1. NUTRITION AND GENE EXPRESSION February 19, 2016 PROMOTER MUTATIONS Mutations in the REGULATORY part of the gene (usually called the PROMOTER) show us the importance of those DNA sequences for the control of gene activity. To study biochemistry, we need tools to study these complex pathways. The data from this study illustrates the effects of promoter mutations, and shows useful methods to study this question.

2. CASE STUDY A mutation that led to low levels of human growth hormone (HGH), and impaired growth. How was the key mutation found, that caused this disorder? In this case, there were low levels of active growth hormone in the bloodstream. What DIFFERENT mutations might lead to low levels of circulating HGH?

3. These findings are taken from: Decreased Expression of the GHRH Receptor Gene Due to a Mutation in a Pit-1 Binding Site ROBERTO SALVATORI, XIAOGUANG FAN, PRIMUS E. MULLIS, AZEB HAILE, AND MICHAEL A. LEVINE Molecular Endocinrology, Vol 16: 450-458, 2002

4. BRIEF SUMMARY: Gene Expression in Eukaryotes This very typical diagram DOES NOT SHOW THE CONTROL REGION OF THE GENE, WHICH LEADS TO CONFUSION!

5. GH Y GHRH Growth hormone is released When growth-hormone-releasing-hormone (GHRH) binds to the receptor on the pituitary cell membrane, growth hormone (GH) is released from a vesicle. If the releasing receptor is absent or defective, GH release can be impaired. This paper examined the gene for the RECEPTOR. PITUITARY CELL

6. The Growth-Hormone-Releasing-Hormone Receptor gene is on chromosome 7 (at location 7p14), as shown in the diagram. The investigators already found that there was a defect in the gene on one chromosome. In the protein made by that gene, there was a glutamic acid (negative charge) instead of lysine (positive charge) at AA position 329, so the receptor was made, but did not work. So what was the defect on the other chromosome? Location of gene for the receptor

7. THE PROMOTER FOR THE GENE FOR THE HUMAN GROWTH HORMONE RELEASING RECEPTOR: it’s about 2600 nucleotides long. The receptor is located on the surface of pituitary cells. It responds to a 44-aa peptide called SOMATOCRININ or HGRH. If this receptor is not present and functional, HGH is not released, and very short stature is the result. The transcription factor Pit-1 binds to the promoter which controls expression of this receptor at the sequence: TATGCAA. Sites in RED bind Pit-1, which is a TF with multiple binding sites on the promoter.

8. It’s been shown that if promoter is normal between –400 and –20, then good expression of the gene will occur. Therefore, investigators looked at the promoter sequence near to the start site for transcription of the receptor gene. REMEMBER: The gene includes the promoter (which regulates expression of the gene) and the sequencewhich codes for the protein itself.

9. NORMAL Pit-1 site: TATGCAA MUTANT Pit-1 site: TATGCCA What did they find? In a child with short stature, they found a key mutation in the PROMOTER for the gene that makes the growth hormone releasing receptor, at position about (-120) from the transcription start site. The change was from an AT base pair to a CG base pair, in the DNA sequence in the promoter, Therefore, this child might not be able to release normal levels of HGH to the bloodstream. How can this be tested?

10. How we assess the SIGNIFICANCE of that mutation from A  C at ( –124) in the promoter? For that purpose, we need to develop many TOOLS to examine how gene expression works. For example: if a person does not release enough HGH, there might be MANY variations in the gene, but majority of those variations are harmless. We need strategies to determine which mutations are really important!

11. If you have transcription factor bound to the promoter, any gene that is downstream will be transcribed. This is the most important research technique to study mechanisms of gene expression.

12. ALL THESE GENES (PROTEINS X, Y, AND Z) HAVE THE SAME REGULATORY DOMAIN ARTIFICIALLY ATACHED UPSTREAM OF THE CODING REGION ATTCG Protein X DNA Protein Y DNA Protein Z DNA RNA for X RNA for Y RNA for Z WITH TRANSCRIPTION FACTOR BOUND UPSTREAM, RNA POL-II WILL MAKE primary RNA transcript FOR ANY DOWNSTREAM DNA

13. LUCIFERASE CONSTRUCTS Small, artificial genes that can be placed inside cells. In the cell, the gene can be activated and make mRNA for luciferase, which is then translated by the ribosomes. The gene has to have a PROMOTER and CODING REGION.

14. Requires the enzyme LUCIFERASE Measured as a burst of light!

15. TOTAL LIGHT GENERATED ADD ATP LIMITED AMOUNT OF LUCIFERASE ABUNDANT LUCIFERASE

16. LUCIFERASE VECTORS: to examine if a TF in a cell activates transcription from a promoter ORI Luc coding domain: will make mRNA for Luc, if transcribed. The mRNA then makes the Luc protein, which is measured Promoters, with different DNA sequences

17. We compare both TATGCAA and mutant TATGCCA in the promoter. ORI Luc coding domain: will make mRNA for Luc, if transcribed Promoters, with different DNA sequences: Pit-1 might bind here PLACE THIS CONSTRUCT IN CELLS THAT MAKE THE PIT-1 PROTEIN

18. TOTAL LIGHT GENERATED CELL EXRACTS ADD ATP MUTANT PROMOTER: TATGCCA NORMAL PROMOTER: TATGCAA

19. These LUCIFERASE-containing artificial genes were placed inside cells that made the Pit-1 transcription factor. But if there were MUTATIONS in the promoter, very small amounts of luciferase were made. The Pit-1 transcription factor was not able to properly bind to the mutant promoter.

20. No promoter in vector ATGCAA TATGCAA in promoter TATGCCA in promoter Mutation elsewhere in promoter LUCIFERASE EXPESSION

21. CONCLUSION : This construct, containing the Luc gene, was placed inside cells that contained the Pit-1 transcription factor. HOWEVER: a small single base-substitution in the promoter stopped Pit-1 from binding, and Luc mRNA was not transcribed from the construct. Correspondingly, the cells made much less luciferase. THIS TECHNIQUE ALLOWED THE INVESTIGATORS TO FIND THE CRITICAL MUTATION THAT CAUSED LOW LEVELS OF HGH TO BE RELEASED. Most of what we know about transcription factor interactions with promoters comes from studies like this with “artificial” chromosomes, with variations in the promoter.