Mechanisms of Gene Regulation in Eukaryotes: Chromatin Remodeling and RNA Processing

1. Key Concepts • Changes in gene expression allow eukaryotic cells to respond to changes in the environment and cause distinct cell types to develop. • Eukaryotic DNA is packaged with proteins into structures that must be opened before transcription can occur. • In eukaryotes, transcription is triggered by regulatory proteins that bind to the promoter and to sequences close to and far from the promoter.

2. Key Concepts • Once transcription is complete, gene expression is controlled by: • Alternative splicing, which allows a single gene to code for several different products. • Molecules that regulate the life span of mRNAs. • Activation or inactivation of protein products. • Cancer can develop when mutations disable genes that regulate cell-cycle control genes.

3. Introduction • The regulation of gene expression is more complex in eukaryotes than in prokaryotes. • Differential gene expression is responsible for creating different cell types, arranging them into tissues, and coordinating their activity to form the multicellular society we call an individual.

4. Mechanisms of Gene Regulation—An Overview • Like prokaryotes, eukaryotes can control gene expression at the levels of transcription, translation, and post-translation. • Three additional levels of control are unique to eukaryotes: • Chromatin remodeling. • RNA processing. • Regulation of mRNA life span or stability.

6. Chromatin Remodeling • In eukaryotes, DNA is wrapped around proteins to create a protein-DNA complex called chromatin. • RNA polymerase cannot access the DNA when it is supercoiled within the nucleus. • The DNA near the promoter must be released from tight interactions with proteins before transcription can begin; this process is called chromatin remodeling.

7. RNA Processing and Control of mRNA Stability • Transcription results in a primary RNA transcript that must undergo RNA processing to produce a mature mRNA. • mRNA stability, or the life span of the mRNA, can also be used to control gene expression.

8. What Is Chromatin’s Basic Structure? • Chromatin has a regular structure with several layers of organization. • Chromatin contains nucleosomes—repeating, beadlike structures. • Nucleosomes consist of negatively charged DNA wrapped twice around eight positively charged histone proteins. • A histone protein called H1 functions to maintain the structure of each nucleosome. • Between each pair of nucleosomes there is a “linker” stretch of DNA.

9. What Is Chromatin’s Basic Structure? • H1 histones also may interact with each other and with histones in other nucleosomes to form a tightly packed structure called a 30-nanometer fiber. • These 30-nanometer fibers in turn may form higher-order structures. • Chromatin’s elaborate structure not only allows the DNA to be packaged in the nucleus, it also plays a key role in regulating gene expression.

11. Chromatin Structure Is Altered in Active Genes • As in bacteria, eukaryotic DNA has sites called promoters where RNA polymerase binds to initiate transcription. • Studies support the idea that chromatin must be relaxed or decondensed for RNA polymerase to bind to the promoter.

12. “Closed” DNA Is Protected from DNase • DNase is an enzyme that cuts DNA at random locations. • The enzyme cannot cut DNA when it is tightly complexed with histones.

14. Histone Mutants • Studies of mutant brewer’s yeast cells led biologists to hypothesize that the lack of histone proteins prevents the assembly of normal chromatin. • These data suggest that the default state of eukaryotic genes is to be turned off. This is a mechanism of negative control. • A form of positive control must be at work to open up DNA at promoter regions for gene expression to occur.

15. How Is Chromatin Altered? • Two major types of protein are involved in modifying chromatin structure: • ATP-dependent chromatin-remodelingcomplexes reshape chromatin. • Other enzymes catalyze the acetylation (addition of acetyl groups) and methylation (addition of methyl groups) of histones. • Acetylation of histones is usually associated with activation of genes. Methylation can be correlated with either activation or inactivation.

16. How Is Chromatin Altered? • One type of acetylation enzyme is called histone acetyl transferases (HATs). They add negatively charged acetyl groups to the positively charged lysine residues in histones. • This acetylation reduces the positive charge on the histones, decondensing the chromatin and allowing gene expression. • Enzymes called histone deacetylases (HDACs) then remove the acetyl groups from histones. This reverses the effects of acetylation and allows chromatin condensation.

18. Chromatin Modifications Can Be Inherited • The pattern of chemical modifications on histones varies from one cell type to another. • The histone code hypothesis contends that precise patterns of chemical modifications of histones contain information that influences whether or not a particular gene is expressed. • Daughter cells inherit patterns of histone modification, and thus patterns of gene expression, from the parent cells. • This is an example of epigenetic inheritance, patterns of inheritance that are not due to differences in gene sequences.

19. Regulatory Sequences and Regulatory Proteins • Eukaryotic promoters are similar to bacterial promoters. There are three conserved sequences and each eukaryotic promoter has two of the three. • The most common sequence is the TATA box. • All eukaryotic promoters are bound by the TATA-binding protein (TBP).

20. Some Regulatory Sequences Are Near the Promoter • Regulatory sequences are sections of DNA that are involved in controlling the activity of genes. When regulatory proteins bind these sequences, they cause gene activity to change. • Some eukaryotic regulatory sequences are similar to those in bacteria, others are very different. • Yeast metabolize the sugar galactose. In the presence of galactose, transcription of the five galactose-utilization genes increases dramatically.

21. Some Regulatory Sequences Are Near the Promoter • Mutant cells fail to produce any of the enzymes required for galactose metabolism, leading to three hypotheses: • The five genes are regulated together even though they are on different chromosomes. • Normal cells have a CAP-like regulatory protein that exerts positive control over the five genes. • The mutant cells have a loss-of-function mutation that completely disables the regulatory protein.

22. Some Regulatory Sequences Are Near the Promoter • Promoter-proximal elements are located just upstream of the promoter and the transcription start site, and have sequences that are unique to specific genes, providing a mechanism for eukaryotic cells to exert precise control over transcription.

24. Some Regulatory Sequences Are Far from the Promoter • While exploring how human immune system cells regulate genes that produce antibodies, Susumu Tonegawa and colleagues discovered that the intron, rather than the exon, contains a regulatory sequence required for transcription to occur. • The results were remarkable because: • The regulatory sequence was far from the promoter. • The regulatory sequence was downstream, rather than upstream, from the promoter. • Regulatory elements that are far from the promoter are termed enhancers.

25. Characteristics of Enhancers • All eukaryotes have enhancers. Enhancers can be more than 100,000 bases away from the promoter. • Enhancers can be located in introns or in untranscribed 5' or 3' sequences flanking the gene they regulate. • There are many types of enhancers and many genes have more than one enhancer. • Enhancers can work even if their normal orientation is flipped or if they are moved to a new location on the same chromosome.

27. Enhancers and Silencers • Enhancers function using positive control—when regulatory proteins bind to enhancers, transcription begins. • Silencers are similar to enhancers but they function in negative control—they repress rather than activate gene expression. • When regulatory proteins bind to silencers, transcription is shut down. • The discovery of enhancers and silencers resulted in redefining the gene as the DNA that codes for a functional polypeptide or RNA molecule and the regulatory sequences required for expression.

28. What Role Do Regulatory Proteins Play? • Different cell types express different genes because they have different histone modifications and contain different regulatory proteins. • Differential gene expression is a result of the production or activation of specific regulatory proteins. Eukaryotic genes are turned on when specific regulatory proteins bind to enhancers and promoter-proximal elements; the genes are turned off when regulatory proteins bind to silencers or when chromatin remains condensed. • This leads to certain proteins being produced only in certain types of cells.

29. The Initiation Complex • Two broad classes of proteins bind to regulatory sequences at the start of transcription: • Regulatory transcription factors bind to enhancers, silencers, and promoter-proximal elements, and are responsible for the expression of particular genes in particular cell types and at particular stages of development. • Basal transcription factors interact with the promoter and are not restricted to particular cell types. Although they are required for transcription, they do not regulate gene expression.

30. The Mediator Complex • Proteins that make up the mediator complex also have a role in starting transcription. • The mediator complex does not bind to DNA. Instead, it creates a physical link between regulatory transcription factors and basal transcription factors.

31. Transcription Initiation in Eukaryotic Cells Step 1: Regulatory transcription factors bind to DNA and recruit the chromatin-remodeling complexes, or HATs. Step 2: Chromatin remodeling results in loosening of the chromatin structure, exposing the promoter region. Step 3: Additional regulatory transcription factors bind enhancers and promoter-proximal elements; they also interact with basal transcription factors that bind the promoter. Step 4: All of the basal transcription factors form the basaltranscriptioncomplex. This complex recruits RNApolymerase so that transcription can begin.

33. Transcription Initiation • Construction of the basal transcription complex depends on interactions among regulatory transcription factors that are bound to enhancers, silencers, and promoter-proximal elements. The result is a large, multimolecular machine that is positioned at the start site and able to start transcription. • The completed complex may contain as many as 60 proteins.

34. Post-Transcriptional Control • Once an mRNA is made, a series of events must occur if the final product is going to affect the cell. • Each of these events offers an opportunity to regulate gene expression. • The three major control points at this stage are as follows: • Splicing mRNAs in various ways. • Altering the rate at which translation is initiated. • Modifying the life span of mRNAs and proteins after translation has occurred.

35. Alternative Splicing of mRNAs • Introns are spliced out of primary RNA transcripts while it is still in the nucleus. • During splicing, changes in gene expression are possible because selected exons may be removed from the primary transcript along with the introns. As a result, the same primary RNA transcript can yield more than one kind of mature, processed mRNA, consisting of different combinations of transcribed exons. • This alternative splicing leads to production of different proteins.

37. Control of Alternative Splicing • Alternative splicing is regulated by proteins that bind to the mRNAs in the nucleus and interact with the spliceosomes. • Over 90% of human sequences for primary mRNA transcripts undergo alternative splicing. • Thus, although humans have just 20,500 genes, it is estimated that we can produce at least 50,000 proteins. • Genes are now considered to be the coding and regulatory sequences that direct the production of one or more related polypeptides or RNAs.

38. mRNA Stability and RNA Interference • In the cytoplasm, additional regulatory mechanisms control gene expression. • The stability of mRNAs in the cytoplasm is highly variable. Some are degraded rapidly, allowing for only a short period of translation, while others are quite stable. • In many cases, the life span of an mRNA is controlled by RNA interference.

39. mRNA Stability and RNA Interference • In the process of RNA interference, specific mRNAs are targeted by tiny, single-stranded RNA molecules called microRNAs (miRNAs). • After binding to proteins called a RNA induced silencing complex (RISC) protein complex, these miRNAs bind to complementary sequences in the mRNA. • Once part of an mRNA becomes double stranded in this way, specific proteins degrade the mRNA or prevent it from being translated into a polypeptide. • About 20–23% of all animal and plant genes are regulated by miRNAs.

41. How Is Translation Controlled? • Many miRNAs interfere directly with translation. • In other cases, mechanisms that do not involved miRNAs are responsible for controlling the timing or rate of translation. • For example, regulatory proteins may bind to mRNAs or ribosomes to regulate translation. • In addition, in response to viral infection, translation can be slowed or stopped by phosphorylation of a certain ribosomal protein.

42. Post-Translational Control • Mechanisms for post-translational regulation allow the cell to respond to new conditions rapidly by activating or inactivating existing proteins. • Regulatory mechanisms occurring late in the flow of information from DNA to RNA to protein involve a trade-off between speed and resource use.

43. Mechanisms of Post-Translational Control • Chaperone proteins can regulate protein folding. • Enzymes may modify proteins by adding carbohydrate groups or cleaving off certain amino acids. • Proteins may be activated or deactivated by phosphorylation. • Targeted protein destruction. • Ubiquitin(UB) tags are added to cyclin proteins and are recognized by a multi-molecular machine called a proteasome, which cuts the proteins into short segments.

44. Comparing Gene Expression in Bacteria and Eukaryotes • There are four primary differences between gene expression in bacteria and eukaryotes: • Packaging: Chromatin structure provides a mechanism of negative control in eukaryotes that does not exist in bacteria. • Alternative splicing: Primary transcripts in eukaryotes must be spliced. This does not occur in bacteria. • Complexity: Transcriptional control is much more complex in eukaryotes than in bacteria. • Coordinated expression: In bacteria, genes may be organized into operons; such operons are rare in eukaryotes.

46. Linking Cancer with Defects in Gene Regulation • Abnormal regulation of gene expression can lead to developmental abnormalities and cancer. • Cancers result from defects in the proteins that control the cell cycle. • All cancers are characterized by uncontrolled cell growth. Cancers become dangerous when they metastasize and stimulate the production of their own vascular supply.

47. Causes of Uncontrolled Cell Growth • Many cancers are associated with mutations in regulatory transcription factors. These mutations lead to cancer when they affect one of two classes of genes: (1) genes that stop or slow the cell cycle, and (2) genes that trigger cell growth and division by initiating specific phases in the cell cycle.

48. Causes of Uncontrolled Cell Growth • Tumor suppressor genes slow or stop the cell cycle. • If a mutation disrupts the normal function of a tumor suppressor gene, cells are released from this negative control of the cell cycle. • Genes that trigger specific phases in the cell cycle are called proto-oncogenes (literally means “early or basic genes that can turn into a cancer”). Code for proteins that help regulate cell growth. • Defects in the regulation of proto-oncogenes cause these genes to stimulate growth at all times. These types of mutations convert proto-oncogenes into oncogenes that promote cancer development, in that proteins (which ones do you think?) for proper division are not produced.

49. p53: A Case Study • The transcription factor p53 acts as a tumor suppressor and has been shown to be abnormal in many types of cancer. • When DNA damage is detected during cell division, the p53 protein binds directly to DNA, arresting the process of division until repairs are made.

50. p53: A Case Study • Mutations that inactivate p53 allow damaged cells to continue through the cell cycle without repairing the DNA damage. • This dramatically elevates the mutation rate and increases the likelihood that a cancer-causing mutation may result. • The role of p53 in preventing cancer is so fundamental that biologists call this gene “the guardian of the genome.”