1. 1 Chapter 15 Lecture Outline Copyright © McGraw-Hill Education. Permission required for reproduction or display. See separate PowerPoint slides for all figures and tables pre- inserted into PowerPoint without notes.

2. INTRODUCTION Eukaryotic organisms derive many benefits from regulating their genes For example They can respond to changes in nutrient availability They can respond to environmental stresses In plants and animals, multicellularity and a more complex cell structure also demand a much greater level of gene regulation 15-2

3. INTRODUCTION Gene regulation is necessary to ensure 1. Expression of genes in an accurate pattern during the various developmental stages of the life cycle Some genes are only expressed during embryonic stages, whereas others are only expressed in the adult 2. Differences among distinct cell types Nerve and muscle cells look so different because of gene regulation rather than differences in DNA content Figure 15.1 describes the steps of gene expression that are regulated in eukaryotes 15-3

4. 15-4 Figure 15.1

5. Transcription factors are proteins that influence the ability of RNA polymerase to transcribe a given gene There are two main types General transcription factors Required for the binding of the RNA pol to the core promoter and its progression to the elongation stage Are necessary for basal transcription Regulatory transcription factors Serve to regulate the rate of transcription of target genes They influence the ability of RNA pol to begin transcription of a particular gene 2-3% of human genes encode transcription factors 15.1 REGULATORY TRANSCRIPTION FACTORS 15-5

6. Regulatory transcription factors recognize cis regulatory elements located near the core promoter These sequences are known as control elements, regulatory elements or regulatory sequences The binding of regulatory transcription factors to control elements affects the transcription of an associated gene A regulatory protein that increases the rate of transcription is termed an activator The sequence it binds is called an enhancer A regulatory protein that decreases the rate of transcription is termed a repressor The sequence it binds is called a silencer Refer to Figure 15.2 15-6

7. Figure 15.2 15-7

8. Most Eukaryotic genes are regulated by many factors This is known as combinatorial control Common factors contributing to combinatorial control are: One or more activator proteins may stimulate transcription One or more repressor proteins may inhibit transcription Activators and repressors may be modulated by: binding of small effector molecules protein-protein interactions covalent modifications Regulatory proteins may alter nucleosomes near the promoter DNA methylation may inhibit transcription prevent binding of an activator protein recruiting proteins that compact the chromatin Various combinations of these factors can contribute to the regulation of a single gene 15-8

9. Transcription factor proteins contain regions, called domains, that have specific functions One domain could be for DNA-binding Another could provide a binding site for effector molecules A motif is a domain, or a portion of a domain, that has a very similar structure in many different proteins Figure 15.3 depicts several different domain structures found in transcription factor proteins Structural Features of Regulatory Transcription Factors 15-9

10. Figure 15.3 15-10 The recognition helix recognizes and makes contact with a base sequence along the major groove of DNA Hydrogen bonding between an  -helix and nucleotide bases is one way a transcription factor can bind to DNA

11. Figure 15.3 15-11 Note: Helix-loop-helix motifs can also mediate protein dimerization Homodimers are formed by two identical transcription factors; Heterodimers are formed by two different transcription factors Two  -helices intertwined due to leucine motifs Alternating leucine residues in both proteins interact (“zip up”), resulting in protein dimerization Composed of one  -helix and two  -sheets held together by a zinc (Zn ++ ) metal ion

12. The binding of a transcription factor to an enhancer increases the rate of transcription This up-regulation can be 10- to 1,000-fold The binding of a transcription factor to a silencer decreases the rate of transcription This is called down-regulation Enhancers and Silencers 15-12

13. Many response elements are orientation independent or bidirectional They can function in the forward or reverse orientation Most response elements are located within a few hundred nucleotides upstream of the promoter However, some are found at various other sites Several thousand (even 100,000) nucleotides away Downstream from the promoter Even within introns! Enhancers and Silencers 15-13

14. Most regulatory transcription factors do not bind directly to RNA polymerase Three common interactions that communicate the effects of regulatory transcription factors are 1. TFIID-direct or through coactivators 2. Mediator 3. recruiting proteins that affect nucleosome composition Refer to Figure 15.4 and 15.5 TFIID and Mediator 15-14

15. Figure 15.4 15-15

16. Figure 15.5 15-16 Transcriptional activator stimulates the function of mediator This enables RNA pol to form a preinitiation complex It then proceeds to the elongation phase of transcription Transcriptional repressor inhibits the function of mediator Transcription is repressed

17. There are three common ways that the function of regulatory transcription factors can be modulated 1. Binding of a small effector molecule 2. Protein-protein interactions 3. Covalent modification Refer to Figure 15.6 Modulation of Regulatory Transcription Factor Functions 15-17

18. Figure 15.6 15-18 The transcription factor can now bind to DNA Formation of homodimers and heterodimers

19. Regulatory transcription factors that respond to steroid hormones are termed steroid receptors The hormone actually binds to the transcription factor The ultimate action of a steroid hormone is to affect gene transcription Steroid hormones are produced by endocrine glands Secreted into the bloodstream Then taken up by cells that respond to the hormone Steroid Hormones and Regulatory Transcription Factors 15-19

20. Cells respond to steroid hormones in different ways Glucocorticoids These influence nutrient metabolism in most cells They promote glucose utilization, fat mobilization and protein breakdown Gonadocorticoids These include estrogen and testosterone They influence the growth and function of the gonads Figure 15.7 shows the stepwise action of glucocorticoid hormones Steroid Hormones and Regulatory Transcription Factors 15-20

21. 15-21 Figure 15.7 Heat shock proteinHeat shock proteins released when hormone binds Nuclear localization signal is exposed Glucocorticoid Response Elements These function as enhancers GREs are located near dozens of different genes, so the hormone can activate many genes Transcription of target gene is activated Formation of a homodimer

22. The CREB protein is another regulatory transcriptional factor CREB is an acronym for cAMP response element-binding CREB protein becomes activated in response to cell- signaling molecules that cause an increase in the cytoplasmic concentration of cAMP Cyclic adenosine monophosphate The CREB protein recognizes a response element with the consensus sequence 5’–TGACGTCA–3’ This has been termed a cAMP response element (CRE) The CREB Protein 15-22

23. 15-23 The activity of the CREB protein Figure 15.8 Could be a hormone, neurotransmitter, growth factor, etc. Acts as a second messenger Activates protein kinase A Phosphorylated CREB binds to DNA and stimulates transcription Unphosphorylated CREB can bind to DNA, but cannot activate RNA pol

24. ATP-dependent chromatin remodeling refers to dynamic changes in chromatin structure These changes range from a few nucleosomes to large scale changes Carried out by diverse multiprotein machines that reposition and restructure nucleosomes 15.2 CHROMATIN REMODELING AND HISTONES 15-24

25. The three-dimensional packing of chromatin is an important parameter affecting gene expression Chromatin is a very dynamic structure that can alternate between two conformations Closed conformation Chromatin is very tightly packed Transcription may be difficult or impossible Open conformation Chromatin is accessible to transcription factors Transcription can take place Chromatin Structure 15-25

26. Nucleosomes have been shown to change position in cells that normally express a particular gene compared with cells in which the gene is inactive For  -globin, nucleosome positioning changes in the promoter region as part of gene activation A key role of some transcriptional activators is to orchestrate changes in chromatin structure Closed conformation Open conformation One way is through ATP-dependent chromatin remodeling. Energy of ATP hydrolysis is used to drive change in location and/or composition of nucleosomes Makes the DNA more or less amenable to transcription 15-26

27. ATP-dependent chromatin remodeling The energy of ATP is used to alter the structure of nucleosomes and thus make the DNA more accessible 15-27 Figure 15.9a Eukaryotes have multiple families of chromatin remodelers; SWI/SNF ISWI INO80 Mi-2 These effects may significantly alter gene expression

28. ATP-dependent chromatin remodeling 15-28 Figure 15.9b and c Creates a gap with no nucleosomes Region with variant histones which affect transcription

29. The five histone genes are moderately repetitive H1, H2A, H2B, H3 and H4 Human genome contains over 70 histone genes Most encode standard histones A few of these genes have accumulated mutations that alters the amino acid sequence These are termed histone variants Some histone variants are incorporated into a subset of nucleosomes to create specialized chromatin See Table 15.1 15-29

30. 15-30

31. Over 50 enzymes have been identified in mammals that selectively modify the amino terminal tails of histones acetylation, methylation and phosphorylation are common (see Figure 15.10) These modifications affect the level of transcription May influence interactions between nucleosomes Occur in patterns that are recognized by proteins Called the histone code The pattern of modifications provide binding sites for proteins that specify alterations to be made to chromatin structure These proteins bind based on the code and affect transcription Histone Code 15-31

32. 15-32 Figure 15.10 Examples of histone modifications p=phosphate ac=acetyl group m=methyl group Histone modifications can change binding to DNA

33. 15-33 Chromatin Immunoprecipitation Sequencing Also known as ChIP-Seq Allows determination of: Where nucleosomes are located Where histone variants are found Where covalent modifications of histones occur See Figure 15.11

34. 15-34 Figure 15.11 Chromatin Immunoprecipitation Sequencing

35. 15-35 Figure 15.11 Chromatin Immunoprecipitation Sequencing

36. 15-36 Figure 15.12 Chromatin Immunoprecipitation Sequencing has revealed a common pattern of nucleosome organization

37. 15-37 Figure 15.13 Transcriptional Activation

38. 15-38 Figure 15.13 Transcriptional Activation

39. DNA methylation is a change in chromatin structure that silences gene expression Carried out by the enzyme DNA methyltransferase It is common in some eukaryotic species, but not all Yeast and Drosophila have little DNA methylation Vertebrates and plants have abundant DNA methylation In mammals, ~ 2 to 7% of the DNA is methylated Refer to Figure 15.14 15.3 DNA Methylation 15-39

40. 15-40 Figure 15.14 Only one strand is methylated Both strands are methylated

41. DNA methylation usually inhibits the transcription of eukaryotic genes Especially when it occurs in the vicinity of the promoter In vertebrates and plants, many genes contain CpG islands near their promoters These CpG islands are 1,000 to 2,000 nucleotides long Contain high number of CpG sites In housekeeping genes The CpG islands are unmethylated Genes tend to be expressed in most cell types In tissue-specific genes The expression of these genes may be silenced by the methylation of CpG islands Methylation may influence the binding of transcription factors Methyl-CpG-binding proteins may recruit factors that lead to compaction of the chromatin 15-41

42. 15-42 Transcriptional silencing via methylation Figure 15.15a Transcriptional activator binds to unmethylated DNA This would inhibit the initiation of transcription

43. 15-43 Transcriptional silencing via methylation Figure 15.15b

44. Methylated DNA sequences are inherited during cell division May explain genomic imprinting (Chapter 5) Specific genes are methylated in gametes from mother or father Pattern of one copy of the gene being methylated and the other not is maintained in the resulting offspring Figure 15.16 illustrates a model explaining how methylation is passed from mother to daughter cell DNA Methylation is Heritable 15-44

45. Figure 15.16 An infrequent and highly regulated event DNA methyltransferase converts hemi- methylated to fully- methylated DNA An efficient and routine event occurring in vertebrate and plant cells 15-45

46. Since eukaryotic gene regulation can occur over long distances, it is important to limit regulation to one particular gene, but not to neighboring genes Insulators are segments of DNA that insulates a gene from the regulatory effects of other genes Some act as barriers to chromatin remodeling or histone-modifying enzymes Others block the effects of enhancers May do this by chromosome looping See Figure 15.17 15.4 INSULATORS 15-46

47. 15-47 Figure 15.17 (a) Insulators as a barrier to changes in chromatin structure Nonacetylated DNA Nonacetylated DNA Proteins that bind to insulators Insulator ac (b) Insulator that blocks the effects of a neighboring enhancer Gene AEnhancerGene B The insulator prevents the enhancer for gene A from activating the expression of gene B. Protein bound to an insulator Insulator Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. Gene within an acetylated region of DNA

48. Encyclopedia of DNA Elements Consortium Isolate and sequence RNA from humans Identify transcription factor binding sites Map DNA methylation sites Identify histone modification sites Map DNase I cleavage sites May lead to a better understanding of human diseases and aid in the development of new drugs and therapies to treat them 15.5 The ENCODE Project 15-48