Gene Regulation and Pathological Development Studies Using Mouse models

1. Gene Regulation and Pathological DevelopmentStudies Using Mouse models Yaowu Zheng Transgenic Research Center zhengyw442@nenu.edu.cn (0431)85098583

2. Materials and Lecturers • Oct. 9: Introduction to Gene regulation, Zheng • Oct. 16: Gene expression and development, Zheng • Oct. 23: Gene expression and coagulation system, Zheng • Oct. 30: Gene expression and immune system, Professor Zhang • Nov. 6: Gene expression and cancer, Zheng • Nov. 13: Gene expression and vascular system, Professor Zhang • Nov. 20: Gene expression and obesity, Dr. Xiaodan Lu • Nov. 27: Gene expression and cardiovascular disease, Zheng • Dec. 4: Gene expression and reproduction, Yan Ji • Dec. 11: Transgenic technology, Zheng Lecturers: • Yan Ji: Ph.D student in Zheng lab. • Xiaodan Lu: Postdoctoral fellow in Jilin University, former Ph.D graduate student from Zheng’s lab • Professor Zhang: Assistant professor, Ph.D graduate from Nanking University • Yaowu Zheng: Professor in Transgenic Research Center, NENU

3. Gene Regulation

4. Introduction to gene regulation

5. Contents • Basic gene regulation • Higher level regulation • Prokaryotic regulation • Eukaryotic regulation • Regulation and development • Gene regulation and disease • Application of gene regulation • Gene regulation studies using transgenic mice

6. CENTRAL DOGMA • Genetic information always goes from DNA to RNA to protein Updated Central Dogma

7. Biological sequence information Primary structure in living organisms • 3 linear biopolymers: • Polydeoxyribonucleotide= DNA • Polyribonucleotide= RNA • Polypeptide=Proteins • 3 classes of information transfer suggested by the dogma: • General case • Replication: DNA → DNA, • Transcription: DNA → RNA, • Translation: RNA → protein (general) • Special case • Reverse transcription: RNA → DNA, • Viral: RNA → RNA, • Non-natural: DNA → protein • Unknown case • protein → DNA, • protein → RNA, • protein → protein? prions

8. Regulation at DNA levels Double helix Structure

9. Prokaryotic Gene Regulation • Combination of a promoter and a gene or genes is called an OPERON • Operon is a cluster of genes encoding related enzymes that are regulated together and performed one related processes • Operon consists of • A promoter site where RNA polyerase binds and begins transcribing the message. • Genes coding protein from the message (mRNA) is called structural genes. • One mRNA codes for more than one protein is called polycistronic. • An upstream region (a gene) that makes a protein called repressor. The gene codes for repressor is called regulatory gene. The repressor is like a transcription factor in eukaryotes. • Active repressor sits a site between promoter and structural genes and blocks transcription, This site is called the operator which works like an enhancer or silencer in eukaryotes.

10. Types of Operons • Inducible Operon • e.g lac operon • Repressible Operon • e.g trp operon

11. Trp Operon • E. coli uses several proteins encoded by a cluster of 5 genes to manufacture the amino acid tryptophan • All 5 genes are transcribed together as a unit called an operon, which produces a single long piece of mRNA for all the proteins • RNA polymerase binds to a promoter located at the beginning of the first gene and transcribe the genes in sequence • A trp repressor protein is made constantly (constitutively). • When tryptophan is present in the cell, it binds to repressor and activates it, so it can binds operator and tell operon to stop making tryptophan. • When tryptophan is absent, the trp repressor is inactive and can not binds to operator, so trp operon is making all the necessary proteins for tryptophan synthesis.

12. Trp Operon

13. Tryptophan Gene Regulation (Negative control)

14. Lac Operon • The lac Operon • Regulates lactose metabolism • It turns on when lactose is present & glucose is absent. • Lactose is a disaccharide, a combination of Galactose & Glucose • To Ferment Lactose E. coli Must: • Transport lactose across cell membrane • Separate the two sugars. To do that it needs three enzymes, it makes them all at once! • 3 Genes Turned On & Off Together: • Lac Z code for b-galactosidase • LacY codes for permease that allows lactose to enter cell • LacA code for enzyme that acetylates lactose

15. Lac Operon • Near the lac operon is another gene, called lacI that codes for the lac repressor protein • The lac repressor gene is expressed “constitutively”, meaning that it is always on. • Just upstream from the transcription start point in the lac operon are two regions called the operator (o) and the promoter (p). • Operator is the DNA sequence that repressor binds. • The promoter is the site where RNA polymerase binds and starts transcription. • Operator and promoter are “cis” or associated to lac operon. • Lac repressor protein is “trans” to lac operon, since the repressor is diffusible and can bind to other lac operator sequences. • In the presence of lactose, the repressor binds to lactose, changes conformation and floats away from the operator. RNA polymerase can bind to the promoter and transcribed z, y and a. Permease allow lactose to enter the cell, b-galactosidase digests the lactose and transacetylase modify galactose. • When level of lactose drops, the released repressor binds to the operator DNA again and blocks lac operon

16. Lac Operon

17. Prokaryotic Translational Control • Shine-Dalgarno sequence is generally located 8 basepairs upstream of the start codon AUG • The six-base consensus sequenceAGGAGG helps to recruit the ribosomes to the mRNA to initiate protein synthesis • The complementary sequence (CCUCCU), called the anti-Shine-Dalgarno sequence, is located at the 3' end of the 16SrRNA in the 30S ribosome subunit.

18. Eukaryotic Gene Regulation • A multi-cell organism may contain millions of cells. • Virtually every cell in your body contains 1 or 2 complete set of genes • But they are not all turned on at all the time or in every tissues • Each cell or tissue in your body expresses only a small subset of genes at any time • During development, different cells express different sets of genes in a precisely regulated fashion • Some genes are expressed only at certain conditions

19. Different morphology, same genome Neuron and lymphocyte

20. Multilevel Gene Regulation in Eukaryotes • Features of Eukaryotic Genomes • Gene structure of eukaryotes • The types of regulation in eukaryotes • Multi-level of gene expression and regulation

21. Chromatin Chromosome

22. Nucleosome: Two of each H2A, H2B, H3 and H4 subunits in the core Average 200 bp 140bp in core area

23. Genome Level Regulation • Epigenetic Modification • Developmental methylation and Acetylation • DNA folding and unfolding • X-chromosomal inactivation • Chromosomal remodeling • Replication

24. Unpredictable Gene Regulation at DNA Levels • Gene Deletion • Gene Duplication • DNA Rearrangement • Gene Amplification • Chemical Modification • Environmental DNA damage, like UV

25. DNA Replication • In the Central Dogma, DNA replication occurs in order to faithfully transmit genetic material to the progeny. • Replication is carried out by a complex group of proteins called the replisome • Replisome consists of a helicase that unwinds the superhelix as well as the double-stranded DNA helix • DNA polymerase and its associated proteins insert new nucleotides in a sequence specific manner, like copy machine. • This process typically takes place during S phase of the cell cycle.

26. Epigenetic Modification • Epigenetics is the study of heritable changes in gene expression or cellular phenotype caused by changes other than underlying DNA sequence • Epigenetic changes are preserved when cells divide. • DNA methylation is important in the control of gene transcription and chromatin structure. • The epigenetic changes in eukaryotic biology is active in the process of cellular differentiation • Methylation of mRNA was demonstrated having a critical role in human energy homeostasis in 2011

27. DNA Methylation • Variation in methylation states of DNA can alter gene expression levels significantly. • Methylation variation usually occurs through the action of DNA methylases. • When the change is heritable, it is considered epigenetic. • When the change in information status is not heritable, it would be a somatic epitype. • The effective information content has been changed by means of the actions of a protein or proteins on DNA, but the primary DNA sequence is not altered. CpG islands • Genomic regions that contain a high frequency of CG dinucleotides. • CpG islands particularly occur at or near the transcription start site of housekeeping genes. • Housekeeping gene:- • A gene is required for basic functions the sustenance of the cell. • Housekeeping genes are constitutively expressed • Luxury gene: • A gene has specialized functions expressed in large amounts in particular cell types or “inducible”.

28. TF RNA pol Unmethylated CpG island TF RNA pol CH3 CH3 CH3 Methylated CpG island Gene regulation by Methylation Active transcription Repressed transcription

29. TF Histone modification  Methylation Acetylation

30. Mechanisms of gene regulation • Histone acetylation directly attracts transcription factors • Histone acetylation further attracts co-activator proteins

31. Eukaryotic Gene Regulation • Key Concept: • Eukaryotic genes are controlled in a group or Individually at different levels • Have many regulatory sequences • Are much more complex than prokaryotic genes

32. Prokaryotes Genes controlled in group by operons 27% of E. coli genes are controlled by operons Housekeeping genes not controlled by operons Simultaneous transcription and translation Eukaryotes No operons, but they still need to coordinate regulation Genes are fragmented into exons and introns More kinds of control elements, many levels of control Separated transcription and translation location and control Epigenetic regulation and Chromatin remodeling Tissue-specific, developmental and inducible regulation Comparisons

33. Basics of eukaryotic gene regulation • Specific transcription factors bind to regulatory elements (enhancers and silencers) and regulate transcription • Regulatory elements may be tissue specific and will activate their gene only in one kind of tissue • Regulatory elements may be developmental and only expressed at certain stages • Regulatory elements may be inducible and expressed only at certain condition • Sometimes the expression of a gene requires the function of two or more different regulatory elements

34. Eukaryotic Gene Regulation • Monocistronic: one mRNA, one protein • Basic promoter: TATA box, CAT box, GC-rich • Enhancers: Distance and orientation independent • Silencers: Distance and orientation independent • Transcription factors: Contain at least 2 domains:_Enhancer specific DNA binding and activation (silencing) of RNA polymerase • Intron and exons: Number and size variable • 3 RNA polymerases in eukaryotes • RNA pol I for ribosomal RNAs • RNA pol II for mRNAs • RNA pol III for small RNAs

35. Mode of Gene Regulation • Developmental: When • Tissue-specific: Where • Event-induced: What • Intensity level: How much Question: How does this happen? What decide tissue-specificity? What decide the developmental stages? What decide the expression level?

36. Eukaryotic Transcription • Promoter strength • Tissue-specific enhancers and silencers • Transcription Factors • Post-transcriptional regulation • RNA stability, RNA degradation and half life

37. Eukaryotic Promoters Core promoter • Determine transcriptional start site • In prokaryote: -10 region • In eukaryote: TATA-box Proximal elements of promoter • in prokaryote: -35 region • In eukaryote: CAAT-box, GC-box Enhancer • A regulatory DNA sequence that greatly enhances the transcription of a gene. They are mostly tissue specific Silencer • A DNA sequence that helps to reduce or shut off the expression of a nearby gene. They are mostly tissue-specific Terminator • A DNA sequence downstream of a gene and recognized by RNA polymerase as a signal to stop transcription. • PolyA signals (AATAAA) tell where to cut and add poly A sequences

38. Transcription factors • Trans-acting factors • Contain at least 2 domains • DNA binding domain • Transcription activation domain 􀂄  Acidic domains,  Glutamine-rich domains 􀂄  Proline-rich domains • They are proteins that bind to the cis-acting elements to control gene expression • Control gene expression in several ways: • may be expressed in a specific tissue, “tissue-specific” • may be expressed at specific time in development, “Developmental” • They may be induced by certain conditions, “inducible” • may be required for protein modification • may be activated by ligand binding, e.g. hormone receptors

39. HTH (helix-turn-helix) α-helix (N-terminus)----DNA-specific α-helix (C-terminus)----non-specific

40. Leu Zipper

41. Three Zinc Finger Motifs forming the recognition site

42. Regulation of Transcription

43. Homeodomain Protein in Drosophila utilizing helix-turn-helix motif to regulate developing patterns

44. Synergistic Regulation

45. INTRONS AND EXONS • Eukaryotic DNA differs from prokaryotic DNA in that the coding sequences are interspersed with noncoding sequences called introns • Sequences retain in mature mRNA are called EXONS. A mature mRNA contains 5’-UT, coding region and 3’UT. • Introns are spliced out, cap and polyA are added before it matures and transported to cytoplasm, where it is ready for translation • Introns can be very large and numerous, so some genes are much bigger than the final processed mRNA • Yeast has 4% genes with introns, Mammals have most genes with introns.

46. Transcriptional Regulation • Three RNA polymerases responsible for all the transcription • Specific transcription factors bind to these regulatory elements and regulate transcription • Regulatory elements may be tissue specific and will activate their gene only in one kind of tissue • Regulatory elements may be developmental and only expressed at certain stages • Regulatory elements may be inducible and expressed only at certain condition • Sometimes the expression of a gene requires the function of two or more different regulatory elements • In eukaryotic cells the primary transcript (pre-mRNA) must be processed further in order to ensure translation. • This normally includes a 5' cap, a poly-A tail and splicing. • Alternative splicing can contributes to the diversity of proteins any single mRNA can produce.

47. Regulation of Eucaryotic Genes

48. Transcription complex

49. Insulator Elements (boundary elements) help to coordinate the regulation

50. Post-Transcriptional Regulation • Gene Regulation through mRNA Processing • Exon shuffling • Alternative splicing • RNA editing • RNA stability • RNA degradation • RNA half life • mRNA Transport Control • RNA Interference (RNAi) • miRNA • siRNA