Publications by authors named "Hillary K Graves"

11 Publications

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Rare deleterious de novo missense variants in RNF2/RING2 are associated with a neurodevelopmental disorder with unique clinical features.

Hum Mol Genet 2021 Apr 16. Epub 2021 Apr 16.

Department of Molecular and Human Genetics, Baylor College of Medicine (BCM), Houston, TX, USA.

The Polycomb group (PcG) gene RNF2 (RING2) encodes a catalytic subunit of the Polycomb Repressive Complex 1 (PRC1), an evolutionarily conserved machinery that post-translationally modifies chromatin to maintain epigenetic transcriptional repressive states of target genes including Hox genes. Here, we describe two individuals, each with rare de novo missense variants in RNF2. Their phenotypes include intrauterine growth retardation, severe intellectual disabilities, behavioral problems, seizures, feeding difficulties and dysmorphic features. Population genomics data suggest that RNF2 is highly constrained for loss-of-function (LoF) and missense variants, and both p.R70H and p.S82R variants have not been reported to date. Structural analyses of the two alleles indicate that these changes likely impact the interaction between RNF2 and BMI1, another PRC1 subunit, or its substrate Histone H2A, respectively. Finally, we provide functional data in Drosophila that these two missense variants behave as LoF alleles in vivo. The evidence provide support for deleterious alleles in RNF2 being associated with a new and recognizable genetic disorder. This tentative gene-disease association in addition to the 12 previously identified disorders caused by PcG genes attests to the importance of these chromatin regulators in Mendelian disorders.
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http://dx.doi.org/10.1093/hmg/ddab110DOI Listing
April 2021

A Genetic Screen for Genes That Impact Peroxisomes in Identifies Candidate Genes for Human Disease.

G3 (Bethesda) 2020 01 7;10(1):69-77. Epub 2020 Jan 7.

Department of Molecular and Human Genetics,

Peroxisomes are subcellular organelles that are essential for proper function of eukaryotic cells. In addition to being the sites of a variety of oxidative reactions, they are crucial regulators of lipid metabolism. Peroxisome loss or dysfunction leads to multi-system diseases in humans that strongly affect the nervous system. In order to identify previously unidentified genes and mechanisms that impact peroxisomes, we conducted a genetic screen on a collection of lethal mutations on the chromosome in Using the number, size and morphology of GFP tagged peroxisomes as a readout, we screened for mutations that altered peroxisomes based on clonal analysis and confocal microscopy. From this screen, we identified eighteen genes that cause increases in peroxisome number or altered morphology when mutated. We examined the human homologs of these genes and found that they are involved in a diverse array of cellular processes. Interestingly, the human homologs from the -chromosome collection are under selective constraint in human populations and are good candidate genes particularly for dominant genetic disease. This screening approach for peroxisome defects allows identification of novel genes that impact peroxisomes in a multicellular organism and is a valuable platform to discover genes potentially involved in dominant disease that could affect peroxisomes.
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http://dx.doi.org/10.1534/g3.119.400803DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6945042PMC
January 2020

Glutathione S-Transferase Regulates Mitochondrial Populations in Axons through Increased Glutathione Oxidation.

Neuron 2019 07 14;103(1):52-65.e6. Epub 2019 May 14.

Department of Neurobiology, University of Massachusetts Medical School, Worcester, MA 01605, USA; Vollum Institute, Oregon Health & Science University, Portland, OR 97239, USA.

Mitochondria are essential in long axons to provide metabolic support and sustain neuron integrity. A healthy mitochondrial pool is maintained by biogenesis, transport, mitophagy, fission, and fusion, but how these events are regulated in axons is not well defined. Here, we show that the Drosophila glutathione S-transferase (GST) Gfzf prevents mitochondrial hyperfusion in axons. Gfzf loss altered redox balance between glutathione (GSH) and oxidized glutathione (GSSG) and initiated mitochondrial fusion through the coordinated action of Mfn and Opa1. Gfzf functioned epistatically with the thioredoxin peroxidase Jafrac1 and the thioredoxin reductase 1 TrxR-1 to regulate mitochondrial dynamics. Altering GSH:GSSG ratios in mouse primary neurons in vitro also induced hyperfusion. Mitochondrial changes caused deficits in trafficking, the metabolome, and neuronal physiology. Changes in GSH and oxidative state are associated with neurodegenerative diseases like Alzheimer's. Our demonstration that GSTs are key in vivo regulators of axonal mitochondrial length and number provides a potential mechanistic link.
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http://dx.doi.org/10.1016/j.neuron.2019.04.017DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6616599PMC
July 2019

Excess free histone H3 localizes to centrosomes for proteasome-mediated degradation during mitosis in metazoans.

Cell Cycle 2016 Aug 1;15(16):2216-2225. Epub 2016 Jun 1.

a Department of Epigenetics and Molecular Carcinogenesis , University of Texas MD Anderson Cancer Center , Houston , TX , USA.

The cell tightly controls histone protein levels in order to achieve proper packaging of the genome into chromatin, while avoiding the deleterious consequences of excess free histones. Our accompanying study has shown that a histone modification that loosens the intrinsic structure of the nucleosome, phosphorylation of histone H3 on threonine 118 (H3 T118ph), exists on centromeres and chromosome arms during mitosis. Here, we show that H3 T118ph localizes to centrosomes in humans, flies, and worms during all stages of mitosis. H3 abundance at the centrosome increased upon proteasome inhibition, suggesting that excess free histone H3 localizes to centrosomes for degradation during mitosis. In agreement, we find ubiquitinated H3 specifically during mitosis and within purified centrosomes. These results suggest that targeting of histone H3 to the centrosome for proteasome-mediated degradation is a novel pathway for controlling histone supply, specifically during mitosis.
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http://dx.doi.org/10.1080/15384101.2016.1192728DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4993543PMC
August 2016

Mutations that prevent or mimic persistent post-translational modifications of the histone H3 globular domain cause lethality and growth defects in Drosophila.

Epigenetics Chromatin 2016 29;9. Epub 2016 Feb 29.

Department of Epigenetics and Molecular Carcinogenesis, University of Texas MD Anderson Cancer Center, Houston, TX 77030 USA ; Department of Pathology and Laboratory Medicine, Weill Cornell Medical College, New York, NY 10065 USA.

Background: Understanding the function of histone post-translational modifications is the key to deciphering how genomic activities are regulated. Among the least well-understood histone modifications in vivo are those that occur on the surface of the globular domain of histones, despite their causing the most profound structural alterations of the nucleosome in vitro. We utilized a Drosophila system to replace the canonical histone genes with mutated histone transgenes.

Results: Mutations predicted to mimic or prevent acetylation on histone H3 lysine (K) 56, K115, K122, and both K115/K122, or to prevent or mimic phosphorylation on H3 threonine (T) 118 and T80, all caused lethality, with the exception of K122R mutants. T118 mutations caused profound growth defects within wing discs, while K115R, K115Q, K56Q, and the K115/K122 mutations caused more subtle growth defects. The H3 K56R and H3 K122R mutations caused no defects in growth, differentiation, or transcription within imaginal discs, indicating that H3 K56 acetylation and K122 acetylation are dispensable for these functions. In agreement, we found the antibody to H3 K122Ac, which was previously used to imply a role for H3 K122Ac in transcription in metazoans, to be non-specific in vivo.

Conclusions: Our data suggest that chromatin structural perturbations caused by acetylation of K56, K115, or K122 and phosphorylation of T80 or T118 are important for key developmental processes.
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http://dx.doi.org/10.1186/s13072-016-0059-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4772521PMC
March 2016

Aurora-A mediated histone H3 phosphorylation of threonine 118 controls condensin I and cohesin occupancy in mitosis.

Elife 2016 Feb 16;5:e11402. Epub 2016 Feb 16.

Department of Epigenetics and Molecular Carcinogenesis, University of Texas MD Anderson Cancer Center, Houston, United States.

Phosphorylation of histone H3 threonine 118 (H3 T118ph) weakens histone DNA-contacts, disrupting the nucleosome structure. We show that Aurora-A mediated H3 T118ph occurs at pericentromeres and chromosome arms during prophase and is lost upon chromosome alignment. Expression of H3 T118E or H3 T118I (a SIN mutation that bypasses the need for the ATP-dependent nucleosome remodeler SWI/SNF) leads to mitotic problems including defects in spindle attachment, delayed cytokinesis, reduced chromatin packaging, cohesion loss, cohesin and condensin I loss in human cells. In agreement, overexpression of Aurora-A leads to increased H3 T118ph levels, causing cohesion loss, and reduced levels of cohesin and condensin I on chromatin. Normal levels of H3 T118ph are important because it is required for development in fruit flies. We propose that H3 T118ph alters the chromatin structure during specific phases of mitosis to promote timely condensin I and cohesin disassociation, which is essential for effective chromosome segregation.
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http://dx.doi.org/10.7554/eLife.11402DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4798946PMC
February 2016

Differential regulation of the Hippo pathway by adherens junctions and apical-basal cell polarity modules.

Proc Natl Acad Sci U S A 2015 Feb 26;112(6):1785-90. Epub 2015 Jan 26.

Departments of Biochemistry and Molecular Biology and Program in Genes and Development, The University of Texas Graduate School of Biomedical Sciences, Houston, TX 77030; Vlaams Instituut voor Biotechnologie Center for the Biology of Disease and Katholieke Universiteit Leuven Center for Human Genetics, University of Leuven, 3000 Leuven, Belgium; and

Adherens junctions (AJs) and cell polarity complexes are key players in the establishment and maintenance of apical-basal cell polarity. Loss of AJs or basolateral polarity components promotes tumor formation and metastasis. Recent studies in vertebrate models show that loss of AJs or loss of the basolateral component Scribble (Scrib) cause deregulation of the Hippo tumor suppressor pathway and hyperactivation of its downstream effectors Yes-associated protein (YAP) and Transcriptional coactivator with PDZ-binding motif (TAZ). However, whether AJs and Scrib act through the same or independent mechanisms to regulate Hippo pathway activity is not known. Here, we dissect how disruption of AJs or loss of basolateral components affect the activity of the Drosophila YAP homolog Yorkie (Yki) during imaginal disc development. Surprisingly, disruption of AJs and loss of basolateral proteins produced very different effects on Yki activity. Yki activity was cell-autonomously decreased but non-cell-autonomously elevated in tissues where the AJ components E-cadherin (E-cad) or α-catenin (α-cat) were knocked down. In contrast, scrib knockdown caused a predominantly cell-autonomous activation of Yki. Moreover, disruption of AJs or basolateral proteins had different effects on cell polarity and tissue size. Simultaneous knockdown of α-cat and scrib induced both cell-autonomous and non-cell-autonomous Yki activity. In mammalian cells, knockdown of E-cad or α-cat caused nuclear accumulation and activation of YAP without overt effects on Scrib localization and vice versa. Therefore, our results indicate the existence of multiple, genetically separable inputs from AJs and cell polarity complexes into Yki/YAP regulation.
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http://dx.doi.org/10.1073/pnas.1420850112DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4330745PMC
February 2015

Mitotic phosphorylation of histone H3 threonine 80.

Cell Cycle 2014 25;13(3):440-52. Epub 2013 Nov 25.

Department of Biochemistry and Molecular Biology; University of Texas; MD Anderson Cancer Center; Houston, TX USA; Department of Molecular and Cellular Biology; Baylor College of Medicine; Houston, Texas USA.

The onset and regulation of mitosis is dependent on phosphorylation of a wide array of proteins. Among the proteins that are phosphorylated during mitosis is histone H3, which is heavily phosphorylated on its N-terminal tail. In addition, large-scale mass spectrometry screens have revealed that histone H3 phosphorylation can occur at multiple sites within its globular domain, yet detailed analyses of the functions of these phosphorylations are lacking. Here, we explore one such histone H3 phosphorylation site, threonine 80 (H3T80), which is located on the nucleosome surface. Phosphorylated H3T80 (H3T80ph) is enriched in metazoan cells undergoing mitosis. Unlike H3S10 and H3S28, H3T80 is not phosphorylated by the Aurora B kinase. Further, mutations of T80 to either glutamic acid, a phosphomimetic, or to alanine, an unmodifiable residue, result in an increase in cells in prophase and an increase in anaphase/telophase bridges, respectively. SILAC-coupled mass spectrometry shows that phosphorylated H3T80 (H3T80ph) preferentially interacts with histones H2A and H4 relative to non-phosphorylated H3T80, and this result is supported by increased binding of H3T80ph to histone octamers in vitro. These findings support a model where H3T80ph, protruding from the nucleosome surface, promotes interactions between adjacent nucleosomes to promote chromatin compaction during mitosis in metazoan cells.
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http://dx.doi.org/10.4161/cc.27269DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3956540PMC
January 2015

De-regulation of JNK and JAK/STAT signaling in ESCRT-II mutant tissues cooperatively contributes to neoplastic tumorigenesis.

PLoS One 2013 13;8(2):e56021. Epub 2013 Feb 13.

Graduate Program in Developmental Biology, Baylor College of Medicine, Houston, Texas, United States of America.

Multiple genes involved in endocytosis and endosomal protein trafficking in Drosophila have been shown to function as neoplastic tumor suppressor genes (nTSGs), including Endosomal Sorting Complex Required for Transport-II (ESCRT-II) components vacuolar protein sorting 22 (vps22), vps25, and vps36. However, most studies of endocytic nTSGs have been done in mosaic tissues containing both mutant and non-mutant populations of cells, and interactions among mutant and non-mutant cells greatly influence the final phenotype. Thus, the true autonomous phenotype of tissues mutant for endocytic nTSGs remains unclear. Here, we show that tissues predominantly mutant for ESCRT-II components display characteristics of neoplastic transformation and then undergo apoptosis. These neoplastic tissues show upregulation of c-Jun N-terminal Kinase (JNK), Notch, and Janus Kinase (JAK)/Signal Transducer and Activator of Transcription (STAT) signaling. Significantly, while inhibition of JNK signaling in mutant tissues partially inhibits proliferation, inhibition of JAK/STAT signaling rescues other aspects of the neoplastic phenotype. This is the first rigorous study of tissues predominantly mutant for endocytic nTSGs and provides clear evidence for cooperation among de-regulated signaling pathways leading to tumorigenesis.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0056021PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3572140PMC
August 2013

Notch signaling activates Yorkie non-cell autonomously in Drosophila.

PLoS One 2012 5;7(6):e37615. Epub 2012 Jun 5.

Department of Biochemistry and Molecular Biology, The University of Texas MD Anderson Cancer Center, Houston, Texas, United States of America.

In Drosophila imaginal epithelia, cells mutant for the endocytic neoplastic tumor suppressor gene vps25 stimulate nearby untransformed cells to express Drosophila Inhibitor-of-Apoptosis-Protein-1 (DIAP-1), conferring resistance to apoptosis non-cell autonomously. Here, we show that the non-cell autonomous induction of DIAP-1 is mediated by Yorkie, the conserved downstream effector of Hippo signaling. The non-cell autonomous induction of Yorkie is due to Notch signaling from vps25 mutant cells. Moreover, activated Notch in normal cells is sufficient to induce non-cell autonomous Yorkie activity in wing imaginal discs. Our data identify a novel mechanism by which Notch promotes cell survival non-cell autonomously and by which neoplastic tumor cells generate a supportive microenvironment for tumor growth.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0037615PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3367968PMC
October 2012

dEHBP1 controls exocytosis and recycling of Delta during asymmetric divisions.

J Cell Biol 2012 Jan 2;196(1):65-83. Epub 2012 Jan 2.

Howard Hughes Medical Institute, Baylor College of Medicine, Houston, TX 77030, USA.

Notch signaling governs binary cell fate determination in asymmetrically dividing cells. Through a forward genetic screen we identified the fly homologue of Eps15 homology domain containing protein-binding protein 1 (dEHBP1) as a novel regulator of Notch signaling in asymmetrically dividing cells. dEHBP1 is enriched basally and at the actin-rich interface of pII cells of the external mechanosensory organs, where Notch signaling occurs. Loss of function of dEHBP1 leads to up-regulation of Sanpodo, a regulator of Notch signaling, and aberrant trafficking of the Notch ligand, Delta. Furthermore, Sec15 and Rab11, which have been previously shown to regulate the localization of Delta, physically interact with dEHBP1. We propose that dEHBP1 functions as an adaptor molecule for the exocytosis and recycling of Delta, thereby affecting cell fate decisions in asymmetrically dividing cells.
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http://dx.doi.org/10.1083/jcb.201106088DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3255984PMC
January 2012