Dnmt3 and G9a cooperate for tissue-specific development in zebrafish.
Although DNA methylation is critical for proper embryonic and tissue-specific development, how different DNA methyltransferases affect tissue-specific development and their targets remains unknown. We address this issue in zebrafish through antisense-based morpholino knockdown of Dnmt3 and Dnmt1. Our data reveal that Dnmt3 is required for proper neurogenesis, and its absence results in profound defects in brain and retina. Interestingly, other organs such as intestine remain unaffected suggesting tissue-specific requirements of Dnmt3. Further, comparison of Dnmt1 knockdown phenotypes with those of Dnmt3 suggested that these two families have distinct functions. Consistent with this idea, Dnmt1 failed to complement Dnmt3 deficiency, and Dnmt3 failed to complement Dnmt1 deficiency. Downstream of Dnmt3 we identify a neurogenesis regulator, lef1, as a Dnmt3-specific target gene that is demethylated and up-regulated in dnmt3 morphants. Knockdown of lef1 rescued neurogenesis defects resulting from Dnmt3 absence. Mechanistically, we show cooperation between Dnmt3 and an H3K9 methyltransferase G9a in regulating lef1. Further, like Dnmt1-Suv39h1 cooperativity, Dnmt3 and G9a seemed to function together for tissue-specific development. G9a knockdown, but not Suv39h1 loss, phenocopied dnmt3 morphants and G9a overexpression provided a striking rescue of dnmt3 morphant phenotypes, whereas Suv39h1 overexpression failed, supporting the notion of specific DNMT-histone methyltransferase networks. Consistent with this model, H3K9me3 levels on the lef1 promoter were reduced in both dnmt3 and g9a morphants, and its knockdown rescued neurogenesis defects in g9a morphants. We propose a model wherein specific DNMT-histone methyltransferase networks are utilized to silence critical regulators of cell fate in a tissue-specific manner.
Rai
Kunal
K
From the Departments of Oncological Sciences, University of Utah, Salt Lake City, Utah 84112; Departments of Huntsman Cancer Institute, University of Utah, Salt Lake City, Utah 84112 and; Departments of Howard Hughes Medical Institute, University of Utah, Salt Lake City, Utah 84112.
Jafri
Itrat F
IF
From the Departments of Oncological Sciences, University of Utah, Salt Lake City, Utah 84112; Departments of Huntsman Cancer Institute, University of Utah, Salt Lake City, Utah 84112 and; Departments of Howard Hughes Medical Institute, University of Utah, Salt Lake City, Utah 84112.
Chidester
Stephanie
S
Departments of Huntsman Cancer Institute, University of Utah, Salt Lake City, Utah 84112 and.
James
Smitha R
SR
the Department of Pharmacology and Therapeutics, Roswell Park Cancer Institute, Buffalo, New York 14263.
Karpf
Adam R
AR
the Department of Pharmacology and Therapeutics, Roswell Park Cancer Institute, Buffalo, New York 14263.
Cairns
Bradley R
BR
From the Departments of Oncological Sciences, University of Utah, Salt Lake City, Utah 84112; Departments of Huntsman Cancer Institute, University of Utah, Salt Lake City, Utah 84112 and; Departments of Howard Hughes Medical Institute, University of Utah, Salt Lake City, Utah 84112. Electronic address: brad.cairns@hci.utah.edu.
Jones
David A
DA
From the Departments of Oncological Sciences, University of Utah, Salt Lake City, Utah 84112; Departments of Huntsman Cancer Institute, University of Utah, Salt Lake City, Utah 84112 and; Departments of Medicinal Chemistry, University of Utah, Salt Lake City, Utah 84112. Electronic address: david.jones@hci.utah.edu.
D000818:Animals; D001921:Brain / Q000166:cytology / Q000196:embryology / Q000378:metabolism; D047369:Chromatin Immunoprecipitation; D000076123:DNA (Cytosine-5-)-Methyltransferase 1; D004248:DNA (Cytosine-5-)-Methyltransferases / Q000235:genetics* / Q000378:metabolism; D004625:Embryo, Nonmammalian / Q000196:embryology / Q000378:metabolism; D020869:Gene Expression Profiling; D015971:Gene Expression Regulation, Enzymologic; D055785:Gene Knockdown Techniques; D011495:Histone-Lysine N-Methyltransferase / Q000235:genetics* / Q000378:metabolism; D017403:In Situ Hybridization; D008954:Models, Biological; D055495:Neurogenesis / Q000235:genetics; D012160:Retina / Q000166:cytology / Q000196:embryology / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism; D015027:Zebrafish / Q000196:embryology / Q000235:genetics* / Q000378:metabolism; D029961:Zebrafish Proteins / Q000235:genetics* / Q000378:metabolism
C507321:LEF1 protein, zebrafish; D014157:Transcription Factors; D029961:Zebrafish Proteins; D000076123:DNA (Cytosine-5-)-Methyltransferase 1; D004248:DNA (Cytosine-5-)-Methyltransferases; C514253:Dnmt1 protein, zebrafish; D011495:Histone-Lysine N-Methyltransferase