Alex Kuo - Academia.edu (original) (raw)

Papers by Alex Kuo

Research paper thumbnail of Improving Usability, Safety and Patient Outcomes with Health Information Technology - From Research to Practice, Information Technology and Communications in Health Conference, ITCH 2019, Victoria, BC, Canada, 14-17 February 2019

Opioid-related harm is a major public health concern in Canada and abroad. There is a growing mar... more Opioid-related harm is a major public health concern in Canada and abroad. There is a growing market of mobile apps that focus on preventing and managing opioid-related harm. The use of mobile health technologies is a promising intervention that can assist with addressing the problem. The aim of this paper is to examine the current state of the mobile app market with respect to prevention and management of opioid-related harm. This will involve a review currently available opioid apps for the major operating systems (iOS, Android, Windows CE and BlackBerry OS) and examine the number of released apps, service providers, operating systems, target user groups, purpose of app, range of features, location, use of evidence, interface, languages, cost and licensing model, and user ratings.

Research paper thumbnail of The Strategic Mobilization of Working-Class Voters: The Implications of Electoral Geography for the Success of the SPD

Abstract will be provided by author.

Research paper thumbnail of Political Origins of AntiLabor Institutions

Abstract will be provided by author.

Research paper thumbnail of A molecular threading mechanism underlies Jumonji lysine demethylase KDM2A regulation of methylated H3K36

Genes & development, Jan 15, 2014

The dynamic reversible methylation of lysine residues on histone proteins is central to chromatin... more The dynamic reversible methylation of lysine residues on histone proteins is central to chromatin biology. Key components are demethylase enzymes, which remove methyl moieties from lysine residues. KDM2A, a member of the Jumonji C domain-containing histone lysine demethylase family, specifically targets lower methylation states of H3K36. Here, structural studies reveal that H3K36 specificity for KDM2A is mediated by the U-shaped threading of the H3K36 peptide through a catalytic groove within KDM2A. The side chain of methylated K36 inserts into the catalytic pocket occupied by Ni(2+) and cofactor, where it is positioned and oriented for demethylation. Key residues contributing to K36me specificity on histone H3 are G33 and G34 (positioned within a narrow channel), P38 (a turn residue), and Y41 (inserts into its own pocket). Given that KDM2A was found to also bind the H3K36me3 peptide, we postulate that steric constraints could prevent α-ketoglutarate from undergoing an "off-lin...

Research paper thumbnail of P-11: Electrical Properties and Stability of Dual-Gate Coplanar Homojunction Amorphous Indium-Gallium-Zinc-Oxide Thin-Film Transistor

SID Symposium Digest of Technical Papers, 2011

The electrical characteristics and stabilities of dual-gate (DG) coplanar homojunction amorphous ... more The electrical characteristics and stabilities of dual-gate (DG) coplanar homojunction amorphous indium-gallium-zinc-oxide thin-film transistors (a-IGZO TFTs) are described. When the gate voltage is applied on top and bottom electrodes, the DG a-IGZO TFT showed an excellent electrical performance with the subthreshold swing of 99 mV/dec, the mobility of 15.1 cm 2 /V·s and the on-off ratio of 10 9 . Under positive bias temperature stress, the device threshold voltage shifts about +4.5V after 10,000 seconds, while its shifts under negative bias temperature stress are very small. The effect of TFT illumination is also discussed.

Research paper thumbnail of The plant homeodomain finger of RAG2 recognizes histone H3 methylated at both lysine-4 and arginine-2

Proceedings of the National Academy of Sciences, 2007

Recombination activating gene (RAG) 1 and RAG2 together catalyze V(D)J gene rearrangement in lymp... more Recombination activating gene (RAG) 1 and RAG2 together catalyze V(D)J gene rearrangement in lymphocytes as the first step in the assembly and maturation of antigen receptors. RAG2 contains a plant homeodomain (PHD) near its C terminus (RAG2-PHD) that recognizes histone H3 methylated at lysine 4 (H3K4me) and influences V(D)J recombination. We report here crystal structures of RAG2-PHD alone and complexed with five modified H3 peptides. Two aspects of RAG2-PHD are unique. First, in the absence of the modified peptide, a peptide N-terminal to RAG2-PHD occupies the substrate-binding site, which may reflect an autoregulatory mechanism. Second, in contrast to other H3K4me3-binding PHD domains, RAG2-PHD substitutes a carboxylate that interacts with arginine 2 (R2) with a Tyr, resulting in binding to H3K4me3 that is enhanced rather than inhibited by dimethylation of R2. Five residues involved in histone H3 recognition were found mutated in severe combined immunodeficiency (SCID) patients. Disruption of the RAG2-PHD structure appears to lead to the absence of T and B lymphocytes, whereas failure to bind H3K4me3 is linked to Omenn Syndrome. This work provides a molecular basis for chromatindependent gene recombination and presents a single protein domain that simultaneously recognizes two distinct histone modifications, revealing added complexity in the read-out of combinatorial histone modifications. analyzed data; and S.R.-M. and W.Y. wrote the paper.

Research paper thumbnail of Aire employs a histone-binding module to mediate immunological tolerance, linking chromatin regulation with organ-specific autoimmunity

Proceedings of the National Academy of Sciences, 2008

Aire induces ectopic expression of peripheral tissue antigens (PTAs) in thymic medullary epitheli... more Aire induces ectopic expression of peripheral tissue antigens (PTAs) in thymic medullary epithelial cells, which promotes immunological tolerance. Beginning with a broad screen of histone peptides, we demonstrate that the mechanism by which this single factor controls the transcription of thousands of genes involves recognition of the amino-terminal tail of histone H3, but not of other histones, by one of Aire's plant homeodomain (PHD) fingers. Certain posttranslational modifications of H3 tails, notably dimethylation or trimethylation at H3K4, abrogated binding by Aire, whereas others were tolerated. Similar PHD finger-H3 tail-binding properties were recently reported for BRAF-histone deacetylase complex 80 and DNA methyltransferase 3L; sequence alignment, molecular modeling, and biochemical analyses showed these factors and Aire to have structure-function relationships in common. In addition, certain PHD1 mutations underlying the polyendocrine disorder autoimmune polyendocrinopathy-candidiasesectodermaldystrophy compromised Aire recognition of H3. In vitro binding assays demonstrated direct physical interaction between Aire and nucleosomes, which was in part buttressed by its affinity to DNA. In vivo Aire interactions with chromosomal regions depleted of H3K4me3 were dependent on its H3 tail-binding activity, and this binding was necessary but not sufficient for the up-regulation of genes encoding PTAs. Thus, Aire's activity as a histone-binding module mediates the thymic display of PTAs that promotes self-tolerance and prevents organ-specific autoimmunity.

Research paper thumbnail of Epigenome Microarray Platform for Proteome-Wide Dissection of Chromatin-Signaling Networks

PLoS ONE, 2009

Knowledge of protein domains that function as the biological effectors for diverse post-translati... more Knowledge of protein domains that function as the biological effectors for diverse post-translational modifications of histones is critical for understanding how nuclear and epigenetic programs are established. Indeed, mutations of chromatin effector domains found within several proteins are associated with multiple human pathologies, including cancer and immunodeficiency syndromes. To date, relatively few effector domains have been identified in comparison to the number of modifications present on histone and non-histone proteins. Here we describe the generation and application of human modified peptide microarrays as a platform for high-throughput discovery of chromatin effectors and for epitope-specificity analysis of antibodies commonly utilized in chromatin research. Screening with a library containing a majority of the Royal Family domains present in the human proteome led to the discovery of TDRD7, JMJ2C, and MPP8 as three new modified histone-binding proteins. Thus, we propose that peptide microarray methodologies are a powerful new tool for elucidating molecular interactions at chromatin.

Research paper thumbnail of RAG2 PHD finger couples histone H3 lysine 4 trimethylation with V(D)J recombination

Nature, 2007

Nuclear processes such as transcription, DNA replication, and recombination are dynamically regul... more Nuclear processes such as transcription, DNA replication, and recombination are dynamically regulated by chromatin structure. Transcription is known to be regulated by chromatin-associated proteins containing conserved protein domains that specifically recognize distinct covalent posttranslational modifications on histones. However, it has been unclear whether similar mechanisms are involved in mammalian DNA recombination. Here, we show that RAG2 -an essential component of the RAG1/2 V(D)J recombinase, that mediates antigen receptor gene assembly 1 -contains a plant homeodomain (PHD) finger that specifically recognizes histone H3 trimethylated at lysine 4 (H3K4me3). The high-resolution crystal structure of the RAG2 PHD finger bound to H3K4me3 reveals the molecular basis of H3K4me3-recognition by RAG2. Mutations that abrogate RAG2's recognition of H3K4me3 severely impair V(D)J recombination in vivo. Reducing the level of H3K4me3 similarly leads to a decrease in V(D)J recombination in vivo. Notably, a conserved tryptophan residue (W453) that constitutes a key structural component of the K4me3-binding surface and is essential for RAG2's recognition of H3K4me3 is mutated in patients with immunodeficiency syndromes. Together our results identify a novel function for histone methylation in mammalian DNA recombination. Furthermore, our results provide the first evidence suggesting that disrupting the read-out of histone modifications can cause an inherited human disease.

Research paper thumbnail of NSD2 Links Dimethylation of Histone H3 at Lysine 36 to Oncogenic Programming

Molecular Cell, 2011

The histone lysine methyltransferase NSD2 (MMSET/WHSC1) is implicated in diverse diseases and com... more The histone lysine methyltransferase NSD2 (MMSET/WHSC1) is implicated in diverse diseases and commonly overexpressed in multiple myeloma due to a recurrent t(4;14) chromosomal translocation. However, the precise catalytic activity of NSD2 is obscure, preventing progress in understanding how this enzyme influences chromatin biology and myeloma pathogenesis. Here we show that dimethylation of histone H3 at lysine 36 (H3K36me2) is the principal chromatinregulatory activity of NSD2. Catalysis of H3K36me2 by NSD2 is sufficient for gene activation. In t(4;14)-positive myeloma cells, the normal genome-wide and gene-specific distribution of H3K36me2 is obliterated, creating a chromatin landscape that selects for a transcription profile favorable for myelomagenesis. Catalytically active NSD2 confers xenograft tumor formation upon t(4;14)-negative cells, and promotes oncogenic transformation of primary cells in an H3K36me2dependent manner. Together our findings establish H3K36me2 as the primary product generated by NSD2, and demonstrate that genomic disorganization of this canonical chromatin mark by NSD2 initiates oncogenic programming.

Research paper thumbnail of ING4 Mediates Crosstalk between Histone H3 K4 Trimethylation and H3 Acetylation to Attenuate Cellular Transformation

Molecular Cell, 2009

Aberrations in chromatin dynamics play a fundamental role in tumorigenesis, yet relatively little... more Aberrations in chromatin dynamics play a fundamental role in tumorigenesis, yet relatively little is known of the molecular mechanisms linking histone lysine methylation to neoplastic disease. ING4 (Inhibitor of Growth 4) is a native subunit of an HBO1 histone acetyltransferase (HAT) complex and a tumor suppressor protein. Here we show a critical role for specific recognition of histone H3 trimethylated at lysine 4 (H3K4me3) by the ING4 PHD finger in mediating ING4 gene expression and tumor suppressor functions. The interaction between ING4 and H3K4me3 augments HBO1 acetylation activity on H3 tails and drives H3 acetylation at ING4 target promoters. Further, ING4 facilitates apoptosis in response to genotoxic stress and inhibits anchorage-independent cell growth, and these functions depend on ING4 interactions with H3K4me3. Together, our results demonstrate a mechanism for brokering crosstalk between H3K4 methylation and H3 acetylation and reveal a molecular link between chromatin modulation and tumor suppressor mechanisms.

Research paper thumbnail of Back channel etch chemistry of advanced a-Si:H TFTs

Microelectronic Engineering, 2011

We report on the effects of back channel etch depth and etchant chemistry on the electrical chara... more We report on the effects of back channel etch depth and etchant chemistry on the electrical characteristics of inverted staggered advanced amorphous silicon thin-film transistors. We found that the optimum amorphous silicon film thickness in the channel is about 800-1100 Å. Three dry etch, HBr + Cl 2 , C 2 F 6 , and CCl 2 F 2 + O 2 , and one wet etch, KOH, chemistries are used for the back channel etch processing. We established that dry etch can be used for the back channel etch of amorphous silicon transistor without degrading its electrical characteristics.

Research paper thumbnail of NMR assignments and histone specificity of the ING2 PHD finger

Magnetic Resonance in Chemistry, 2009

The ING2 plant homeodomain (PHD) finger is recruited to the nucleosome through specific binding t... more The ING2 plant homeodomain (PHD) finger is recruited to the nucleosome through specific binding to histone H3 trimethylated at lysine 4 (H3K4me3). Here, we describe backbone and side chain assignments of the ING2 PHD finger, analyze its binding to the unmodified and modified histone and p53 peptides, and map the histone H3 and H3K4me3 binding sites based on chemical shift perturbation analysis.

Research paper thumbnail of Histone H3K4me3 Binding Is Required for the DNA Repair and Apoptotic Activities of ING1 Tumor Suppressor

Journal of Molecular Biology, 2008

Inhibitor of growth 1 (ING1) is implicated in oncogenesis, DNA damage repair and apoptosis. Mutat... more Inhibitor of growth 1 (ING1) is implicated in oncogenesis, DNA damage repair and apoptosis. Mutations within the ING1 gene and altered expression levels of ING1 are found in multiple human cancers. Here, we show that both DNA repair and apoptotic activities of ING1 require the interaction of the C-terminal plant homeodomain (PHD) finger with trimethylated at Lys 4 histone H3 (H3K4me3). The ING1 PHD finger recognizes methylated H3K4 but not other histone modifications as revealed by the peptide microarrays. The molecular mechanism of the histone recognition is elucidated based on a 2.1 Å resolution crystal structure of the PHD-H3K4me3 complex. The K4me3 occupies a deep hydrophobic pocket formed by the conserved Y212 and W235 residues that make cation-π contacts with the trimethylammonium group. Both aromatic residues are essential in the H3K4me3 recognition, as substitution of these residues with Ala disrupts the interaction. Unlike the wild type ING1, the W235A mutant, overexpressed in the stable clones of melanoma cells or in HT1080 cells, was unable to stimulate DNA repair after UV irradiation or promote DNA-damage induced apoptosis, indicating that H3K4me3 binding is necessary for these biological functions of ING1. Furthermore, N216S, V218I and G221V mutations, found in human malignances, impair the ability of ING1 to associate with H3K4me3 or to induce nucleotide repair and cell death, linking the tumorigenic activity of ING1 with epigenetic regulation. Together, our findings reveal the critical role of the H3K4me3 interaction in mediating cellular responses to genotoxic stresses and offer new insight into the molecular mechanism underlying the tumor suppressive activity of ING1.

Research paper thumbnail of Binding of the MLL PHD3 Finger to Histone H3K4me3 Is Required for MLL-Dependent Gene Transcription

Journal of Molecular Biology, 2010

The MLL (Mixed Lineage Leukemia) proto-oncogene encodes a histone methyltransferase that creates ... more The MLL (Mixed Lineage Leukemia) proto-oncogene encodes a histone methyltransferase that creates the methylated histone H3K4 epigenetic marks, commonly associated with actively transcribed genes. In addition to its canonical histone methyltransferase SET domain, the MLL protein contains three plant homeodomain (PHD) fingers that are well conserved between species but whose potential roles and requirements for MLL function are unknown. Here we demonstrate that the third PHD domain of MLL (PHD3) binds histone H3 trimethylated at lysine 4 (H3K4me3) with high affinity and specificity, and H3K4me2 with eight-fold lower affinity. Biochemical and structural analysis using NMR and fluorescence spectroscopy identified key amino acids essential for the interaction with H3K4me3. Site-directed mutations of the residues involved in recognition of H3K4me3 compromised in vitro H3K4me3 binding but not in vivo localization of full-length MLL to chromatin sites in target promoters of MEIS1 and HOXA genes. Whereas intact PHD3 finger was necessary for MLL occupancy at these promoters, H3K4me3 binding was critical for MLL transcriptional activity. These results demonstrate that MLL occupancy and target gene activation can be functionally separated. Furthermore, these findings reveal that MLL not only "writes" the H3K4me3 mark, but also binds the mark as well, and this binding is required for the transcriptional maintenance functions of MLL.

Research paper thumbnail of The Target of the NSD Family of Histone Lysine Methyltransferases Depends on the Nature of the Substrate

Journal of Biological Chemistry, 2009

The NSD (nuclear receptor SET domain-containing) family of histone lysine methyltransferases is a... more The NSD (nuclear receptor SET domain-containing) family of histone lysine methyltransferases is a critical participant in chromatin integrity as evidenced by the number of human diseases associated with the aberrant expression of its family members. Yet, the specific targets of these enzymes are not clear, with marked discrepancies being reported in the literature. We demonstrate that NSD2 can exhibit disparate target preferences based on the nature of the substrate provided. The NSD2 complex purified from human cells and recombinant NSD2 both exhibit specific targeting of histone H3 lysine 36 (H3K36) when provided with nucleosome substrates, but histone H4 lysine 44 is the primary target in the case of octamer substrates, irrespective of the histones being native or recombinant. This disparity is negated when NSD2 is presented with octamer targets in conjunction with short single- or double-stranded DNA. Although the octamers cannot form nucleosomes, the target is nonetheless nucleosome-specific as is the product, dimethylated H3K36. This study clarifies in part the previous discrepancies reported with respect to NSD targets. We propose that DNA acts as an allosteric effector of NSD2 such that H3K36 becomes the preferred target.

Research paper thumbnail of Advanced Multilayer Amorphous Silicon Thin-Film Transistor Structure: Film Thickness Effect on Its Electrical Performance and Contact Resistance

Japanese Journal of Applied Physics, 2008

We report the intrinsic and extrinsic electrical characteristics of advanced multilayer amorphous... more We report the intrinsic and extrinsic electrical characteristics of advanced multilayer amorphous silicon (a-Si:H) thin-film transistor (TFT) with dual amorphous silicon nitride (a-SiN X :H) and a-Si:H layers. The thickness effect of the high electronic quality a-Si:H film on the transistor's electrical property was investigated; with increasing film thickness, both field-effect mobility and subthreshold swing show improvement and the threshold voltage remain unchanged. However, the contact resistance increases with the a-Si:H film thickness. Using the two-step plasma enhanced chemical vapor deposition process, we fabricated TFT's with acceptable field-effect mobility ($1 cm 2 V À1 s À1 ) and threshold voltage (<1:5 V) with enhanced throughput.

Research paper thumbnail of Advanced Amorphous Silicon Thin-Film Transistors for AM-OLEDs: Electrical Performance and Stability

IEEE Transactions on Electron Devices, 2000

We fabricated and characterized the advanced amorphous silicon thin-film transistors with a bilay... more We fabricated and characterized the advanced amorphous silicon thin-film transistors with a bilayer structure for both the active and gate dielectric films. The electrical field across the gate insulator has a significant influence on the device threshold voltage electrical stability. We show that high thin-film transistor stability can be achieved even under the presence of a high channel current. Its electrical and high-temperature stability improves up to a factor of five when the TFT biasing condition changes from the linear to the saturation region of operation.

Research paper thumbnail of Electrical Properties and Stability of Dual-Gate Coplanar Homojunction DC Sputtered Amorphous Indium–Gallium–Zinc–Oxide Thin-Film Transistors and Its Application to AM-OLEDs

IEEE Transactions on Electron Devices, 2000

The electrical characteristics and stability of dualgate (DG) coplanar homojunction amorphous ind... more The electrical characteristics and stability of dualgate (DG) coplanar homojunction amorphous indium-galliumzinc-oxide thin-film transistors (a-IGZO TFTs) on glass substrates are described herein. In this device structure, both top gate (TG) and bottom gate are defined by lithography, allowing independent biasing when adjacent TFTs are present. The DG a-IGZO TFT demonstrates excellent electrical performance with subthreshold swing (SS) of 99 mV/dec, field-effect mobility of 15.1 cm 2 /V · s, and ON-OFF current ratio of 10 9 . By applying various bias voltages on the TG electrode, it is found that the TFT threshold voltage can be controlled without any change of the SS and off current. Under conditions of negative bias temperature stress (BTS), the transfer curves of the TFT exhibit negligible shifts after 10 000 s. Larger shifts are observed under conditions of a positive BTS. Finally, the application of this DG device to active-matrix organic light-emitting displays is suggested.

Research paper thumbnail of Lysine methylation of the NF-κB subunit RelA by SETD6 couples activity of the histone methyltransferase GLP at chromatin to tonic repression of NF-κB signaling

Nature immunology, 2011

Signaling via the methylation of lysine residues in proteins has been linked to diverse biologica... more Signaling via the methylation of lysine residues in proteins has been linked to diverse biological and disease processes, yet the catalytic activity and substrate specificity of many human protein lysine methyltransferases (PKMTs) are unknown. We screened over 40 candidate PKMTs and identified SETD6 as a methyltransferase that monomethylated chromatin-associated transcription factor NF-κB subunit RelA at Lys310 (RelAK310me1). SETD6-mediated methylation rendered RelA inert and attenuated RelA-driven transcriptional programs, including inflammatory responses in primary immune cells. RelAK310me1 was recognized by the ankryin repeat of the histone methyltransferase GLP, which under basal conditions promoted a repressed chromatin state at RelA target genes through GLP-mediated methylation of histone H3 Lys9 (H3K9). NF-κB-activation-linked phosphorylation of RelA at Ser311 by protein kinase C-ζ (PKC-ζ) blocked the binding of GLP to RelAK310me1 and relieved repression of the target gene. O...

Research paper thumbnail of Improving Usability, Safety and Patient Outcomes with Health Information Technology - From Research to Practice, Information Technology and Communications in Health Conference, ITCH 2019, Victoria, BC, Canada, 14-17 February 2019

Opioid-related harm is a major public health concern in Canada and abroad. There is a growing mar... more Opioid-related harm is a major public health concern in Canada and abroad. There is a growing market of mobile apps that focus on preventing and managing opioid-related harm. The use of mobile health technologies is a promising intervention that can assist with addressing the problem. The aim of this paper is to examine the current state of the mobile app market with respect to prevention and management of opioid-related harm. This will involve a review currently available opioid apps for the major operating systems (iOS, Android, Windows CE and BlackBerry OS) and examine the number of released apps, service providers, operating systems, target user groups, purpose of app, range of features, location, use of evidence, interface, languages, cost and licensing model, and user ratings.

Research paper thumbnail of The Strategic Mobilization of Working-Class Voters: The Implications of Electoral Geography for the Success of the SPD

Abstract will be provided by author.

Research paper thumbnail of Political Origins of AntiLabor Institutions

Abstract will be provided by author.

Research paper thumbnail of A molecular threading mechanism underlies Jumonji lysine demethylase KDM2A regulation of methylated H3K36

Genes & development, Jan 15, 2014

The dynamic reversible methylation of lysine residues on histone proteins is central to chromatin... more The dynamic reversible methylation of lysine residues on histone proteins is central to chromatin biology. Key components are demethylase enzymes, which remove methyl moieties from lysine residues. KDM2A, a member of the Jumonji C domain-containing histone lysine demethylase family, specifically targets lower methylation states of H3K36. Here, structural studies reveal that H3K36 specificity for KDM2A is mediated by the U-shaped threading of the H3K36 peptide through a catalytic groove within KDM2A. The side chain of methylated K36 inserts into the catalytic pocket occupied by Ni(2+) and cofactor, where it is positioned and oriented for demethylation. Key residues contributing to K36me specificity on histone H3 are G33 and G34 (positioned within a narrow channel), P38 (a turn residue), and Y41 (inserts into its own pocket). Given that KDM2A was found to also bind the H3K36me3 peptide, we postulate that steric constraints could prevent α-ketoglutarate from undergoing an "off-lin...

Research paper thumbnail of P-11: Electrical Properties and Stability of Dual-Gate Coplanar Homojunction Amorphous Indium-Gallium-Zinc-Oxide Thin-Film Transistor

SID Symposium Digest of Technical Papers, 2011

The electrical characteristics and stabilities of dual-gate (DG) coplanar homojunction amorphous ... more The electrical characteristics and stabilities of dual-gate (DG) coplanar homojunction amorphous indium-gallium-zinc-oxide thin-film transistors (a-IGZO TFTs) are described. When the gate voltage is applied on top and bottom electrodes, the DG a-IGZO TFT showed an excellent electrical performance with the subthreshold swing of 99 mV/dec, the mobility of 15.1 cm 2 /V·s and the on-off ratio of 10 9 . Under positive bias temperature stress, the device threshold voltage shifts about +4.5V after 10,000 seconds, while its shifts under negative bias temperature stress are very small. The effect of TFT illumination is also discussed.

Research paper thumbnail of The plant homeodomain finger of RAG2 recognizes histone H3 methylated at both lysine-4 and arginine-2

Proceedings of the National Academy of Sciences, 2007

Recombination activating gene (RAG) 1 and RAG2 together catalyze V(D)J gene rearrangement in lymp... more Recombination activating gene (RAG) 1 and RAG2 together catalyze V(D)J gene rearrangement in lymphocytes as the first step in the assembly and maturation of antigen receptors. RAG2 contains a plant homeodomain (PHD) near its C terminus (RAG2-PHD) that recognizes histone H3 methylated at lysine 4 (H3K4me) and influences V(D)J recombination. We report here crystal structures of RAG2-PHD alone and complexed with five modified H3 peptides. Two aspects of RAG2-PHD are unique. First, in the absence of the modified peptide, a peptide N-terminal to RAG2-PHD occupies the substrate-binding site, which may reflect an autoregulatory mechanism. Second, in contrast to other H3K4me3-binding PHD domains, RAG2-PHD substitutes a carboxylate that interacts with arginine 2 (R2) with a Tyr, resulting in binding to H3K4me3 that is enhanced rather than inhibited by dimethylation of R2. Five residues involved in histone H3 recognition were found mutated in severe combined immunodeficiency (SCID) patients. Disruption of the RAG2-PHD structure appears to lead to the absence of T and B lymphocytes, whereas failure to bind H3K4me3 is linked to Omenn Syndrome. This work provides a molecular basis for chromatindependent gene recombination and presents a single protein domain that simultaneously recognizes two distinct histone modifications, revealing added complexity in the read-out of combinatorial histone modifications. analyzed data; and S.R.-M. and W.Y. wrote the paper.

Research paper thumbnail of Aire employs a histone-binding module to mediate immunological tolerance, linking chromatin regulation with organ-specific autoimmunity

Proceedings of the National Academy of Sciences, 2008

Aire induces ectopic expression of peripheral tissue antigens (PTAs) in thymic medullary epitheli... more Aire induces ectopic expression of peripheral tissue antigens (PTAs) in thymic medullary epithelial cells, which promotes immunological tolerance. Beginning with a broad screen of histone peptides, we demonstrate that the mechanism by which this single factor controls the transcription of thousands of genes involves recognition of the amino-terminal tail of histone H3, but not of other histones, by one of Aire's plant homeodomain (PHD) fingers. Certain posttranslational modifications of H3 tails, notably dimethylation or trimethylation at H3K4, abrogated binding by Aire, whereas others were tolerated. Similar PHD finger-H3 tail-binding properties were recently reported for BRAF-histone deacetylase complex 80 and DNA methyltransferase 3L; sequence alignment, molecular modeling, and biochemical analyses showed these factors and Aire to have structure-function relationships in common. In addition, certain PHD1 mutations underlying the polyendocrine disorder autoimmune polyendocrinopathy-candidiasesectodermaldystrophy compromised Aire recognition of H3. In vitro binding assays demonstrated direct physical interaction between Aire and nucleosomes, which was in part buttressed by its affinity to DNA. In vivo Aire interactions with chromosomal regions depleted of H3K4me3 were dependent on its H3 tail-binding activity, and this binding was necessary but not sufficient for the up-regulation of genes encoding PTAs. Thus, Aire's activity as a histone-binding module mediates the thymic display of PTAs that promotes self-tolerance and prevents organ-specific autoimmunity.

Research paper thumbnail of Epigenome Microarray Platform for Proteome-Wide Dissection of Chromatin-Signaling Networks

PLoS ONE, 2009

Knowledge of protein domains that function as the biological effectors for diverse post-translati... more Knowledge of protein domains that function as the biological effectors for diverse post-translational modifications of histones is critical for understanding how nuclear and epigenetic programs are established. Indeed, mutations of chromatin effector domains found within several proteins are associated with multiple human pathologies, including cancer and immunodeficiency syndromes. To date, relatively few effector domains have been identified in comparison to the number of modifications present on histone and non-histone proteins. Here we describe the generation and application of human modified peptide microarrays as a platform for high-throughput discovery of chromatin effectors and for epitope-specificity analysis of antibodies commonly utilized in chromatin research. Screening with a library containing a majority of the Royal Family domains present in the human proteome led to the discovery of TDRD7, JMJ2C, and MPP8 as three new modified histone-binding proteins. Thus, we propose that peptide microarray methodologies are a powerful new tool for elucidating molecular interactions at chromatin.

Research paper thumbnail of RAG2 PHD finger couples histone H3 lysine 4 trimethylation with V(D)J recombination

Nature, 2007

Nuclear processes such as transcription, DNA replication, and recombination are dynamically regul... more Nuclear processes such as transcription, DNA replication, and recombination are dynamically regulated by chromatin structure. Transcription is known to be regulated by chromatin-associated proteins containing conserved protein domains that specifically recognize distinct covalent posttranslational modifications on histones. However, it has been unclear whether similar mechanisms are involved in mammalian DNA recombination. Here, we show that RAG2 -an essential component of the RAG1/2 V(D)J recombinase, that mediates antigen receptor gene assembly 1 -contains a plant homeodomain (PHD) finger that specifically recognizes histone H3 trimethylated at lysine 4 (H3K4me3). The high-resolution crystal structure of the RAG2 PHD finger bound to H3K4me3 reveals the molecular basis of H3K4me3-recognition by RAG2. Mutations that abrogate RAG2's recognition of H3K4me3 severely impair V(D)J recombination in vivo. Reducing the level of H3K4me3 similarly leads to a decrease in V(D)J recombination in vivo. Notably, a conserved tryptophan residue (W453) that constitutes a key structural component of the K4me3-binding surface and is essential for RAG2's recognition of H3K4me3 is mutated in patients with immunodeficiency syndromes. Together our results identify a novel function for histone methylation in mammalian DNA recombination. Furthermore, our results provide the first evidence suggesting that disrupting the read-out of histone modifications can cause an inherited human disease.

Research paper thumbnail of NSD2 Links Dimethylation of Histone H3 at Lysine 36 to Oncogenic Programming

Molecular Cell, 2011

The histone lysine methyltransferase NSD2 (MMSET/WHSC1) is implicated in diverse diseases and com... more The histone lysine methyltransferase NSD2 (MMSET/WHSC1) is implicated in diverse diseases and commonly overexpressed in multiple myeloma due to a recurrent t(4;14) chromosomal translocation. However, the precise catalytic activity of NSD2 is obscure, preventing progress in understanding how this enzyme influences chromatin biology and myeloma pathogenesis. Here we show that dimethylation of histone H3 at lysine 36 (H3K36me2) is the principal chromatinregulatory activity of NSD2. Catalysis of H3K36me2 by NSD2 is sufficient for gene activation. In t(4;14)-positive myeloma cells, the normal genome-wide and gene-specific distribution of H3K36me2 is obliterated, creating a chromatin landscape that selects for a transcription profile favorable for myelomagenesis. Catalytically active NSD2 confers xenograft tumor formation upon t(4;14)-negative cells, and promotes oncogenic transformation of primary cells in an H3K36me2dependent manner. Together our findings establish H3K36me2 as the primary product generated by NSD2, and demonstrate that genomic disorganization of this canonical chromatin mark by NSD2 initiates oncogenic programming.

Research paper thumbnail of ING4 Mediates Crosstalk between Histone H3 K4 Trimethylation and H3 Acetylation to Attenuate Cellular Transformation

Molecular Cell, 2009

Aberrations in chromatin dynamics play a fundamental role in tumorigenesis, yet relatively little... more Aberrations in chromatin dynamics play a fundamental role in tumorigenesis, yet relatively little is known of the molecular mechanisms linking histone lysine methylation to neoplastic disease. ING4 (Inhibitor of Growth 4) is a native subunit of an HBO1 histone acetyltransferase (HAT) complex and a tumor suppressor protein. Here we show a critical role for specific recognition of histone H3 trimethylated at lysine 4 (H3K4me3) by the ING4 PHD finger in mediating ING4 gene expression and tumor suppressor functions. The interaction between ING4 and H3K4me3 augments HBO1 acetylation activity on H3 tails and drives H3 acetylation at ING4 target promoters. Further, ING4 facilitates apoptosis in response to genotoxic stress and inhibits anchorage-independent cell growth, and these functions depend on ING4 interactions with H3K4me3. Together, our results demonstrate a mechanism for brokering crosstalk between H3K4 methylation and H3 acetylation and reveal a molecular link between chromatin modulation and tumor suppressor mechanisms.

Research paper thumbnail of Back channel etch chemistry of advanced a-Si:H TFTs

Microelectronic Engineering, 2011

We report on the effects of back channel etch depth and etchant chemistry on the electrical chara... more We report on the effects of back channel etch depth and etchant chemistry on the electrical characteristics of inverted staggered advanced amorphous silicon thin-film transistors. We found that the optimum amorphous silicon film thickness in the channel is about 800-1100 Å. Three dry etch, HBr + Cl 2 , C 2 F 6 , and CCl 2 F 2 + O 2 , and one wet etch, KOH, chemistries are used for the back channel etch processing. We established that dry etch can be used for the back channel etch of amorphous silicon transistor without degrading its electrical characteristics.

Research paper thumbnail of NMR assignments and histone specificity of the ING2 PHD finger

Magnetic Resonance in Chemistry, 2009

The ING2 plant homeodomain (PHD) finger is recruited to the nucleosome through specific binding t... more The ING2 plant homeodomain (PHD) finger is recruited to the nucleosome through specific binding to histone H3 trimethylated at lysine 4 (H3K4me3). Here, we describe backbone and side chain assignments of the ING2 PHD finger, analyze its binding to the unmodified and modified histone and p53 peptides, and map the histone H3 and H3K4me3 binding sites based on chemical shift perturbation analysis.

Research paper thumbnail of Histone H3K4me3 Binding Is Required for the DNA Repair and Apoptotic Activities of ING1 Tumor Suppressor

Journal of Molecular Biology, 2008

Inhibitor of growth 1 (ING1) is implicated in oncogenesis, DNA damage repair and apoptosis. Mutat... more Inhibitor of growth 1 (ING1) is implicated in oncogenesis, DNA damage repair and apoptosis. Mutations within the ING1 gene and altered expression levels of ING1 are found in multiple human cancers. Here, we show that both DNA repair and apoptotic activities of ING1 require the interaction of the C-terminal plant homeodomain (PHD) finger with trimethylated at Lys 4 histone H3 (H3K4me3). The ING1 PHD finger recognizes methylated H3K4 but not other histone modifications as revealed by the peptide microarrays. The molecular mechanism of the histone recognition is elucidated based on a 2.1 Å resolution crystal structure of the PHD-H3K4me3 complex. The K4me3 occupies a deep hydrophobic pocket formed by the conserved Y212 and W235 residues that make cation-π contacts with the trimethylammonium group. Both aromatic residues are essential in the H3K4me3 recognition, as substitution of these residues with Ala disrupts the interaction. Unlike the wild type ING1, the W235A mutant, overexpressed in the stable clones of melanoma cells or in HT1080 cells, was unable to stimulate DNA repair after UV irradiation or promote DNA-damage induced apoptosis, indicating that H3K4me3 binding is necessary for these biological functions of ING1. Furthermore, N216S, V218I and G221V mutations, found in human malignances, impair the ability of ING1 to associate with H3K4me3 or to induce nucleotide repair and cell death, linking the tumorigenic activity of ING1 with epigenetic regulation. Together, our findings reveal the critical role of the H3K4me3 interaction in mediating cellular responses to genotoxic stresses and offer new insight into the molecular mechanism underlying the tumor suppressive activity of ING1.

Research paper thumbnail of Binding of the MLL PHD3 Finger to Histone H3K4me3 Is Required for MLL-Dependent Gene Transcription

Journal of Molecular Biology, 2010

The MLL (Mixed Lineage Leukemia) proto-oncogene encodes a histone methyltransferase that creates ... more The MLL (Mixed Lineage Leukemia) proto-oncogene encodes a histone methyltransferase that creates the methylated histone H3K4 epigenetic marks, commonly associated with actively transcribed genes. In addition to its canonical histone methyltransferase SET domain, the MLL protein contains three plant homeodomain (PHD) fingers that are well conserved between species but whose potential roles and requirements for MLL function are unknown. Here we demonstrate that the third PHD domain of MLL (PHD3) binds histone H3 trimethylated at lysine 4 (H3K4me3) with high affinity and specificity, and H3K4me2 with eight-fold lower affinity. Biochemical and structural analysis using NMR and fluorescence spectroscopy identified key amino acids essential for the interaction with H3K4me3. Site-directed mutations of the residues involved in recognition of H3K4me3 compromised in vitro H3K4me3 binding but not in vivo localization of full-length MLL to chromatin sites in target promoters of MEIS1 and HOXA genes. Whereas intact PHD3 finger was necessary for MLL occupancy at these promoters, H3K4me3 binding was critical for MLL transcriptional activity. These results demonstrate that MLL occupancy and target gene activation can be functionally separated. Furthermore, these findings reveal that MLL not only "writes" the H3K4me3 mark, but also binds the mark as well, and this binding is required for the transcriptional maintenance functions of MLL.

Research paper thumbnail of The Target of the NSD Family of Histone Lysine Methyltransferases Depends on the Nature of the Substrate

Journal of Biological Chemistry, 2009

The NSD (nuclear receptor SET domain-containing) family of histone lysine methyltransferases is a... more The NSD (nuclear receptor SET domain-containing) family of histone lysine methyltransferases is a critical participant in chromatin integrity as evidenced by the number of human diseases associated with the aberrant expression of its family members. Yet, the specific targets of these enzymes are not clear, with marked discrepancies being reported in the literature. We demonstrate that NSD2 can exhibit disparate target preferences based on the nature of the substrate provided. The NSD2 complex purified from human cells and recombinant NSD2 both exhibit specific targeting of histone H3 lysine 36 (H3K36) when provided with nucleosome substrates, but histone H4 lysine 44 is the primary target in the case of octamer substrates, irrespective of the histones being native or recombinant. This disparity is negated when NSD2 is presented with octamer targets in conjunction with short single- or double-stranded DNA. Although the octamers cannot form nucleosomes, the target is nonetheless nucleosome-specific as is the product, dimethylated H3K36. This study clarifies in part the previous discrepancies reported with respect to NSD targets. We propose that DNA acts as an allosteric effector of NSD2 such that H3K36 becomes the preferred target.

Research paper thumbnail of Advanced Multilayer Amorphous Silicon Thin-Film Transistor Structure: Film Thickness Effect on Its Electrical Performance and Contact Resistance

Japanese Journal of Applied Physics, 2008

We report the intrinsic and extrinsic electrical characteristics of advanced multilayer amorphous... more We report the intrinsic and extrinsic electrical characteristics of advanced multilayer amorphous silicon (a-Si:H) thin-film transistor (TFT) with dual amorphous silicon nitride (a-SiN X :H) and a-Si:H layers. The thickness effect of the high electronic quality a-Si:H film on the transistor's electrical property was investigated; with increasing film thickness, both field-effect mobility and subthreshold swing show improvement and the threshold voltage remain unchanged. However, the contact resistance increases with the a-Si:H film thickness. Using the two-step plasma enhanced chemical vapor deposition process, we fabricated TFT's with acceptable field-effect mobility ($1 cm 2 V À1 s À1 ) and threshold voltage (<1:5 V) with enhanced throughput.

Research paper thumbnail of Advanced Amorphous Silicon Thin-Film Transistors for AM-OLEDs: Electrical Performance and Stability

IEEE Transactions on Electron Devices, 2000

We fabricated and characterized the advanced amorphous silicon thin-film transistors with a bilay... more We fabricated and characterized the advanced amorphous silicon thin-film transistors with a bilayer structure for both the active and gate dielectric films. The electrical field across the gate insulator has a significant influence on the device threshold voltage electrical stability. We show that high thin-film transistor stability can be achieved even under the presence of a high channel current. Its electrical and high-temperature stability improves up to a factor of five when the TFT biasing condition changes from the linear to the saturation region of operation.

Research paper thumbnail of Electrical Properties and Stability of Dual-Gate Coplanar Homojunction DC Sputtered Amorphous Indium–Gallium–Zinc–Oxide Thin-Film Transistors and Its Application to AM-OLEDs

IEEE Transactions on Electron Devices, 2000

The electrical characteristics and stability of dualgate (DG) coplanar homojunction amorphous ind... more The electrical characteristics and stability of dualgate (DG) coplanar homojunction amorphous indium-galliumzinc-oxide thin-film transistors (a-IGZO TFTs) on glass substrates are described herein. In this device structure, both top gate (TG) and bottom gate are defined by lithography, allowing independent biasing when adjacent TFTs are present. The DG a-IGZO TFT demonstrates excellent electrical performance with subthreshold swing (SS) of 99 mV/dec, field-effect mobility of 15.1 cm 2 /V · s, and ON-OFF current ratio of 10 9 . By applying various bias voltages on the TG electrode, it is found that the TFT threshold voltage can be controlled without any change of the SS and off current. Under conditions of negative bias temperature stress (BTS), the transfer curves of the TFT exhibit negligible shifts after 10 000 s. Larger shifts are observed under conditions of a positive BTS. Finally, the application of this DG device to active-matrix organic light-emitting displays is suggested.

Research paper thumbnail of Lysine methylation of the NF-κB subunit RelA by SETD6 couples activity of the histone methyltransferase GLP at chromatin to tonic repression of NF-κB signaling

Nature immunology, 2011

Signaling via the methylation of lysine residues in proteins has been linked to diverse biologica... more Signaling via the methylation of lysine residues in proteins has been linked to diverse biological and disease processes, yet the catalytic activity and substrate specificity of many human protein lysine methyltransferases (PKMTs) are unknown. We screened over 40 candidate PKMTs and identified SETD6 as a methyltransferase that monomethylated chromatin-associated transcription factor NF-κB subunit RelA at Lys310 (RelAK310me1). SETD6-mediated methylation rendered RelA inert and attenuated RelA-driven transcriptional programs, including inflammatory responses in primary immune cells. RelAK310me1 was recognized by the ankryin repeat of the histone methyltransferase GLP, which under basal conditions promoted a repressed chromatin state at RelA target genes through GLP-mediated methylation of histone H3 Lys9 (H3K9). NF-κB-activation-linked phosphorylation of RelA at Ser311 by protein kinase C-ζ (PKC-ζ) blocked the binding of GLP to RelAK310me1 and relieved repression of the target gene. O...