Alzheimer's Disease-Like Neurodegeneration in Porphyromonas gingivalis Infected Neurons with Persistent Expression of Active Gingipains.

BACKGROUND: Porphyromonas gingivalis (P. gingivalis) and its gingipain virulence factors have been identified as pathogenic effectors in Alzheimer's disease (AD). In a recent study we demonstrated the presence of gingipains in over 90% of postmortem AD brains, with gingipains localizing to the cytoplasm of neurons. However, infection of neurons by P. gingivalis has not been previously reported. OBJECTIVE: To demonstrate intraneuronal P. gingivalis and gingipain expression in vitro after infecting neurons derived from human inducible pluripotent stem cells (iPSC) with P. gingivalis for 24, 48, and 72 h. METHODS: Infection was characterized by transmission electron microscopy, confocal microscopy, and bacterial colony forming unit assays. Gingipain expression was monitored by immunofluorescence and RT-qPCR, and protease activity monitored with activity-based probes. Neurodegenerative endpoints were assessed by immunofluorescence, western blot, and ELISA. RESULTS: Neurons survived the initial infection and showed time dependent, infection induced cell death. P. gingivalis was found free in the cytoplasm or in lysosomes. Infected neurons displayed an accumulation of autophagic vacuoles and multivesicular bodies. Tau protein was strongly degraded, and phosphorylation increased at T231. Over time, the density of presynaptic boutons was decreased. CONCLUSION: P. gingivalis can invade and persist in mature neurons. Infected neurons display signs of AD-like neuropathology including the accumulation of autophagic vacuoles and multivesicular bodies, cytoskeleton disruption, an increase in phospho-tau/tau ratio, and synapse loss. Infection of iPSC-derived mature neurons by P. gingivalis provides a novel model system to study the cellular mechanisms leading to AD and to investigate the potential of new therapeutic approaches.

Treatment of Porphyromonas gulae infection and downstream pathology in the aged dog by lysine-gingipain inhibitor COR388.

COR388, a small-molecule lysine-gingipain inhibitor, is currently being investigated in a Phase 2/3 clinical trial for Alzheimer's disease (AD) with exploratory endpoints in periodontal disease. Gingipains are produced by two species of bacteria, Porphyromonas gingivalis and Porphyromonas gulae, typically associated with periodontal disease and systemic infections in humans and dogs, respectively. P. gulae infection in dogs is associated with periodontal disease, which provides a physiologically relevant model to investigate the pharmacology of COR388. In the current study, aged dogs with a natural oral infection of P. gulae and periodontal disease were treated with COR388 by oral administration for up to 90 days to assess lysine-gingipain target engagement and reduction of bacterial load and downstream pathology. In a 28-day dose-response study, COR388 inhibited the lysine-gingipain target and reduced P. gulae load in saliva, buccal cells, and gingival crevicular fluid. The lowest effective dose was continued for 90 days and was efficacious in continuous reduction of bacterial load and downstream periodontal disease pathology. In a separate histology study, dog brain tissue showed evidence of P. gulae DNA and neuronal lysine-gingipain, demonstrating that P. gulae infection is systemic and spreads beyond its oral reservoir, similar to recent observations of P. gingivalis in humans. Together, the pharmacokinetics and pharmacodynamics of COR388 lysine-gingipain inhibition, along with reduction of bacterial load and periodontal disease in naturally occurring P. gulae infection in the dog, support the use of COR388 in targeting lysine-gingipain and eliminating P. gingivalis infection in humans.

Porphyromonas gingivalis in Alzheimer's disease brains: Evidence for disease causation and treatment with small-molecule inhibitors.

Porphyromonas gingivalis, the keystone pathogen in chronic periodontitis, was identified in the brain of Alzheimer's disease patients. Toxic proteases from the bacterium called gingipains were also identified in the brain of Alzheimer's patients, and levels correlated with tau and ubiquitin pathology. Oral P. gingivalis infection in mice resulted in brain colonization and increased production of Aß1-42, a component of amyloid plaques. Further, gingipains were neurotoxic in vivo and in vitro, exerting detrimental effects on tau, a protein needed for normal neuronal function. To block this neurotoxicity, we designed and synthesized small-molecule inhibitors targeting gingipains. Gingipain inhibition reduced the bacterial load of an established P. gingivalis brain infection, blocked Aß1-42 production, reduced neuroinflammation, and rescued neurons in the hippocampus. These data suggest that gingipain inhibitors could be valuable for treating P. gingivalis brain colonization and neurodegeneration in Alzheimer's disease.

The cancer stem cell marker aldehyde dehydrogenase is required to maintain a drug-tolerant tumor cell subpopulation.

Selective kinase inhibitors have emerged as an important class of cancer therapeutics, and several such drugs are now routinely used to treat advanced-stage disease. However, their clinical benefit is typically short-lived because of the relatively rapid acquisition of drug resistance following treatment response. Accumulating preclinical and clinical data point to a role for a heterogeneous response to treatment within a subpopulation of tumor cells that are intrinsically drug-resistant, such as cancer stem cells. We have previously described an epigenetically determined reversibly drug-tolerant subpopulation of cancer cells that share some properties with cancer stem cells. Here, we define a requirement for the previously established cancer stem cell marker ALDH (aldehyde dehydrogenase) in the maintenance of this drug-tolerant subpopulation. We find that ALDH protects the drug-tolerant subpopulation from the potentially toxic effects of elevated levels of reactive oxygen species (ROS) in these cells, and pharmacologic disruption of ALDH activity leads to accumulation of ROS to toxic levels, consequent DNA damage, and apoptosis specifically within the drug-tolerant subpopulation. Combining ALDH inhibition with other kinase-directed treatments delayed treatment relapse in vitro and in vivo, revealing a novel combination treatment strategy for cancers that might otherwise rapidly relapse following single-agent therapy.

Comparative annotation of functional regions in the human genome using epigenomic data.

Epigenetic regulation is dynamic and cell-type dependent. The recently available epigenomic data in multiple cell types provide an unprecedented opportunity for a comparative study of epigenetic landscape. We developed a machine-learning method called ChroModule to annotate the epigenetic states in eight ENCyclopedia Of DNA Elements cell types. The trained model successfully captured the characteristic histone-modification patterns associated with regulatory elements, such as promoters and enhancers, and showed superior performance on identifying enhancers compared with the state-of-art methods. In addition, given the fixed number of epigenetic states in the model, ChroModule allows straightforward illustration of epigenetic variability in multiple cell types. Using this feature, we found that invariable and variable epigenetic states across cell types correspond to housekeeping functions and stimulus response, respectively. Especially, we observed that enhancers, but not the other regulatory elements, dictate cell specificity, as similar cell types share common enhancers, and cell-type-specific enhancers are often bound by transcription factors playing critical roles in that cell type. More interestingly, we found some genomic regions are dormant in cell type but primed to become active in other cell types. These observations highlight the usefulness of ChroModule in comparative analysis and interpretation of multiple epigenomes.

Accurate identification and analysis of human mRNA isoforms using deep long read sequencing.

Precise identification of RNA-coding regions and transcriptomes of eukaryotes is a significant problem in biology. Currently, eukaryote transcriptomes are analyzed using deep short-read sequencing experiments of complementary DNAs. The resulting short-reads are then aligned against a genome and annotated junctions to infer biological meaning. Here we use long-read complementary DNA datasets for the analysis of a eukaryotic transcriptome and generate two large datasets in the human K562 and HeLa S3 cell lines. Both data sets comprised at least 4 million reads and had median read lengths greater than 500 bp. We show that annotation-independent alignments of these reads provide partial gene structures that are very much in-line with annotated gene structures, 15% of which have not been obtained in a previous de novo analysis of short reads. For long-noncoding RNAs (i.e., lncRNA) genes, however, we find an increased fraction of novel gene structures among our alignments. Other important aspects of transcriptome analysis, such as the description of cell type-specific splicing, can be performed in an accurate, reliable and completely annotation-free manner, making it ideal for the analysis of transcriptomes of newly sequenced genomes. Furthermore, we demonstrate that long read sequence can be assembled into full-length transcripts with considerable success. Our method is applicable to all long read sequencing technologies.

A major epigenetic programming mechanism guided by piRNAs.

A central enigma in epigenetics is how epigenetic factors are guided to specific genomic sites for their function. Previously, we reported that a Piwi-piRNA complex associates with the piRNA-complementary site in the Drosophila genome and regulates its epigenetic state. Here, we report that Piwi-piRNA complexes bind to numerous piRNA-complementary sequences throughout the genome, implicating piRNAs as a major mechanism that guides Piwi and Piwi-associated epigenetic factors to program the genome. To test this hypothesis, we demonstrate that inserting piRNA-complementary sequences to an ectopic site leads to Piwi, HP1a, and Su(var)3-9 recruitment to the site as well as H3K9me2/3 enrichment and reduced RNA polymerase II association, indicating that piRNA is both necessary and sufficient to recruit Piwi and epigenetic factors to specific genomic sites. Piwi deficiency drastically changed the epigenetic landscape and polymerase II profile throughout the genome, revealing the Piwi-piRNA mechanism as a major epigenetic programming mechanism in Drosophila.

Architecture of the human regulatory network derived from ENCODE data.

Transcription factors bind in a combinatorial fashion to specify the on-and-off states of genes; the ensemble of these binding events forms a regulatory network, constituting the wiring diagram for a cell. To examine the principles of the human transcriptional regulatory network, we determined the genomic binding information of 119 transcription-related factors in over 450 distinct experiments. We found the combinatorial, co-association of transcription factors to be highly context specific: distinct combinations of factors bind at specific genomic locations. In particular, there are significant differences in the binding proximal and distal to genes. We organized all the transcription factor binding into a hierarchy and integrated it with other genomic information (for example, microRNA regulation), forming a dense meta-network. Factors at different levels have different properties; for instance, top-level transcription factors more strongly influence expression and middle-level ones co-regulate targets to mitigate information-flow bottlenecks. Moreover, these co-regulations give rise to many enriched network motifs (for example, noise-buffering feed-forward loops). Finally, more connected network components are under stronger selection and exhibit a greater degree of allele-specific activity (that is, differential binding to the two parental alleles). The regulatory information obtained in this study will be crucial for interpreting personal genome sequences and understanding basic principles of human biology and disease.

A highly integrated and complex PPARGC1A transcription factor binding network in HepG2 cells.

PPARGC1A is a transcriptional coactivator that binds to and coactivates a variety of transcription factors (TFs) to regulate the expression of target genes. PPARGC1A plays a pivotal role in regulating energy metabolism and has been implicated in several human diseases, most notably type II diabetes. Previous studies have focused on the interplay between PPARGC1A and individual TFs, but little is known about how PPARGC1A combines with all of its partners across the genome to regulate transcriptional dynamics. In this study, we describe a core PPARGC1A transcriptional regulatory network operating in HepG2 cells treated with forskolin. We first mapped the genome-wide binding sites of PPARGC1A using chromatin-IP followed by high-throughput sequencing (ChIP-seq) and uncovered overrepresented DNA sequence motifs corresponding to known and novel PPARGC1A network partners. We then profiled six of these site-specific TF partners using ChIP-seq and examined their network connectivity and combinatorial binding patterns with PPARGC1A. Our analysis revealed extensive overlap of targets including a novel link between PPARGC1A and HSF1, a TF regulating the conserved heat shock response pathway that is misregulated in diabetes. Importantly, we found that different combinations of TFs bound to distinct functional sets of genes, thereby helping to reveal the combinatorial regulatory code for metabolic and other cellular processes. In addition, the different TFs often bound near the promoters and coding regions of each other's genes suggesting an intricate network of interdependent regulation. Overall, our study provides an important framework for understanding the systems-level control of metabolic gene expression in humans.

Ubiquitous heterogeneity and asymmetry of the chromatin environment at regulatory elements.

Gene regulation at functional elements (e.g., enhancers, promoters, insulators) is governed by an interplay of nucleosome remodeling, histone modifications, and transcription factor binding. To enhance our understanding of gene regulation, the ENCODE Consortium has generated a wealth of ChIP-seq data on DNA-binding proteins and histone modifications. We additionally generated nucleosome positioning data on two cell lines, K562 and GM12878, by MNase digestion and high-depth sequencing. Here we relate 14 chromatin signals (12 histone marks, DNase, and nucleosome positioning) to the binding sites of 119 DNA-binding proteins across a large number of cell lines. We developed a new method for unsupervised pattern discovery, the Clustered AGgregation Tool (CAGT), which accounts for the inherent heterogeneity in signal magnitude, shape, and implicit strand orientation of chromatin marks. We applied CAGT on a total of 5084 data set pairs to obtain an exhaustive catalog of high-resolution patterns of histone modifications and nucleosome positioning signals around bound transcription factors. Our analyses reveal extensive heterogeneity in how histone modifications are deposited, and how nucleosomes are positioned around binding sites. With the exception of the CTCF/cohesin complex, asymmetry of nucleosome positioning is predominant. Asymmetry of histone modifications is also widespread, for all types of chromatin marks examined, including promoter, enhancer, elongation, and repressive marks. The fine-resolution signal shapes discovered by CAGT unveiled novel correlation patterns between chromatin marks, nucleosome positioning, and sequence content. Meta-analyses of the signal profiles revealed a common vocabulary of chromatin signals shared across multiple cell lines and binding proteins.

ChIP-seq guidelines and practices of the ENCODE and modENCODE consortia.

Chromatin immunoprecipitation (ChIP) followed by high-throughput DNA sequencing (ChIP-seq) has become a valuable and widely used approach for mapping the genomic location of transcription-factor binding and histone modifications in living cells. Despite its widespread use, there are considerable differences in how these experiments are conducted, how the results are scored and evaluated for quality, and how the data and metadata are archived for public use. These practices affect the quality and utility of any global ChIP experiment. Through our experience in performing ChIP-seq experiments, the ENCODE and modENCODE consortia have developed a set of working standards and guidelines for ChIP experiments that are updated routinely. The current guidelines address antibody validation, experimental replication, sequencing depth, data and metadata reporting, and data quality assessment. We discuss how ChIP quality, assessed in these ways, affects different uses of ChIP-seq data. All data sets used in the analysis have been deposited for public viewing and downloading at the ENCODE (http://encodeproject.org/ENCODE/) and modENCODE (http://www.modencode.org/) portals.

Extensive promoter-centered chromatin interactions provide a topological basis for transcription regulation.

Higher-order chromosomal organization for transcription regulation is poorly understood in eukaryotes. Using genome-wide Chromatin Interaction Analysis with Paired-End-Tag sequencing (ChIA-PET), we mapped long-range chromatin interactions associated with RNA polymerase II in human cells and uncovered widespread promoter-centered intragenic, extragenic, and intergenic interactions. These interactions further aggregated into higher-order clusters, wherein proximal and distal genes were engaged through promoter-promoter interactions. Most genes with promoter-promoter interactions were active and transcribed cooperatively, and some interacting promoters could influence each other implying combinatorial complexity of transcriptional controls. Comparative analyses of different cell lines showed that cell-specific chromatin interactions could provide structural frameworks for cell-specific transcription, and suggested significant enrichment of enhancer-promoter interactions for cell-specific functions. Furthermore, genetically-identified disease-associated noncoding elements were found to be spatially engaged with corresponding genes through long-range interactions. Overall, our study provides insights into transcription regulation by three-dimensional chromatin interactions for both housekeeping and cell-specific genes in human cells.

A high-resolution whole-genome map of key chromatin modifications in the adult Drosophila melanogaster.

Epigenetic research has been focused on cell-type-specific regulation; less is known about common features of epigenetic programming shared by diverse cell types within an organism. Here, we report a modified method for chromatin immunoprecipitation and deep sequencing (ChIP-Seq) and its use to construct a high-resolution map of the Drosophila melanogaster key histone marks, heterochromatin protein 1a (HP1a) and RNA polymerase II (polII). These factors are mapped at 50-bp resolution genome-wide and at 5-bp resolution for regulatory sequences of genes, which reveals fundamental features of chromatin modification landscape shared by major adult Drosophila cell types: the enrichment of both heterochromatic and euchromatic marks in transposons and repetitive sequences, the accumulation of HP1a at transcription start sites with stalled polII, the signatures of histone code and polII level/position around the transcriptional start sites that predict both the mRNA level and functionality of genes, and the enrichment of elongating polII within exons at splicing junctions. These features, likely conserved among diverse epigenomes, reveal general strategies for chromatin modifications.

AlleleSeq: analysis of allele-specific expression and binding in a network framework.

To study allele-specific expression (ASE) and binding (ASB), that is, differences between the maternally and paternally derived alleles, we have developed a computational pipeline (AlleleSeq). Our pipeline initially constructs a diploid personal genome sequence (and corresponding personalized gene annotation) using genomic sequence variants (SNPs, indels, and structural variants), and then identifies allele-specific events with significant differences in the number of mapped reads between maternal and paternal alleles. There are many technical challenges in the construction and alignment of reads to a personal diploid genome sequence that we address, for example, bias of reads mapping to the reference allele. We have applied AlleleSeq to variation data for NA12878 from the 1000 Genomes Project as well as matched, deeply sequenced RNA-Seq and ChIP-Seq data sets generated for this purpose. In addition to observing fairly widespread allele-specific behavior within individual functional genomic data sets (including results consistent with X-chromosome inactivation), we can study the interaction between ASE and ASB. Furthermore, we investigate the coordination between ASE and ASB from multiple transcription factors events using a regulatory network framework. Correlation analyses and network motifs show mostly coordinated ASB and ASE.

A large gene network in immature erythroid cells is controlled by the myeloid and B cell transcriptional regulator PU.1.

PU.1 is a hematopoietic transcription factor that is required for the development of myeloid and B cells. PU.1 is also expressed in erythroid progenitors, where it blocks erythroid differentiation by binding to and inhibiting the main erythroid promoting factor, GATA-1. However, other mechanisms by which PU.1 affects the fate of erythroid progenitors have not been thoroughly explored. Here, we used ChIP-Seq analysis for PU.1 and gene expression profiling in erythroid cells to show that PU.1 regulates an extensive network of genes that constitute major pathways for controlling growth and survival of immature erythroid cells. By analyzing fetal liver erythroid progenitors from mice with low PU.1 expression, we also show that the earliest erythroid committed cells are dramatically reduced in vivo. Furthermore, we find that PU.1 also regulates many of the same genes and pathways in other blood cells, leading us to propose that PU.1 is a multifaceted factor with overlapping, as well as distinct, functions in several hematopoietic lineages.

Genome-wide chromatin occupancy analysis reveals a role for ASH2 in transcriptional pausing.

An important mechanism for gene regulation involves chromatin changes via histone modification. One such modification is histone H3 lysine 4 trimethylation (H3K4me3), which requires histone methyltranferase complexes (HMT) containing the trithorax-group (trxG) protein ASH2. Mutations in ash2 cause a variety of pattern formation defects in the Drosophila wing. We have identified genome-wide binding of ASH2 in wing imaginal discs using chromatin immunoprecipitation combined with sequencing (ChIP-Seq). Our results show that genes with functions in development and transcriptional regulation are activated by ASH2 via H3K4 trimethylation in nearby nucleosomes. We have characterized the occupancy of phosphorylated forms of RNA Polymerase II and histone marks associated with activation and repression of transcription. ASH2 occupancy correlates with phosphorylated forms of RNA Polymerase II and histone activating marks in expressed genes. Additionally, RNA Polymerase II phosphorylation on serine 5 and H3K4me3 are reduced in ash2 mutants in comparison to wild-type flies. Finally, we have identified specific motifs associated with ASH2 binding in genes that are differentially expressed in ash2 mutants. Our data suggest that recruitment of the ASH2-containing HMT complexes is context specific and points to a function of ASH2 and H3K4me3 in transcriptional pausing control.

ChIP-Seq: a method for global identification of regulatory elements in the genome.

This unit describes ChIP-Seq methodology, which involves chromatin immunoprecipitation (ChIP) followed by high-throughput sequencing (Seq), and enables the genome-wide identification of binding sites of transcription factors (TFs) and other DNA-binding proteins. The process is initiated by cross-linking DNA and DNA-bound proteins. Subsequently, chromatin is isolated from nuclei and subjected to sonication. An antibody against a specific TF or DNA-binding protein is then used to immunoprecipitate specific DNA-TF complexes. ChIP DNA is purified, sequencing adapters are ligated, and 30- to 35-nucleotide (nt) sequence reads are generated. The sequence of the DNA fragments is mapped back to the reference genome for determination of the binding sites.

Genomic binding profiles of functionally distinct RNA polymerase III transcription complexes in human cells.

Genome-wide occupancy profiles of five components of the RNA polymerase III (Pol III) machinery in human cells identified the expected tRNA and noncoding RNA targets and revealed many additional Pol III-associated loci, mostly near short interspersed elements (SINEs). Several genes are targets of an alternative transcription factor IIIB (TFIIIB) containing Brf2 instead of Brf1 and have extremely low levels of TFIIIC. Strikingly, expressed Pol III genes, unlike nonexpressed Pol III genes, are situated in regions with a pattern of histone modifications associated with functional Pol II promoters. TFIIIC alone associates with numerous ETC loci, via the B box or a novel motif. ETCs are often near CTCF binding sites, suggesting a potential role in chromosome organization. Our results suggest that human Pol III complexes associate preferentially with regions near functional Pol II promoters and that TFIIIC-mediated recruitment of TFIIIB is regulated in a locus-specific manner.

Dynamic transcriptomes during neural differentiation of human embryonic stem cells revealed by short, long, and paired-end sequencing.

To examine the fundamental mechanisms governing neural differentiation, we analyzed the transcriptome changes that occur during the differentiation of hESCs into the neural lineage. Undifferentiated hESCs as well as cells at three stages of early neural differentiation-N1 (early initiation), N2 (neural progenitor), and N3 (early glial-like)-were analyzed using a combination of single read, paired-end read, and long read RNA sequencing. The results revealed enormous complexity in gene transcription and splicing dynamics during neural cell differentiation. We found previously unannotated transcripts and spliced isoforms specific for each stage of differentiation. Interestingly, splicing isoform diversity is highest in undifferentiated hESCs and decreases upon differentiation, a phenomenon we call isoform specialization. During neural differentiation, we observed differential expression of many types of genes, including those involved in key signaling pathways, and a large number of extracellular receptors exhibit stage-specific regulation. These results provide a valuable resource for studying neural differentiation and reveal insights into the mechanisms underlying in vitro neural differentiation of hESCs, such as neural fate specification, neural progenitor cell identity maintenance, and the transition from a predominantly neuronal state into one with increased gliogenic potential.

Genome-wide identification of binding sites defines distinct functions for Caenorhabditis elegans PHA-4/FOXA in development and environmental response.

Transcription factors are key components of regulatory networks that control development, as well as the response to environmental stimuli. We have established an experimental pipeline in Caenorhabditis elegans that permits global identification of the binding sites for transcription factors using chromatin immunoprecipitation and deep sequencing. We describe and validate this strategy, and apply it to the transcription factor PHA-4, which plays critical roles in organ development and other cellular processes. We identified thousands of binding sites for PHA-4 during formation of the embryonic pharynx, and also found a role for this factor during the starvation response. Many binding sites were found to shift dramatically between embryos and starved larvae, from developmentally regulated genes to genes involved in metabolism. These results indicate distinct roles for this regulator in two different biological processes and demonstrate the versatility of transcription factors in mediating diverse biological roles.