Andrew J. Kueh

Mof (MYST1 or KAT8) is essential for progression of embryonic development past the blastocyst stage and required for normal chromatin architecture.

ABSTRACT Acetylation of histone tails is a hallmark of transcriptionally active chromatin. Mof (males absent on the first; also called MYST1 or KAT8) is a member of the MYST family of histone acetyltransferases and was originally discovered as an essential component of the X chromosome dosage compensation system in Drosophila. In order to examine the role of Mof in mammals in vivo, we generated mice carrying a null mutation of the Mof gene. All Mof-deficient embryos fail to develop beyond the expanded blastocyst stage and die at implantation in vivo. Mof-deficient cell lines cannot be derived from Mof−/− embryos in vitro. Mof−/− embryos fail to acetylate histone 4 lysine 16 (H4K16) but have normal acetylation of other N-terminal histone lysine residues. Mof−/− cell nuclei exhibit abnormal chromatin aggregation preceding activation of caspase 3 and DNA fragmentation. We conclude that Mof is functionally nonredundant with the closely related MYST histone acetyltransferase Tip60. Our results show that Mof performs a different role in mammals from that in flies at the organism level, although the molecular function is conserved. We demonstrate that Mof is required specifically for the maintenance of H4K16 acetylation and normal chromatin architecture of all cells of early male and female embryos.

HBO1 Is Required for H3K14 Acetylation and Normal Transcriptional Activity during Embryonic Development

ABSTRACT We report here that the MYST histone acetyltransferase HBO1 (histone acetyltransferase bound to ORC; MYST2/KAT7) is essential for postgastrulation mammalian development. Lack of HBO1 led to a more than 90% reduction of histone 3 lysine 14 (H3K14) acetylation, whereas no reduction of acetylation was detected at other histone residues. The decrease in H3K14 acetylation was accompanied by a decrease in expression of the majority of genes studied. However, some genes, in particular genes regulating embryonic patterning, were more severely affected than “housekeeping” genes. Development of HBO1-deficient embryos was arrested at the 10-somite stage. Blood vessels, mesenchyme, and somites were disorganized. In contrast to previous studies that reported cell cycle arrest in HBO1-depleted cultured cells, no defects in DNA replication or cell proliferation were seen in Hbo1 mutant embryo primary fibroblasts or immortalized fibroblasts. Rather, a high rate of cell death and DNA fragmentation was observed in Hbo1 mutant embryos, resulting initially in the degeneration of mesenchymal tissues and ultimately in embryonic lethality. In conclusion, the primary role of HBO1 in development is that of a transcriptional activator, which is indispensable for H3K14 acetylation and for the normal expression of essential genes regulating embryonic development.

DR5 and caspase-8 are dispensable in ER stress-induced apoptosis

The endoplasmic reticulum (ER) stress response constitutes cellular reactions triggered by a wide variety of stimuli that disturb folding of proteins, often leading to apoptosis. ER stress-induced apoptotic cell death is thought to be an important contributor to many human pathological conditions. The molecular mechanism of this apoptosis process has been highly controversial with both the receptor and the mitochondrial pathways being implicated. Using knockout mouse models and RNAi-mediated gene silencing in cell lines, our group and others had demonstrated the importance of the mitochondrial apoptotic pathway in ER stress-induced cell death, particularly the role of the pro-apoptotic BH3-only BCL-2 family members, BIM and PUMA. However, a recent report suggested a central role for the death receptor, DR5, activated in a ligand-independent manner, and the initiator caspase, caspase-8, in ER stress-induced cell death. This prompted us to re-visit our previous observations and attempt to reproduce the newly published findings. Here we report that the mitochondrial apoptotic pathway, activated by BH3-only proteins, is essential for ER stress-induced cell death and that, in contrast to the previous report, DR5 as well as caspase-8 are not required for this process.

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

Cellular senescence is an important mechanism that restricts tumour growth. The Ink4a-Arf locus (also known as Cdkn2a), which encodes p16INK4A and p19ARF, 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.

A non‐canonical function of Ezh2 preserves immune homeostasis

Enhancer of zeste 2 (Ezh2) mainly methylates lysine 27 of histone‐H3 (H3K27me3) as part of the polycomb repressive complex 2 (PRC2) together with Suz12 and Eed. However, Ezh2 can also modify non‐histone substrates, although it is unclear whether this mechanism has a role during development. Here, we present evidence for a chromatin‐independent role of Ezh2 during T‐cell development and immune homeostasis. T‐cell‐specific depletion of Ezh2 induces a pronounced expansion of natural killer T (NKT) cells, although Ezh2‐deficient T cells maintain normal levels of H3K27me3. In contrast, removal of Suz12 or Eed destabilizes canonical PRC2 function and ablates NKT cell development completely. We further show that Ezh2 directly methylates the NKT cell lineage defining transcription factor PLZF, leading to its ubiquitination and subsequent degradation. Sustained PLZF expression in Ezh2‐deficient mice is associated with the expansion of a subset of NKT cells that cause immune perturbation. Taken together, we have identified a chromatin‐independent function of Ezh2 that impacts on the development of the immune system.

Excessive versus Physiologically Relevant Levels of Retinoic Acid in Embryonic Stem Cell Differentiation

Over the past two decades, embryonic stem cells (ESCs) have been established as a valuable system to study the complex molecular events that underlie the collinear activation of Hox genes during development. When ESCs are induced to differentiate in response to retinoic acid (RA), Hox genes are transcriptionally activated in their chromosomal order, with the most 3′ Hox genes activated first, sequentially followed by more 5′ Hox genes. In contrast to the low levels of RA detected during gastrulation (∼33 nM), a time when Hox genes are induced during embryonic development, high levels of RA are used to study Hox gene activation in ESCs in vitro (1–10 µM). This compelled us to compare RA‐induced ESC differentiation in vitro with Hox gene activation in vivo. In this study, we show that treatment of ESCs for 2 days with RA best mimics activation of Hox genes during embryonic development. Furthermore, we show that defects in Hox gene expression known to occur in embryos lacking the histone acetyltransferase MOZ (also called MYST3 or KAT6A) were masked in Moz‐deficient ESCs when excessive RA (0.5–5 µM) was used. The role of MOZ in Hox gene activation was only evident when ESCs were differentiated at low concentrations of RA, namely 20 nM, which is similar to RA levels in vivo. Our results demonstrate that using RA at physiologically relevant levels to study the activation of Hox genes, more accurately reflects the molecular events during the early phase of Hox gene activation in vivo. Stem Cells 2014;32:1451–1458

Chromatin immunoprecipitation of mouse embryos.

During prenatal development, a large number of different cell types are formed, the vast majority of which contain identical genetic material. The basis of the great variety in cell phenotype and function is the differential expression of the approximately 25,000 genes in the mammalian genome. Transcriptional activity is regulated at many levels by proteins, including members of the basal transcriptional apparatus, DNA-binding transcription factors, and chromatin-binding proteins. Importantly, chromatin structure dictates the availability of a specific genomic locus for transcriptional activation as well as the efficiency, with which transcription can occur. Chromatin immunoprecipitation (ChIP) is a method to assess if chromatin modifications or proteins are present at a specific locus. ChIP involves the cross linking of DNA and associated proteins and immunoprecipitation using specific antibodies to DNA-associated proteins followed by examination of the co-precipitated DNA sequences or proteins. In the last few years, ChIP has become an essential technique for scientists studying transcriptional regulation and chromatin structure. Using ChIP on mouse embryos, we can document the presence or absence of specific proteins and chromatin modifications at genomic loci in vivo during mammalian development. Here, we describe a ChIP technique adapted for mouse embryos.

MOZ and BMI1 play opposing roles during Hox gene activation in ES cells and in body segment identity specification in vivo

Significance The body is patterned in the anterior–posterior axis by the correct spatial and temporal expression of Hox genes during embryonic development. One mechanism critical for the precise spatiotemporal expression of Hox genes is the chromatin state. While changes in chromatin conformation during the activation of Hox genes have been well described, the importance of the factors that in turn regulate chromatin remains enigmatic and controversial. In the current study, we investigate the role of two critical chromatin regulators, MOZ and BMI1, during Hox gene activation and in specifying body segment identity. We establish the importance of MOZ and BMI1 during the initial activation of Hox genes in ES cells and in correctly specifying body segment identity during embryonic development. Hox genes underlie the specification of body segment identity in the anterior–posterior axis. They are activated during gastrulation and undergo a dynamic shift from a transcriptionally repressed to an active chromatin state in a sequence that reflects their chromosomal location. Nevertheless, the precise role of chromatin modifying complexes during the initial activation phase remains unclear. In the current study, we examined the role of chromatin regulators during Hox gene activation. Using embryonic stem cell lines lacking the transcriptional activator MOZ and the polycomb-family repressor BMI1, we showed that MOZ and BMI1, respectively, promoted and repressed Hox genes during the shift from the transcriptionally repressed to the active state. Strikingly however, MOZ but not BMI1 was required to regulate Hox mRNA levels after the initial activation phase. To determine the interaction of MOZ and BMI1 in vivo, we interrogated their role in regulating Hox genes and body segment identity using Moz;Bmi1 double deficient mice. We found that the homeotic transformations and shifts in Hox gene expression boundaries observed in single Moz and Bmi1 mutant mice were rescued to a wild type identity in Moz;Bmi1 double knockout animals. Together, our findings establish that MOZ and BMI1 play opposing roles during the onset of Hox gene expression in the ES cell model and during body segment identity specification in vivo. We propose that chromatin-modifying complexes have a previously unappreciated role during the initiation phase of Hox gene expression, which is critical for the correct specification of body segment identity.

Hepatocyte growth factor renders BRAF mutant human melanoma cell lines resistant to PLX4032 by downregulating the pro-apoptotic BH3-only proteins PUMA and BIM.

A large proportion of melanomas harbour the activating BRAFV600E mutation that renders these cells dependent on MAPK signalling for their survival. Although the highly specific and clinically approved BRAFV600E kinase inhibitor, PLX4032, induces apoptosis of melanoma cells bearing this mutation, the underlying molecular mechanisms are not fully understood. Here, we reveal that PLX4032-induced apoptosis depends on the induction of the pro-apoptotic BH3-only protein PUMA with a minor contribution of its relative BIM. Apoptosis could be significantly augmented when PLX4032 was combined with an inhibitor of the pro-survival protein BCL-XL, whereas neutralization of the pro-survival family member BCL-2 caused no additional cell death. Although the initial response to PLX4032 in melanoma patients is very potent, resistance to the drug eventually develops and relapse occurs. Several factors can cause melanoma cells to develop resistance to PLX4032; one of them is the activation of the receptor tyrosine kinase cMET on melanoma cells by its ligand, hepatocyte growth factor (HGF), provided by the tumour microenvironment or the cancer cells themselves. We found that HGF mediates resistance of cMET-expressing BRAF mutant melanoma cells to PLX4032-induced apoptosis through downregulation of PUMA and BIM rather than by increasing the expression of pro-survival BCL-2-like proteins. These results suggest that resistance to PLX4032 may be overcome by specifically increasing the levels of PUMA and BIM in melanoma cells through alternative signalling cascades or by blocking pro-survival BCL-2 family members with suitable BH3 mimetic compounds.

Cortical Layer Inversion and Deregulation of Reelin Signaling in the Absence of SOCS6 and SOCS7.

Abstract Mutations of the reelin gene cause severe defects in cerebral cortex development and profound intellectual impairment. While many aspects of the reelin signaling pathway have been identified, the molecular and ultimate cellular consequences of reelin signaling remain unknown. Specifically, it is unclear if termination of reelin signaling is as important for normal cortical neuron migration as activation of reelin signaling. Using mice that are single or double deficient, we discovered that combined loss of the suppressors of cytokine signaling, SOCS6 and SOCS7, recapitulated the cortical layer inversion seen in mice lacking reelin and led to a dramatic increase in the reelin signaling molecule disabled (DAB1) in the cortex. The SRC homology domains of SOCS6 and SOCS7 bound DAB1 ex vivo. Mutation of DAB1 greatly diminished binding and protected from degradation by SOCS6. Phosphorylated DAB1 was elevated in cortical neurons in the absence of SOCS6 and SOCS7. Thus, constitutive activation of reelin signaling was observed to be equally detrimental as lack of activation. We hypothesize that, by terminating reelin signaling, SOCS6 and SOCS7 may allow new cycles of reelin signaling to occur and that these may be essential for cortical neuron migration.