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Crystal structure of Streptococcus pyogenes EndoS, an immunomodulatory endoglycosidase specific for human IgG antibodies

Sun, 06/28/2015 - 5:06pm

To evade host immune mechanisms, many bacteria secrete immunomodulatory enzymes. Streptococcus pyogenes, one of the most common human pathogens, secretes a large endoglycosidase, EndoS, which removes carbohydrates in a highly specific manner from IgG antibodies. This modification renders antibodies incapable of eliciting host effector functions through either complement or Fc gamma receptors, providing the bacteria with a survival advantage. On account of this antibody-specific modifying activity, EndoS is being developed as a promising injectable therapeutic for autoimmune diseases that rely on autoantibodies. Additionally, EndoS is a key enzyme used in the chemoenzymatic synthesis of homogenously glycosylated antibodies with tailored Fc gamma receptor-mediated effector functions. Despite the tremendous utility of this enzyme, the molecular basis of EndoS specificity for, and processing of, IgG antibodies has remained poorly understood. Here, we report the X-ray crystal structure of EndoS and provide a model of its encounter complex with its substrate, the IgG1 Fc domain. We show that EndoS is composed of five distinct protein domains, including glycosidase, leucine-rich repeat, hybrid Ig, carbohydrate binding module, and three-helix bundle domains, arranged in a distinctive V-shaped conformation. Our data suggest that the substrate enters the concave interior of the enzyme structure, is held in place by the carbohydrate binding module, and that concerted conformational changes in both enzyme and substrate are required for subsequent antibody deglycosylation. The EndoS structure presented here provides a framework from which novel endoglycosidases could be engineered for additional clinical and biotechnological applications.

The HP1 homolog rhino anchors a nuclear complex that suppresses piRNA precursor splicing

Sun, 06/28/2015 - 5:06pm

piRNAs guide an adaptive genome defense system that silences transposons during germline development. The Drosophila HP1 homolog Rhino is required for germline piRNA production. We show that Rhino binds specifically to the heterochromatic clusters that produce piRNA precursors, and that binding directly correlates with piRNA production. Rhino colocalizes to germline nuclear foci with Rai1/DXO-related protein Cuff and the DEAD box protein UAP56, which are also required for germline piRNA production. RNA sequencing indicates that most cluster transcripts are not spliced and that rhino, cuff, and uap56 mutations increase expression of spliced cluster transcripts over 100-fold. LacI::Rhino fusion protein binding suppresses splicing of a reporter transgene and is sufficient to trigger piRNA production from a trans combination of sense and antisense reporters. We therefore propose that Rhino anchors a nuclear complex that suppresses cluster transcript splicing and speculate that stalled splicing differentiates piRNA precursors from mRNAs.

Regulation of histone H3K4 methylation in brain development and disease

Sun, 06/28/2015 - 5:06pm

The growing list of mutations implicated in monogenic disorders of the developing brain includes at least seven genes (ARX, CUL4B, KDM5A, KDM5C, KMT2A, KMT2C, KMT2D) with loss-of-function mutations affecting proper regulation of histone H3 lysine 4 methylation, a chromatin mark which on a genome-wide scale is broadly associated with active gene expression, with its mono-, di- and trimethylated forms differentially enriched at promoter and enhancer and other regulatory sequences. In addition to these rare genetic syndromes, dysregulated H3K4 methylation could also play a role in the pathophysiology of some cases diagnosed with autism or schizophrenia, two conditions which on a genome-wide scale are associated with H3K4 methylation changes at hundreds of loci in a subject-specific manner. Importantly, the reported alterations for some of the diseased brain specimens included a widespread broadening of H3K4 methylation profiles at gene promoters, a process that could be regulated by the UpSET(KMT2E/MLL5)-histone deacetylase complex. Furthermore, preclinical studies identified maternal immune activation, parental care and monoaminergic drugs as environmental determinants for brain-specific H3K4 methylation. These novel insights into the epigenetic risk architectures of neurodevelopmental disease will be highly relevant for efforts aimed at improved prevention and treatment of autism and psychosis spectrum disorders.

Principles of regulatory information conservation between mouse and human

Sun, 06/28/2015 - 5:06pm

To broaden our understanding of the evolution of gene regulation mechanisms, we generated occupancy profiles for 34 orthologous transcription factors (TFs) in human-mouse erythroid progenitor, lymphoblast and embryonic stem-cell lines. By combining the genome-wide transcription factor occupancy repertoires, associated epigenetic signals, and co-association patterns, here we deduce several evolutionary principles of gene regulatory features operating since the mouse and human lineages diverged. The genomic distribution profiles, primary binding motifs, chromatin states, and DNA methylation preferences are well conserved for TF-occupied sequences. However, the extent to which orthologous DNA segments are bound by orthologous TFs varies both among TFs and with genomic location: binding at promoters is more highly conserved than binding at distal elements. Notably, occupancy-conserved TF-occupied sequences tend to be pleiotropic; they function in several tissues and also co-associate with many TFs. Single nucleotide variants at sites with potential regulatory functions are enriched in occupancy-conserved TF-occupied sequences.

piPipes: a set of pipelines for piRNA and transposon analysis via small RNA-seq, RNA-seq, degradome- and CAGE-seq, ChIP-seq and genomic DNA sequencing

Sun, 06/28/2015 - 5:06pm

MOTIVATION: PIWI-interacting RNAs (piRNAs), 23-36 nt small silencing RNAs, repress transposon expression in the metazoan germ line, thereby protecting the genome. Although high-throughput sequencing has made it possible to examine the genome and transcriptome at unprecedented resolution, extracting useful information from gigabytes of sequencing data still requires substantial computational skills. Additionally, researchers may analyze and interpret the same data differently, generating results that are difficult to reconcile. To address these issues, we developed a coordinated set of pipelines, 'piPipes', to analyze piRNA and transposon-derived RNAs from a variety of high-throughput sequencing libraries, including small RNA, RNA, degradome or 7-methyl guanosine cap analysis of gene expression (CAGE), chromatin immunoprecipitation (ChIP) and genomic DNA-seq. piPipes can also produce figures and tables suitable for publication. By facilitating data analysis, piPipes provides an opportunity to standardize computational methods in the piRNA field.

SUPPLEMENTARY INFORMATION: Supplementary information, including flowcharts and example figures for each pipeline, are available at Bioinformatics online.

AVAILABILITY AND IMPLEMENTATION: piPipes is implemented in Bash, C++, Python, Perl and R. piPipes is free, open-source software distributed under the GPLv3 license and is available at http://bowhan.github.io/piPipes/.

CONTACT: Phillip.Zamore@umassmed.edu or Zhiping.Weng@umassmed.edu

SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Epigenetic dysregulation of hairy and enhancer of split 4 (HES4) is associated with striatal degeneration in postmortem Huntington brains

Sun, 06/28/2015 - 5:06pm

To investigate epigenetic contributions to Huntington's disease (HD) pathogenesis, we carried out genome-wide mapping of the transcriptional mark, trimethyl-histone H3-lysine 4 (H3K4me3) in neuronal nuclei extracted from prefrontal cortex of HD cases and controls using chromatin immunoprecipitation followed by deep-sequencing. Neuron-specific mapping of the genome-wide distribution of H3K4me3 revealed 136 differentially enriched loci associated with genes implicated in neuronal development and neurodegeneration, including GPR3, TMEM106B, PDIA6 and the Notch signaling genes hairy and enhancer of split 4 (HES4) and JAGGED2, supporting the view that the neuronal epigenome is affected in HD. Importantly, loss of H3K4me3 at CpG-rich sequences on the HES4 promoter was associated with excessive DNA methylation, reduced binding of nuclear proteins to the methylated region and altered expression of HES4 and HES4 targeted genes MASH1 and P21 involved in striatal development. Moreover, hypermethylation of HES4 promoter sequences was strikingly correlated with measures of striatal degeneration and age-of-onset in a cohort of 25 HD brains (r = 0.56, P = 0.006). Lastly, shRNA knockdown of HES4 in human neuroblastoma cells altered MASH1 and P21 mRNA expression and markedly increased mutated HTT-induced aggregates and cell death. These findings, taken together, suggest that epigenetic dysregulation of HES4 could play a critical role in modifying HD disease pathogenesis and severity.

Comparative functional characterization of the CSR-1 22G-RNA pathway in Caenorhabditis nematodes

Sun, 06/28/2015 - 5:06pm

As a champion of small RNA research for two decades, Caenorhabditis elegans has revealed the essential Argonaute CSR-1 to play key nuclear roles in modulating chromatin, chromosome segregation and germline gene expression via 22G-small RNAs. Despite CSR-1 being preserved among diverse nematodes, the conservation and divergence in function of the targets of small RNA pathways remains poorly resolved. Here we apply comparative functional genomic analysis between C. elegans and Caenorhabditis briggsae to characterize the CSR-1 pathway, its targets and their evolution. C. briggsae CSR-1-associated small RNAs that we identified by immunoprecipitation-small RNA sequencing overlap with 22G-RNAs depleted in cbr-csr-1 RNAi-treated worms. By comparing 22G-RNAs and target genes between species, we defined a set of CSR-1 target genes with conserved germline expression, enrichment in operons and more slowly evolving coding sequences than other genes, along with a small group of evolutionarily labile targets. We demonstrate that the association of CSR-1 with chromatin is preserved, and show that depletion of cbr-csr-1 leads to chromosome segregation defects and embryonic lethality. This first comparative characterization of a small RNA pathway in Caenorhabditis establishes a conserved nuclear role for CSR-1 and highlights its key role in germline gene regulation across multiple animal species.

Glycolytic enzymes localize to ribonucleoprotein granules in Drosophila germ cells, bind Tudor and protect from transposable elements

Sun, 06/28/2015 - 5:05pm

Germ cells give rise to all cell lineages in the next-generation and are responsible for the continuity of life. In a variety of organisms, germ cells and stem cells contain large ribonucleoprotein granules. Although these particles were discovered more than 100 years ago, their assembly and functions are not well understood. Here we report that glycolytic enzymes are components of these granules in Drosophila germ cells and both their mRNAs and the enzymes themselves are enriched in germ cells. We show that these enzymes are specifically required for germ cell development and that they protect their genomes from transposable elements, providing the first link between metabolism and transposon silencing. We further demonstrate that in the granules, glycolytic enzymes associate with the evolutionarily conserved Tudor protein. Our biochemical and single-particle EM structural analyses of purified Tudor show a flexible molecule and suggest a mechanism for the recruitment of glycolytic enzymes to the granules. Our data indicate that germ cells, similarly to stem cells and tumor cells, might prefer to produce energy through the glycolytic pathway, thus linking a particular metabolism to pluripotency.

Pitfalls of mapping high-throughput sequencing data to repetitive sequences: Piwi's genomic targets still not identified

Sun, 06/28/2015 - 5:05pm

Huang et al. (2013) recently reported that chromatin immunoprecipitation sequencing (ChIP-seq) reveals the genome-wide sites of occupancy by Piwi, a piRNA-guided Argonaute protein central to transposon silencing in Drosophila. Their study also reported that loss of Piwi causes widespread rewiring of transcriptional patterns, as evidenced by changes in RNA polymerase II occupancy across the genome. Here we reanalyze their data and report that the underlying deep-sequencing dataset does not support the authors' genome-wide conclusions.

miR-10b-5p expression in Huntington's disease brain relates to age of onset and the extent of striatal involvement

Sun, 06/28/2015 - 5:05pm

BACKGROUND: MicroRNAs (miRNAs) are small non-coding RNAs that recognize sites of complementarity of target messenger RNAs, resulting in transcriptional regulation and translational repression of target genes. In Huntington's disease (HD), a neurodegenerative disease caused by a trinucleotide repeat expansion, miRNA dyregulation has been reported, which may impact gene expression and modify the progression and severity of HD.

METHODS: We performed next-generation miRNA sequence analysis in prefrontal cortex (Brodmann Area 9) from 26 HD, 2 HD gene positive, and 36 control brains. Neuropathological information was available for all HD brains, including age at disease onset, CAG-repeat size, Vonsattel grade, and Hadzi-Vonsattel striatal and cortical scores, a continuous measure of the extent of neurodegeneration. Linear models were performed to examine the relationship of miRNA expression to these clinical features, and messenger RNA targets of associated miRNAs were tested for gene ontology term enrichment. RESULTS: We identified 75 miRNAs differentially expressed in HD brain (FDR q-value < 0.05). Among the HD brains, nine miRNAs were significantly associated with Vonsattel grade of neuropathological involvement and three of these, miR-10b-5p, miR-10b-3p, and miR-302a-3p, significantly related to the Hadzi-Vonsattel striatal score (a continuous measure of striatal involvement) after adjustment for CAG length. Five miRNAs (miR-10b-5p, miR-196a-5p, miR-196b-5p, miR-10b-3p, and miR-106a-5p) were identified as having a significant relationship to CAG length-adjusted age of onset including miR-10b-5p, the mostly strongly over-expressed miRNA in HD cases. Although prefrontal cortex was the source of tissue profiled in these studies, the relationship of miR-10b-5p expression to striatal involvement in the disease was independent of cortical involvement. Correlation of miRNAs to the clinical features clustered by direction of effect and the gene targets of the observed miRNAs showed association to processes relating to nervous system development and transcriptional regulation.

CONCLUSIONS: These results demonstrate that miRNA expression in cortical BA9 provides insight into striatal involvement and support a role for these miRNAs, particularly miR-10b-5p, in HD pathogenicity. The miRNAs identified in our studies of postmortem brain tissue may be detectable in peripheral fluids and thus warrant consideration as accessible biomarkers for disease stage, rate of progression, and other important clinical characteristics of HD.

Providing Information across Multiple Devices to the Public Health Workforce: Challenges and Opportunities

Fri, 06/26/2015 - 4:47pm

Public health workers are increasingly using mobile technology to access information. PHPartners.org, the web portal of the Partners in Information Access for the Public Health Workforce, has implemented a responsively designed website to allow users to access and easily view the same information across multiple devices including mobile phones, tablets, and desktop computers. This webinar will present an overview of the benefits of responsive web design, the challenges to implementation, and future developments.

Required Data Management Training for Graduate Students in an Earth and Environmental Sciences Department

Wed, 06/24/2015 - 2:51pm

The increasing importance of data management in the sciences has led the Department of Earth & Environmental Sciences at a research intensive university to work closely with the Physical Sciences Librarian and Data Services Librarian on campus to provide mandatory training to its graduate students. Although integrating data management training into the graduate program curriculum may not be possible, there are still opportunities to ensure students learn such skills prior to graduating. This article describes the four approaches taken thus far – a seminar about basic data management during the department’s weekly seminar series, creation of a Data Profile form that students were asked to complete, an interactive workshop during the department’s annual retreat, and assistance with writing data management plans. Buy-in for requiring data management training was essential from both faculty and students and was possible because both groups understood the value of research data management skills. Also vital to the success of these approaches was how the subject specialist and data librarians leveraged their respective areas of expertise in a complementary fashion to address disciplinary as well as broader data-related concerns.

Differences in the Data Practices, Challenges, and Future Needs of Graduate Students and Faculty Members

Wed, 06/24/2015 - 2:51pm

Objective: In light of academic libraries expanding the data services they offer, this article hopes to improve the understanding of data practices, challenges and future needs of academic library user groups.

Setting, Design and Method: In the fall of 2013, librarians and campus grant specialist conducted an institution wide web-based survey at the University of Kansas, a major public research institution, with several hundred respondents.

Results: Graduate students and faculty members report differences in their data practices, research challenges and data-related needs.

Conclusions: Academic libraries should target data services to the interests and needs of their distinct user groups.

The bone-specific Runx2-P1 promoter displays conserved three-dimensional chromatin structure with the syntenic Supt3h promoter

Wed, 06/24/2015 - 11:13am

Three-dimensional organization of chromatin is fundamental for transcriptional regulation. Tissue-specific transcriptional programs are orchestrated by transcription factors and epigenetic regulators. The RUNX2 transcription factor is required for differentiation of precursor cells into mature osteoblasts. Although organization and control of the bone-specific Runx2-P1 promoter have been studied extensively, long-range regulation has not been explored. In this study, we investigated higher-order organization of the Runx2-P1 promoter during osteoblast differentiation. Mining the ENCODE database revealed interactions between Runx2-P1 and Supt3h promoters in several non-mesenchymal human cell lines. Supt3h is a ubiquitously expressed gene located within the first intron of Runx2. These two genes show shared synteny across species from humans to sponges. Chromosome conformation capture analysis in the murine pre-osteoblastic MC3T3-E1 cell line revealed increased contact frequency between Runx2-P1 and Supt3h promoters during differentiation. This increase was accompanied by enhanced DNaseI hypersensitivity along with RUNX2 and CTCF binding at the Supt3h promoter. Furthermore, interplasmid-3C and luciferase reporter assays showed that the Supt3h promoter can modulate Runx2-P1 activity via direct association. Taken together, our data demonstrate physical proximity between Runx2-P1 and Supt3h promoters, consistent with their syntenic nature. Importantly, we identify the Supt3h promoter as a potential regulator of the bone-specific Runx2-P1 promoter. Acids Research.

Uniform nomenclature for the mitochondrial contact site and cristae organizing system

Wed, 06/24/2015 - 11:13am

The mitochondrial inner membrane contains a large protein complex that functions in inner membrane organization and formation of membrane contact sites. The complex was variably named the mitochondrial contact site complex, mitochondrial inner membrane organizing system, mitochondrial organizing structure, or Mitofilin/Fcj1 complex. To facilitate future studies, we propose to unify the nomenclature and term the complex "mitochondrial contact site and cristae organizing system" and its subunits Mic10 to Mic60.

HDAC4 integrates PTH and sympathetic signaling in osteoblasts

Wed, 06/24/2015 - 11:13am

Parathyroid hormone (PTH) and the sympathetic tone promote Rankl expression in osteoblasts and osteoclast differentiation by enhancing cyclic adenosine monophosphate production through an unidentified transcription factor for PTH and through ATF4 for the sympathetic tone. How two extracellular cues using the same second messenger in the same cell elicit different transcriptional events is unknown. In this paper, we show that PTH favors Rankl expression by triggering the ubiquitination of HDAC4, a class II histone deacetylase, via Smurf2. HDAC4 degradation releases MEF2c, which transactivates the Rankl promoter. Conversely, sympathetic signaling in osteoblasts favors the accumulation of HDAC4 in the nucleus and its association with ATF4. In this context, HDAC4 increases Rankl expression. Because of its ability to differentially connect two extracellular cues to the genome of osteoblasts, HDAC4 is a critical regulator of osteoclast differentiation.

The Dynamics of Morphogenesis in the Early Mouse Embryo

Wed, 06/24/2015 - 11:13am

Over the past two decades, our understanding of mouse development from implantation to gastrulation has grown exponentially with an upsurge of genetic, molecular, cellular, and morphogenetic information. New discoveries have exalted the role of extraembryonic tissues in orchestrating embryonic patterning and axial specification. At the same time, the identification of unexpected morphogenetic processes occurring during mouse gastrulation has challenged established dogmas and brought new insights into the mechanisms driving germ layer formation. In this article, we summarize the key findings that have reinvigorated the contemporary view of early postimplantation mammalian development.

Cooperative binding of the outer arm-docking complex underlies the regular arrangement of outer arm dynein in the axoneme

Wed, 06/24/2015 - 11:13am

Outer arm dynein (OAD) in cilia and flagella is bound to the outer doublet microtubules every 24 nm. Periodic binding of OADs at specific sites is important for efficient cilia/flagella beating; however, the molecular mechanism that specifies OAD arrangement remains elusive. Studies using the green alga Chlamydomonas reinhardtii have shown that the OAD-docking complex (ODA-DC), a heterotrimeric complex present at the OAD base, functions as the OAD docking site on the doublet. We find that the ODA-DC has an ellipsoidal shape approximately 24 nm in length. In mutant axonemes that lack OAD but retain the ODA-DC, ODA-DC molecules are aligned in an end-to-end manner along the outer doublets. When flagella of a mutant lacking ODA-DCs are supplied with ODA-DCs upon gamete fusion, ODA-DC molecules first bind to the mutant axonemes in the proximal region, and the occupied region gradually extends toward the tip, followed by binding of OADs. This and other results indicate that a cooperative association of the ODA-DC underlies its function as the OAD-docking site and is the determinant of the 24-nm periodicity.

Characterization of THB1, a Chlamydomonas reinhardtii truncated hemoglobin: linkage to nitrogen metabolism and identification of lysine as the distal heme ligand

Wed, 06/24/2015 - 11:13am

The nuclear genome of the model organism Chlamydomonas reinhardtii contains genes for a dozen hemoglobins of the truncated lineage. Of those, THB1 is known to be expressed, but the product and its function have not yet been characterized. We present mutagenesis, optical, and nuclear magnetic resonance data for the recombinant protein and show that at pH near neutral in the absence of added ligand, THB1 coordinates the heme iron with the canonical proximal histidine and a distal lysine. In the cyanomet state, THB1 is structurally similar to other known truncated hemoglobins, particularly the heme domain of Chlamydomonas eugametos LI637, a light-induced chloroplastic hemoglobin. Recombinant THB1 is capable of binding nitric oxide (NO(*)) in either the ferric or ferrous state and has efficient NO(*) dioxygenase activity. By using different C. reinhardtii strains and growth conditions, we demonstrate that the expression of THB1 is under the control of the NIT2 regulatory gene and that the hemoglobin is linked to the nitrogen assimilation pathway.

One to only two: a short history of the centrosome and its duplication

Wed, 06/24/2015 - 11:13am

This review discusses some of the history of the fundamental, but not fully solved problem of how the centrosome duplicates from one to only two as the cell prepares for mitosis. We start with some of the early descriptions of the centrosome and the remarkably prescient but then controversial inferences drawn concerning its function in the cell. For more than 100 years, one of the most difficult issues for the concept of the centrosome has been to integrate observations that centrosomes appear to be important for spindle assembly in animal cells yet are not evident in higher plant cells and some animal cells. This stirred debate over the existence of centrosomes and their importance. A parallel debate concerned the role of the centrioles in organizing centrosomes. The relatively recent elucidation of bipolar spindle assembly around chromatin allows a re-examination of the role of centrioles in controlling centrosome duplication in animal cells. The problem of how centrosomes precisely double in preparation for mitosis in animal cells has now moved to the mystery of how only one procentriole is assembled at each mother centriole.