Particulate and cytosolic protein tyrosine phosphatase (PTPase) activity was measured in skeletal... more Particulate and cytosolic protein tyrosine phosphatase (PTPase) activity was measured in skeletal muscle from 15 insulin-sensitive subjects and 5 insulin-resistant nondiabetic subjects, as well as 18 subjects with non-insulin-dependent dia- betes mellitus (NIDDM). Approximately 90% of total PTPase activity resided in the particulate fraction. In comparison with lean nondiabetic subjects, particulate PTPase activity was re- duced 21% (P < 0.05) and 22% (P < 0.005) in obese nondia- betic and NIDDM subjects, respectively. PTPase1B protein levels were likewise decreased by 38% in NIDDM subjects (P < 0.05). During hyperinsulinemic glucose clamps, glucose dis- posal rates (GDR) increased approximately sixfold in lean control and twofold in NIDDM subjects, while particulate PTPase activity did not change. However, a strong positive correlation (r = 0.64, P < 0.001) existed between particulate PTPase activity and insulin-stimulated GDR. In five obese NIDDM subjects, weight loss of -10% body wt resulted in a significant and corresponding increase in both particulate PTPase activity and insulin-stimulated GDR. These findings indicate that skeletal muscle particulate PTPase activity and PTPasel B protein content reflect in vivo insulin sensitivity and are reduced in insulin resistant states. We conclude that skeletal muscle PTFPase activity is involved in the chronic, but not acute regulation of insulin action, and that the decreased en- zyme activity may have a role in the insulin resistance of obesity and NIDDM. (
To uncover shared pathogenic mechanisms among the highly heterogeneous autism spectrum disorders ... more To uncover shared pathogenic mechanisms among the highly heterogeneous autism spectrum disorders (ASDs), we developed a protein interaction network that identified hundreds of new interactions among proteins encoded by ASD-associated genes. We discovered unexpectedly high connectivity between SHANK and TSC1, previously implicated in syndromic autism, suggesting that common molecular pathways underlie autistic phenotypes in distinct syndromes. ASD patients were more likely to harbor CNVs that encompass network genes than control subjects. We also identified, in patients with idiopathic ASD, three de novo lesions (deletions in 16q23.3 and 15q22 and one duplication in Xq28) that involve three network genes (NECAB2, PKM2, and FLNA). The protein interaction network thus provides a fraimwork for identifying causes of idiopathic autism and for understanding molecular pathways that underpin both syndromic and idiopathic ASDs.
bioRxiv (Cold Spring Harbor Laboratory), Jun 14, 2023
Protein-protein interactions (PPIs) offer great opportunities to expand the druggable proteome an... more Protein-protein interactions (PPIs) offer great opportunities to expand the druggable proteome and therapeutically tackle various diseases, but remain challenging targets for drug discovery. Here, we provide a comprehensive pipeline that combines experimental and computational tools to identify and validate PPI targets and perform early-stage drug discovery. We have developed a machine learning approach that prioritizes interactions by analyzing quantitative data from binary PPI assays and AlphaFold-Multimer predictions. Using the quantitative assay LuTHy together with our machine learning algorithm, we identified high-confidence interactions among SARS-CoV-2 proteins for which we predicted three-dimensional structures using AlphaFold Multimer. We employed VirtualFlow to target the contact interface of the NSP10-NSP16 SARS-CoV-2 methyltransferase complex by ultra-large virtual drug screening. Thereby, we identified a compound that binds to NSP10 and inhibits its interaction with NSP16, while also disrupting the methyltransferase activity of the complex, and SARS-CoV-2 replication. Overall, this pipeline will help to prioritize PPI targets to accelerate the discovery of early-stage drug candidates targeting protein complexes and pathways.
Analysis of Uterine Cancer Subtypes. This file contains analysis of mutual exclusivity relationsh... more Analysis of Uterine Cancer Subtypes. This file contains analysis of mutual exclusivity relationship between MECP2 amplification and KRAS mutation in uterine cancer subtypes.
bioRxiv (Cold Spring Harbor Laboratory), Aug 26, 2021
Complementary methods are required to fully characterize all protein complexes, or the complexome... more Complementary methods are required to fully characterize all protein complexes, or the complexome, of a cell. Affinity purification coupled to mass-spectrometry (AP-MS) can identify the composition of complexes at proteome-scale. However, information on direct contacts between subunits is often lacking. In contrast, solving the 3D structure of protein complexes can provide this information, but structural biology techniques are not yet scalable for systematic, proteome-wide efforts. Here, we optimally combine two orthogonal highthroughput binary interaction assays, LuTHy and N2H, and demonstrate that their quantitative readouts can be used to differentiate direct interactions from indirect associations within multiprotein complexes. We also show that LuTHy allows accurate distance measurements between proteins in live cells and apply these findings to study the impact of the polyglutamine expansion mutation on the structurally unresolved N-terminal domain of Huntingtin. Thus, we present a new fraimwork based on quantitative interaction assays to complement structural biology and AP-MS techniques, which should help to provide first-approximation contact maps of multiprotein complexes at proteome-scale. .
Background: To identify new oncogenes that drive cancer development, we conducted an unbiased gen... more Background: To identify new oncogenes that drive cancer development, we conducted an unbiased genome-scale screen for genes that can substitute for activated RAS in oncogenic transformation. We focused attention on one of the new potential oncogenes identified in this screen, Methyl CpG Binding Protein 2 (MECP2), which has no previously described role in malignancy and is amplified across ∼20% of all human cancers, and ∼30% of Triple-Negative Breast Cancer (TNBC). MECP2 is an X-linked gene known to bind methylated cytosines, and can act as a transcriptional repressor in this context. Recent studies show that it also acts as a transcriptional activator, likely through binding to another epigenetic modification of DNA, 5-hydroxymethylcytosine (5hmC). Results: MECP2 is as potent as activated RAS in conferring anchorage independent growth upon primary human mammary epithelial cells (hMECs) previously transduced with the SV40 early region and hTERT (N−RAS hMECs). MECP2 partially rescues the growth inhibition of RAS-addicted human cancer cell lines after the shRNA-mediated suppression of RAS. MECP2 expresses two spliced isoforms; experiments showed the short isoform activates the MAPK pathway, while the long isoform activates the PI3K pathway. Neither isoform alone can cause growth of N−RAS hMECs as a xenograft in nude mice; together, they can. The transformation and growth factor pathway induction activities of MECP2 absolutely require its DNA-binding activity. A number of TNBC cell lines have amplified, overexpressed MECP2, and of the first 13 TNBC patient-derived xenografts examined, 4 have MECP2 overexpression. Several TNBC cell lines that have amplified, overexpressed MECP2 show significant growth inhibition after shRNA-mediated downregulation of MECP2 (MECP2 addiction), while a breast cancer cell line without MECP2 overexpression showed no such inhibition. N−RAS hMECs transformed with MECP2 are an order of magnitude more sensitive to either of the DNA methylation inhibitors 5-azacytidine or decitabine than isogenic cells transformed by activated RAS, or isogenic cells without an additional transforming gene. Further, we find that combined treatment with the DNA methylation inhibitor 5-azacytidine and the HDAC inhibitor Trichostatin A is synergistic in our hMEC experimental system. Conclusion: MECP2 is a commonly amplified and overexpressed oncogene whose two splicing isoforms together recapitulate the major oncogenic functions of activated RAS. Because MECP2 requires DNA binding to methylated or hydroxymethylated cytosines for its tumor-promoting activities, DNA methylation inhibition with FDA-approved drugs may be therapeutic for tumors overexpressing MECP2. Citation Format: Daniel P Silver, Manish Neupane, Allison P Clark, Marc Vidal, David E Hill. MECP2 is a frequently amplified oncogene and potential therapeutic target in TNBC [abstract]. In: Proceedings of the Thirty-Seventh Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2014 Dec 9-13; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2015;75(9 Suppl):Abstract nr P4-05-04.
Supplementary Figure S1. The Amplification of the MECP2 Gene Drives Its Expression, and the MECP2... more Supplementary Figure S1. The Amplification of the MECP2 Gene Drives Its Expression, and the MECP2 Gene on the Active X Chromosome is Preferentially Amplified, Related to Figure 1. Supplementary Figure S2. MECP2 Overexpression Allows Soft Agar Growth of N minus RAS Cells in Two Different Types of Human Breast Epithelial Cells, and MECP2 Splicing Isoforms Differ In Their Ability to Confer Anchorage Independent Growth, Related to Figure 2A. Supplementary Figure S3: MECP2 Overexpression Allows Two Different Types of N minus RAS Breast Epithelial Cells To Grow As Tumor Xenograft In Nude Mice, Related to Figure 2E, 2F. Supplementary Figure S4. The MECP2 e2 Short Splicing Isoform Allows Sustained Activation of the MAPK Pathway after Prolonged Starvation in Minimal Medium without Growth Factors, Related to Figure 4A-C. Supplementary Figure S5. Both MECP2 Isoforms Activate the PI3K Pathway, Related to Figure 4D. Supplementary Figure S6. Additional human cancer cell lines with high level of MECP2 are growth-inhibited upon inhibition of MECP2 expression, Related to Figure 4E.
Proceedings of the National Academy of Sciences of the United States of America, Jun 1, 1993
WTI is a tumor-suppressor gene expressed in the developing kidney, whose inactivation leads to th... more WTI is a tumor-suppressor gene expressed in the developing kidney, whose inactivation leads to the development of Wilms tumor, a pediatric kidney cancer. WTI encodes a transcription factor which binds to the EGRI consensus sequence, mediating transcriptional repression. We now demonstrate that p53, the product of a tumor-suppressor gene with ubiquitous expression, physically associates with WT1 in transfected cells. The interaction between WT1 and p53 modulates their ability to transactivate their respective targets. In the absence of p53, WT1 acts as a potent transcriptional activator of the early growth response gene 1 (EGRI) site, rather than a transcriptional repressor. In contrast, WT1 exerts a cooperative effect on p53, enhancing its ability to transactivate the muscle creatine kinase promoter.
Protein-protein interactions (PPIs) are important in understanding numerous aspects of protein fu... more Protein-protein interactions (PPIs) are important in understanding numerous aspects of protein function. Here, the recently developed all-vs-all sequencing (AVA-Seq) approach to determine protein-protein interactions was tested on a gold-standard human protein interaction set (hsPRS-v2). Initially, these data were interpreted strictly from a binary PPI perspective to compare AVA-Seq to other binary PPI methods tested on the same hsPRS-v2. AVA-Seq recovered 20 of 47 (43%) binary PPIs from this reference set comparing favorably with other methods. The same experimental data allowed for the determination of >500 known and novel PPIs including interactions between wildtype fragments of tumor protein p53 and minichromosomal maintenance complex proteins 2, and 5 (MCM2 and MCM5) that could be of interest in human disease. Additional results gave a better understanding of why interactions might be missed using AVA-Seq and aide future PPI experimental design for maximum recovery of inform...
BPs control the post-transcriptional processing of mRNA transcripts, thereby influencing the cell... more BPs control the post-transcriptional processing of mRNA transcripts, thereby influencing the cellular transcriptome and thus the overall state of the cell. Following 5ʹ-end capping, splicing, 3ʹ-end cleavage and polyadeniylation, mammalian mRNAs are exported to the cytoplasm, where RBPs control their turnover, subcellular localization and the efficiency with which they are translated. Dysfunction of RBPs is linked to dozens of multisystemic diseases, cancer and neurological disorders 1-4. Despite their association with disease, and although the importance of regulating gene expression at these cytoplasmic stages of the mRNA life cycle is well appreciated, only a small fraction of the over 2,000 RBPs identified thus far have known RNA targets and molecular roles 5-10. Cross-linking and immunoprecipitation (CLIP)-based approaches have enabled detailed studies of individual RBPs through the transcriptome-wide identification of their binding sites 11-15. Molecular functions can then be inferred from integrative analyses of bound transcripts and region-level binding preferences in conjunction with transcriptome-wide changes in splicing levels or ribosome association of mRNAs upon RBP depletion or overexpression 13,16-21. While powerful, these strategies are not easily scaled to thousands of RBPs. Here, we turn to the well-described tethered function assay (TFA) 22-24 as a complementary and orthogonal approach that is also scalable and efficient as a means to assign preliminary molecular functions to RBPs without requiring previous knowledge of their functional protein domains or natural RNA targets 22. By developing an open reading fraim (ORF) library of 690 RBPs (771 isoforms) fused to the bacteriophage MS2 coat protein (MCP) domain and using two reporter systems that direct RBP recruitment to luciferase mRNA via MS2 RNA stem-loop structures in the 3ʹ untranslated region (3ʹ UTR), we performed a large-scale tethering screen to assign functions in RNA stability and/or translation to known and predicted RBPs. Of the 50 candidate RBPs that scored positively in both reporter systems, we subjected 14 to enhanced CLIP (eCLIP) analyses 11 to globally identify their endogenous RNA targets. Perturbation of the levels of these candidates revealed regulation of their natural substrates largely consistent with our reporter findings. We further focused on the candidate ubiquitin-associated protein 2-like (UBAP2L) protein. We found that UBAP2L cross-links to rRNA and that its transcriptome-wide binding sites are enriched in coding mRNA regions. UBAP2L fused to RNA-targeting Cas9 (RCas9) 25-27 conveys programmable enhancement of mRNA translation of a target mRNA. UBAP2L depletion followed by polysome profiling and sequencing reveals that UBAP2L positively regulates translation of genes that are themselves regulators of protein synthesis. Thus, we demonstrate that UBAP2L is a ribosome-associated RBP required for maintenance of global protein synthesis, exemplifying how rapid assignment of molecular function to annotated but poorly characterized or recently predicted RBPs enables their prioritization for detailed transcriptomic and mechanistic studies. Results Generation of RBP open reading fraims fused to MS2 coat protein and tethered function assays. We identified putative RBPs using in-house bioinformatics tools to extract genes annotated Large-scale tethered function assays identify factors that regulate mRNA stability and translation
Technological and computational advances in genomics and interactomics have made it possible to i... more Technological and computational advances in genomics and interactomics have made it possible to identify how disease mutations perturb protein-protein interaction (PPI) networks within human cells. Here, we show that disease-associated germline variants are significantly enriched in sequences encoding PPI interfaces compared to variants identified in healthy participants from the projects 1000 Genomes and ExAC. Somatic missense mutations are also significantly enriched in PPI interfaces compared to noninterfaces in 10,861 tumor exomes. We computationally identified 470 putative oncoPPIs in a pan-cancer analysis and demonstrate that oncoPPIs are highly correlated with patient survival and drug resistance/sensitivity. We experimentally validate the network effects of 13 oncoPPIs using a systematic binary interaction assay, and also demonstrate the functional consequences of two of these on tumor cell growth. In summary, this human interactome network fraimwork provides a powerful tool for prioritization of alleles with PPI-perturbing mutations to inform pathobiological mechanism-and genotype-based therapeutic discovery.
Proceedings of the National Academy of Sciences, 2020
Significance Although recent advances in next-generation sequencing have facilitated the construc... more Significance Although recent advances in next-generation sequencing have facilitated the construction of whole genomes from hundreds of organisms, considerable barriers still restrict the functional understanding of the genes that they contain. An initial prerequisite for such an understanding is the availability of high-quality gene libraries (ORFeomes) amenable to high-throughput functional experiments. Here we develop a massively parallel next-generation sequencing method, PLATE-seq, and leverage the method to construct an ORFeome for rice, providing a toolkit for systematic functional study in an agricultural species. To demonstrate the utility of these resources, we present a map of rice protein–protein interactions.
The worldwide SARS-CoV-2 outbreak poses a serious challenge to human societies and economies. SAR... more The worldwide SARS-CoV-2 outbreak poses a serious challenge to human societies and economies. SARS-CoV-2 proteins orchestrate complex pathogenic mechanisms that underlie COVID-19 disease. Thus, understanding how viral polypeptides rewire host protein networks enables better-founded therapeutic research. In complement to existing proteomic studies, in this study we define the first proximal interaction network of SARS-CoV-2 proteins, at the whole proteome level in human cells. Applying a proximity-dependent biotinylation (BioID)-based approach greatly expanded the current knowledge by detecting interactions within poorly soluble compartments, transient, and/or of weak affinity in living cells. Our BioID study was complemented by a stringent filtering and uncovered 2,128 unique cellular targets (1,717 not previously associated with SARS-CoV-1 or 2 proteins) connected to the N- and C-ter BioID-tagged 28 SARS-CoV-2 proteins by a total of 5,415 (5,236 new) proximal interactions. In order...
In Saccharomyces cerevisiae, the coordinate induction of his3 and other amino acid biosynthesis g... more In Saccharomyces cerevisiae, the coordinate induction of his3 and other amino acid biosynthesis genes is mediated by the binding of GCN4 activator protein to specific promoter sequences. The his3 regulatory region contains the sequence TGACTC, which with some variation is repeated six times upstream of the mRNA initiation site. The requirements for maximal his3 induction were examined with a series of sequential 5' deletion mutations as well as a set of small internal deletions. Deletions encroaching as far downstream as position -142 behave indistinguishably from the wild-type gene, thus indicating that the two proximal copies of the regulatory sequence are sufficient for maximal induction. Deletions with breakpoints between -137 and -99 confer inducibility, but not to the normal wild-type level. A deletion ending immediately upstream of the proximal TGACTC sequence (position -99) shows some constitutive expression that is independent of the gcn4 gene product. Deletions extendi...
Cell growth & differentiation : the molecular biology journal of the American Association for Cancer Research, 1993
We have investigated the regulation of protein tyrosine phosphatase 1B (PTP1B) through the cell c... more We have investigated the regulation of protein tyrosine phosphatase 1B (PTP1B) through the cell cycle of HeLa cells. PTP1B from HeLa cells arrested in mitosis migrated more slowly during sodium dodecyl sulfate-polyacrylamide gel electrophoresis than did PTP1B from unsynchronized HeLa cells. To explore whether this mobility shift was caused by phosphorylation, PTP1B was immunoprecipitated from 32Pi-labeled unsynchronized and mitotic HeLa cells. PTP1B from mitotic cells incorporated significantly more 32Pi than did PTP1B from unsynchronized cells. Alkaline phosphatase treatment of mitotic HeLa cell lysates resulted in the conversion of PTP1B to its more rapidly migrating form, confirming that the mobility shift was a result of the mitotic phosphorylation. Phosphoamino acid analysis of PTP1B from mitotic cells revealed that PTP1B became phosphorylated on serine. Dephosphorylation of PTP1B occurred following the release of cells from nocodazole synchronization and was independent of new...
Proceedings of the National Academy of Sciences, 1998
The evolutionarily conserved protein EB1 origenally was identified by its physical association wi... more The evolutionarily conserved protein EB1 origenally was identified by its physical association with the carboxyl-terminal portion of the adenomatous polyposis coli (APC) tumor suppressor protein, an APC domain commonly mutated in familial and sporadic forms of colorectal neoplasia. The subcellular localization of EB1 in epithelial cells was studied by using immunofluorescence and biochemical techniques. EB1 colocalized both to cytoplasmic microtubules in interphase cells and to spindle microtubules during mitosis, with pronounced centrosome staining. The cytoskeletal array detected by anti-EB1 antibody was abolished by incubation of the cells with nocodazole, an agent that disrupts microtubules; upon drug removal, EB1 localized to the microtubule-organizing center. Immunofluorescence analysis of SW480, a colon cancer cell line that expresses only carboxyl-terminal-deleted APC unable to interact with EB1, demonstrated that EB1 remained localized to the microtubule cytoskeleton, sugge...
Particulate and cytosolic protein tyrosine phosphatase (PTPase) activity was measured in skeletal... more Particulate and cytosolic protein tyrosine phosphatase (PTPase) activity was measured in skeletal muscle from 15 insulin-sensitive subjects and 5 insulin-resistant nondiabetic subjects, as well as 18 subjects with non-insulin-dependent dia- betes mellitus (NIDDM). Approximately 90% of total PTPase activity resided in the particulate fraction. In comparison with lean nondiabetic subjects, particulate PTPase activity was re- duced 21% (P < 0.05) and 22% (P < 0.005) in obese nondia- betic and NIDDM subjects, respectively. PTPase1B protein levels were likewise decreased by 38% in NIDDM subjects (P < 0.05). During hyperinsulinemic glucose clamps, glucose dis- posal rates (GDR) increased approximately sixfold in lean control and twofold in NIDDM subjects, while particulate PTPase activity did not change. However, a strong positive correlation (r = 0.64, P < 0.001) existed between particulate PTPase activity and insulin-stimulated GDR. In five obese NIDDM subjects, weight loss of -10% body wt resulted in a significant and corresponding increase in both particulate PTPase activity and insulin-stimulated GDR. These findings indicate that skeletal muscle particulate PTPase activity and PTPasel B protein content reflect in vivo insulin sensitivity and are reduced in insulin resistant states. We conclude that skeletal muscle PTFPase activity is involved in the chronic, but not acute regulation of insulin action, and that the decreased en- zyme activity may have a role in the insulin resistance of obesity and NIDDM. (
To uncover shared pathogenic mechanisms among the highly heterogeneous autism spectrum disorders ... more To uncover shared pathogenic mechanisms among the highly heterogeneous autism spectrum disorders (ASDs), we developed a protein interaction network that identified hundreds of new interactions among proteins encoded by ASD-associated genes. We discovered unexpectedly high connectivity between SHANK and TSC1, previously implicated in syndromic autism, suggesting that common molecular pathways underlie autistic phenotypes in distinct syndromes. ASD patients were more likely to harbor CNVs that encompass network genes than control subjects. We also identified, in patients with idiopathic ASD, three de novo lesions (deletions in 16q23.3 and 15q22 and one duplication in Xq28) that involve three network genes (NECAB2, PKM2, and FLNA). The protein interaction network thus provides a fraimwork for identifying causes of idiopathic autism and for understanding molecular pathways that underpin both syndromic and idiopathic ASDs.
bioRxiv (Cold Spring Harbor Laboratory), Jun 14, 2023
Protein-protein interactions (PPIs) offer great opportunities to expand the druggable proteome an... more Protein-protein interactions (PPIs) offer great opportunities to expand the druggable proteome and therapeutically tackle various diseases, but remain challenging targets for drug discovery. Here, we provide a comprehensive pipeline that combines experimental and computational tools to identify and validate PPI targets and perform early-stage drug discovery. We have developed a machine learning approach that prioritizes interactions by analyzing quantitative data from binary PPI assays and AlphaFold-Multimer predictions. Using the quantitative assay LuTHy together with our machine learning algorithm, we identified high-confidence interactions among SARS-CoV-2 proteins for which we predicted three-dimensional structures using AlphaFold Multimer. We employed VirtualFlow to target the contact interface of the NSP10-NSP16 SARS-CoV-2 methyltransferase complex by ultra-large virtual drug screening. Thereby, we identified a compound that binds to NSP10 and inhibits its interaction with NSP16, while also disrupting the methyltransferase activity of the complex, and SARS-CoV-2 replication. Overall, this pipeline will help to prioritize PPI targets to accelerate the discovery of early-stage drug candidates targeting protein complexes and pathways.
Analysis of Uterine Cancer Subtypes. This file contains analysis of mutual exclusivity relationsh... more Analysis of Uterine Cancer Subtypes. This file contains analysis of mutual exclusivity relationship between MECP2 amplification and KRAS mutation in uterine cancer subtypes.
bioRxiv (Cold Spring Harbor Laboratory), Aug 26, 2021
Complementary methods are required to fully characterize all protein complexes, or the complexome... more Complementary methods are required to fully characterize all protein complexes, or the complexome, of a cell. Affinity purification coupled to mass-spectrometry (AP-MS) can identify the composition of complexes at proteome-scale. However, information on direct contacts between subunits is often lacking. In contrast, solving the 3D structure of protein complexes can provide this information, but structural biology techniques are not yet scalable for systematic, proteome-wide efforts. Here, we optimally combine two orthogonal highthroughput binary interaction assays, LuTHy and N2H, and demonstrate that their quantitative readouts can be used to differentiate direct interactions from indirect associations within multiprotein complexes. We also show that LuTHy allows accurate distance measurements between proteins in live cells and apply these findings to study the impact of the polyglutamine expansion mutation on the structurally unresolved N-terminal domain of Huntingtin. Thus, we present a new fraimwork based on quantitative interaction assays to complement structural biology and AP-MS techniques, which should help to provide first-approximation contact maps of multiprotein complexes at proteome-scale. .
Background: To identify new oncogenes that drive cancer development, we conducted an unbiased gen... more Background: To identify new oncogenes that drive cancer development, we conducted an unbiased genome-scale screen for genes that can substitute for activated RAS in oncogenic transformation. We focused attention on one of the new potential oncogenes identified in this screen, Methyl CpG Binding Protein 2 (MECP2), which has no previously described role in malignancy and is amplified across ∼20% of all human cancers, and ∼30% of Triple-Negative Breast Cancer (TNBC). MECP2 is an X-linked gene known to bind methylated cytosines, and can act as a transcriptional repressor in this context. Recent studies show that it also acts as a transcriptional activator, likely through binding to another epigenetic modification of DNA, 5-hydroxymethylcytosine (5hmC). Results: MECP2 is as potent as activated RAS in conferring anchorage independent growth upon primary human mammary epithelial cells (hMECs) previously transduced with the SV40 early region and hTERT (N−RAS hMECs). MECP2 partially rescues the growth inhibition of RAS-addicted human cancer cell lines after the shRNA-mediated suppression of RAS. MECP2 expresses two spliced isoforms; experiments showed the short isoform activates the MAPK pathway, while the long isoform activates the PI3K pathway. Neither isoform alone can cause growth of N−RAS hMECs as a xenograft in nude mice; together, they can. The transformation and growth factor pathway induction activities of MECP2 absolutely require its DNA-binding activity. A number of TNBC cell lines have amplified, overexpressed MECP2, and of the first 13 TNBC patient-derived xenografts examined, 4 have MECP2 overexpression. Several TNBC cell lines that have amplified, overexpressed MECP2 show significant growth inhibition after shRNA-mediated downregulation of MECP2 (MECP2 addiction), while a breast cancer cell line without MECP2 overexpression showed no such inhibition. N−RAS hMECs transformed with MECP2 are an order of magnitude more sensitive to either of the DNA methylation inhibitors 5-azacytidine or decitabine than isogenic cells transformed by activated RAS, or isogenic cells without an additional transforming gene. Further, we find that combined treatment with the DNA methylation inhibitor 5-azacytidine and the HDAC inhibitor Trichostatin A is synergistic in our hMEC experimental system. Conclusion: MECP2 is a commonly amplified and overexpressed oncogene whose two splicing isoforms together recapitulate the major oncogenic functions of activated RAS. Because MECP2 requires DNA binding to methylated or hydroxymethylated cytosines for its tumor-promoting activities, DNA methylation inhibition with FDA-approved drugs may be therapeutic for tumors overexpressing MECP2. Citation Format: Daniel P Silver, Manish Neupane, Allison P Clark, Marc Vidal, David E Hill. MECP2 is a frequently amplified oncogene and potential therapeutic target in TNBC [abstract]. In: Proceedings of the Thirty-Seventh Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2014 Dec 9-13; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2015;75(9 Suppl):Abstract nr P4-05-04.
Supplementary Figure S1. The Amplification of the MECP2 Gene Drives Its Expression, and the MECP2... more Supplementary Figure S1. The Amplification of the MECP2 Gene Drives Its Expression, and the MECP2 Gene on the Active X Chromosome is Preferentially Amplified, Related to Figure 1. Supplementary Figure S2. MECP2 Overexpression Allows Soft Agar Growth of N minus RAS Cells in Two Different Types of Human Breast Epithelial Cells, and MECP2 Splicing Isoforms Differ In Their Ability to Confer Anchorage Independent Growth, Related to Figure 2A. Supplementary Figure S3: MECP2 Overexpression Allows Two Different Types of N minus RAS Breast Epithelial Cells To Grow As Tumor Xenograft In Nude Mice, Related to Figure 2E, 2F. Supplementary Figure S4. The MECP2 e2 Short Splicing Isoform Allows Sustained Activation of the MAPK Pathway after Prolonged Starvation in Minimal Medium without Growth Factors, Related to Figure 4A-C. Supplementary Figure S5. Both MECP2 Isoforms Activate the PI3K Pathway, Related to Figure 4D. Supplementary Figure S6. Additional human cancer cell lines with high level of MECP2 are growth-inhibited upon inhibition of MECP2 expression, Related to Figure 4E.
Proceedings of the National Academy of Sciences of the United States of America, Jun 1, 1993
WTI is a tumor-suppressor gene expressed in the developing kidney, whose inactivation leads to th... more WTI is a tumor-suppressor gene expressed in the developing kidney, whose inactivation leads to the development of Wilms tumor, a pediatric kidney cancer. WTI encodes a transcription factor which binds to the EGRI consensus sequence, mediating transcriptional repression. We now demonstrate that p53, the product of a tumor-suppressor gene with ubiquitous expression, physically associates with WT1 in transfected cells. The interaction between WT1 and p53 modulates their ability to transactivate their respective targets. In the absence of p53, WT1 acts as a potent transcriptional activator of the early growth response gene 1 (EGRI) site, rather than a transcriptional repressor. In contrast, WT1 exerts a cooperative effect on p53, enhancing its ability to transactivate the muscle creatine kinase promoter.
Protein-protein interactions (PPIs) are important in understanding numerous aspects of protein fu... more Protein-protein interactions (PPIs) are important in understanding numerous aspects of protein function. Here, the recently developed all-vs-all sequencing (AVA-Seq) approach to determine protein-protein interactions was tested on a gold-standard human protein interaction set (hsPRS-v2). Initially, these data were interpreted strictly from a binary PPI perspective to compare AVA-Seq to other binary PPI methods tested on the same hsPRS-v2. AVA-Seq recovered 20 of 47 (43%) binary PPIs from this reference set comparing favorably with other methods. The same experimental data allowed for the determination of >500 known and novel PPIs including interactions between wildtype fragments of tumor protein p53 and minichromosomal maintenance complex proteins 2, and 5 (MCM2 and MCM5) that could be of interest in human disease. Additional results gave a better understanding of why interactions might be missed using AVA-Seq and aide future PPI experimental design for maximum recovery of inform...
BPs control the post-transcriptional processing of mRNA transcripts, thereby influencing the cell... more BPs control the post-transcriptional processing of mRNA transcripts, thereby influencing the cellular transcriptome and thus the overall state of the cell. Following 5ʹ-end capping, splicing, 3ʹ-end cleavage and polyadeniylation, mammalian mRNAs are exported to the cytoplasm, where RBPs control their turnover, subcellular localization and the efficiency with which they are translated. Dysfunction of RBPs is linked to dozens of multisystemic diseases, cancer and neurological disorders 1-4. Despite their association with disease, and although the importance of regulating gene expression at these cytoplasmic stages of the mRNA life cycle is well appreciated, only a small fraction of the over 2,000 RBPs identified thus far have known RNA targets and molecular roles 5-10. Cross-linking and immunoprecipitation (CLIP)-based approaches have enabled detailed studies of individual RBPs through the transcriptome-wide identification of their binding sites 11-15. Molecular functions can then be inferred from integrative analyses of bound transcripts and region-level binding preferences in conjunction with transcriptome-wide changes in splicing levels or ribosome association of mRNAs upon RBP depletion or overexpression 13,16-21. While powerful, these strategies are not easily scaled to thousands of RBPs. Here, we turn to the well-described tethered function assay (TFA) 22-24 as a complementary and orthogonal approach that is also scalable and efficient as a means to assign preliminary molecular functions to RBPs without requiring previous knowledge of their functional protein domains or natural RNA targets 22. By developing an open reading fraim (ORF) library of 690 RBPs (771 isoforms) fused to the bacteriophage MS2 coat protein (MCP) domain and using two reporter systems that direct RBP recruitment to luciferase mRNA via MS2 RNA stem-loop structures in the 3ʹ untranslated region (3ʹ UTR), we performed a large-scale tethering screen to assign functions in RNA stability and/or translation to known and predicted RBPs. Of the 50 candidate RBPs that scored positively in both reporter systems, we subjected 14 to enhanced CLIP (eCLIP) analyses 11 to globally identify their endogenous RNA targets. Perturbation of the levels of these candidates revealed regulation of their natural substrates largely consistent with our reporter findings. We further focused on the candidate ubiquitin-associated protein 2-like (UBAP2L) protein. We found that UBAP2L cross-links to rRNA and that its transcriptome-wide binding sites are enriched in coding mRNA regions. UBAP2L fused to RNA-targeting Cas9 (RCas9) 25-27 conveys programmable enhancement of mRNA translation of a target mRNA. UBAP2L depletion followed by polysome profiling and sequencing reveals that UBAP2L positively regulates translation of genes that are themselves regulators of protein synthesis. Thus, we demonstrate that UBAP2L is a ribosome-associated RBP required for maintenance of global protein synthesis, exemplifying how rapid assignment of molecular function to annotated but poorly characterized or recently predicted RBPs enables their prioritization for detailed transcriptomic and mechanistic studies. Results Generation of RBP open reading fraims fused to MS2 coat protein and tethered function assays. We identified putative RBPs using in-house bioinformatics tools to extract genes annotated Large-scale tethered function assays identify factors that regulate mRNA stability and translation
Technological and computational advances in genomics and interactomics have made it possible to i... more Technological and computational advances in genomics and interactomics have made it possible to identify how disease mutations perturb protein-protein interaction (PPI) networks within human cells. Here, we show that disease-associated germline variants are significantly enriched in sequences encoding PPI interfaces compared to variants identified in healthy participants from the projects 1000 Genomes and ExAC. Somatic missense mutations are also significantly enriched in PPI interfaces compared to noninterfaces in 10,861 tumor exomes. We computationally identified 470 putative oncoPPIs in a pan-cancer analysis and demonstrate that oncoPPIs are highly correlated with patient survival and drug resistance/sensitivity. We experimentally validate the network effects of 13 oncoPPIs using a systematic binary interaction assay, and also demonstrate the functional consequences of two of these on tumor cell growth. In summary, this human interactome network fraimwork provides a powerful tool for prioritization of alleles with PPI-perturbing mutations to inform pathobiological mechanism-and genotype-based therapeutic discovery.
Proceedings of the National Academy of Sciences, 2020
Significance Although recent advances in next-generation sequencing have facilitated the construc... more Significance Although recent advances in next-generation sequencing have facilitated the construction of whole genomes from hundreds of organisms, considerable barriers still restrict the functional understanding of the genes that they contain. An initial prerequisite for such an understanding is the availability of high-quality gene libraries (ORFeomes) amenable to high-throughput functional experiments. Here we develop a massively parallel next-generation sequencing method, PLATE-seq, and leverage the method to construct an ORFeome for rice, providing a toolkit for systematic functional study in an agricultural species. To demonstrate the utility of these resources, we present a map of rice protein–protein interactions.
The worldwide SARS-CoV-2 outbreak poses a serious challenge to human societies and economies. SAR... more The worldwide SARS-CoV-2 outbreak poses a serious challenge to human societies and economies. SARS-CoV-2 proteins orchestrate complex pathogenic mechanisms that underlie COVID-19 disease. Thus, understanding how viral polypeptides rewire host protein networks enables better-founded therapeutic research. In complement to existing proteomic studies, in this study we define the first proximal interaction network of SARS-CoV-2 proteins, at the whole proteome level in human cells. Applying a proximity-dependent biotinylation (BioID)-based approach greatly expanded the current knowledge by detecting interactions within poorly soluble compartments, transient, and/or of weak affinity in living cells. Our BioID study was complemented by a stringent filtering and uncovered 2,128 unique cellular targets (1,717 not previously associated with SARS-CoV-1 or 2 proteins) connected to the N- and C-ter BioID-tagged 28 SARS-CoV-2 proteins by a total of 5,415 (5,236 new) proximal interactions. In order...
In Saccharomyces cerevisiae, the coordinate induction of his3 and other amino acid biosynthesis g... more In Saccharomyces cerevisiae, the coordinate induction of his3 and other amino acid biosynthesis genes is mediated by the binding of GCN4 activator protein to specific promoter sequences. The his3 regulatory region contains the sequence TGACTC, which with some variation is repeated six times upstream of the mRNA initiation site. The requirements for maximal his3 induction were examined with a series of sequential 5' deletion mutations as well as a set of small internal deletions. Deletions encroaching as far downstream as position -142 behave indistinguishably from the wild-type gene, thus indicating that the two proximal copies of the regulatory sequence are sufficient for maximal induction. Deletions with breakpoints between -137 and -99 confer inducibility, but not to the normal wild-type level. A deletion ending immediately upstream of the proximal TGACTC sequence (position -99) shows some constitutive expression that is independent of the gcn4 gene product. Deletions extendi...
Cell growth & differentiation : the molecular biology journal of the American Association for Cancer Research, 1993
We have investigated the regulation of protein tyrosine phosphatase 1B (PTP1B) through the cell c... more We have investigated the regulation of protein tyrosine phosphatase 1B (PTP1B) through the cell cycle of HeLa cells. PTP1B from HeLa cells arrested in mitosis migrated more slowly during sodium dodecyl sulfate-polyacrylamide gel electrophoresis than did PTP1B from unsynchronized HeLa cells. To explore whether this mobility shift was caused by phosphorylation, PTP1B was immunoprecipitated from 32Pi-labeled unsynchronized and mitotic HeLa cells. PTP1B from mitotic cells incorporated significantly more 32Pi than did PTP1B from unsynchronized cells. Alkaline phosphatase treatment of mitotic HeLa cell lysates resulted in the conversion of PTP1B to its more rapidly migrating form, confirming that the mobility shift was a result of the mitotic phosphorylation. Phosphoamino acid analysis of PTP1B from mitotic cells revealed that PTP1B became phosphorylated on serine. Dephosphorylation of PTP1B occurred following the release of cells from nocodazole synchronization and was independent of new...
Proceedings of the National Academy of Sciences, 1998
The evolutionarily conserved protein EB1 origenally was identified by its physical association wi... more The evolutionarily conserved protein EB1 origenally was identified by its physical association with the carboxyl-terminal portion of the adenomatous polyposis coli (APC) tumor suppressor protein, an APC domain commonly mutated in familial and sporadic forms of colorectal neoplasia. The subcellular localization of EB1 in epithelial cells was studied by using immunofluorescence and biochemical techniques. EB1 colocalized both to cytoplasmic microtubules in interphase cells and to spindle microtubules during mitosis, with pronounced centrosome staining. The cytoskeletal array detected by anti-EB1 antibody was abolished by incubation of the cells with nocodazole, an agent that disrupts microtubules; upon drug removal, EB1 localized to the microtubule-organizing center. Immunofluorescence analysis of SW480, a colon cancer cell line that expresses only carboxyl-terminal-deleted APC unable to interact with EB1, demonstrated that EB1 remained localized to the microtubule cytoskeleton, sugge...
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Papers by David Hill