ABSTRACT
Histone acetyltransferases (HATs) are important to gene activation, altering chromatin structures to facilitate association of transcription proteins with gene promoters. The functions of individual HATs in mammalian developmental are not well defined. Our previous studies demonstrated that Gcn5, a prototypical HAT, is required for mesodermal maintenance in early embryos. Homozygous Gcn5 null embryos die soon after gastrulation, preventing determination of Gcn5 functions later during development. We report here the creation of a Gcn5(flox(neo)) allele, which is only partially functional and gives rise to a hypomorphic phenotype. Mice homozygous for this allele had an increased risk of cranial neural tube closure defects (NTDs) and exencephaly. These defects were found at an even greater penetrance in Gcn5(flox(neo)/Delta) embryos. These results indicate that normal levels of Gcn5 expression are critical for neural tube closure in mice and predict that mutations in this HAT may be associated with increased risk of NTDs in humans.
Subject(s)
Gene Expression Regulation, Developmental , Gene Expression Regulation, Enzymologic , Neural Tube Defects/enzymology , Neural Tube Defects/genetics , Neural Tube/physiology , p300-CBP Transcription Factors/metabolism , Alleles , Animals , Body Patterning , Cell Death , Cell Proliferation , Female , Folic Acid/metabolism , Genotype , Hedgehog Proteins/genetics , Hedgehog Proteins/metabolism , Humans , Mice , Mice, Inbred C57BL , Mice, Knockout , Neural Tube Defects/pathology , Phenotype , Signal Transduction/physiology , Transcriptional Activation , p300-CBP Transcription Factors/geneticsABSTRACT
Gcn5 is a prototypical histone acetyltransferase (HAT) that serves as a coactivator for multiple DNA-bound transcription factors. We previously determined that deletion of Gcn512 (hereafter referred to as Gcn5) causes embryonic lethality in mice. Gcn5 null embryos undergo gastrulation but exhibit high levels of apoptosis, leading to loss of mesodermal lineages. To further define the functions of Gcn5 during development, we created Gcn5(-/-) mouse embryonic stem (ES) cells. These cells survived in vitro and formed embryoid bodies (EBs) that expressed markers for ectodermal, mesodermal, and endodermal lineages. Gcn5(-/-) EBs were misshapen and smaller than wild-type EBs by day 6, with an increased proportion of cells in G2/M. Expression of Oct 4 and Nodal was prematurely curtailed in Gcn5(-/-) EBs, indicating early loss of pluripotent ES cells. Gcn5(-/-) EBs differentiated efficiently into skeletal and cardiac muscle, which derive from mesoderm. High percentage Gcn5(-/-) chimeric embryos created by injection of Gcn5(-/-) ES cells into wild-type blastocysts were delayed in development and died early. Interestingly, elevated levels of apoptosis were observed specifically in Gcn5 null cells within the chimeric embryos. Collectively, these data indicate that Gcn5 may be required to maintain pluripotent states and that loss of Gcn5 invokes a cell-autonomous pathway of cell death in vivo.
Subject(s)
Cell Cycle Proteins/metabolism , Embryonic Stem Cells/metabolism , Histone Acetyltransferases/deficiency , Histone Acetyltransferases/metabolism , Transcription Factors/deficiency , Transcription Factors/metabolism , Acetylation , Animals , Apoptosis , Cell Cycle Proteins/genetics , Cell Differentiation , Cell Lineage , Cells, Cultured , Embryo, Mammalian/cytology , Embryo, Mammalian/embryology , Embryo, Mammalian/metabolism , Embryonic Stem Cells/cytology , Gene Expression Regulation, Developmental , Histone Acetyltransferases/genetics , Mice , Mice, Inbred C57BL , Mice, Knockout , Muscle, Skeletal/cytology , Muscle, Skeletal/embryology , Muscle, Skeletal/metabolism , Proteins/genetics , Proteins/metabolism , RNA, Untranslated , Transcription Factors/genetics , p300-CBP Transcription FactorsABSTRACT
Previous studies revealed that deletion of genes encoding the histone acetyltransferases GCN5, p300, or CBP results in embryonic lethality in mice. PCAF and GCN5 physically interact with p300 and CBP in vitro. To determine whether these two groups of histone acetyltransferases interact functionally in vivo, we created mice lacking one or more alleles of p300, GCN5, or PCAF. As expected, we found that mice heterozygous for any single null allele are viable. The majority of GCN5(+/-)p300(+/-) mice also survive to adulthood with no apparent abnormalities. However, approximately 25% of these mice die before birth. These embryos are developmentally stunted and exhibit increased apoptosis compared with wild-type or single GCN5(+/-) or p300(+/-) littermates at embryonic day 8.5. In contrast, no abnormalities were observed in PCAF(-/-) p300(+/-) mice. Of interest, we find that p300 protein levels vary in different mouse genetic backgrounds, which likely contributes to the incomplete penetrance of the abnormal phenotype of GCN5(+/-) p300(+/-) mice. Our data indicate that p300 cooperates specifically with GCN5 to provide essential functions during early embryogenesis.
