ADDITION BY ME CHAPTER 20 (GENES X) PROMOTERS AND ENHANCERS

1. ADDITION BY ME CHAPTER 20 (GENES X) PROMOTERS AND ENHANCERS

2. Eukaryotic gene control: purposes and general principles • Unlike bacterial cells and most single cell eukaryotes, cells in multicellular organisms have relatively few genes that are reversibly regulated by environmental conditions • However, gene control in multicellular organisms is important for development and differentiation, and is generally not reversible

3. Regulatory elements in eukaryotic DNA often are many kilobases from start sites • The basic principles that control transcription in bacteria also apply to eukaryotic organisms: transcription is initiated at a specific base pair and is controlled by the binding of trans-acting proteins (transcription factors) to cis-acting regulatory DNA sequences • However, eukaryotic cis-acting elements are often much further from the promoter they regulate, and transcription from a single promoter may be regulated by binding of multiple transcription factors to alternative control elements.

4. Promoter sequence components can be • identified by: • Transcription control sequences can be identified by analysis of a 5’-deletion series. • Finding precise consensus sequence that is bound by a particular factor in multiple promoters. • Introduction of point mutations at particular base pairs and testing the promoter function. • Finding the proteins which bound to promoters by footprinting and mutation.

5. Transcription control sequences can be identified by analysis of a 5’-deletion series.

6. REPORTER GENES The primary objective is to identify gene regulatory sequences by testing a large number of deletion and point mutants in an in vivo cell transfection assays. Promoter mapping studies can be done by creating artificial “reporter gene” by fusing regulatory region of the test gene to a heterologous gene coding sequences that direct the synthesis of a readily detectable protein product. The steady state level of the reporter protein in the transfected cell is directly correlated to the steady state level of reporter mRNA, then it is possible to use protein based reporter assays for promoter mapping experiments.

7. Repoter gene constructs • When trying to determine what are the cis-acting regulatory regions that control the expression (transcription) levels of a gene, is usually routine to clone a large piece of DNA containing these sequences and hook it to a downstream sequence that encodes an easy-to-measure protein called a reporter. • This is a lot more convenient and accurate than trying to estimate the absolute levels of mRNA. • In vivo transgene reporter expression driven by the promoter of interest will reveal the temporal/spatial pattern of expression of the gene, measured by an easier method than the alternative in situ hybridization.

8. Reporter genes: • 1. must encode a protein activity that is similar to one already present in the cell. • 2. The protein assay should be sensitive enough, reproducible and easy to perform. • 3. The reporter protein function should not interfere with host cellular processes in a way that will alter intracellular signaling pathways or metabolic rates.

9. Repoter gene constructs: • The most used reporters are: • bacterial b-galactosidase, • chloramfenicol acetyl transferase (CAT), • fire-fly luciferase • and most recently, the green fluorescent protein (GFP) which allows measurements in living cells or organisms. • Part of the whole of a protein rather than a promoter can also be fused in frame to this reporters to find out where it localizes. Reporter gene must not being expressed endogenously in the cell types that you are working on, so their background should be null.

13. reporter gene (e.g. luciferase) How does one identify transciptional control mechanisms? promoter transcriptional unit • promoter & transcriptional unit are independent

14. reporter gene (e.g. luciferase) luciferase activity 1 2 3 4 5 Use reporter to identify promoter sequence 1 promoter? transcriptional unit 2 promoter? 3 promoter? 4 promoter? 5 promoter?

15. luciferase activity 1 2 3 4 5 Use reporter to identify promoter sequence = AAAATCACCCCACTGCAA promoter

16. Purify Transcription Factor Affinity chromatography AAAATCACCCCACTGCAA pour nuclear extract allow to bind elute transcriptionfactor affinity column

17. Transcriptional Reporter Gene Assay Start site of Transcription -2000 +1 Gene Promoter Reporter Gene ATG LDL Receptor Promoter -141-- +36 Translational Start Site Chloramphenicol Acetyltransferase Luciferase

18. Construction and analysis of a 5-deletion series

19. Identification of transcription-control elements with linker mutants

20. Using reporter protein to determine the pattern of a gene’s expression. In this example the coding sequence for protein X is replaced by the coding sequence for Y protein. B) Various fragments of DNA containing candidate regulatory sequences are added in combinations. The recombinant DNA molecules are then tested for expression after their transfection into a variety of different cell types of mammalian cells, and the result is summarized in C. For experiments in eukaryotic cells, two commonly used reporter proteins are enzymes b- galactosidase (b-gal) and green fluorescent protein or GFP. as

21. METHODS FOR STUDYING DNA-PROTEIN INTERACTIONS DNA footprinting Electrophoretic mobility shift assay C. Chromatin immunoprecipitation assay

22. DNA FOOTPRINTING identify protein-DNA interactions

23. DNase I footprinting assays identify protein-DNA interactions

26. Electrophoretic mobility shift assayidentify protein-DNA interactions

28. Gel-shift assays identify protein-DNA interactions Fragment bound to proteins are retarded The free DNA fragment migrate rapidly to the bottom of the gel

29. ChIP (Chromatin ImmunoPrecipitation) Chromatin immunoprecipitation, or ChIP, refers to a procedure used to determine whether a given protein binds to a specific DNA sequencein vivo

31. Chromatin immunoprecipitation This methodology allows the identification of sites in a genome that are occupied in vivo by a gene regulatory protein. The identities of the precipitated, amplified DNA fragments can be determined by hybridizing the mixture of fragments to DNA microarrays.

32. ChIP: Chromatin immunoprecipitation Shearing of DNA (formaldehyde) Antibody against acetyl’ed H3 & H4 Gene promoter is amplified using primers specific for that region

35. From DNA to RNA Genes can be expressed with different efficiencies.Gene A is transcribed and translated much more efficiently than gene B. This allows the amount of protein A in the cell to be much greater than that of protein B.

36. DNA is transcribed by the enzyme RNA polymerase. The RNA polymerase (pale blue) moves stepwise along the DNA, unwinding the DNA helix at its active site. As it progresses, the polymerase adds nucleotides (here, small “T” shapes) one by one to the RNA chain at the polymerization site using an exposed DNA strand as a template. The RNA transcript is thus a single-stranded complementary copy of one of the two DNA strands. The polymerase has a rudder that displaces the newly formed RNA, allowing the two strands of DNA behind the polymerase to rewind. A short region of DNA/RNA helix (approximately nine nucleotides in length) is therefore formed only transiently, and a “window” of DNA/RNA helix therefore moves along the DNA with the polymerase. The incoming nucleotides are in the form of ribonucleoside triphosphates (ATP, UTP, CTP, and GTP), and the energy stored in their phosphate–phosphate bonds provides the driving force for the polymerization reaction (see Figure 5–4).

40. Cells produce several types of RNA

41. RNA polymerase I rRNA transcription • RNA Polymerase II mRNA transcription • RNA Polymerase III tRNA and other small RNAs • Promoters • RNA polymerase I and RNA Polymerase II are mostly upstream of the start point. • RNA Polymerase III lie downstream of the start point. • Each promoter contains characteristic sets of short conserved sequences that are recognized by the appropriate class of factors. RNA pol I and III recognize restricted set of promoters, rely upon a small number of accessory factors. • RNA pol II show more variation in sequence, cis acting elements are spread out over a region of >200bp.

42. Mammalian gene Gene Enhancer Promoter Start Exon Intron Exon Termination Promoter: DNA sequence located 5’ to a gene that is the sites where transcription is initiated. Example: TATA box. It is the binding sites for transcription factors. Enhancer: Eukaryotic control element that can increase expression of a gene. Located some distance from the gene either up- or downstream. Human gene number estimate: 28,000–34,000 genes.

43. Figure 24.01: Transcription is controlled by a promoter and enhancer.

44. Figure 24.02: RNA polymerase has >10 subunits.

45. RNA POLYMERASE I PROMOTER • It transcribes genes for rRNA from a single type of promoter • It consists of a bipartite sequence • The core promoter and upstream control element (UCE) • The core promoter: • Surrounds the start point (-45 to +20) • It is sufficient to start transcription • UCE: -180 to -170 • Both regions are rich in G.C bases • They are ~85% identical

46. RNA polymerase I requires two factors: • The factor UBF1 wraps DNA around a protein structure to bring the core and UPE into proximity. • SL1 includes the factor TBP that is involved in initiation by all three RNA polymerases. • RNA polymerase binds to the UBF1-SL1 complex at the core promoter. • SL1 consists of 4 proteins: • 3 TBP-associated factors (TAF1) + 1 TBP (TATA box binding protein) • TBP is necessary to start transcription for RNA polymerase II and III as well. • TBP is well conserved between species • TBP does not directly bind to G.C rich DNA. Other factors are also necessary.

47. Figure 24.05: Pol I promoters have two sequence components.

48. RNA Polymerase III promoter: The promoters have two general classes Promoters for 5S and tRNA genes are internal. They lie downstream of the start point between positions +55 and +80 within the gene. The promoters for snRNA genes lie upstream of the start point. There are two types of internal promoters: Type I and Type II Type I Box A and Box C Type II Box A and Box B

49. There are three accessory factors: TFIIIA Zinc finger protein TFIIIB TBP and two proteins (B” and BRF proteins) TFIIIC contains 5 subunit, >500 kD • TFIIIA and TFIIIC do not fully affect the initiation reaction (assembly factors), their role is to bind TFIIIB at the right location. • TFIIIB is crucial. It allows RNA pol III to bind at the start site point (initiation factor or positioning factor).

50. Internal promoters: • have short consensus sequences located within the transcription unit • cause initiation to occur a fixed distance upstream • Upstream promoters contain three short consensus sequences upstream of the startpoint that are bound by transcription factors. Figure 24.06: Pol III promoters may be downstream of startpoint.