobic small molecules activate transcription factors. Corticoids and steroid sex hormones are synthesized from cholesterol, vitamin D is a steroid, thyroid hormones are synthesized from tyrosine, and retinoic acid is synthesized from isoprene (in fish liver). 21.5 Steroid receptors have several independent domains

13. Figure 21.7 Glucocorticoids regulate gene transcription by causing their receptor to bind to an enhancer whose action is needed for promoter function. 21.5 Steroid receptors have several independent domains

14. Figure 21.8 Receptors for many steroid and thyroid hormones have a similar organization, with an individual N-terminal region, conserved DNA-binding region, and a C-terminal hormone-binding region. 21.5 Steroid receptors have several independent domains

15. Figure 21.8 Receptors for many steroid and thyroid hormones have a similar organization, with an individual N-terminal region, conserved DNA-binding region, and a C-terminal hormone-binding region. 21.5 Steroid receptors have several independent domains

16. Figure 21.5 The first finger of a steroid receptor controls specificity of DNA-binding (positions shown in red); the second finger controls specificity of dimerization (positions shown in blue). The expanded view of the first finger shows that discrimination between GRE and ERE target sequences rests on two amino acids at the base. 21.5 Steroid receptors have several independent domains

17. Figure 21.19 Coactivators may have HAT activities that acetylate the tails of nucleosomal histones. 21.5 Steroid receptors have several independent domains

18. Figure 21.20 A repressor complex contains three components: a DNA binding subunit, a corepressor, and a histone deacetylase. 21.5 Steroid receptors have several independent domains

19. Figure 21.9 TR and RAR bind the SMRT corepressor in the absence of ligand. The promoter is not expressed. When SMRT is displaced by binding of ligand, the receptor binds a coactivator complex. This leads to activation of transcription by the basal apparatus. 21.5 Steroid receptors have several independent domains

20. Figure 21.10 The homeodomain may be the sole DNA-binding motif in a transcriptional regulator or may be combined with other motifs. It represents a discrete (60 residue) part of the protein. 21.6 Homeodomains bind related targets in DNA

21. Figure 21.11 The homeodomain of the Antennapedia gene represents the major group of genes containing homeoboxes in Drosophila; engrailed (en) represents another type of homeotic gene; and the mammalian factor Oct-2 represents a distantly related group of transcription factors. The homeodomain is conventionally numbered from 1 to 60. It starts with the N-terminal arm, and the three helical regions occupy residues 10-22,28-38, and 42- 58. 21.6 Homeodomains bind related targets in DNA

22. Figure 21.12 Helix 3 of the homeodomain binds in the major groove of DNA, with helices 1 and 2 lying outside the double helix. Helix 3 contacts both the phosphate backbone and specific bases. The N- terminal arm lies in the minor groove, and makes additional contacts. 21.6 Homeodomains bind related targets in DNA

23. Figure 29.8 The posterior pathway has two branches, responsible for abdominal development and germ cell formation. 21.6 Homeodomains bind related targets in DNA

24. Figure 21.13 All HLH proteins have regions corresponding to helix 1 and helix 2, separated by a loop of 10-24 residues. Basic HLH proteins have a region with conserved positive charges immediately adjacent to helix 1. 21.7 Helix-loop-helix proteins interact by combinatorial association

25. Figure 21.14 An HLH dimer in which both subunits are of the bHLH type can bind DNA, but a dimer in which one subunit lacks the basic region cannot bind DNA. 21.7 Helix-loop-helix proteins interact by combinatorial association

26. Figure 21.15 The basic regions of the bZIP motif are held together by the dimerization at the adjacent zipper region when the hydrophobic faces of two leucine zippers interact in parallel orientation. 21.8 Leucine zippers are involved in dimer formation

27. Figure 20.19 An enhancer contains several structural motifs. The histogram plots the effect of all mutations that reduce enhancer function to <75% of wild type. Binding sites for proteins are indicated below the histogram. 21.8 Leucine zippers are involved in dimer formation

28. Chromatin remodeling describes the energy-dependent displacement or reorganization of nucleosomes that occurs in conjunction with activation of genes for transcription. 21.9 Chromatin remodeling is an active process

29. Figure 21.16 The pre- emptive model for transcription of chromatin proposes that if nucleosomes form at a promoter, transcription factors (and RNA polymerase) cannot bind. If transcription factors (and RNA polymerase) bind to the promoter to establish a stable complex for initiation, histones are excluded. 21.9 Chromatin remodeling is an active process

30. Figure 21.17 The dynamic model for transcription of chromatin relies upon factors that can use energy provided by hydrolysis of ATP to displace nucleosomes from specific DNA sequences. 21.9 Chromatin remodeling is an active process

31. Figure 21.18 Hormone receptor and NF1 cannot bind simultaneously to the MMTV promoter in the form of linear DNA, but can bind when the DNA is presented on a nucleosomal surface. 21.9 Chromatin remodeling is an active process

32. Figure 21.18 Hormone receptor and NF1 cannot bind simultaneously to the MMTV promoter in the form of linear DNA, but can bind when the DNA is presented on a nucleosomal surface. 21.9 Chromatin remodeling is an active process

33. HAT (histone acetyltransferase) enzymes modify histones by addition of acetyl groups; some transcriptional coactivators have HAT activity. HDAC (histone deacetyltransferase) enzymes remove acetyl groups from histones; they may be associated with repressors of transcription. 21.10 Histone acetylation and deacetylation control chromatin activity

34. Figure 20.26 An upstream transcription factor may bind a coactivator that contacts the basal apparatus. 21.10 Histone acetylation and deacetylation control chromatin activity

35. Figure 21.19 Coactivators may have HAT activities that acetylate the tails of nucleosomal histones. 21.10 Histone acetylation and deacetylation control chromatin activity

36. Figure 21.20 A repressor complex contains three components: a DNA binding subunit, a corepressor, and a histone deacetylase. 21.10 Histone acetylation and deacetylation control chromatin activity

37. Figure 21.21 Pc-G proteins do not initiate repression, but are responsible for maintaining it. 21.11 Polycomb and trithorax are antagonistic repressors and activators

38. Figure 19.45 Extension of heterochromatin inactivates genes. The probability that a gene will be inactivated depends on its distance from the heterochromatin region. 21.11 Polycomb and trithorax are antagonistic repressors and activators

39. Domain of a chromosome may refer either to a discrete structural entity defined as a region within which supercoiling is independent of other domains; or to an extensive region including an expressed gene that has heightened sensitivity to degradation by the enzyme DNAase I. MAR (matrix attachment site; also known as SAR for scaffold attachment site) is a region of DNA that attaches to the nuclear matrix. 21.12 Long range regulation and insulation of domains

40. Figure 4.1 Each of the  -like and  -like globin gene families is organized into a single cluster that includes functional genes and pseudogenes ( . 21.12 Long range regulation and insulation of domains

41. Figure 4.1 Each of the  -like and  -like globin gene families is organized into a single cluster that includes functional genes and pseudogenes ( . 21.12 Long range regulation and insulation of domains

42. Figure 21.22 A globin domain is marked by hypersensitive sites at either end. The group of sites at the 5 ￠ side constitutes the LCR and is essential for the function of all genes in the cluster. 21.12 Long range regulation and insulation of domains

43. Figure 19.42 Sensitivity to DNAase I can be measured by determining the rate of disappearance of the material hybridizing with a particular probe. 21.12 Long range regulation and insulation of domains

44. Figure 21.23 Specialized chromatin structures that include hypersensitive sites mark the ends of a domain in the D. melanogaster genome and insulate genes between them from the effects of surrounding sequences. 21.12 Long range regulation and insulation of domains

45. Figure 21.24 A protein that binds to the insulator scs ￠ is localized at interbands in Drosophila polytene chromosomes. Red staining identifies the DNA (the bands) on both the upper and lower samples; green staining identifies BEAF32 (often at interbands) on the upper sample. Yellow shows coincidence of the two labels. Some of the more prominent stained interbands are marked by white lines. Photograph kindly provided by Uli Laemmli. 21.12 Long range regulation and insulation of domains

46. Figure 21.25 The insulator of the gypsy transposon blocks the action of an enhancer when it is placed between the enhancer and the promoter. 21.12 Long range regulation and insulation of domains

47. Figure 29.32 The homeotic genes of the ANT-C complex confer identity on the most anterior segments of the fly. The genes vary in size, and are interspersed with other genes. The antp gene is very large and has alternative forms of expression. 21.12 Long range regulation and insulation of domains

48. Figure 21.26 Fab-7 is a boundary element that is necessary for the independence of regulatory elements iab-6 and iab-7. 21.12 Long range regulation and insulation of domains

49. Figure 21.27 Domains may possess three types of sites: insulators to prevent effects from spreading between domains; MARs to attach the domain to the nuclear matrix; and LCRs that are required for initiation of transcription. 21.12 Long range regulation and insulation of domains

50. Figure 21.28 The restriction enzyme MspI cleaves all CCGG sequences whether or not they are methylated at the second C, but HpaII cleaves only nonmethylated CCGG tetramers. 21.13 Gene expression is associated with demethylation

51. Figure 21.29 The results of MspI and HpaII cleavage are compared by gel electrophoresis of the fragments. 21.13 Gene expression is associated with demethylation

52. Figure 13.30 Replication of methylated DNA gives hemimethylated DNA, which maintains its state at GATC sites until the Dam methylase restores the fully methylated condition. 21.13 Gene expression is associated with demethylation

53. Figure 21.30 The typical density of CpG doublets in mammalian DNA is ~1/100 bp, as seen for a  - globin gene. In a CpG- rich island, the density is increased to >10 doublets/100 bp. The island in the APRT gene starts ~100 bp upstream of the promoter and extends ~400 bp into the gene. Each vertical line represents a CpG doublet. 21.13 Gene expression is associated with demethylation

54. Figure 21.20 A repressor complex contains three components: a DNA binding subunit, a corepressor, and a histone deacetylase. 21.13 Gene expression is associated with demethylation

55. 1. Some regulatory promoter elements are present in many genes and are recognized by ubiquitous factors; others are present in a few genes and are recognized by tissue-specific factors. 2. Several groups of transcription factors have been identified by sequence homologies. 3. Another motif involved in DNA-binding is the zinc finger, which is found in proteins that bind DNA or RNA (or sometimes both). 4. Steroid receptors were the first members identified of a group of transcription factors in which the protein is activated by binding a small hydrophobic hormone. 5. The leucine zipper contains a stretch of amino acids rich in leucine that are involved in dimerization of transcription factors. Summary

56. 6. HLH (helix-loop-helix) proteins have amphipathic helices that are responsible for dimerization, adjacent to basic regions that bind to DNA. 7. Many transcription factors function as dimers, and it is common for there to be multiple members of a family that form homodimers and heterodimers. 8. The existence of a preinitiation complex signals that the gene is in an "active" state, ready to be transcribed. 9. The variety of situations in which hypersensitive sites occur suggests that their existence reflects a general principle. 10. Genes whose control regions are organized in nucleosomes usually are not expressed. Summary

57. 11. Acetylation of histones occurs at both replication and transcription and could be necessary to form a less compact chromatin structure. 12. Active chromatin and inactive chromatin are not in equilibrium. 13. A group of hypersensitive sites upstream of the cluster of -globin genes forms a locus control region (LCR) that is required for expression of all of the genes in the domain. 14. CpG islands contain concentrations of CpG doublets and often surround the promoters of constitutively expressed genes, although they are also found at the promoters of regulated genes. 15. The formation of heterochromatin occurs by proteins that bind to specific chromosomal regions (such as telomeres) and that interact with histones. Summary