Publications by authors named "Hannah K Vanyai"

13 Publications

  • Page 1 of 1

Control of skeletal morphogenesis by the Hippo-YAP/TAZ pathway.

Development 2020 11 12;147(21). Epub 2020 Nov 12.

The Francis Crick Institute, 1 Midland Rd, St Pancras, NW1 1AT London, UK

The Hippo-YAP/TAZ pathway is an important regulator of tissue growth, but can also control cell fate or tissue morphogenesis. Here, we investigate the function of the Hippo pathway during the development of cartilage, which forms the majority of the skeleton. Previously, YAP was proposed to inhibit skeletal size by repressing chondrocyte proliferation and differentiation. We find that, , / double knockout impairs murine chondrocyte proliferation, whereas constitutively nuclear accelerates proliferation, in line with the canonical role of this pathway in most tissues. However, , cartilage-specific knockout of / does not prevent chondrocyte proliferation, differentiation or skeletal growth, but rather results in various skeletal deformities including cleft palate. Cartilage-specific expression of or knockout of / do not increase cartilage growth, but instead lead to catastrophic malformations resembling chondrodysplasia or achondrogenesis. Physiological YAP target genes in cartilage include , and several matrix remodelling enzymes. Thus, YAP/TAZ activity controls chondrocyte proliferation , possibly reflecting a regenerative response, but is dispensable for chondrocyte proliferation , and instead functions to control cartilage morphogenesis via regulation of the extracellular matrix.
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http://dx.doi.org/10.1242/dev.187187DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7673359PMC
November 2020

CRISPR-Mediated Protein Tagging with Nanoluciferase to Investigate Native Chemokine Receptor Function and Conformational Changes.

Cell Chem Biol 2020 05 12;27(5):499-510.e7. Epub 2020 Feb 12.

Cell Signalling and Pharmacology Research Group, Division of Physiology, Pharmacology & Neuroscience, School of Life Sciences, University of Nottingham, Queens Medical Centre, Nottingham NG7 2UH, UK; Centre of Membrane Proteins and Receptors, University of Birmingham and University of Nottingham, The Midlands, UK; Harry Perkins Institute of Medical Research and Centre for Medical Research, The University of Western Australia, QEII Medical Centre, Nedlands, WA 6009, Australia. Electronic address:

G protein-coupled receptors are a major class of membrane receptors that mediate physiological and pathophysiological cellular signaling. Many aspects of receptor activation and signaling can be investigated using genetically encoded luminescent fusion proteins. However, the use of these biosensors in live cell systems requires the exogenous expression of the tagged protein of interest. To maintain the normal cellular context here we use CRISPR/Cas9-mediated homology-directed repair to insert luminescent tags into the endogenous genome. Using NanoLuc and bioluminescence resonance energy transfer we demonstrate fluorescent ligand binding at genome-edited chemokine receptors. We also demonstrate that split-NanoLuc complementation can be used to investigate conformational changes and internalization of CXCR4 and that recruitment of β-arrestin2 to CXCR4 can be monitored when both proteins are natively expressed. These results show that genetically encoded luminescent biosensors can be used to investigate numerous aspects of receptor function at native expression levels.
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http://dx.doi.org/10.1016/j.chembiol.2020.01.010DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7242902PMC
May 2020

MOZ directs the distal-less homeobox gene expression program during craniofacial development.

Development 2019 07 24;146(14). Epub 2019 Jul 24.

Walter and Eliza Hall Institute of Medical Research, Melbourne, Parkville, VIC 3052, Australia

Oral clefts are common birth defects. Individuals with oral clefts who have identical genetic mutations regularly present with variable penetrance and severity. Epigenetic or chromatin-mediated mechanisms are commonly invoked to explain variable penetrance. However, specific examples of these are rare. Two functional copies of the (, ) gene, encoding a MYST family lysine acetyltransferase chromatin regulator, are essential for human craniofacial development, but the molecular role of MOZ in this context is unclear. Using genetic interaction and genomic studies, we have investigated the effects of loss of MOZ on the gene expression program during mouse development. Among the more than 500 genes differentially expressed after loss of MOZ, 19 genes had previously been associated with cleft palates. These included four distal-less homeobox (DLX) transcription factor-encoding genes, , , and and DLX target genes (including , , and ). MOZ occupied the locus and was required for normal levels of histone H3 lysine 9 acetylation. MOZ affected Dlx gene expression cell-autonomously within neural crest cells. Our study identifies a specific program by which the chromatin modifier MOZ regulates craniofacial development.
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http://dx.doi.org/10.1242/dev.175042DOI Listing
July 2019

A citizen science model for implementing statewide educational DNA barcoding.

PLoS One 2019 10;14(1):e0208604. Epub 2019 Jan 10.

Museums Victoria, Carlton Gardens, Victoria, Australia.

Our aim was to develop a widely available educational program in which students conducted authentic research that met the expectations of both the scientific and educational communities. This paper describes the development and implementation of a citizen science project based on DNA barcoding of reptile specimens obtained from the Museums Victoria frozen tissue collection. The student program was run by the Gene Technology Access Centre (GTAC) and was delivered as a "one day plus one lesson" format incorporating a one-day wet laboratory workshop followed by a single lesson at school utilising online bioinformatics tools. The project leveraged the complementary resources and expertise of the research and educational partners to generate robust scientific data that could be analysed with confidence, meet the requirements of the Victorian state education curriculum, and provide participating students with an enhanced learning experience. During two 1-week stints in 2013 and 2014, 406 students mentored by 44 postgraduate university students participated in the project. Students worked mainly in pairs to process ~200 tissue samples cut from 53 curated reptile specimens representing 17 species. A total of 27 novel Cytochrome Oxidase subunit 1 (CO1) sequences were ultimately generated for 8 south-east Australian reptile species of the families Scincidae and Agamidae.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0208604PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6328199PMC
September 2019

Subtle Changes in the Levels of BCL-2 Proteins Cause Severe Craniofacial Abnormalities.

Cell Rep 2018 09;24(12):3285-3295.e4

The Walter and Eliza Hall Institute of Medical Research, Melbourne, VIC 3052, Australia; Department of Medical Biology, The University of Melbourne, Melbourne, VIC 3052, Australia. Electronic address:

Apoptotic cell death removes unwanted cells and is regulated by interactions between pro-survival and pro-apoptotic members of the BCL-2 protein family. The regulation of apoptosis is thought to be crucial for normal embryonic development. Accordingly, complete loss of pro-survival MCL-1 or BCL-XL (BCL2L1) causes embryonic lethality. However, it is not known whether minor reductions in pro-survival proteins could cause developmental abnormalities. We explored the rate-limiting roles of MCL-1 and BCL-XL in development and show that combined loss of single alleles of Mcl-1 and Bcl-x causes neonatal lethality. Mcl-1;Bcl-x mice display craniofacial anomalies, but additional loss of a single allele of pro-apoptotic Bim (Bcl2l11) restores normal development. These findings demonstrate that the control of cell survival during embryogenesis is finely balanced and suggest that some human craniofacial defects, for which causes are currently unknown, may be due to subtle imbalances between pro-survival and pro-apoptotic BCL-2 family members.
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http://dx.doi.org/10.1016/j.celrep.2018.08.048DOI Listing
September 2018

Inhibitors of histone acetyltransferases KAT6A/B induce senescence and arrest tumour growth.

Nature 2018 08 1;560(7717):253-257. Epub 2018 Aug 1.

The Walter and Eliza Hall Institute of Medical Research, Parkville, Melbourne, Victoria, Australia.

Acetylation of histones by lysine acetyltransferases (KATs) is essential for chromatin organization and function. Among the genes coding for the MYST family of KATs (KAT5-KAT8) are the oncogenes KAT6A (also known as MOZ) and KAT6B (also known as MORF and QKF). KAT6A has essential roles in normal haematopoietic stem cells and is the target of recurrent chromosomal translocations, causing acute myeloid leukaemia. Similarly, chromosomal translocations in KAT6B have been identified in diverse cancers. KAT6A suppresses cellular senescence through the regulation of suppressors of the CDKN2A locus, a function that requires its KAT activity. Loss of one allele of KAT6A extends the median survival of mice with MYC-induced lymphoma from 105 to 413 days. These findings suggest that inhibition of KAT6A and KAT6B may provide a therapeutic benefit in cancer. Here we present highly potent, selective inhibitors of KAT6A and KAT6B, denoted WM-8014 and WM-1119. Biochemical and structural studies demonstrate that these compounds are reversible competitors of acetyl coenzyme A and inhibit MYST-catalysed histone acetylation. WM-8014 and WM-1119 induce cell cycle exit and cellular senescence without causing DNA damage. Senescence is INK4A/ARF-dependent and is accompanied by changes in gene expression that are typical of loss of KAT6A function. WM-8014 potentiates oncogene-induced senescence in vitro and in a zebrafish model of hepatocellular carcinoma. WM-1119, which has increased bioavailability, arrests the progression of lymphoma in mice. We anticipate that this class of inhibitors will help to accelerate the development of therapeutics that target gene transcription regulated by histone acetylation.
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http://dx.doi.org/10.1038/s41586-018-0387-5DOI Listing
August 2018

Embryogenesis and Adult Life in the Absence of Intrinsic Apoptosis Effectors BAX, BAK, and BOK.

Cell 2018 05;173(5):1217-1230.e17

The Walter and Eliza Hall Institute of Medical Research, Parkville, Victoria 3052, Australia; Department of Medical Biology, The University of Melbourne, Melbourne, Victoria 3052, Australia. Electronic address:

Intrinsic apoptosis, reliant on BAX and BAK, has been postulated to be fundamental for morphogenesis, but its precise contribution to this process has not been fully explored in mammals. Our structural analysis of BOK suggests close resemblance to BAX and BAK structures. Notably, BokBaxBak animals exhibited more severe defects and died earlier than BaxBak mice, implying that BOK has overlapping roles with BAX and BAK during developmental cell death. By analyzing BokBaxBak triple-knockout mice whose cells are incapable of undergoing intrinsic apoptosis, we identified tissues that formed well without this process. We provide evidence that necroptosis, pyroptosis, or autophagy does not substantially substitute for the loss of apoptosis. Albeit very rare, unexpected attainment of adult BokBaxBak mice suggests that morphogenesis can proceed entirely without apoptosis mediated by these proteins and possibly without cell death in general.
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http://dx.doi.org/10.1016/j.cell.2018.04.036DOI Listing
May 2018

Using nanoBRET and CRISPR/Cas9 to monitor proximity to a genome-edited protein in real-time.

Sci Rep 2017 06 9;7(1):3187. Epub 2017 Jun 9.

Molecular Endocrinology and Pharmacology, Harry Perkins Institute of Medical Research, QEII Medical Centre, Nedlands, Western Australia, 6009, Australia.

Bioluminescence resonance energy transfer (BRET) has been a vital tool for understanding G protein-coupled receptor (GPCR) function. It has been used to investigate GPCR-protein and/or -ligand interactions as well as GPCR oligomerisation. However the utility of BRET is limited by the requirement that the fusion proteins, and in particular the donor, need to be exogenously expressed. To address this, we have used CRISPR/Cas9-mediated homology-directed repair to generate protein-Nanoluciferase (Nluc) fusions under endogenous promotion, thus allowing investigation of proximity between the genome-edited protein and an exogenously expressed protein by BRET. Here we report BRET monitoring of GPCR-mediated β-arrestin2 recruitment and internalisation where the donor luciferase was under endogenous promotion, in live cells and in real time. We have investigated the utility of CRISPR/Cas9 genome editing to create genome-edited fusion proteins that can be used as BRET donors and propose that this strategy can be used to overcome the need for exogenous donor expression.
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http://dx.doi.org/10.1038/s41598-017-03486-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5466623PMC
June 2017

De Novo Mutations in DENR Disrupt Neuronal Development and Link Congenital Neurological Disorders to Faulty mRNA Translation Re-initiation.

Cell Rep 2016 06 26;15(10):2251-2265. Epub 2016 May 26.

EMBL Australia, The Australian Regenerative Medicine Institute, Monash University, Clayton, VIC 3800, Australia; The Harry Perkins Institute of Medical Research, QEII Medical Centre and Centre for Medical Research, the University of Western Australia, Nedlands, WA 6009, Australia. Electronic address:

Disruptions to neuronal mRNA translation are hypothesized to underlie human neurodevelopmental syndromes. Notably, the mRNA translation re-initiation factor DENR is a regulator of eukaryotic translation and cell growth, but its mammalian functions are unknown. Here, we report that Denr influences the migration of murine cerebral cortical neurons in vivo with its binding partner Mcts1, whereas perturbations to Denr impair the long-term positioning, dendritic arborization, and dendritic spine characteristics of postnatal projection neurons. We characterized de novo missense mutations in DENR (p.C37Y and p.P121L) detected in two unrelated human subjects diagnosed with brain developmental disorder to find that each variant impairs the function of DENR in mRNA translation re-initiation and disrupts the migration and terminal branching of cortical neurons in different ways. Thus, our findings link human brain disorders to impaired mRNA translation re-initiation through perturbations in DENR (OMIM: 604550) function in neurons.
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http://dx.doi.org/10.1016/j.celrep.2016.04.090DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4906373PMC
June 2016

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

Proc Natl Acad Sci U S A 2015 Apr 14;112(17):5437-42. Epub 2015 Apr 14.

Development and Cancer Division, The Walter and Eliza Hall Institute of Medical Research, Melbourne, VIC 3052, Australia; Department of Medical Biology, University of Melbourne, Melbourne, VIC 3052, Australia;

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.
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http://dx.doi.org/10.1073/pnas.1422872112DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4418851PMC
April 2015

Mesodermal expression of Moz is necessary for cardiac septum development.

Dev Biol 2015 Jul 23;403(1):22-9. Epub 2015 Apr 23.

Walter and Eliza Hall Institute of Medical Research, Melbourne 3052, Victoria, Australia; Department of Medical Biology, The University of Melbourne, Melbourne 3052, Victoria, Australia. Electronic address:

Ventricular septal defects (VSDs) are the most commonly occurring congenital heart defect. They are regularly associated with complex syndromes, including DiGeorge syndrome and Holt-Oram syndrome, which are characterised by haploinsufficiency for the T-box transcription factors TBX1 and TBX5, respectively. The histone acetyltransferase monocytic leukaemia zinc finger protein, MOZ (MYST3/KAT6A), is required for the expression of the Tbx1 and Tbx5 genes. Homozygous loss of MOZ results in DiGeorge syndrome-like defects including VSD. The Moz gene is expressed in the ectodermal, mesodermal and endodermal aspects of the developing pharyngeal apparatus and heart; however it is unclear in which of these tissues MOZ is required for heart development. The role of MOZ in the activation of Tbx1 would suggest a requirement for MOZ in the mesoderm, because deletion of Tbx1 in the mesoderm causes VSDs. Here, we investigated the tissue-specific requirements for MOZ in the mesoderm. We demonstrate that Mesp1-cre-mediated deletion of Moz results in high penetrance of VSDs and overriding aorta and a significant decrease in MOZ-dependant Tbx1 and Tbx5 expression. Together, our data suggest that the molecular pathogenesis of VSDs in Moz germline mutant mice is due to loss of MOZ-dependant activation of mesodermal Tbx1 and Tbx5 expression.
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http://dx.doi.org/10.1016/j.ydbio.2015.04.011DOI Listing
July 2015

MOZ regulates B-cell progenitors and, consequently, Moz haploinsufficiency dramatically retards MYC-induced lymphoma development.

Blood 2015 Mar 20;125(12):1910-21. Epub 2015 Jan 20.

The Walter and Eliza Hall Institute of Medical Research, Melbourne, Victoria, Australia; and Department of Medical Biology, and.

The histone acetyltransferase MOZ (MYST3, KAT6A) is the target of recurrent chromosomal translocations fusing the MOZ gene to CBP, p300, NCOA3, or TIF2 in particularly aggressive cases of acute myeloid leukemia. In this study, we report the role of wild-type MOZ in regulating B-cell progenitor proliferation and hematopoietic malignancy. In the Eμ-Myc model of aggressive pre-B/B-cell lymphoma, the loss of just one allele of Moz increased the median survival of mice by 3.9-fold. MOZ was required to maintain the proliferative capacity of B-cell progenitors, even in the presence of c-MYC overexpression, by directly maintaining the transcriptional activity of genes required for normal B-cell development. Hence, B-cell progenitor numbers were significantly reduced in Moz haploinsufficient animals. Interestingly, we find a significant overlap in genes regulated by MOZ, mixed lineage leukemia 1, and mixed lineage leukemia 1 cofactor menin. This includes Meis1, a TALE class homeobox transcription factor required for B-cell development, characteristically upregulated as a result of MLL1 translocations in leukemia. We demonstrate that MOZ localizes to the Meis1 locus in pre-B-cells and maintains Meis1 expression. Our results suggest that even partial inhibition of MOZ may reduce the proliferative capacity of MEIS1, and HOX-driven lymphoma and leukemia cells.
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http://dx.doi.org/10.1182/blood-2014-08-594655DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4440887PMC
March 2015

MOZ regulates the Tbx1 locus, and Moz mutation partially phenocopies DiGeorge syndrome.

Dev Cell 2012 Sep 23;23(3):652-63. Epub 2012 Aug 23.

The Walter and Eliza Hall Institute of Medical Research, Melbourne, Victoria 3052, Australia.

DiGeorge 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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http://dx.doi.org/10.1016/j.devcel.2012.07.010DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3442180PMC
September 2012