Understanding and Predicting Transcription Factor Specificities

Richard S. MannC2B2/MAGNet Center Third Annual Retreat

April 11, 2008

Signal Transduction

Transcriptional control by multiprotein complexes

p50 p65

Signal integration

Vast combinatorial ensembles with a min number of factors

Evolvability

from Wolberger C., 1999

Dave Kosman, UCSD

Drosophila Hox genes

lab

DfdScr Antp

Ubx

abd-A

Abd-B

Hox factors: molecular architects of morphological diversity

lab pb Dfd

zen bcd

Scr Antp

ftz

Ubx abd-A abd-B

D. melanogaster

Hoxd1 Hoxd4 Hoxd8 Hoxd9 Hoxd10 Hoxd11 Hoxd12 Hoxd13Hoxd3

Hoxa1 Hoxa2 Hoxa4 Hoxa5 Hoxa6 Hoxa7 Hoxa9 Hoxa10 Hoxa11 Hoxa13Hoxa3

Hoxb1 Hoxb2 Hoxb4 Hoxb5 Hoxb6 Hoxb7 Hoxb8 Hoxb9 Hoxa13Hoxb3

Hoxc4 Hoxc5 Hoxc6 Hoxc8 Hoxc9 Hoxc10 Hoxc11 Hoxc12 Hoxc13

Mus musculus

A P

Adapted from Perason J.C. et al., 2005

head thorax abdomen

Problem of Hox specificity: Paradox 1

identical residues make DNA contacts

YPWM helix 1 helix 2 helix 3

Homeodomain

linker N-term arm

1 10 20 30 40 50 60Lab TYKWMQ(109)NNSGRTNFTNKQLTELEKEFHFNRYLTRARRIEIANTLQLNETQVKIWFQNRRMKQKKRVPb EYPWMK(28) PRRLRTAYTNTQLLELEKEFHFNKYLCRPRRIEIAASLDLTERQVKVWFQNRRMKHKRQTDfd IYPWMK(17) PKRQRTAYTRHQILELEKEFHYNRYLTRRRRIEIAHTLVLSERQIKIWFQNRRMKWKKDNScr IYPWMK(14) TKRQRTSYTRYQTLELEKEFHFNRYLTRRRRIEIAHALCLTERQIKIWFQNRRMKWKKEHAntp LYPWMR (8) RKRGRQTYTRYQTLELEKEFHFNRYLTRRRRIEIAHALCLTERQIKIWFQNRRMKWKKENUbx FYPWMA (7) RRRGRQTYTRYQTLELEKEFHTNHYLTRRRRIEMAHALCLTERQIKIWFQNRRMKLKKEIAbd-A RYPWMT(24) RRRGRQTYTRFQTLELEKEFHFNHYLTRRRRIEIAHALCLTERQIKIWFQNRRMKLKKELAbd-B LHEWTG (3) VRKKRKPYSKFQTLELEKEFLFNAYVSKQKRWELARNLQLTERQVKIWFQNRRMKNKKNS

Paradox 2: most Hox proteins bind to very similar ‘AT’ rich binding sites

Passner, Aggarwal

Ubx

Exd

YPWM

Paradox 3: residues important for specificity are usually disordered

}Salivary Gland

D. Andrew; BioEssays 23:901-911

A

P

Fkh

Distinct properties of Hox-Exd binding sites

fkh250 fkh250con

Ryoo et al., 1999

Exd Scr

AGATTAATCG

paralogspecific

Exd Hox

AGATTTATGG

shared

fkh250

Passner, Jain, Aggarwal

fkh250con

His–12

Arg3

fkh250 has two minor groove width minima that dictate electrostatic potential

Rohs, Sosinsky, Honig

YPWM helix 1 helix 2 helix 3

homeodomainlinker N-term

arm

‘General’ Hox-DNA contacts

Base-specific hydrogen bonds

‘Specific’ Hox-DNA contacts

Recognition of DNA SHAPE

YPWM helix 1 helix 2 helix 3

homeodomainlinker N-term

arm

‘General’ Hox-DNA contacts‘Specific’ Hox-DNA contacts

YPWM helix 1 helix 2 helix 3

homeodomainlinker N-term

arm

‘General’ Hox-DNA contacts‘Specific’ Hox-DNA contacts

What are theglobal DNA

binding specificities?How general is

this mechanism?

What are therange of DNA

recognition modes?

YPWM helix 1 helix 2 helix 3

homeodomainlinker N-term

arm

‘General’ Hox-DNA contacts‘Specific’ Hox-DNA contacts

What are theglobal DNA

binding specificities?How general is

this mechanism?

What are therange of DNA

recognition modes?

His–12 and Arg3 are among Scr’s ‘signature’ residues

HOMEODOMAINLINKERYPWM

His–12 Arg3

Lab SSIPTYKWMQLKRNVPXD1 SYVSTFDWMKVKRNPPmA1 SPAQTFDWMKVKRNPPhB1 PTARTFDWMKVKRNPPxA1 GPTQTFDWMKVKRNPPCB1 SRARTFDWMKVKRNPPMB1 LTPRTFDWMKVKRNPPPB DSVPEYPWMKEKKTSRHB2 PPAPEFPWMKEKKSAKHB3 LTKQIFPWMKESRQTSHD3 ISKQIFPWMKESRQNSMB3 LTKQIFPWMKESRQTSDFD GERIIYPWMKKIHVAGCD4 QPAVVYPWMKKVHVNSMD4 QPAVVYPWMKKVHVNSHD4 QPAVVYPWMKKVHVNSMA4 KEPVVYPWMKKIHVSAHA4 KEPVVYPWMKKIHVSACA4 KEPVVYPWMKKIHVSTCB4 KEPVVYPWMKKVHVSTMB4 KEPVVYPWMRKVHVSTHC4 KQPIVYPWMKKIHVSTMC4 KQPIVYPWMKKIHVSTXB4 QDPVVYPWMKKAHISKScr NPPQIYPWMKRVHLGTMA5 AQPQIYPWMRKLHISHHA5 AQPQIYPWMRKLHISHMB5 QTPQIFPWMRKLHISHHB5 QSPQIFPWMRKLHINHXB5 QSPQIFPWMRKLHINHHC5 QPPQIYPWMTKLHMSHANT MPSPLYPWMRSQPGKCCB8 SPTQLFPWMRPQAAAGMB8 SPTQLFPWMRPQAAAGXB8 SPTQLFPWMRPQAAGRCD8 SPAQMFPWMRPQAAPGMD8 SPSQMFPWMRPQAAPGMC8 SPSLMFPWMRPHAPGRUBX SNHTFYPWMAIAGECPXB7 ANLRIYPWMRSAGADRHB7 SNFRIYPWMRSSGTDRMB7 SNFRIYPWMRSSGPDRXA7 SHFRIYPWMRSSGPDRCA7 ANFRIYPWMRSSGPDRMA7 ASFRIYPWMRSSGPDRABA ADLPRYPWMTLTDWMGMC6 ASIQIYPWMQRMNSHSHC6 ASIQIYPWMQRMNSHSXC6 GSIQIYPWMQRMNSHSMB6 CSTPVYPWMQRMNSCNHB6 CSTPVYPWMQRMNSCN

Lab

Pb

Dfd

Scr

Antp

UbxAbdA

Hox6

Paralog-specific‘signature’ residues

surrounding the YPWM motif

Fkh250conFkh250

Ubx

Kd >300nM Kd ~30nM

Dfd

Kd ~40nMKd ~20nM

ScrKd ~10nM Kd ~12nM

Fkh250DfdFkh250

Dfd is a repressor of fkh250

Hox

Act

Rep

Hox binding site

Hox binding sitecofactors

DNA binding

Regulation

Two steps in Hox specificity

YPWM helix 1 helix 2 helix 3

homeodomainlinker N-term

arm

‘General’ Hox-DNA contacts‘Specific’ Hox-DNA contacts

What are theglobal DNA

binding specificities?How general is

this mechanism?

What are therange of DNA

recognition modes?

Cognate Sequence Identifier (CSI)

Aseem Ansari

Correlation between Exd and Dfd binding

Karl Haucshild and Aseem Ansari

Exd+Scr Exd+Ubx

Sequences that preferUbx-Exd

Sequences that preferScr-Exd

Exd Dfd

Karl Haucshild and Aseem Ansari

YPWM helix 1 helix 2 helix 3

homeodomainlinker N-term

arm

‘General’ Hox-DNA contacts‘Specific’ Hox-DNA contacts

What are theglobal DNA

binding specificities?How general is

this mechanism?

What are therange of DNA

recognition modes?

Scr

Hox cofactor DNA

Exd fkh250

Lab Exd Lab48/95

Ubx Exd DllR

AbdA Exd DllR

AbdA En DllR

Dfd Exd fkh250

?

Xiangshu Jin

Remo Rohs, Barry Honig

Fkh250con

All Hox?

Fkh250

Scr, Dfd

DllR

Ubx, AbdA

Lab48/95

Lab

DNA shape varies among Hox binding sites

Ebner

Lab

Lab

YPWM helix 1 helix 2 helix 3

homeodomainlinker N-term

arm

‘General’ Hox-DNA contacts‘Specific’ Hox-DNA contacts

What are theglobal DNA

binding specificities?

How general is this mechanism?

What are therange of DNA

recognition modes?

Rohit Joshi

Jonathan PassnerRinku JainAneel Aggarwal

Remo RohsAlona SosinskyBarry Honig

Xiangshu Jin

Karl HauschildAseem Ansari

Andrea Califano