Abstract
BackgroundMithramycin (MTR), a clinically approved DNA-bindingantitumor antibiotic has been found to cross the blood-brain barrier and is in its preclinical trials in Huntington sdisease (HD) [1]. It improved altered nucleosome homeos-tasis in HD mice, normalizing the chromatin pattern. Ithas the ability to rebalance epigenetic histone modifica-tions. The binding properties of MTR with histone pro-teins have been examined from the perspective of thecurrent proposition from our laboratory to classify smallDNA binding molecules in terms of their ability to bindchromosomal DNA alone (single binding mode) or bothhistones and chromosomal DNA (dual binding mode)[2,3].Materials and methodsChemicals were from Sigma, histones from New Eng-land Biolabs, antibodies from Abacm, Active Motif.Spectrofluorimetry, circular dichroism (CD) studies, invitro histone acetyl transferase (HAT) assay and westernblot analysis were employed as experimental tools.ResultsSpectrofluorimetric measurements and CD studies showthat MTR binds with micromolar dissociation constant tocore histone assembly and its individual histone compo-nents. A single isoelliptic point in the CD spectra indicatesformation of one type of complex between MTR and his-tones. Complex formation leads to decrease in molar ellip-ticity in the far UV CD spectra of core histones. Theseresults led us to investigate the role of MTR as a potentialepigenetic modulator. In vitro HAT assays using CREB-binding protein (CBP) HAT domain, a transcriptionalcoactivator, showed that MTR inhibits H3 K18 acetylation(specifically mediated mark by CBP/p300 in vivo ) in H3.3recombinant histone whereas it induces H3 K18 hyperace-tylation in H3.1/H4 tetramer as well as core histones.ConclusionsMTR exhibits dual binding mode since it binds to histoneproteins present in chromatin besides DNA [4]. It altersCBP induced H3 K18 acetylation. In contrast to MTR-DNA interaction, association of MTR with histones doesnot require obligatory presence of bivalent metal ions. Theinteraction of MTR with histones hints at a different sce-nario in MTR-chromatin interaction. Experiments are inprogress to address the effect of MTR on other epigeneticmarks.Thesignificanceofthesein vitro experimentswould also be examined by performing cell line basedexperiments. These data would be useful in understandingthe mechanism of action of anticancer drugs as well as indesigning new-generation therapeutics.
Highlights
Mithramycin (MTR), a clinically approved DNA-binding antitumor antibiotic has been found to cross the bloodbrain barrier and is in its preclinical trials in Huntington’s disease (HD) [1]
Materials and methods Chemicals were from Sigma, histones from New England Biolabs, antibodies from Abacm, Active Motif
Spectrofluorimetry, circular dichroism (CD) studies, in vitro histone acetyl transferase (HAT) assay and western blot analysis were employed as experimental tools
Summary
Mithramycin (MTR), a clinically approved DNA-binding antitumor antibiotic has been found to cross the bloodbrain barrier and is in its preclinical trials in Huntington’s disease (HD) [1]. It improved altered nucleosome homeostasis in HD mice, normalizing the chromatin pattern. The binding properties of MTR with histone proteins have been examined from the perspective of the current proposition from our laboratory to classify small DNA binding molecules in terms of their ability to bind chromosomal DNA alone (single binding mode) or both histones and chromosomal DNA (dual binding mode) [2,3]
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