Differential Gene Expression

Transcription

1 Biology 4361 Developmental Biology Differential Gene Expression September 28, 2006

2 Chromatin Structure ~140 bp ~60 bp Transcriptional Regulation: 1. Packing prevents access CH 3 2. Acetylation ( C O ) state of histones controls DNA binding histone acetyltransferases allow transcription histone deacetylases repression 3. Histone methylation (CH 3 ) further repression

3 Chromatin configuration John H. Frenster heterochromatin remains tightly condensed throughout most of the cell cycle replicates later euchromatin active chromatin

4 Anatomy of the Gene core promoter transcription initiation site 5 region transcribed region 3 TATAT +1 upstream downstream

5 Human β globin Gene Sequence

6 Production of β globin 5

7 Promoters and Enhancers RNA polymerase binds to promoter at the TATA box however, Pol II cannot initiate transcription alone various proteins bind to regulatory sequences upstream and downstream of transcription initiation site sequences in the promoter region bind Basal Transcription Factors promoter region 5 3 basal transcription factors facilitate Pol II binding and activity

8 Eukaryotic Transcription Initiation Complex basal transcription factors: sequential binding binding mediated by small proteins TBP associated factors (TAFs) mediator complex 1. TFIID complex binds to the TATA box through its TATA Binding Protein (TBP) subunit 2. TFIID is stabilized by TFIIA 3. TFIIB and TFIIH join the complex on the TATA box; TFIIE and TFIIF associate with RNA polymerase II

9 Eukaryotic Transcription Initiation Complex 2 4. RNA polymerase II is positioned by TFIIB, and its carboxy terminal domain (CTD) is bound by TFIID 5. The CTD is phosphorylated by TFIIH and is released by TFIID; RNA polymerase II can now transcribe mrna

10 Stabilization of Transcriptional Initiation Complex by TAFs TBP TATA binding protein TAF(s) TBP associated factors

11 Enhancers 1 Enhancers are cis acting regulatory elements cis (same or same side); elements that reside on the same DNA stand; DNA sequences trans (other side); elements that originate from another DNA strand, e.g. regulatory proteins DNA sequences that regulate gene expression by affecting the transcription initiation complex on the promoter. Function bind specific regulatory proteins = transcription factors Enhancer locations can be highly variable, with respect to the transcribed portion of the gene. upstream (5 ), downstream (3 ), within transcribed region close proximity or as many as 106 bp away Enhancers are modular; e.g. mouse Pax6 gene expressed in multiple tissues, each under the influence of a different enhancer

12 Enhancers differ from promoters: 1) need a promoter to work 2) can work at a distance 3) can work in reverse orientation Enhancers 2

13 Enhancer Generalizations 1. Most gene transcription requires enhancers. 2. Enhancers are the major determinants of differential transcription in space (cell type) and time (developmental stage). 3. There can be multiple signals (e.g. multiple enhancer sites) for a given gene, and each enhancer can be bound by more than one transcription factor (not at the same time). 4. Transcription is regulated by the interaction of transcription factors bound to enhancers and the transcription initiation complex assembled at the promoter. 5. Enhancers are combinatorial. Various DNA sequences regulate temporal and spatial gene expression; these can be mixed and matched. 6. Enhancers are modular. A gene can have several enhancer elements, each of which may turn it on in different sets of cells. 7. Enhancers generally activate transcription by remodeling chromatin to expose the promoter, or by facilitating the binding of RNA polymerase to the promoter by stabilizing TAFs. 8. Enhancers can also inhibit transcription (aka Silencers).

14 Methods Reporter Genes Reporter gene construct: enhancer elements for Pax6 attached to structural genes for reporter molecules. exons for Pax6 mouse Pax6 LacZ β galactosidase gene for reporter protein, e.g. β galactosidase (blue) luciferase (produces light) green fluorescent protein (GFP) Drosophila

15 Transcription Factors Proteins that bind to enhancer or promoter regions activate or repress transcription Most bind to specific DNA sequences (e.g. enhancers) Transcription factors are grouped together in families, based on structural similarities families share common framework in DNA binding sites slight differences in binding sites cause differences in recognition

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17 estrogen receptor zinc finger domain

18 Transcription Factor Domains Three major domains: 1. DNA binding recognizes particular DNA sequence transcription factor engrailed

19 Transcription Factor Domains Three major domains: 1. DNA binding recognizes particular DNA sequence 2. trans activation activates or represses transcription often involved with proteins involved in binding RNA polymerase II; e.g. TFIIB, TFIIE often involved with enzymes that modify histones 3. protein protein interaction domain promotes dimerization allows it to be modulated by TAFs or other transcription factors

20 Transcription Factor Domains Transcription factor MITF basic helix loop helix homodimer is the functional protein The trans activating domain is contained in the center of the protein. when bound to a promoter or enhancer, the protein is able to bind a TAF (p300/cbp) TAF p300/cbp is a histone acetyltranferase

21 Enhancer Modules Enhancers are modular: e.g. Pax6 gene expressed in the eye, pancreas, nervous system. also expressed in different genes within these tissues Within these modules, transcription factors work in a combinatorial fashion. Transcription factors operate in cascades: one stimulates the production of several others.

22 DNA Methylation vertebrates; not Drosophila, nematodes, inverts methylation stabilizes nucleosome; stable nucleosome = transcriptional repression degree of methylation is proportional to degree of transcription absence of methylation correlates with tissue specific expression methylation patterns maintained throughout cell division by DNA (cytosine 5) methyltransferase

23 Genomic Imprinting Special case of DNA methylation Alleles from maternal and paternal genome are differentially methylated. Methylation patterns can be distinguished based on resulting phenotypes.

24 Dosage Compensation Mammals, Drosophila, nematodes XX = female, XY = male; result is too much X product Drosophila transcription rate of male X is doubled C. elegans both Xs partially repressed ( = hermaphrodite) Mammals Inactivation of a single X chromosome in mammalian XX cells XX cell early embryo both active later embryo only one active Barr bodies

25 X Chromosome inactivation Lyon hypothesis: 1. Both X chromosomes active in very early female development. 2. One X is inactivated in each cell 3. Inactivation is random 4. The process is irreversible. All progeny cells will retain the same inactivation pattern calico cat: heterozygous for coat color genes contained on X chromosome early late X chromosome inactivation