Transcription Regulation And Gene Expression in Eukaryotes

FS 2016 Graduate Course G2P Matthias and RG Clerc

Pharmazentrum Hörsaal 2 16h15-18h00

MODULAR STRUCTURE OF UPSTREAM TRANSCRIPTION FACTORS

•Experimental approaches

•DNA binding domains (DBD)

•Transcription regulation domains (TAD, TRD)

•Ligand binding domains

RG Clerc. March 16, 2016

TFIIB

RNAPII

TFIIATFIID

TFIIH TFIIFTFIIE

The Players in Transcription Regulation• DNA-binding transcription factors (upstreamfactors)

• Chromatin regulators• Coactivators and corepressors: Mediator, etc..• Basal Machinery: RNA PolII, GTFs

Gene

BasalMachinery

DNA-binding TFs

Chromatin regulatorsMediator

Coactivator

MODULAR STRUCTURE OF UPSTREAM TRANSCRIPTION FACTORS

•Experimental approaches “gel shift assay” (EMSA)

•DNA binding domains (DBD)

(modular highly ordered structure, classification of upstream TF)

•Transcription regulation domains (TAD, TRD):

(induced structure, not ordered on their own)

•Hormone binding domains

(structured modules, pharmacological tractable)

Typical gene and components involved in gene activation and inactivation: the players in transcription regulation

Assembly of the RNA Pol II preinitiation complex (PIC) and the major players in transcription regulation

DBDs are responsible for localizing a TAD domain in the proximity of the basal apparatus, recruiting co-factors specificity

S. Buratowski. 2012. Nature 483:286-287

Modular structure of upstream transcription factors

C

DBD

Structural domain

Transcription regulation (TAD, TRD)

GENEDNA binding site (RE)

TADStructural domain DBDN

N Structural domain DBD CTRD

Modular structure of upstream transcription factors

C

CStructural domain DBD 2 TAD 1

“domain swapping experiment”

N

TAD 1Structural domain DBD 1N

Structural domain DBD 2 CN TAD 2X X

Electrophoresis mobility shift assay (EMSA): a corner stone in deciphering DNA binding specificities

EMSA competition experiment

DNA methylation interference assay

DNase I footprinting assay

Affinity chromatography to purify DNA:protein and protein:protein interactions

Southwestern: Expression cloning of DNA binding protein eg. cloning of POU domain Oct-2

Staudt L. Clerc RG and Baltimore D. Science 241:577-581

TRX Factor Binding Assay on Random DNA oligomers: PCR Binding Site Selection

Bertolino E. Clerc RG. J Biol Chem 270:31178-31188

TRX Factor Binding Assay on Random DNA oligomers: PCR Binding Site Selection

Bertolino E. Clerc RG. J. Biol Chem.270:31178-31188

•DNA binding domain: the “motif mindset” concept: no one had anticipated that basic stuctural scaffolds would be used over and over again to recognize DNA and other proteins.

•Surprisingly large variety of protein familiy motifs involved in DNA sequence -specific recognition (promoter/enhancer contain matrices of short consensus sequences (RE)

•The role of DNA binding domains is to specifically deliver the transcription activation domain into the vincinity of the PIC

DNA Binding Domains (DBD)

•When a new protein was discovered, its gene cloned, the first questions were and to some extends still is: does it has any motifs we can understand? A helix-turn-helix? A zinc finger? An HMG domain? A β sheet DNA Motif?

•Uptream Trx factors are grouped with respect to their respective highly conserved DNA binding domains (DBD)

•In the case of DNA binding transcription factors the initial concept came from major stuctural determinations of bacterial regulators (lambda phage repressor, lambda Cro, Trp, 434 repressor, and CAP (catabolite activator protein from E. coli)

DNA Binding Domains (DBD)

A proteome-wide classification of DNA-binding protein DBD families

helix-turn-helix

zinc finger

helix-loop-helix

leucine zipper

Number of Swissprot (SW) entries for the respective DNA binding folds

Helix-Turn-Helix Motif (HTH) Domain: high degree of homology between bacterial and eukaryotes DBD

Typically relatively short motifs are responsible for specific binding to DNA

Homeodomain-DNA Complex (PDB 1hdd)

Gln50

Asn51Ile47

Arg3

Arg5

5’

T

A

A

T

T

3’

Computer drawing of the tridimensional structure as determined by X-Ray crystallography of Engrailed homeodomain

Vollmer JY and Clerc RG J. Neurochem. 71:1-20

Homeodomain protein classes implicated in the mouse brain development

Vollmer JY and Clerc RG J. Neurochem. 71:1-20

posterior late anterior early

low RA response high RA response

trunk hindbrain

Body plan is constructed through interaction of the developmentally regulated homeotic genes

Duboule D. editor Guidebook Homeoboxgenes. Oxford Press 1994

The POU domain: a large conserved region in the mammalian pit-1, oct1,2 and C. elegans unc96 gene products

Herr W. Clerc RG and Baltimore D. Genes & Dev 12:1513-1516

POU domain DNA interaction

5’ATGCAAAT 3’ (Octamer)Müller-Immerglück M, Matthias P. EMBO J. 9:1625-1631.

A zinc finger domain is a DBD

Originally identified in TFIIIA (5S rRNA), Zinc fingers form alpha helices that insert into the major groove associated with stabilizing beta sheets

A

TFIIIA contains 9 sequential CysHis zinc fingers with distinct DNA binding and TRX functions

DNA binding

in vitro transcription

recruitment of TFIIIC

Rothfels K et al. (2007) Nucl Acid Res 35:4869-81

TFIIIA-DNA

TFIIIA-TFIIIC-DNA

DNA

Originally identified in TFIIIA (5S rRNA), Zinc fingers form alpha helices that insert into the major groove associated with stabilizing beta sheets

Individual zinc finger domains that recognize DNA triplets with high specificity and affinity are fused to nonspecific nuclease domain to create designer TRX F for gene targeting

Targeted genome editing using zinc finger nucleases: custom DBD for transcription factors and nucleases (http://www.zincfingertools.org)

Tumor-specific missense mutations within CTCF transcriptional repressor/activator zinc finger domains

Filippova GN et al. 2002. Cancer Research 62:48-52

Tumor-specific mutations within CTCF Zf selectively alter CTCF DNA binding selectivity to different genome target sites

A distinct form of Zn finger motifs in Nuclear Receptors

Glucocorticoid Receptor-DNA Complex (PDB 1glu)

Zn finger

K461 V462 R466

5’A G A A C A NNN T G T T C T3’

T C T T G T NNN A C AA G A

Generation of DNA binding specificity mutants: glucocorticoid receptor ZnF DBD acquires estradiol specificity (ERE)

bZIP domain: basic “leucine zipper” (leucine residues spaced by 7 AA residues)

bZIP domain: prototypical amphipathic coiled-coil alpha helix

c-Fos-c-Jun-DNA Complex (PDB 1fos)

5’ T G A G T C A 3’

NF kappa B system pathways mediate cellular responses to a wide variety of stimuli including TNFalpha and lymphotoxine beta

Only one of the two DNA-bound orientations of AP-1 (fos-jun) found in solution cooperates with NFATp

Oakley M. and Verdine GL. Curr Biol 5:882 (1995)

Oakley M. and Verdine GL. Curr Biol 5:882 (1995)

Only one of the two DNA-bound orientations of AP-1 found in solution cooperates with NFATp

Helix-Loop-Helix Domains: Prototypical TRXF dimerizers

Each amphipathic helix presents a face of hydrophobic residues on one side and charged residues on the other side

bHLH proteins typically bind to a consensus sequence called an E-box, CANNTG

HMG “High Mobility Group” Motif (eg.lymphoid (T-cell) enhancer binding factor LEF-1)

DNA bending analysis by EMSA

LEF1 HMG Domain-DNA Complex (PDB 2lef)

L6

M13M10

E28

HELIX 2

HELIX 1

HELIX 3BASIC C-TERM

5’

C

C

T

T

T

G

A

A

G

C

3’

Y76

LEF1 HMG Domain-DNA Complex (PDB 2lef)

5’

C

C

T

T

T

G

A

A

G

C

3’

TBP : TATAA – Binding protein

• Acidic domains (DDD, EEE), VP16, GAL4, GCN4, GR

• Glutamine-rich domain (QQQXXXQQQ), SP1

• Proline-rich domains (PPPXXXPPP), CTCF, AP2

• Isoleucine-rich domain IIXXII), NTF

Several motifs involved in transactivation

Transcription Activation Domains -TADs

protein motives commonly referred to as “negative noodles, acid blobs” ! (Sigler P. Nature 333:210-212)

Transcription Activation Domains -TADs

• in contrary to DBDs, few structural data on TADs available

• TADs are intrinsically disordered unfolded proteins (resistant to crystal packing)

• TADs indirectly recruit Pol II by recruiting co-activators that serve as physical bridge between TADs and PIC/RNA Pol II holoenzyme

• TADs were reported to become structured (eg p53 helical TAD) upon binding to co-activators

Wells et al. PNAS 105:5762-5767

Ser/Thr and Gln Transcription activation domains -SP1, a prototypical constitutive TAD

5'-(G/T)GGGCGG(G/A)(G/A)(C/T)-

SP1 contains two glutamine-serine/threonine-rich TADs which interact with TAF110 (Gill g., Tjian R. PNAS 91:192-196)

(Gill G., Tjian R. PNAS 91:192-196)

Prototypical ligand mediated transcription activation domain (eg acidic negatively charged GR TAD)

unliganded liganded

co-repressor binding co-activator binding

“mouse trap”

HPV transactivation domain TAD forms a dimer

Harris SF, Botchan M. Science 284: 1673-1677

Antson AA et al. Nature 403: 805-809

HPV transactivation domain

Antson AA et al. Nature 403: 805-809Knight JD, Botchan M. PNAS 88:3204-3208

E2 binding site

E2 binding site

Canonical transrepression domains in upstream transcription factorseg. “Krüppel associated box” (KRAB) containing zinc-finger repressor proteins are associated with strong transcriptional repression of RNA polymerase I, II and III

Transrepression Domain (TRD)

Transcriptional repression of eukaryotic promoters by direct, (recruitment of co-repressors), or indirect, eg. HDAC’s recruitment to form heterochromatin, other chromatin modifications

Urrutia R. (2007) Genome Biology 4:231