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C2B2/MAGNet Center Third Annual Retreat
Understanding and Predicting Transcription Factor Specificities Richard S. Mann C2B2/MAGNet Center Third Annual Retreat April 11, 2008
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Signal Transduction
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Transcriptional control by multiprotein complexes
Signal integration Vast combinatorial ensembles with a min number of factors Evolvability from Wolberger C., 1999
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Drosophila Hox genes Abd-B lab abd-A Dfd Ubx Scr Antp
Dave Kosman, UCSD
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Hox factors: molecular architects of morphological diversity
lab pb Dfd zen bcd Scr Antp ftz Ubx abd-A abd-B D. melanogaster head thorax abdomen A P Hoxd1 Hoxd4 Hoxd8 Hoxd9 Hoxd10 Hoxd11 Hoxd12 Hoxd13 Hoxd3 Hoxa1 Hoxa2 Hoxa4 Hoxa5 Hoxa6 Hoxa7 Hoxa9 Hoxa10 Hoxa11 Hoxa13 Hoxa3 Hoxb1 Hoxb2 Hoxb4 Hoxb5 Hoxb6 Hoxb7 Hoxb8 Hoxb9 Hoxb3 Hoxc4 Hoxc5 Hoxc6 Hoxc8 Hoxc9 Hoxc10 Hoxc11 Hoxc12 Hoxc13 Mus musculus The Hox factors in flies are eight and they are organized in a split cluster. The ANTP-C comprises Lab, Pb and Dfd, which sculpt the head, as well as the thorax specific genes Scr and Antp. The BX-C members Ubx, Abd-A and Abd-B are involved in the specification of the abdominal segments. Vertebrates have a complement of 39 Hox genes organized in four clusters, with eight paralog groups corresponding to the fly counterparts, even though not all eight groups are represented in each cluster. Adapted from Perason J.C. et al., 2005
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Problem of Hox specificity: Paradox 1
Homeodomain linker N-term arm YPWM helix 1 helix 2 helix 3 Lab TYKWMQ(109)NNSGRTNFTNKQLTELEKEFHFNRYLTRARRIEIANTLQLNETQVKIWFQNRRMKQKKRV Pb EYPWMK(28) PRRLRTAYTNTQLLELEKEFHFNKYLCRPRRIEIAASLDLTERQVKVWFQNRRMKHKRQT Dfd IYPWMK(17) PKRQRTAYTRHQILELEKEFHYNRYLTRRRRIEIAHTLVLSERQIKIWFQNRRMKWKKDN Scr IYPWMK(14) TKRQRTSYTRYQTLELEKEFHFNRYLTRRRRIEIAHALCLTERQIKIWFQNRRMKWKKEH Antp LYPWMR (8) RKRGRQTYTRYQTLELEKEFHFNRYLTRRRRIEIAHALCLTERQIKIWFQNRRMKWKKEN Ubx FYPWMA (7) RRRGRQTYTRYQTLELEKEFHTNHYLTRRRRIEMAHALCLTERQIKIWFQNRRMKLKKEI Abd-A RYPWMT(24) RRRGRQTYTRFQTLELEKEFHFNHYLTRRRRIEIAHALCLTERQIKIWFQNRRMKLKKEL Abd-B LHEWTG (3) VRKKRKPYSKFQTLELEKEFLFNAYVSKQKRWELARNLQLTERQVKIWFQNRRMKNKKNS Slide shows the homeodomain seq for various hox proteins and Shows the residues of the homeodomain which contact DNA sequence in red identical residues make DNA contacts Paradox 2: most Hox proteins bind to very similar ‘AT’ rich binding sites
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Paradox 3: residues important for specificity are usually disordered
Ubx YPWM Exd Passner, Aggarwal
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} D. Andrew; BioEssays 23: Salivary Gland A P Fkh
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Distinct properties of Hox-Exd binding sites
fkh250 fkh250con Exd Scr Exd Hox AGATTAATCG AGATTTATGG paralog specific shared Ryoo et al., 1999
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fkh250 fkh250con His–12 Arg3 Passner, Jain, Aggarwal
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fkh250 has two minor groove width minima that dictate electrostatic potential
It is thought that part of the specificity information may lie within the target DNA itself. Rohs, Sosinsky, Honig
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‘Specific’ Hox-DNA contacts Recognition of DNA SHAPE
Base-specific hydrogen bonds ‘General’ Hox-DNA contacts homeodomain linker N-term arm YPWM helix 1 helix 2 helix 3
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‘General’ Hox-DNA contacts ‘Specific’ Hox-DNA contacts
YPWM helix 1 helix 2 helix 3 homeodomain linker N-term arm ‘General’ Hox-DNA contacts ‘Specific’ Hox-DNA contacts
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binding specificities? What are the range of DNA recognition modes?
‘Specific’ Hox-DNA contacts ‘General’ Hox-DNA contacts homeodomain linker N-term arm YPWM helix 1 helix 2 helix 3 What are the global DNA binding specificities? What are the range of DNA recognition modes? How general is this mechanism?
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binding specificities? What are the range of DNA recognition modes?
‘Specific’ Hox-DNA contacts ‘General’ Hox-DNA contacts homeodomain linker N-term arm YPWM helix 1 helix 2 helix 3 What are the global DNA binding specificities? What are the range of DNA recognition modes? How general is this mechanism?
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His–12 and Arg3 are among Scr’s ‘signature’ residues
HOMEODOMAIN LINKER YPWM His–12 Arg3
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Paralog-specific ‘signature’ residues surrounding the YPWM motif Lab
Lab SSIPTYKWMQLKRNVP XD1 SYVSTFDWMKVKRNPP mA1 SPAQTFDWMKVKRNPP hB1 PTARTFDWMKVKRNPP xA1 GPTQTFDWMKVKRNPP CB1 SRARTFDWMKVKRNPP MB1 LTPRTFDWMKVKRNPP PB DSVPEYPWMKEKKTSR HB2 PPAPEFPWMKEKKSAK HB3 LTKQIFPWMKESRQTS HD3 ISKQIFPWMKESRQNS MB3 LTKQIFPWMKESRQTS DFD GERIIYPWMKKIHVAG CD4 QPAVVYPWMKKVHVNS MD4 QPAVVYPWMKKVHVNS HD4 QPAVVYPWMKKVHVNS MA4 KEPVVYPWMKKIHVSA HA4 KEPVVYPWMKKIHVSA CA4 KEPVVYPWMKKIHVST CB4 KEPVVYPWMKKVHVST MB4 KEPVVYPWMRKVHVST HC4 KQPIVYPWMKKIHVST MC4 KQPIVYPWMKKIHVST XB4 QDPVVYPWMKKAHISK Scr NPPQIYPWMKRVHLGT MA5 AQPQIYPWMRKLHISH HA5 AQPQIYPWMRKLHISH MB5 QTPQIFPWMRKLHISH HB5 QSPQIFPWMRKLHINH XB5 QSPQIFPWMRKLHINH HC5 QPPQIYPWMTKLHMSH ANT MPSPLYPWMRSQPGKC CB8 SPTQLFPWMRPQAAAG MB8 SPTQLFPWMRPQAAAG XB8 SPTQLFPWMRPQAAGR CD8 SPAQMFPWMRPQAAPG MD8 SPSQMFPWMRPQAAPG MC8 SPSLMFPWMRPHAPGR UBX SNHTFYPWMAIAGECP XB7 ANLRIYPWMRSAGADR HB7 SNFRIYPWMRSSGTDR MB7 SNFRIYPWMRSSGPDR XA7 SHFRIYPWMRSSGPDR CA7 ANFRIYPWMRSSGPDR MA7 ASFRIYPWMRSSGPDR ABA ADLPRYPWMTLTDWMG MC6 ASIQIYPWMQRMNSHS HC6 ASIQIYPWMQRMNSHS XC6 GSIQIYPWMQRMNSHS MB6 CSTPVYPWMQRMNSCN HB6 CSTPVYPWMQRMNSCN Lab Pb Dfd Scr Antp Ubx AbdA Hox6
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Fkh250 Fkh250con Scr Dfd Ubx Kd ~10nM Kd ~12nM Kd ~40nM Kd ~20nM
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Dfd is a repressor of fkh250
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Two steps in Hox specificity
Hox binding site DNA binding cofactors Hox binding site We are trying to understand what determines the specificity of Hox family of transcription factors. Over the years its been known that there are many aspects to this problem. Hox activity can be interpreted in two parts 1-Recognition and recruitment of the Hox and its cofactors to the cognate binding 2-regulation of the target gene by activator or repressors. Regulation Act Rep
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binding specificities? What are the range of DNA recognition modes?
‘Specific’ Hox-DNA contacts ‘General’ Hox-DNA contacts homeodomain linker N-term arm YPWM helix 1 helix 2 helix 3 What are the global DNA binding specificities? What are the range of DNA recognition modes? How general is this mechanism?
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Cognate Sequence Identifier (CSI)
Aseem Ansari
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Correlation between Exd and Dfd binding
Karl Haucshild and Aseem Ansari
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Exd+Scr Exd+Ubx Sequences that prefer Scr-Exd Sequences that prefer Ubx-Exd
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Exd Dfd Karl Haucshild and Aseem Ansari
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binding specificities? What are the range of DNA recognition modes?
‘Specific’ Hox-DNA contacts ‘General’ Hox-DNA contacts homeodomain linker N-term arm YPWM helix 1 helix 2 helix 3 What are the global DNA binding specificities? What are the range of DNA recognition modes? How general is this mechanism?
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Hox cofactor DNA Scr Exd fkh250 Lab Exd Lab48/95 Ubx DllR AbdA En Dfd
? Xiangshu Jin
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Lab DNA shape varies among Hox binding sites Fkh250con Fkh250 All Hox?
Scr, Dfd Ebner Lab DllR Lab48/95 Ubx, AbdA Lab Remo Rohs, Barry Honig
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binding specificities? What are the range of DNA recognition modes?
‘Specific’ Hox-DNA contacts ‘General’ Hox-DNA contacts homeodomain linker N-term arm YPWM helix 1 helix 2 helix 3 What are the global DNA binding specificities? What are the range of DNA recognition modes? How general is this mechanism?
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Rohit Joshi Jonathan Passner Rinku Jain Aneel Aggarwal Remo Rohs Alona Sosinsky Barry Honig Xiangshu Jin Karl Hauschild Aseem Ansari Andrea Califano
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