A high-throughput small molecule screen identifies synergism between DNA methylation and Aurora kinase pathways for X reactivation.
X-chromosome inactivation is a mechanism of dosage compensation in which one of the two X chromosomes in female mammals is transcriptionally silenced. Once established, silencing of the inactive X (Xi) is robust and difficult to reverse pharmacologically. However, the Xi is a reservoir of >1,000 functional genes that could be potentially tapped to treat X-linked disease. To identify compounds that could reactivate the Xi, here we screened ∼367,000 small molecules in an automated high-content screen using an Xi-linked GFP reporter in mouse fibroblasts. Given the robust nature of silencing, we sensitized the screen by "priming" cells with the DNA methyltransferase inhibitor, 5-aza-2'-deoxycytidine (5azadC). Compounds that elicited GFP activity include VX680, MLN8237, and 5azadC, which are known to target the Aurora kinase and DNA methylation pathways. We demonstrate that the combinations of VX680 and 5azadC, as well as MLN8237 and 5azadC, synergistically up-regulate genes on the Xi. Thus, our work identifies a synergism between the DNA methylation and Aurora kinase pathways as being one of interest for possible pharmacological reactivation of the Xi.
Howard Hughes Medical Institute, Massachusetts General Hospital, Boston, MA 02114.|Department of Molecular Biology, Massachusetts General Hospital, Boston, MA 02114.|Department of Genetics, Harvard Medical School, Boston, MA 02115.
Derek
D
Lessing
Howard Hughes Medical Institute, Massachusetts General Hospital, Boston, MA 02114.|Department of Molecular Biology, Massachusetts General Hospital, Boston, MA 02114.|Department of Genetics, Harvard Medical School, Boston, MA 02115.
Thomas O
TO
Dial
Howard Hughes Medical Institute, Massachusetts General Hospital, Boston, MA 02114.|Department of Molecular Biology, Massachusetts General Hospital, Boston, MA 02114.|Department of Genetics, Harvard Medical School, Boston, MA 02115.
Chunyao
C
Wei
Centre for Genomic Regulation, 08003 Barcelona, Spain.
Bernhard
B
Payer
Howard Hughes Medical Institute, Massachusetts General Hospital, Boston, MA 02114.|Department of Molecular Biology, Massachusetts General Hospital, Boston, MA 02114.|Department of Genetics, Harvard Medical School, Boston, MA 02115.|Center for Medical Genetics, Ghent University, 9000 Ghent, Belgium.
Lieselot L G
LL
Carrette
Howard Hughes Medical Institute, Massachusetts General Hospital, Boston, MA 02114.|Department of Molecular Biology, Massachusetts General Hospital, Boston, MA 02114.|Department of Genetics, Harvard Medical School, Boston, MA 02115.
Barry
B
Kesner
Howard Hughes Medical Institute, Massachusetts General Hospital, Boston, MA 02114.|Department of Molecular Biology, Massachusetts General Hospital, Boston, MA 02114.|Department of Genetics, Harvard Medical School, Boston, MA 02115.
Attila
A
Szanto
Division of Preclinical Innovation, National Center for Advancing Translational Sciences, National Institutes of Health, Rockville, MD 20850.
Ajit
A
Jadhav
Division of Preclinical Innovation, National Center for Advancing Translational Sciences, National Institutes of Health, Rockville, MD 20850.
David J
DJ
Maloney
Division of Preclinical Innovation, National Center for Advancing Translational Sciences, National Institutes of Health, Rockville, MD 20850.
Anton
A
Simeonov
Broad Institute, Cambridge, MA 02142.
Jimmy
J
Theriault
Broad Institute, Cambridge, MA 02142.
Thomas
T
Hasaka
Clinical Research Division, Fred Hutchinson Cancer Research Center, Seattle, WA 98109.
Antonio
A
Bedalov
Department of Cell and Developmental Biology, University of Pennsylvania School of Medicine, Philadelphia, PA 19104.
Marisa S
MS
Bartolomei
Howard Hughes Medical Institute, Massachusetts General Hospital, Boston, MA 02114; lee@molbio.mgh.harvard.edu.|Department of Molecular Biology, Massachusetts General Hospital, Boston, MA 02114.|Department of Genetics, Harvard Medical School, Boston, MA 02115.
Jeannie T
JT
Lee
D000818:Animals; D064096:Aurora Kinase A / Q000037:antagonists & inhibitors / Q000235:genetics; D064107:Aurora Kinase B / Q000037:antagonists & inhibitors / Q000235:genetics; D064127:Aurora Kinases / Q000037:antagonists & inhibitors* / Q000235:genetics; D001374:Azacitidine / Q000008:administration & dosage / Q000031:analogs & derivatives; D001381:Azepines / Q000008:administration & dosage; D002460:Cell Line; D019175:DNA Methylation / Q000187:drug effects*; D000077209:Decitabine; D004353:Drug Evaluation, Preclinical; D004357:Drug Synergism; D005260:Female; D055785:Gene Knockdown Techniques; D050172:Genes, X-Linked; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D057166:High-Throughput Screening Assays; D051379:Mice; D008822:Mice, Transgenic; D010879:Piperazines / Q000008:administration & dosage; D011743:Pyrimidines / Q000008:administration & dosage; D014960:X Chromosome / Q000187:drug effects / Q000235:genetics; D049951:X Chromosome Inactivation / Q000187:drug effects*; D003521:Cyclopropanes; D011720:Pyrazoles
D064096:Aurora Kinase A; D064107:Aurora Kinase B; D064127:Aurora Kinases; D001374:Azacitidine; D001381:Azepines; D000077209:Decitabine; D049452:Green Fluorescent Proteins; D010879:Piperazines; D011743:Pyrimidines; C550258:MLN 8237; C484810:tozasertib; D003521:Cyclopropanes; D011720:Pyrazoles; C576918:Aurka protein, mouse; C576924:Aurkb protein, mouse
Aurora kinase; DNA methyltransferase; X reactivation; high-throughput screen; small molecules