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Genome Biology and Biotechnology. 9. The localizome. Prof. M. Zabeau Department of Plant Systems Biology Flanders Interuniversity Institute for Biotechnology (VIB) University of Gent International course 2005. Summary. DNA localizome or DNA interactome

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Genome biology and biotechnology

Genome Biology and Biotechnology

9. The localizome

Prof. M. Zabeau

Department of Plant Systems Biology

Flanders Interuniversity Institute for Biotechnology (VIB)

University of Gent

International course 2005


Summary

Summary

  • DNA localizome or DNA interactome

    • Genome-wide mapping of DNA binding proteins

      • Transcription factor binding sites

      • Localization of replication origins

  • Protein localizome

    • High throughput localization of proteins in cellular compartments


Functional maps or omes

Functional Mapsor “-omes”

Genes or proteins

1 2 3 4 5 n

“Conditions”

Genes

ORFeome

Mutational phenotypes

Phenome

Transcriptome

Expression profiles

DNA Interactome

Protein-DNA interactions

Localizome

Cellular, tissue location

Interactome

Protein interactions

Proteome

proteins

After: Vidal M., Cell, 104, 333 (2001)


Genome wide analysis of regulatory sequences

Genome-wide Analysis of Regulatory Sequences

  • Gene expression is regulated by transcription factors selectively binding to regulatory regions

    • protein–DNA interactions involve sequence-specific recognition

    • Other factors, such as chromatin structuremay be involved

  • Sequence-specific DNA-binding proteins from eukaryotes generally

    • recognize degenerate motifs of 5–10 base pairs

    • Consequently, potential recognition sequences for transcription factors occur frequently throughout the genome

  • Genome-wide surveys of in vivo DNA binding proteins

    • provides a platform to answer these questions


Genome wide analysis of regulatory sequences1

Genome-wide Analysis of Regulatory Sequences

  • Methods combine

    • Large-scale analysis of in vivo protein–DNA crosslinking

    • microarray technology

  • ChIP-on-chip

    • Chromatin Immuno-Precipitation on DNA chips

Reprinted from: Biggin M., Nature Genet.28, 303 (2001)


Genome wide location and function of dna binding proteins

Genome-Wide Location and Function of DNA Binding Proteins

  • Paper presents

    • proof of principle for microarray-based approaches to determine the genome-widelocation of DNA-bound proteins

      • Study of thebinding sites of a couple of well known gene-specific transcription activators in yeast: Gal4 and Ste12

    • Combines data from

      • in vivo DNA binding analysis with

      • expression analysis

      • to identifygenes whose expression is directly controlled by these transcription factors

Ren et. al., Science, 290, 2306 (2000)


Chromatin immuno precipitation chip procedure

ChromatinImmuno Precipitation (Chip) Procedure

  • Cells arefixed with formaldehyde, harvested, and sonicated

  • DNA fragments cross-linked to a protein of interestare enriched by immunoprecipitation with a specific antibody

  • Immuno-precipitated DNA is amplifiedandlabeled with the fluorescent dye Cy5

  • Control DNA not enrichedby immunoprecipitation is amplifiedandlabeled with thedifferent fluorophore Cy3

  • DNAs are mixed and hybridized to a microarray of intergenic sequences

  • The relative binding of theprotein of interest to each sequence is calculated from the IP-enriched/unenriched ratioof fluorescence from 3 experiments

Reprinted from: Ren et. al., Science, 290, 2306 (2000)


Modified chromatin immuno precipitation chip procedure

Modified ChromatinImmuno Precipitation (Chip) Procedure

Close-up of a scanned image of a micro-array containing 6361 intergenic region DNA fragments of the yeast genome

ChIP-enriched DNA fragment

Reprinted from: Ren et. al., Science, 290, 2306 (2000)


Proof of concept gal4 transcription factor

Proof of concept: Gal4 transcription factor

  • Identification of sites bound by the transcriptionalactivator Gal4 in the yeast genome and genes induced by galactose

    • Gal4 activates genes necessaryfor galactose metabolism

      • The best characterized transcription factor in yeast

    • 10 genes werebound by Gal4 and induced in galactose

      • 7 genes in the Gal pathway, previously reported to be regulated by Gal4

      • 3 novel genes: MTH1, PCL10, and FUR4

Reprinted from: Ren et. al., Science, 290, 2306 (2000)


Genome wide location of gal4 protein

Genome-wide location of Gal4 protein

Genes whose promoter regions are bound by Gal4 and whose expression levels were induced at least twofold by galactose

Reprinted from: Ren et. al., Science, 290, 2306 (2000)


Role of gal4 in galactose dependent cellular regulation

Fur4

Pcl10

MTH1

Role of Gal4 in Galactose-dependent Cellular Regulation

The identification of MTH1, PCL10, and FUR4 as Gal4-regulated genes explains how regulation of several different metabolic pathways can be coordinated

increases intracellular pools of uracil

reduces levels of glucose transporter

Reprinted from: Ren et. al., Science, 290, 2306 (2000)


Conclusions

Conclusions

  • The genes whoseexpression is controlled directly by transcriptional activatorsin vivo

    • Areidentified bya combination of genome-wide location and expression analysis

  • Genome-wide location analysisprovides information

    • On the binding sites at which proteins residein the genome under in vivo conditions


Genomic binding sites of the yeast cell cycle transcription factors sbf and mbf

Genomic Binding Sites of the Yeast Cell-cycle Transcription Factors SBF and MBF

  • Paper presents

    • The use of CHIP and DNA microarrays to define the genomic binding sites of the SBF and MBF transcription factors in vivo

    • The SBF and MBF transcription factors are active in the initiation of the cell division cycle (G1/S) in yeast

      • A few target genes of SBF and MBF are known but the precise roles of these two transcription factors are unknown

      • The two transcription factors are heterodimers containing the same Swi6 subunit and a DNA binding subunit

        • MBF is a heterodimer of Mbp1 and Swi6

        • SBF is a heterodimer of Swi4 and Swi6

Iyer et al., Nature 409: 533 (2001)


Genomic targets of sbf and mbf

Genomic targets of SBF and MBF

Reprinted from: Iyer et al., Nature 409: 533 (2001)


In vivo targets of sbf and mbf

In Vivo Targets of SBF and MBF

  • The CHIP experiments identified

    • 163 possible targets of SBF

    • 87 possible targets of MBF

    • 43 possible targets of both factors

  • Support for the possible in vivo targets

    • Most of the genes downstream of the putative binding sites peak in G1/S

    • Target genes are highly enriched for functions related to DNA replication, budding and the cell cycle

    • In vivo binding sites are highly enriched for sequences matching the defined consensus binding sites

Reprinted from: Iyer et al., Nature 409: 533 (2001)


Expression profiles of sbf and mbf targets

Transcriptome data for synchronized cell cultures

Expression Profiles of SBF and MBF Targets

Reprinted from: Iyer et al., Nature 409: 533 (2001)


Expression profiles of sbf and mbf targets1

Expression Profiles of SBF and MBF Targets

  • Why are two different transcription factors used to mediate identical transcriptional programmes during the cell-division cycle in yeast?

    • A possible answer is suggested by differences in the functions of the genes that they regulate

      • Many of the targets of SBF have roles in cell-wall biogenesis and budding

      • 25% of the MBF target genes have known roles in DNA replication, recombination and repair

    • The results support a model in which

      • SBF is the principal controller of membrane and cell-wall formation

      • MBF primarily controls DNA replication

  • The need for DNA replication and membrane / cell-wall biogenesis may be different in the mitotic and meiotic cell cycle

Reprinted from: Iyer et al., Nature 409: 533 (2001)


A high resolution map of active promoters in the human genome

A high-resolution map of active promoters in the human genome

Kim et. al., Nature 436: 876-880 (2005)

  • Paper presents

    • a genome-wide map of active promoters in human fibroblast cells

      • determined by experimentally locating the sites of RNA polymerase II preinitiation complex (PIC) binding

      • map defines 10,567 active promoters corresponding to

        • 6,763 known genes

        • >1,196 un-annotated transcriptional units

    • Global view of functional relationships in human cells between

      • transcriptional machinery

      • chromatin structure

      • gene expression


Identification of active promoters in the human genome

Identification of active promoters in the human genome

  • Microarrays cover

    • All non-repeat DNA at 100 bp resolution

  • Pol II preinitiation complex (PIC)

    • RNA polymerase II

    • transcription factor IID

    • general transcription factors

  • ChIP of PIC-bound DNA

    • monoclonal antibody against TAF1 subunit of the complex (TBP associated factor 1 )

Reprinted from: Kim et. al., Nature 436: 876-880 (2005)


Results from tfiid chip on chip analysis

Results from TFIID ChIP-on-chip analysis

Reprinted from: Kim et. al., Nature 436: 876-880 (2005)


Characterization of active promoters

Characterization of active promoters

  • Matched the 12,150 TFIID-binding sites to

    • the 5' end of known transcripts in transcript databases

    • 87% of the PIC-binding sites were within 2.5 kb of annotated 5' ends of known messenger RNAs

  • 8,960 promoters were mapped

    • within annotated boundaries of 6,763 known genes in the EnsEMBL genes

Reprinted from: Kim et. al., Nature 436: 876-880 (2005)


The chromatin modification features of the active promoters

The chromatin-modification features of the active promoters

  • Validation of active promoters

    • ChIP-on-chip using an anti-RNAP antibody

    • ChIP-on-chip analysis using

      • anti-acetylated histone H3 (AcH3) antibodies

      • anti-dimethylated lysine 4 on histone H3 (MeH3K4) antibodies

      • known epigenetic markers of active genes

Reprinted from: Kim et. al., Nature 436: 876-880 (2005)


Tfiid rnap ach3 and meh3k4 profiles on the promoter of rps24 gene

TFIID, RNAP, AcH3 and MeH3K4 profiles on the promoter of RPS24 gene

Reprinted from: Kim et. al., Nature 436: 876-880 (2005)


Additional findings

Additional findings

  • Promoters of non-coding transcripts

    • Are very similar to promoters of protein coding genes

  • Promoters of novel genes

    • Estimate 13% of human genes remain to be annotated in the genome

  • Clustering of active promoters

    • co-regulated genes tend to be organized into coordinately regulated domains

  • Genes using multiple promoters

Reprinted from: Kim et. al., Nature 436: 876-880 (2005)


Multiple promoters in human genes

Multiple promoters in human genes

  • WEE1 gene locus

    • Two different transcripts with alternative 5’ends

      • Encoding different proteins

    • Two different TFIID-binding sites- two promoters

    • Differential transcription during the cell cycle

Reprinted from: Kim et. al., Nature 436: 876-880 (2005)


The transcriptome of a cell line

The transcriptome of a cell line

  • Functional relationship between transcription machinery and gene expression

    • correlated genome-wide expression profiles with PIC promoter occupancy

  • Four general classes of promoters

    • Actively transcribed genes

    • Weakly expressed genes

    • Weakly PIC bound genes

    • Inactive genes

Reprinted from: Kim et. al., Nature 436: 876-880 (2005)


Genome biology and biotechnology

Genome-Wide Distribution of ORC and MCM Proteins in yeast: High-Resolution Mapping of Replication Origins

  • Paper presents

    • Genome-wide location analysis to maptheDNA replication origins in the 16 yeast chromosomes by determining the binding sites of prereplicative complex proteins

Wyrick et. al., Science, 294, 2357 (2001)


Chromosome replication in eukaryotic cells

Chromosome Replication In Eukaryotic Cells

  • Chromosome replication

    • initiates from origins of replication distributed along chromosomes

    • Origins of replication comprise autonomously replicating sequences (ARS)

      • ARS contain an 11-bp ARS consensus sequence (ACS)

        • Essential for replication initiation

        • Recognized by the Origin Recognition Complex (ORC)

      • The majority of sequence matches to the ACS in the genome do not have ARS activity

  • Prereplicative complexes at replication origins comprise

    • Origin Recognition Complex (ORC) proteins

    • Minichromosome Maintenance (MCM) proteins

Reprinted from: Wyrick et. al., Science, 294, 2357 (2001)


Prereplicative complexes at origins of replication

Prereplicative Complexes At Origins Of Replication

Reprinted from: Stillman, Science, 294, 2301(2001)


Orc and mcm binding sites compared with known arss

High degree of correlation between MCM and ORC binding sites and known ARSs

Correct identification of 88% known ARSs

The method can accurately identify the position of ARSs to a resolution of 1 kb or less

ORC- and MCM-binding sites compared with known ARSs

Reprinted from: Wyrick et. al., Science, 294, 2357 (2001)


Genome wide location of potential replication origins

Genome-wide Location Of Potential Replication Origins

Identification of 429 potential origins on the entire genome

Reprinted from: Wyrick et. al., Science, 294, 2357 (2001)


Conclusions1

Conclusions

  • The ChIP-based method identified the majority of origins found in the analysis of genome-wide replication timing in yeast

    • and provides direct, high-resolution mapping of potential origins

  • Similar approaches identified origins in other organisms

    • For example: Coordination of replication and transcription along a Drosophila chromosome

      • MacAlpine et al., Genes & Dev. 18: 3094-3105 (2004)

Reprinted from: Wyrick et. al., Science, 294, 2357 (2001)


Functional maps or omes1

Functional Mapsor “-omes”

Genes or proteins

1 2 3 4 5 n

“Conditions”

Genes

ORFeome

Mutational phenotypes

Phenome

Transcriptome

Expression profiles

DNA Interactome

Protein-DNA interactions

Localizome

Cellular, tissue location

Interactome

Protein interactions

Proteome

proteins

After: Vidal M., Cell, 104, 333 (2001)


Global analysis of protein localization in budding yeast

Global analysis of protein localization in budding yeast

Huh et. al., Nature425, 686 - 691(2004)

  • Paper presents

    • An approach to define the organization of proteins in the context of cellular compartments involving

    • the construction and analysis of a collection of yeast strains expressing full-length, chromosomally tagged green fluorescent protein fusion proteins


Experimental strategy

Experimental Strategy

  • Systematic tagging of yeast ORFs with green fluorescent protein (GFP)

    • GFP is fused to the carboxy terminus of each ORF

    • Full length fusion proteins are expressed from their native promoters and chromosomal location

  • The collection of yeast strains expressing GFP fusions was analyzed by

    • fluorescence microscopy to determine the primary subcellular localization of the fusion proteins

      • Defines 12 categories

    • co-localization with red fluorescent protein (RFP) markers to refine the subcellular localization

      • Defines 11 additional categories

Reprinted from: Huh et. al., Nature 425, 686 - 691 (2004)


Construction of gfp fusion proteins

Construction of GFP fusion proteins

  • For each ORF a pair of PCR primers was designed

    • Homologous to the chromosomal insertion site

    • Matching a GFP – selectable marker construct

  • Yeast was transformed with the PCR products to generate

    • Strains expressing chromosomally tagged ORFs

Reprinted from: Huh et. al., Nature 425, 686 - 691 (2004)


Representative gfp images

Representative GFP Images

Nucleus

ER

Nuclear periphery

Bud neck

Lipid particle

mitochondrion

Reprinted from: Huh et. al., Nature 425, 686 - 691 (2004)


Gfp and rfp co localization images

GFP and RFP Co-localization Images

Nucleolar marker

Reprinted from: Huh et. al., Nature 425, 686 - 691 (2004)


Global results

Global results

22 categories

  • Constructed ~6.000 ORF-GFP fusions

    • 4.156 had localizable GFP signals (~75% of the yeast proteome)

    • Good concordance with data from earlier studies

      • GFP does not affect the location

      • Localized 70% of the new proteins

    • Major compartments: cytoplasm (30%) and the nucleus (25%)

    • 20 other compartments: 44% of the proteins

  • Most the proteins can be located in discrete cellular compartments

Reprinted from: Huh et. al., Nature 425, 686 - 691 (2004)


The proteome of the nucleolus

The proteome of the nucleolus

  • Detected 164 proteins in the nucleolus

    • Plus 45 identified in other studies

  • Data are consistent with MS analysis of human Nucleolar proteins

    • Allows identification of yeast-human orthologs

Reprinted from: Huh et. al., Nature 425, 686 - 691 (2004)


Transcriptional co regulation and subcellular localization are correlated

Transcriptional co-regulation and subcellular localization are correlated

subcellular localization

33 transcription modules

Co-regulated genes

Reprinted from: Huh et. al., Nature 425, 686 - 691 (2004)


Conclusion

Conclusion

  • The high-resolution, high-coverage localization data set

    • represents 75% of the yeast proteome

      • classified into 22 distinct subcellular localization categories,

  • Analysis of these proteins

    • in the context of transcriptional, genetic, and protein–protein interaction data

      • provides a comprehensive view of interactions within and between organelles in eukaryotic cells.

      • helps reveal the logic of transcriptional co-regulation

Reprinted from: Huh et. al., Nature 425, 686 - 691 (2004)


Recommended reading

Recommended reading

  • DNA-interactome

    • Genome-Wide Location of DNA Binding Proteins

      • Ren et. al., Science, 290, 2306 (2000)

    • Map of active promoters in the human genome

      • Kim et. al., Nature 436: 876-880 (2005)

  • Global analysis of protein localization in yeast

    • Huh et. al., Nature425, 686 - 691(2004)


Further reading

Further reading

  • Genome-Wide Location of DNA Binding Proteins

    • Genomic Binding Sites of the Yeast Cell-cycle Transcription Factors SBF and MBF

      • Iyer et al., Nature 409: 533 (2001)

    • High-Resolution Mapping of Replication Origins

      • Wyrick et. al., Science, 294, 2357 (2001)


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