Mechanism of Histone H3K4me3 Recognition by the Plant Homeodomain of Inhibitor of Growth 3.
Aberrant access to genetic information disrupts cellular homeostasis and can lead to cancer development. One molecular mechanism that regulates access to genetic information includes recognition of histone modifications, which is carried out by protein modules that interact with chromatin and serve as landing pads for enzymatic activities that regulate gene expression. The ING3 tumor suppressor protein contains a plant homeodomain (PHD) that reads the epigenetic code via recognition of histone H3 tri-methylated at lysine 4 (H3K4me3), and this domain is lost or mutated in various human cancers. However, the molecular mechanisms targeting ING3 to histones and the role of this interaction in the cell remain elusive. Thus, we employed biochemical and structural biology approaches to investigate the interaction of the ING3 PHD finger (ING3PHD) with the active transcription mark H3K4me3. Our results demonstrate that association of the ING3PHD with H3K4me3 is in the sub-micromolar range (KD ranging between 0.63 and 0.93 μm) and is about 200-fold stronger than with the unmodified histone H3. NMR and computational studies revealed an aromatic cage composed of Tyr-362, Ser-369, and Trp-385 that accommodate the tri-methylated side chain of H3K4. Mutational analysis confirmed the critical importance of Tyr-362 and Trp-385 in mediating the ING3PHD-H3K4me3 interaction. Finally, the biological relevance of ING3PHD-H3K4me3 binding was demonstrated by the failure of ING3PHD mutant proteins to enhance ING3-mediated DNA damage-dependent cell death. Together, our results reveal the molecular mechanism of H3K4me3 selection by the ING3PHD and suggest that this interaction is important for mediating ING3 tumor suppressive activities.
From the Department of Pharmaceutical Sciences, Albany College of Pharmacy and Health Sciences, Colchester, Vermont 05446.
Sophia
S
Kim
From the Department of Pharmaceutical Sciences, Albany College of Pharmacy and Health Sciences, Colchester, Vermont 05446.
Senthil
S
Natesan
the National Magnetic Resonance Facility at Madison and Department of Biochemistry, University of Wisconsin-Madison, Madison, Wisconsin 53706, and.
Gabriel
G
Cornilescu
From the Department of Pharmaceutical Sciences, Albany College of Pharmacy and Health Sciences, Colchester, Vermont 05446.
Samuel
S
Carlson
the National Magnetic Resonance Facility at Madison and Department of Biochemistry, University of Wisconsin-Madison, Madison, Wisconsin 53706, and.
Marco
M
Tonelli
the Newcastle Cancer Centre at the Northern Institute for Cancer Research, Newcastle University, Newcastle Upon Tyne NE2 4HH, United Kingdom.
Urszula L
UL
McClurg
the Newcastle Cancer Centre at the Northern Institute for Cancer Research, Newcastle University, Newcastle Upon Tyne NE2 4HH, United Kingdom.
Olivier
O
Binda
the Newcastle Cancer Centre at the Northern Institute for Cancer Research, Newcastle University, Newcastle Upon Tyne NE2 4HH, United Kingdom.
Craig N
CN
Robson
the National Magnetic Resonance Facility at Madison and Department of Biochemistry, University of Wisconsin-Madison, Madison, Wisconsin 53706, and.
John L
JL
Markley
From the Department of Pharmaceutical Sciences, Albany College of Pharmacy and Health Sciences, Colchester, Vermont 05446.
Stefan
S
Balaz
From the Department of Pharmaceutical Sciences, Albany College of Pharmacy and Health Sciences, Colchester, Vermont 05446, karen.glass@acphs.edu.
Karen C
0000-0002-2761-733X
KC
Glass
D019943:Amino Acid Substitution; D016923:Cell Death; D004249:DNA Damage; D044127:Epigenesis, Genetic; D006657:Histones / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D018398:Homeodomain Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D006801:Humans; D008745:Methylation; D020125:Mutation, Missense; D019906:Nuclear Magnetic Resonance, Biomolecular; D054829:RING Finger Domains; D025521:Tumor Suppressor Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism
D006657:Histones; D018398:Homeodomain Proteins; C459806:ING3 protein, human; D025521:Tumor Suppressor Proteins
Inhibitor of Growth 3; PHD finger; epigenetics; histone acetyltransferase; histone methylation; isothermal titration calorimetry (ITC); nuclear magnetic resonance (NMR); plant homeodomain