Chromatin Immunoprecipitation of Mouse Embryos

Voss, Anne K.; Dixon, Mathew P.; McLennan, Tamara J.; Kueh, Andrew J.; Thomas, Tim

doi:10.1007/978-1-61779-376-9_23

Cited by 14 publications

(7 citation statements)

References 8 publications

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“…ChIP followed by qPCR was carried out as described previously (Voss et al., 2009, 2012) using primers described in Supplemental Experimental Procedures. Antibodies used for ChIP, immunoblotting, and immunofluorescence were directed against ING5 (Abnova; H00084289-B01), FOXA2 (HNF-3b [M-20]: sc-6554; Santa Cruz Biotechnology), H3K9ac (Millipore; Upstate 07-352), H3K9me3 (Abcam; ab8898), and H3K14ac (Millipore; Upstate 07-353).…”

Section: Methodsmentioning

confidence: 99%

MOZ Regulates the Tbx1 Locus, and Moz Mutation Partially Phenocopies DiGeorge Syndrome

et al. 2012

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SummaryDiGeorge syndrome, caused by a 22q11 microdeletion or mutation of the TBX1 gene, varies in severity greatly, even among monozygotic twins. Epigenetic phenomena have been invoked to explain phenotypic differences in individuals of identical genetic composition, although specific chromatin modifications relevant to DiGeorge syndrome are elusive. Here we show that lack of the histone acetyltransferase MOZ (MYST3/KAT6A) phenocopies DiGeorge syndrome, and the MOZ complex occupies the Tbx1 locus, promoting its expression and histone 3 lysine 9 acetylation. Importantly, DiGeorge syndrome-like anomalies are present in mice with homozygous mutation of Moz and in heterozygous Moz mutants when combined with Tbx1 haploinsufficiency or oversupply of retinoic acid. Conversely, a Tbx1 transgene rescues the heart phenotype in Moz mutants. Our data reveal a molecular mechanism for a specific chromatin modification of the Tbx1 locus intersecting with an environmental determinant, modeling variability in DiGeorge syndrome.

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Section: Methodsmentioning

confidence: 99%

MOZ Regulates the Tbx1 Locus, and Moz Mutation Partially Phenocopies DiGeorge Syndrome

et al. 2012

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“…ChIP was performed essentially as described (Voss et al, 2012) on chromatin pooled from 25 E10.5 PA1s. Cross-linked chromatin was incubated with 5 μg Grhl2 antibody (HPA004820, batch B96161, Sigma-Aldrich) or 5 μg normal rabbit IgG (2729, batch 09/2009.…”

Section: Chipmentioning

confidence: 99%

Inactivation of Zeb1 in GRHL2-deficient mouse embryos rescues mid-gestation viability and secondary palate closure

Carpinelli

Vries

Auden

et al. 2020

Disease Models &Amp; Mechanisms

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Cleft lip and palate are common birth defects resulting from failure of the facial processes to fuse during development. The mammalian grainyhead-like (Grhl1-3) genes play key roles in a number of tissue fusion processes including neurulation, epidermal wound healing and eyelid fusion. One family member, Grhl2, is expressed in the epithelial lining of the first pharyngeal arch in mice at embryonic day (E)10.5, prompting analysis of the role of this factor in palatogenesis. Grhl2null mice die at E11.5 with neural tube defects and a cleft face phenotype, precluding analysis of palatal fusion at a later stage of development. However, in the first pharyngeal arch of Grhl2-null embryos, dysregulation of transcription factors that drive epithelialmesenchymal transition (EMT) occurs. The aberrant expression of these genes is associated with a shift in RNA-splicing patterns that favours the generation of mesenchymal isoforms of numerous regulators. Driving the EMT perturbation is loss of expression of the EMT-suppressing transcription factors Ovol1 and Ovol2, which are direct GRHL2 targets. The expression of the miR-200 family of microRNAs, also GRHL2 targets, is similarly reduced, resulting in a 56-fold upregulation of Zeb1 expression, a major driver of mesenchymal cellular identity. The critical role of GRHL2 in mediating cleft palate in Zeb1 −/− mice is evident, with rescue of both palatal and facial fusion seen in Grhl2 −/− ;Zeb1 −/− embryos. These findings highlight the delicate balance between GRHL2/ZEB1 and epithelial/mesenchymal cellular identity that is essential for normal closure of the palate and face. Perturbation of this pathway may underlie cleft palate in some patients.

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“…ChIP was carried out as previously described 75 with two million subconfluent MEFs per sample. Immunoprecipitation was carried out using antibodies against H3K9ac (Cell Signaling, Danvers, MA, USA, #9649 S), H3K14ac (Cell Signaling, #7627), H3K23ac (Merck, 07-355), H3K27ac (Abcam, ab4729), MOZ (Abcam, ab41718) and agarose-bead bound V5 antibody (Sigma, A7345).…”

Section: Chromatin Immunoprecipitationmentioning

confidence: 99%

MOZ (MYST3, KAT6A) inhibits senescence via the INK4A-ARF pathway

et al. 2015

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Cellular senescence is an important mechanism that restricts tumour growth. The Ink4a-Arf locus (also known as Cdkn2a), which encodes p16(INK4A) and p19(ARF), has a central role in inducing and maintaining senescence. Given the importance of cellular senescence in restraining tumour growth, great emphasis is being placed on the identification of novel factors that can modulate senescence. The MYST-family histone acetyltransferase MOZ (MYST3, KAT6A), first identified in recurrent translocations in acute myeloid leukaemia, has been implicated in both the promotion and inhibition of senescence. In this study, we investigate the role of MOZ in cellular senescence and show that MOZ is a potent inhibitor of senescence via the INK4A-ARF pathway. Primary mouse embryonic fibroblasts (MEFs) isolated from Moz-deficient embryos exhibit premature senescence, which was rescued on the Ink4a-Arf(-/-) background. Importantly, senescence resulting from the absence of MOZ was not accompanied by DNA damage, suggesting that MOZ acts independently of the DNA damage response. Consistent with the importance of senescence in cancer, expression profiling revealed that genes overexpressed in aggressive and highly proliferative cancers are expressed at low levels in Moz-deficient MEFs. We show that MOZ is required to maintain normal levels of histone 3 lysine 9 (H3K9) and H3K27 acetylation at the transcriptional start sites of at least four genes, Cdc6, Ezh2, E2f2 and Melk, and normal mRNA levels of these genes. CDC6, EZH2 and E2F2 are known inhibitors of the INK4A-ARF pathway. Using chromatin immunoprecipitation, we show that MOZ occupies the Cdc6, Ezh2 and Melk loci, thereby providing a direct link between MOZ, H3K9 and H3K27 acetylation, and normal transcriptional levels at these loci. This work establishes that MOZ is an upstream inhibitor of the INK4A-ARF pathway, and suggests that inhibiting MOZ may be one way to induce senescence in proliferative tumour cells.

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Chromatin Immunoprecipitation of Mouse Embryos

Cited by 14 publications

References 8 publications

MOZ Regulates the Tbx1 Locus, and Moz Mutation Partially Phenocopies DiGeorge Syndrome

MOZ Regulates the Tbx1 Locus, and Moz Mutation Partially Phenocopies DiGeorge Syndrome

Inactivation of Zeb1 in GRHL2-deficient mouse embryos rescues mid-gestation viability and secondary palate closure

MOZ (MYST3, KAT6A) inhibits senescence via the INK4A-ARF pathway

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