Transcriptional regulation and promoter analysis Ju-Ming Wang Ins.of Biosignal Transduction (IBT),NCKU
Transcriptional regulation and promoter analysis Ju-Ming Wang Ins. of Biosignal Transduction (IBT), NCKU
.Transcriptional regulation Promoter and transcription factors Promoter identification Transcription factor-binding analysis 2
2 •Transcriptional regulation Promoter and transcription factors • Promoter identification • Transcription factor-binding analysis
ntro Gene Expression analysis Gene Expression analysis DNA Messenger RNA Protein Transcription Translation cDNA libraries construction Express sequence tags(ESTs) 0 cDNA Microarray experiments Differential display Subtractive hybridization SAGE Microarray 3
3 DNA Messenger RNA Protein Transcription Translation cDNA libraries construction cDNA Microarray experiments Express sequence tags (ESTs) Gene Expression analysis 0 0.5 1 1.5 2 2.5 3.5 4 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 WSB1 WSB1 KNSL2 KNSL2 KIAA0013 KIAA0013 PIF1 PIF1 KNSL1 KNSL1 FLJ22624 FLJ22624 ESPL1 ESPL1 Differential display Subtractive hybridization SAGE Microarray Introduction- Gene Expression analysis
The organization of genes on a human chromosome. human chromosome 22-48x 10 nucleotide pairs of DNA eterochromatin 10%of chromosome arm-40 genes 10 1%of chrom me containing 4 genes 10 lanking region Intron one gene of 3.4 x 10 regulatory DNA folded protein With the publication of the"first draft"of the entire human genome in 2001 and the "finished DNA sequence"in 2004
4 The organization of genes on a human chromosome. With the publication of the “first draft” of the entire human genome in 2001 and the “finished DNA sequence” in 2004. Exon Intron Flanking region
● criptional regulation-Promoter and transcription factors Transcriptional regulation Promoter and transcription factors A transcription unit Terminator -35-10-1+1+10 proximal distal Upstream<Downstreg wwwergito.com 5
5 Transcriptional regulation Promoter and transcription factors A transcription unit Transcriptional regulation- Promoter and transcription factors
criptional regulation-Promoter and transcription factors Promoter Gene structure Translation Transcription Poly A start site Transcription start site s立gmal terminatin site 食 Promoter region Cap ORF Ply A tail mRNA ☐AAAAAA 3'UTR
6 Promoter Transcriptional regulation- Promoter and transcription factors
◆ criptional regulation-Promoter and transcription factors P76 p53-binding NLS NES ACIDIC ZF RF NoLS Promoter in intron ☐441 P1 P2 ATG Hdm2 gene 可可四而)网网网☒X网☐ P53RE ATG 491 p53-binding NLS NES ACIDIC ZF RF NoLS Unbiased mapping of transcription factor binding sites along human chromosomes 21 and 22 points to widespread regulation of noncoding RNAs Ce2004,1164499-509 This mapping reveals an unexpectedly large number of transcription factor binding site (TFBS)regions,with a minimal estimate of 12,000 for Sp1,25,000 for cMyc,and 1600 for p53 when extrapolated to the full genome.Only 22%of these TFBS regions are locatedat the 5'termini of protein-coding genes while 36%lie within or immediately 3'to well- characterized genes and are significantly correlated with noncoding RNAs
7 Promoter in intron Transcriptional regulation- Promoter and transcription factors Unbiased mapping of transcription factor binding sites along human chromosomes 21 and 22 points to widespread regulation of noncoding RNAs Cell. 2004;116(4):499-509 This mapping reveals an unexpectedly large number of transcription factor binding site (TFBS) regions, with a minimal estimate of 12,000 for Sp1, 25,000 for cMyc, and 1600 for p53 when extrapolated to the full genome. Only 22% of these TFBS regions are located at the 5' termini of protein-coding genes while 36% lie within or immediately 3' to wellcharacterized genes and are significantly correlated with noncoding RNAs
◆ criptional regulation-Promoter and transcription factors Chromatin remodeling GCN5- -SWI/SNF SWI5 Mediato 3 6 Preinitiation complex Mediato RNA Pol II TATA BP GENERAL TFs Lodish et al.Molecular Biology of the Cell(5th ed.).W.H.Freeman Co..2003. Remodeling complex:use energy from ATP hydrolysis to alter nucleosome structure or to move nucleosomes around
8 SWI/SNF SWI5 RNA Pol II TATA BP GENERAL TFs Lodish et al. Molecular Biology of the Cell (5th ed.). W.H. Freeman & Co., 2003. Transcriptional regulation- Promoter and transcription factors Chromatin remodeling Remodeling complex: use energy from ATP hydrolysis to alter nucleosome structure or to move nucleosomes around
criptional regulation-Promoter and transcription factors gene regulatory sequences spacer DNA eneral transcription RNA polymerase ATA box upstream promoter transcription Graphics from the book of "Essential Cell Biology"Alberts,1997,p.p.268
9 Transcriptional regulation- Promoter and transcription factors Graphics from the book of “Essential Cell Biology” Alberts, 1997,p.p. 268
◆ criptional requlation-Promoter and transcription factors How to dissect the transcriptional regulation? Primer B -197TCTCGATCGATCTOOCCOCCTOGGCCTCCCAAAGTGCT AP-1 Age of One transcription Factor on one gene CREBI CCAAT.BOx #l CCAAT.Box =2 -397GGGCTGCAGCGCTGCC cRd cRd 10
10 Transcriptional regulation- Promoter and transcription factors How to dissect the transcriptional regulation? Age of One transcription Factor on one gene