1. Gene Regulation in Eukaryotes
Chapter 17
Gene Regulation in Eukaryotes

2. In eukaryotic cells, expression of a gene can be regulated at all those steps we saw in bacteria, and a few additional ones besides.
In many cases, a given transcript can be spliced in alternative ways to generate different products, and this too can be regulated.
The eukaryotic transcriptional machinery is more elaborate than its bacterial counterpart.

3. Nucleosomes and their modifier influence access to genes.
Many eukaryotic genes have more regulatory binding sites and are controlled by more regulatory proteins than are typical bacterial genes.
Promoter
Regulatory binding sites
Regulatory sequences: the stretch of DNA encompassing the complete collection of regulator binding sites for a given gene.

4. In multicellular organisms , regulatory sequences can spread thousands of nucleotides from the promoter-both upstream and downstream –and can be made up of tens of regulator binding sites. Often these binding sites are grouped in unites called enhancers, and a given enhancer binds regulators responsible for activating the gene at a given time and place. Alternative enhancers bind different groups of regulators and control expression of the same gene at different times and places in response to different signals. (Figure 17-1)

5. FIGURE 17-1 The regulatory elements of a bacterial, yeast, and human gene.

6. OUTLINE
Conserved Mechanisms of Transcriptional Regulation from Yeast to Mammals
Recruitment of Protein Complexes to Genes by Eukaryotic Activators
Signal Integration and Combinatorial Control
Transcriptional Repressors

7. Signal Transduction and the Control of Transcriptional Regulators
Gene “Silencing” by Modification of Histones and DNA
Eukaryotic Gene Regulation at Steps after Transcription Initiation
RNAs in Gene Regulation

8. CONSERVED MECHANISMS OF TRANSCRIPTIONAL REGULATION FROM YEAST TO MAMMALS

9. Many of the basic features of gene regulation are the same in all eukaryotes.
This is tested using a reporter gene. The reporter gene consists of binding sites for the yeast activator inserted upstream of the promoter of a gene whose expression level is readily measured.

10. The typical eukaryotic activator works in a manner similar to the simplest bacterial case.
In contrast , repressors work in a variety of ways ,some different from anything we encountered in bacteria.
Gene silencing, in which modification to regions of chromatin keep genes in sometimes large stretches of DNA switched off.

11. Activators Have Separate DNA Binding and Activation Functions
Eukaryotic activators have separate DNA binding and activating regions as well. The two surfaces are very often in separate domains of the protein. (Figure 17-2)

12. FIGURE 17-2 Gal4 bound to its site on DNA.

13. One such gene is called GAL1
One such gene is called GAL1. GAL4 binds to four sites located 275bp upstream of GAL1(Figure 17-3).
The separate DNA binding and activating regions of Gal4 were revealed in two complementary experiments.

14. FIGURE 17-3 The regulatory sequences of the yeast GAL1 gene.

15. FIGURE17-4 Domain swap experiment.

16. Eukaryotic Regulators Use a Range of DNA-Binding Domains, but DNA Recognition Involves the Same Principles as Found in Bacteria

17. One class of eukaryotic regulatory protein presents the recognition helix as part of a structure very like the helic0turn-helix domain; others present the recognition helix within quite different domain structures.
In eukaryotes bind DNA as heterodimers, and in some cases even as monomers.

18. Homeodomain proteins. The homeodomain is a class of helix-turn-helix DNA-binding domain and recognizes DNA in essentially the same way as those bacterial proteins (Figure 17-5).

19. FIGURE 17-5 DNA recognition by a homeodomain.

20. FIGURE 17-6 Zinc finger domain.
Zinc containing DNA-Binding Domains Zinc finger proteins, Zinc cluster domain.
FIGURE 17-6 Zinc finger domain.

21. FIGURE 17-7 Leucine zipper bound to DNA.
Leucine zipper motif. This motif combines dimerization and DNA-binding surfaces within a single structural unit.
FIGURE 17-7 Leucine zipper bound to DNA.

22. Helix-loop-helix proteins
Helix-loop-helix proteins. An extended αhelical region from each of two monomers insets into the major groove of the DNA. Leucine zipper and HLH proteins are often called basic zipper and basic HLH proteins : this is because the region of the αhelix that binds DNA contains basic amino acid residues (Figure 17-8).

23. FIGURE 17-8 Helix-loop-helix motif.

24. Activating Regions Are Not Well-Defined Structures
It is believed that activating regions consist of reiterated small units, each of which has a weak activating capacity on its own. Each unit is a short sequence of amino acids, the greater the number of units, and the more acidic each unit, the stronger the resulting activating region.

25. RECRUITMENT OF PROTEIN COMPLEXES TO GENES BY EUKARYOTIC ACTIVATORS

26. Activators Recruit the Transcriptional Machinery to the Gene

27. Eukaryotic activators rarely, if ever, through a direct interaction between the activator and RNA polymerase. First, the activator can interact with parts of the transcription machinery other than polymerase, and, by recruiting them, recruit polymerase as well. Second, activators can recruit nucleosome modifiers that alter chromatin in the vicinity of a gene and thereby help polymerase bind.

28. According to one view, most of the machinery comes to the gene in a single , very large complex called the holoenzyme, which contains the mediator , RNA polymerase , and some of the general transcription factors (Figure 17-9).

29. FIGURE 17-9 Activation of transcription initiation in eukaryotes by recruitment of the transcription machinery.

30. Recruitment can be visualized using the technique called chromatin immunoprecipitation (ChIP ).
In activator bypass experiments, activation is observed when RNA polymerase is recruited to the promoter without using the natural activator –polymerase interaction (Figure 17-10).

31. FIGURE 17-10 Activation of transcription through direct tethering of mediator to DNA.

32. Activators also Recruit Nucleosome Modifiers that Help the Transcription Machinery Bind at the Promoter

33. In activator bypass experiments, activation is observed when RNA polymerase is recruited to the promoter without using the natural activator –polymerase interaction (Figure 17-10).

34. FIGURE 17-11 Local alterations in chromatin structure directed by activators.

35. Action at a Distance: Loops and Insulators
Various models have been proposed to explain how proteins binding in between enhancers and promoters might help activation in the cells of higher eukaryotes. In Drosophila, a protein called Chip aids communication between enhancer and gene.

36. In eukaryotes, chromatin may in some places form special structures that actively bring enhancers and promoters closer together.
If an enhancer activates a specific gene 50kb away , what stops it from activating other gene whose promoters are within that range? Specific elements called insulator control the actions of activators (Figure 17-12).

37. FIGURE 17-12 Insulators block activation by enhancers.

38. In other assays, insulators also seem able to inhibit the spread of chromatin modifications.
Silencing is a specialized form of repression that can spread along chromatin, switching off multiple genes without the need for each to bear binding sites for specific repressors. Insulator elements can block this spreading, so insulators protect genes from both indiscriminate activation and repression.

39. Appropriate Regulation of Some Groups of Genes Requires Locus Control Regions

40. There are five different globin genes in humans (Figure 17-13a)
There are five different globin genes in humans (Figure 17-13a). Although clustered, these genes are not all expressed at the same time. Rather, the different genes are expressed at different stages of development.
A group of regulatory elements collectively called the locus control region, or LCR, is found kb upstream of the whole cluster of globin genes (Figure 17-13).

41. FIGURE 17-13 Regulation by LCRs.

42. SIGNAL INTEGRATION AND COMBINATORIAL CONTROL

43. Activators Work Together Synergistically to Integrate Signals

44. When multiple activators work together, they do so synergistically
When multiple activators work together, they do so synergistically. Two activators can recruit a single complex.
Cooperativity: Synergy can also result from activators helping each other bind under conditions where the binding of one depends on binding of the other (Figure 17-14).
Synergy is critical for signal integration by activators.

45. FIGURE 17-14 Cooperative binding of activators.

46. Signal Integration: the HO Gene Is Controlled by Two Regulators; One Recruits Nucleosome Modifiers and the Other Recruits Mediator
The yeast S. cerebisiae divides by budding. We will focus here on the expression of a gene called HO. The HO gene is expressed only in mother cells and only at a certain point in the cell cycle.

47. FIGURE 17-15 Control of the HO gene.

48. Signal Integration: Cooperative Binding of Activators at the Human β-Interferon Gene
The human β-interferon gene is activated in cells upon viral infection. Infection triggers three activators: NFkB, IRF, Jun/ATF. The structure formed by these regulators bound to the enhancer is called an enhanceosome (Figure 17-16).

49. FIGURE 17-16 The human β-interferon enhanceosome.

50. The binding of the activators is cooperative for two reasons
The binding of the activators is cooperative for two reasons. First, the activators interact with each other. second, an additional protein, called HMG-I, binds within the enhancer and aids biding of the activators by bending the DNA in a way that facilitates the interactions among them.

51. Combinatorial Control Lies at the Heart of the Complexity and Diversity of Eukaryotes
We encountered simple cases of combinatorial control in bacteria, such as CAP. There is extensive combinatorial control in eukaryotes. we first consider a generic case (Figure 17-17).

52. FIGURE 17-17 Combinatorial control.

53. A given activator can interact with multiple targets
A given activator can interact with multiple targets. This provides an explanation for why different regulators can work together in so many combinations: because each can use any of an array of targets, the combinations that work together are unrestricted.

54. Combinatorial Control of the Mating-Type Genes from Saccharomyces cerevisiae

55. The yeast S. cerevisiae exists in three forms: two haploid cells of different mating type-α and a- and the diploid formed when an α and an a cell mate and fuse. a cells make the regulatory protein a1; α cells make the proteins α1 and α2. A fourth regulatory protein, called Mcm1, is present in both cell types (Figure 17-18).

56. FIGURE 17-18 Control of cell-type specific genes in yeast.

57. TRANSCRIPTIONAL REPRESSORS

58. In bacteria we saw that many repressors work by binding to sites that overlap the promoter and thus block binding of RNA polymerase. But we also saw other ways they can work: they can bind to sites adjacent to promoters and, by interacting with polymerase bound there, inhibit the enzyme from initiating transcription. They can also interfere with the action of activator.

59. In eukaryotes we see all these except the first
In eukaryotes we see all these except the first. We also see anther form of repression, perhaps the most common in eukaryotes, repressors can recruit nucleosome modifiers, they compact the chromatin or remove groups recognized by the transcriptional machinery, such as, histone deacetylases.
These various examples of repression are shown schematically in Figure

60. FIGURE 17-19 Ways in which eukaryotic repressors work.

61. In the presence of glucose, Mig1 binds and switches off the GAL genes (Figure 17-20).
Two mechanisms have been proposed to explain the repressing effect of Tup1. First, Tup1 recruits histone deaxetylases. Second, Tup1 interacts dirxtly with the transcription machinery at the promoter and inhibits initiation.

62. FIGURE 17-20 Repression of the GAL1 gene in yeast.

63. Signal Transduction and the Control of Transcriptional Regulators

64. Signals Are Often Communicated to Transcriptional Regulators through Signal Transduction Pathways

65. In the case of NtrC, the signal induces a kinase that phosphorylates NtrC. This type of indirect signaling is an example of a signal transduction pathway.
In a signal transduction pathway, the initiating ligand is typically detected by a specific cell surface receptor: the ligand binds to an extracellular domain of the receptor and this binding is communicated to the intracellular domain.

66. Figure 17-21 show two examples of signal transduction pathways
Figure show two examples of signal transduction pathways. First is the SATA pathway (Figure 17-21a). The MAP kinase pathway that controls activators such as Jun(Figure 17-21b).

67. FIGURE 17-21 Two signal transduction pathways from mammalian cells.

68. Signals Control the Activities of Eukaryotic Transcriptional Regulators in a Variety of Ways
In eukaryotes, transcriptional regulators are not typically controlled at the level of DNA binding. Regulators are instead usually controlled in one of two basic ways:

69. Unmasking an Activation Region
Unmasking an Activation Region. This is done either by a conformational changes in the DNA bound activator, revealing a previously buried activating region; or by release of a masking protein that previously interacted with, and eclipsed, an activating region (Figure 17-22).

70. FIGURE 17-22 The yeast activator Gal4 is regulated by the Gal80 protein.

71. Transport Into and Out of the Nucleus
Transport Into and Out of the Nucleus. When not active, many activators and repressors are held in the cytoplasm. The signaling ligand causes them to move to the nucleus where they act.

72. Activators and Repressors Sometimes Come in Pieces

73. For example, the activator can come in pieces: the DNA-binding domain and activating region can be on separate polypeptides, which come tighter on DNA to form the activator. Protein complexes formingon DNA, and the nature of the complexcan determine whether the DNA-binding protein activates or represses nearby genes, such as glucocorticoid receptor (GR).

74. GENE “SILENCING” BY MODIFICATION OF HISTONES AND DNA

75. We now turn to mechanisms of gene silencing
We now turn to mechanisms of gene silencing. Silencing is a position effect—a gene is silenced because of where it is located, not in response to a specific environmental signal. Also, silencing can “spread” over large stretches of DNA, switching off multiple genes, even ones quite distant from the initiation event.

76. The most common form of silencing is associated with a dense form of chromatin called heterochromatin.
Telomeres and centromeres and other regions of the chromosome are in a heterochromatic state, and in which genes are found, such as in the silent mating-type locus in yeast. And in mammalian cells, about 50% of the genomes is estimated to be in some form of heterochromatin.

77. Transcription can also be silenced by methylation of DNA by enzymes called DNA methylases. This kind of silencing is not found in yeast but is common in mammalian cells.

78. Silencing in Yeast Is Mediated by Deacetylation and Methylation of Histones
We consider the telomere as an example (Figure 17-23).

79. FIGURE 17-23 Silencing at the yeast telomere.

80. Histone Modifications and the Histone Code Hypothesis
It has been proposed that a histone code exists. According to this idea, different patterns of modifications on histone tails can be “read” to mean different things.

81. The particular pattern of modifications at any given location would recruit specific proteins, the particular set depending on the number, type, and disposition of recognition domains those proteins carry.
Consider one simple case—lysine 9 on the tail of histone H3.

82. DNA Methylation Is Associated with Silenced Genes in Mammalian Cells
Some mammalian genes are kept silent by methylation of nearby DNA sequences. In fact, large regions of the mammalian genome are marked in this way, and often DNA methylation is seen in regions that are also heterochromatic (Figure 17-24).

83. FIGURE 17-24 Switching a gene off through DNA methylation and histone modification.

84. DNA methylation lies at the heart of a phenomenon called imprinting
DNA methylation lies at the heart of a phenomenon called imprinting. In a diploid cell, there are two copies of mast genes. In most cases, the two alleles are expressed at comparable levels. But there are a few cases where one copy of gene is expressed while the other is silent.Two well-studied examples are the human H19 and Igf2 genes (Figure 17-25).

85. FIGURE Imprinting.

86. Some States of Gene Expression Are Inherited through Cell Division even when the Initiation Signal Is No Longer Present

87. The inheritance of gene expression patterns, in the absence of either mutation or the initiation signal is called epigenetic regulation.
Nucleosome and DNA modifications can provide the basis for epigenetic inheritance. Consider a gene switched off by methylation of local histones.

88. DNA methylation is even more reliably inherited, as shown in Figure Thus, certain DNA methylases can methylate, at low frequency, previously unmodified DNA; but far more efficiently, so-called maintenance methylases modify hemimethylated DNA

89. FIGURE 17-26 Patterns of DNA methylation can be maintained through cell division.

90. EUKARYOTIC GENE REGULATION AT STEPS AFTER TRANSCRIPTION INITIATION

91. Some Activators Control Transcriptional Elongation rather than Initiation

92. At some genes there are sequences downstream of the promoter that cause pausing or stalling of the polymerase soon after initiation. At those genes, the presence or absence of certain elongation factors greatly influences the level at which the genes expressed.
One example is the HSP70 gene from Drosophila. This gene, activates by heat shock, is controlled by two activators working together.

93. The Regulation of Alternative mRNA Splicing Can Produce Different Protein Products in Different Cell Types
The sex of a fly is determined by the ratio of X chromosomes to autosomes. A femal results from a ratio of 1, and a male from a ratio of 0.5 (Figure 17-27).

94. FIGURE 17-27 Early Transcriptional regulation of Sxl in male and female flies.

95. It is Sxl protein itself, present in the female that directs splicing of the RNA made from Pm and ensures the inhibitory exon is spliced out. Functional Sxl protein continues to be made in females. That protein regulates the splicing of other RNAs in the female as well as its own (Figure 17-28).

96. FIGURE 17-28 A cascade of alternative splicing events determines the sex of a fly.

97. Expression of the Yeast Transcriptional Activator Gcn4 Is Controlled at the Level of Translation

98. Although it is a transcriptional activator, Gcn4 is itself regulated at the level of translation. In the presence of low levels of amino aids, the Gcn4 mRNA is translated. In the presence of high levels of amino acids, the Gcn4 mRNA is not translated (Figure 17-29).
The mRNA encoding the Gc4 protein contains four small open reading frames (called uORFs) upstream of the coding sequence for Gcn4 (Figure 17-29).

99. FIGURE 17-29 Translational control of Gcn4 in response to amino acid starvation.

100. RANs IN GENE REGULATION

101. Short RNAs can direct repression of genes with homology to those short RNAs. This repression, called RNA interference (RNAi), can manifest as translational inhibition of the mRNA, destruction of the mRNA or transcriptional silencing of the promoter that directs expression of that mRNA.

102. Double-Stranded RNA Inhibits Expression of Genes Homologous to that RNA
The discovery that simply introducing double-stranded RNA (dsRNA) into a cell can repress genes containing sequences identical to that dsRNA was remarkable in 1998 when it was reported.

103. Short Interfering RNAs(siRNAs) Are Produced from dsRNA and Direct Machinery that Switches Off Genes in Various Ways

104. Dicer is an RNAselⅢ-like enzyme that recognizes and digests long dsRNA
Dicer is an RNAselⅢ-like enzyme that recognizes and digests long dsRNA. These short RNAs inhibit expression of a homologous gene in three ways: they trigger destruction of its mRNA; they inhibit translation of its mRNA; or they induce chromatin modifications within the promoter that silence the gene. RISC

105. complex contains, in addition to the siRNAs themselves, various proteins including members of the Argonaut family, which are believed to interact with the RNA component.

106. There is another feature of RNAi silencing worth nothing—its extreme efficiency. Thus, very small amounts of dsRNA are enough to induce complete shutdown of target genes. While it remains unclear why the effect is so strong, it might involve an RNA-dependent RNA polymerase which is required in many cases of RNAi.

107. MicroRNAs Control the Expression of some Genes during Development

108. There is another class of naturally occurring RNAs, called microRNAs (miRNA), that direct repression of genes in the sane way as siRNAs. The miRNAs, typically 21 or 22 nts long, arise from larger precursors transcribed from non-protein encoding genes. These transcripts contain sequences that form stem loop structures, which are processed by Dicer. The miRNAs they produce lead to the destruction or translational repression of target mRNAs with homolog to the miRNA.

109. It is estimated that there are about 120 genes that encode miRNA precursors in worms, and 250 in humans. Often these miRNAs are expressed in developmentally regulated patterns, and, where characterized, their targets are typically mRNAs that encode regulatory proteins with important roles in the development of the organism in question.

110. Despites this, the mechanism of RNAi may have evolved originally to protect cells from any infectious, or otherwise disruptive, element that employs a dsRNA intermediate in its replicative cycle.