Contact information Blaine Bartholomew Office: Neckers Bldg., Rm. 211 Phone:

Transcription

1 Contact information Blaine Bartholomew Office: Neckers Bldg., Rm. 211 Phone: Class notes Other references Principles of Biochemistry by Lehninger 4th edition, pp , , , And for more in depth information (A) Thompson & Thompson, Genetics in Medicine, 1991 (B) Gelehrter, Collins, & Ginsburg, Principles of Medical Genetics, Second ed., 1998 (C)Connor & Ferguson-Smith, Medical Genetics, Fifth ed., 1996, (D) Jorde, Carey, Bamshad, & White, Medical Genetics, 2nd ed., 1999

2 Developmental Expression of Human Globin Genes ζ2ε2 α2ε2 ζ2γ2 α2γ2 from Gelehrter, Collins, & Ginsburg, Principles of Medical Genetics, Second Ed., p. 93, 1998 α2β2 α2δ2

3 Genomic Organization Single copy: most common type with one copy/haploid Duplicated genes: 2 or more copies e.g. histones Gene families: closely related genes with similar but nonidentical function e.g. beta globin family Pseudogenes: nonfunctional copies of functional genes that arise by gene duplication e.g. b globin family

4 β Globin Gene Family Functional Genes (expressed at different times during development) ε, Gγ, Aγ, δ, β Nonfunctional Pseudogenes ψβ2, ψβ1 Lodish, Fig. 10-3b

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6 Human Globin Genes and Hemoglobins Produced During Development ζ ζψ ψα ψα α2 α1 Chromosome Chromosome 11 5 LCR ε Gγ Aγ ψβ δ β α- like genes 3 β-like genes α β HbA α β Heme Developmental Period Embryonic Fetal Adult Hb Gowe r 1 ζ2 ε2 Hb Gowe r 2 α2 ε2 Hb Port land ζ2 γ2 adapted from Thompson & Thompson Genetics in Medicine, p. 249, 1991 Hb F α2 γ2 HbA2 α2 δ2 minor HbA α2 β2

7 Sickle cell diseases Sickle cell anemia Caused by a single point mutation Sickle cell-hemoglobin C Sickle cell beta-thalassemia Caused by mutations in the β globin gene locus Decreased or absent adult β globin synthesis

8 One other version of these mutations Individuals with defective adult β globin genes can be phenotypically normally if There are compensatory mutations that cause expression of fetal β-like genes after birth ( G γ and A γ globin). This condition is called HPFH or hereditary persistence of fetal hemoglobin

9 How is the expression of these genes regulated? The important underlying DNA sequences and associated factors

10 Eukaryotic & Prokaryotic Transcription RNA polymerases

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12 RNA Polymerases A. E. coli RNA polymerase 1. core enzyme = ββ'(α)2 has catalytic activity but cannot recognize start site of transcription ~500,000 daltons dimensions: 100 X 100 X 160 angstroms requires Mg2+ for activity b' binds 2 Zn atoms 2. holoenzyme = core enzyme + sigma factor (s) carries out four functions: (i) template binding (ii) RNA chain initiation (iii) chain elongation (iv) chain termination

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15 RNA Polymerases B. Eukaryotic RNA polymerases (RNAP) 1. 3 nuclear RNA polymerases a. RNAP I- transcribes rrna genes b. RNAP II - transcribes mrna genes c. RNAP III - transcribes trna, 5S rrna, and other small RNA genes d. have different subunits, large multisubunit complexes are functionally similar to E. coli RNA polymerase e. cannot bind to their respective promoters alone, but requires transcription factor for promoter specific recruitment

16 RNA Polymerases 2. organelle specific RNA polymerases more prokaryotic-like 1. chloroplast 2. mitochondria

17 RNA Polymerases 3. RNAP II a. core subunits - have sequence similarity to the core subunits of E. coli core RNA polymerase or subunits of other eukaryotic RNA polymerases b. shared or common subunits same subunits found in RNAP III and II or in RNAP I and RNAP II c. unique subunits - no similar homologs found anywhere else

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19 How does RNA polymerase II find its correct binding site?

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22 Promoter Structure B. mrna genes transcribed by RNAP II 1. TATA box element - located between -30 and -20 bps 2. Initiator region or In: centered on the start site of transcription 3. DPE: downstream promoter element 4. Response elements (RE) a. upstream of the TATA box b. many different kinds - help respond to signals c. multiple RE present - synergy

23 How is transcription of particular genes get turned on in response to external stimuli such as stress (heat, starvation, and so on), hormones and other small molecule effectors?

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27 Promoter Structure B. mrna genes transcribed by RNAP II 5. enhancers a. can be located at great distances (>1000 bps) from start site of transcription either from the 5' or 3' end of gene b. stimulates transcription (~100 times) c. orientation independent

28 Promoter Structure B. mrna genes transcribed by RNAP II 5. enhancers d. two models of how enhancers might work i. entry point of RNAP II by preventing nucleosomes from binding or an altered DNA conformation that promotes RNAP II recognition ii. transcription factors bound to enhancer will stimulate binding of RNAP II to promoter regions closer to the start site of transcription

29 Transcription Factors General versus promoter specific transcription factors. Factors that are required for all mrna genes and others that are required for only a small subset of genes

30 Promoter Specific Transcription Factors General Transcription Factors

31 General Transcription Factors

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33 Regulation of Transcription I. Basal vs. activated transcription for mrna genes A. General transcription factor (TF) vs. promoter-specific 1. general TFs are required by all mrna genes a. an absolute requirement b. transcription can occur alone with these factors and is by definition the basal level of transcription 2. promoter-specific TFs are different for each gene 3. the promoter-specific TFs are required for maximal level of transcription or for activated transcription (induction)

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38 β globin gene transcription factors

39 Erythroid specific Transcription Factors PROTEIN MOTIF CLASS INTERACTS WITH EKLF CACCC Kruppel-like C2H2 zinc finger GATA-1, CBP/p300, TAFII130 GATA-1 (A/T)GATA(A/G GATA-like C4 zinc fingers Sp1, GATA-1, FOG, EKLF, CBP/p300 NF-E2 TGCTGA(C/G)TCA basic leucine zipper TAFII130, CBP/p

40 Regulation of Transcription II. Question of Activation A. diversity of response - combinatorial effect 1. properties of response elements (RE) 2. relatedness of RE and enhancers 3. trans acting factors induction: heat shock, heavy metals, viral infection, growth factors, steroids 4. greater multiplicity with combinatorial approach B. Master gene regulatory proteins 1. response elements shared 2. example of homeodomains

41 What is the difference between basal and activated transcription?

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45 What s wrong with this picture?

46 DNA is packaged into compact structures inside the cell

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51 Higher order structure folding of chromatin fibers Differing extents of compaction Also this facilitates in DNA fitting inside the nucleus

52 β Globin Gene Cluster: Chromatin Structure

53 Gaining Access to the DNA Large multi-subunit protein complexes alter the state of chromatin

54 What is chromatin remodeling? The process of making DNA more or less accessible in the eukaryotic genome using a series of specialized proteins

55 Accessibility is Key Regulatory Step

56 ATP-dependent chromatin remodeling machines Different classes of chromatin remodeling complexes

57 Different classes of ATP-dependent chromatin remodeling complexes A. SWI/SNF or SWI-SNF like complexes required for activation of a small subset of genes found in humans, flies, and yeast at least two different forms of the complex in yeast or humans in yeast it has 11 or 17 subunits total complex size is Megadaltons

58 SWI/SNF or SWI/SNF-like Complexes Are Found in All Eukaryotes

59 Activities associated with ATPdependent chromatin remodeling Translate nucleosomes along DNA Change translational position (general) Nucleosome spacing (ISWI) Displace or dissemble nucleosomes (SWI/SNF and RSC) Octamer transfer Loss of H2A/H2B dimer Histone exchange Example of H2A exchanged for H2AZ by SWR1

60 Bulge Diffusion Model for chromatin remodeling Initiates from outside the Nucleosome Initially breaks contacts at edge of Nucleosome These two point are also true for other model(s)

61 DNA can be cut by nucleases (LCR) Site 3 has SWI/SNF bound at this region

62 Chromatin Remodeling ATP-dependent chromatin remodeling Moves nucleosomes along DNA Involved in displacing nucleosomes from DNA Exchanges parts of the nucleosome Covalent modification of histone proteins ACETYLATION METHYLATION PHOSPHORYLATION UBIQUITINATION Poly ADP RIBOSYLATION SUMOLYATION

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64 Sites of Histone Modification

65 Histone acetyltransferases (HATs) A. cytoplasmic HATs (Type B) i. acetylate newly synthesized H3 and H4 histones before depositing on to DNA ii. after binding to DNA the acetyl groups are removed B. nuclear HATs (Type A) i. have been found to be required for gene activation ii. part of TFIID has HAT activity (TAFII250) iii. these HAT proteins are usually large multisubunit complexes

66 Mapping sites of histone modifications Have developed a technique called ChIP (Chromatin Immunoprecipitation) Identify the genomic location where different histone modifications reside Is dependent on antibodies that recognize the particular modification Can be used to examine the entire genome

67 Pattern of histone acetylation on the β globin gene

68 Chromatin Euchromatin - dispersed (acetylated) chromatin, gene rich regions of the genome Heterochromatin - chromatin is condensed (deacetylated), gene poor regions of the genome human genome 3 x 10 9 bp with an estimated 20-25,000 genes only 2-4% of the DNA codes for proteins

69 Histone acetylation generally associated with gene activation

70 Summary of regulatory elements What is an insulator element (which is similar to what is also called a boundary element)?

71 Insulators: Two models

72 Nuclear Matrix or Scaffold

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74 DNA Loops/ Domains

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76 Chromosome Territories Individual chromosomes occupy distinct regions in the nucleus

77 Chromosome Territories Gene rich chromosomal regions Tend to be oriented toward the nuclear interior Gene poor chromosomal regions Tend to be associated with the with nuclear periphery Dynamic reorganization

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