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bioRxiv2017

A DNA Contact Map for the Mouse Runx1 Gene Identifies Novel Hematopoietic Enhancers

The transcription factor Runx1 is essential for definitive hematopoiesis, and the RUNX1 gene is frequently translocated or mutated in leukemia. Runx1 is transcribed from two promoters, P1 and P2, to give rise to different protein isoforms. Although the expression of Runx1 must be tightly regulated for normal blood development, the mechanisms that regulate Runx1 isoform expression during hematopoiesis remain poorly understood. Gene regulatory elements located in non-coding DNA are likely to be important for Runx1 transcription. Here we use circular chromosome conformation capture sequencing to identify DNA interactions with the P1 and P2 promoters of Runx1, and the previously identified +24 enhancer, in the mouse multipotent hematopoietic progenitor cell line HPC-7. The active promoter, P1, interacts with nine non-coding regions that are occupied by transcription factors within a 1 Mb topologically associated domain. Eight of nine regions function as blood-specific enhancers in zebrafish. Interestingly, the +24 enhancer interacted with multiple distant regions on chromosome 16, indicating it may regulate the expression of additional genes. The Runx1 DNA contact map identifies connections with multiple novel hematopoietic enhancers that are likely to be involved in regulating Runx1 expression in hematopoietic progenitor cells.

molecular biology↗

bioRxiv2017

DRUID: A pipeline for reproducible transcriptome-wide measurements of mRNA stability

Control of messenger RNA (mRNA) stability is an important aspect of gene regulation. The gold standard for measuring mRNA stability transcriptome-wide uses metabolic labeling, biochemical isolation of labeled RNA populations, and high-throughput sequencing. However, difficult normalization procedures and complex computational algorithms have inhibited widespread adoption of this approach. Here, we present DRUID (for Determination of Rates Using Intron Dynamics), a computational pipeline that is robust, easy to use, and freely available. Our pipeline uses endogenous introns to normalize time course data and yields more reproducible half-lives than other methods, even with datasets that were otherwise unusable. DRUID can handle datasets from a variety of organisms, spanning yeast to humans, and we even applied it retroactively on published datasets. We anticipate that DRUID will allow broad application of metabolic labeling for studies of mRNA stability.

molecular biology↗

bioRxiv2017

Splicing-dependent NMD requires Prp17 in Saccharomyces cerevisiae

Nonsense mediated mRNA decay (NMD) is regarded as the function of a specialized cytoplasmic translation-coupled mRNA decay pathway in eukaryotes, however, whether a premature translation termination codon (PTC) will lead to NMD often depends on splicing a downstream intron in the nucleus. Deposition of the exon junction complex (EJC) on mRNA is understood to mediate such splicing-dependent NMD in mammalian cells. The budding yeast, Saccharomyces cerevisiae, which has introns in only 5% of its genes, characteristically at the start of the coding region, and lacks proteins essential for EJC assembly, is not expected to undergo splicing-dependent NMD. However, we found that the presence of an intron near a PTC can also enhance NMD in this organism, regardless of whether it is downstream or upstream. These data provide evidence for a hitherto unsuspected EJC-independent mechanism linking translation and pre-mRNA in S. cerevisiae.

molecular biology↗

bioRxiv2017

Epigenetic maintenance of DNA methylation after evolutionary loss of the de novo methyltransferase

Cytosine methylation of DNA is a widespread modification of DNA that plays numerous critical roles, yet has been lost many times in diverse eukaryotic lineages. In the yeast Cryptococcus neoformans, CG methylation occurs in transposon-rich repeats and requires the DNA methyltransferase, Dnmt5. We show that Dnmt5 displays exquisite maintenance-type specificity in vitro and in vivo and utilizes similar in vivo cofactors as the metazoan maintenance methylase Dnmt1. Remarkably, phylogenetic and functional analysis revealed that the ancestral species lost the gene for a de novo methylase, DnmtX, between 50-150 MYA. We examined how methylation has persisted since the ancient loss of DnmtX. Experimental and comparative studies reveal efficient replication of methylation patterns in C. neoformans, rare stochastic methylation loss and gain events, and the action of natural selection. We propose that an epigenome has been propagated for >50 MY through a process analogous to Darwinian evolution of the genome.

molecular biology↗

bioRxiv2017

Leukocyte counts based on site-specific DNA methylation analysis

The composition of white blood cells is usually assessed by histomorphological parameters or flow cytometric measurements. Alternatively, leukocyte differential counts (LDCs) can be estimated by deconvolution algorithms for genome-wide DNA methylation (DNAm) profiles. We identified cell-type specific CG dinucleotides (CpGs) that facilitate relative quantification of leukocyte subsets. Site-specific analysis of DNAm levels by pyrosequencing provides similar precision of LDCs as conventional methods, whereas it is also applicable to frozen samples and requires only very small volumes of blood. Furthermore, we describe a new approach for absolute quantification of cell numbers based on a non-methylated reference DNA. Our \"Epi-Blood-Count\" facilitates robust and cost effective analysis of blood counts for clinical application.

molecular biology↗

bioRxiv2017

Inducible, tunable and multiplex human gene regulation using CRISPR-Cpf1-based transcription factors

Targeted and inducible regulation of mammalian gene expression is a broadly important research capability that may also enable development of novel therapeutics for treating human diseases. Here we demonstrate that a catalytically inactive RNA-guided CRISPR-Cpf1 nuclease fused to transcriptional activation domains can up-regulate endogenous human gene expression. We engineered drug-inducible Cpf1-based activators and show how this system can be used to tune the regulation of endogenous gene transcription in human cells. Leveraging the simpler multiplex capability of the Cpf1 platform, we show that we can induce both synergistic and combinatorial gene expression in human cells. Our work should enable the creation of other Cpf1-based gene regulatory fusion proteins and the development of multiplex gene perturbation library screens for understanding complex cellular phenotypes.

molecular biology↗

bioRxiv2017

Suppression of non-homologous end joining does not rescue DNA repair defects in Fanconi anemia patient cells

Severe cellular sensitivity and aberrant chromosomal rearrangements in response to DNA interstrand crosslink (ICL) inducing agents are hallmarks of Fanconi anemia (FA) deficient cells. These phenotypes have previously been ascribed to inappropriate activity of non-homologous end joining (NHEJ) rather than a direct consequence of DNA ICL repair defects. Here we used chemical inhibitors, RNAi, and Clusterd Regularly Interspaced Short Palindromic Repeat (CRISPR)-Cas9 to inactivate various components of NHEJ in cells from FA patients. We show that suppression of DNA-PKcs, DNA Ligase IV and 53BP1 is not capable of rescuing ICL-induced proliferation defects and only 53BP1 knockout partially suppresses the chromosomal abnormalities of FA patient cells.

molecular biology↗

bioRxiv2017

Architecture and dynamics of overlapped RNA regulatory networks

A single protein can bind and regulate many mRNAs. Multiple proteins with similar specificities often bind and control overlapping sets of mRNAs. Yet, little is known about the architecture or dynamics of overlapped networks. We focused on three proteins with similar structures and related RNA-binding specificities - Puf3p, Puf4p, and Puf5p of S. cerevisiae. Using RNA Tagging, we identified a \"super-network\" comprised of four sub-networks: Puf3p, Puf4p, and Puf5p sub-networks, and one controlled by both Puf4p and Puf5p. The architecture of individual subnetworks, and thus the super-network, are determined by competition among particular PUF proteins to bind mRNAs, their affinities for binding elements, and the abundances of the proteins. The super-network responds dramatically: the remaining network can either expand or contract. These strikingly opposite outcomes are determined by an interplay between the relative abundance of the RNAs and proteins, and their affinities for one another. The diverse interplay between overlapping RNA-protein networks provides versatile opportunities for regulation and evolution.

molecular biology↗

bioRxiv2017

Viral apoptosis evasion via the MAPK pathway by use of a host long noncoding RNA

An emerging realisation of infectious disease is the high incidence of genetic instability resulting from pathogen-induced DNA lesions, often leading to classical hallmarks of cancer such as evasion of apoptosis. The Human Immunodeficiency Virus type 1 (HIV-1) induces apoptosis in CD4+ T cells but is largely non-cytopathic in macrophages, thereby leading to long-term dissemination of the pathogen specifically by these host cells. Apoptosis is triggered by double-strand breaks (DSBs), such as those induced by integrating retroviruses, and is coordinated by the p53-regulated long noncoding RNA lincRNA-p21, in a complex with its protein binding partners HuR and hnRNP-K. Here, we monitor the cellular response to infection to determine how HIV-1 induces DSBs in macrophages yet evades apoptosis in these cells. We show that the virus does so by securing the pro-survival MAP2K1/ERK2 cascade early upon entry, in a gp120-dependent manner, to orchestrate a complex dysregulation of lincRNA-p21. By sequestering HuR in the nucleus, HIV-1 enables lincRNA-p21 degradation. Simultaneously, the virus permits transcription of pro-survival genes by sequestering hnRNP-K in the cytoplasm via the MAP2K1/ERK2 pathway. Notably, this pro-survival cascade is unavailable for similar viral manipulation in CD4+ T cells. The introduction of MAP2K1, ERK2 or HDM2 inhibitors in HIV-infected macrophages results in apoptosis providing strong evidence that the viral-mediated apoptotic block can be released, specifically by restoring the nuclear interaction of lincRNA-p21 and hnRNP-K. These results reveal pathogenic control of apoptosis and DNA damage via a host long noncoding RNA, and present MAP2K1/ERK2 inhibitors as a novel therapeutic intervention strategy for HIV-1 infection in macrophages.

molecular biology↗

bioRxiv2017

Coral Symbiodinium community composition across the Belize Mesoamerican Barrier Reef System is driven by host species and environmental variability

Reef-building corals maintain a symbiotic relationship with dinoflagellate algae of the genus Symbiodinium and this symbiosis is vital for the survival of the coral holobiont. Symbiodinium community composition within the coral host has been shown to influence a corals ability to resist and recover from stress. A multitude of stressors including ocean warming, ocean acidification, and eutrophication have been linked to global scale decline in coral health and cover in recent decades. Three distinct thermal regimes (highTP, modTP, and lowTP) following an inshore-offshore gradient of declining average temperatures and thermal variation were identified on the Belize Mesoamerican Barrier Reef System (MBRS). Quantitative metabarcoding of the ITS-2 locus was employed to investigate differences and similarities in Symbiodinium genetic diversity of the Caribbean corals Siderastrea siderea, S. radians, and Pseudodiploria strigosa between the three thermal regimes. A total of ten Symbiodinium lineages were identified across the three coral host species. Siderastrea siderea associated with distinct Symbiodinium communities, however Symbiodinium communities of its congener, S. radians, and P. strigosa, were more similar to one another. Thermal regime played a role in defining Symbiodinium communities in S. siderea but not S. radians or P. strigosa. Against expectations, Symbiodinium trenchii, a symbiont known to confer thermal tolerance, was dominant only in S. siderea at one sampled offshore site and was rare inshore, suggesting that coral thermal tolerance in more thermally variable inshore habitats is achieved through alternative mechanisms. Overall, thermal parameters alone were likely not the only primary drivers of Symbiodinium community composition, suggesting that environmental variables unrelated to temperature (i.e., light availability, or nutrients) may play key roles in structuring coral-algal communities in Belize and that the relative importance of these environmental variables may vary by coral host species.

molecular biology↗

bioRxiv2017

Histone demethylation and c-MYC activation enhance translational capacity in response to amino acid restriction

Nutrient limitation may elicit adaptive epigenetic changes but the nature and mechanisms of the cellular response to specific nutrient deficiencies are incompletely understood. We report that depriving human cells of amino acids (AAs) induces specific loss of H4K20me1 from gene bodies and elevated binding of c-MYC at promoters genome-wide. These effects are most pronounced at ribosomal protein and translation initiation genes, which are upregulated, leading to enhanced protein synthetic capacity. Combination of H4K20 methyltransferase depletion and c-MYC over-expression in rich media is required and sufficient to recapitulate the effects of AA restriction. Our data reveal an unexpected and epigenetically implemented increase in translational capacity when AAs are limiting, likely to safeguard the proteome by making effective use of limited resources.\n\nOne Sentence Summary: Combination of H4K20me1 demethylation and c-MYC activation enhance translational capacity in response to amino acid restriction.

molecular biology↗

bioRxiv2017

Head-on replication-transcription collisions lead to formation of life threatening R-loops

Encounters between transcription and DNA replication machineries lead to conflicts that shape genomes, influence evolution, and lead to genetic diseases in humans. Although unclear why, head-on transcription (lagging strand genes) is especially disruptive to replication, increases DNA breaks, and promotes mutagenesis. Here, we show that head-on replication-transcription conflicts lead to pervasive RNA:DNA hybrid formation in Bacillus subtilis. We find that replication beyond head-on conflict regions requires the activity of a RNA:DNA hybrid processing enzyme, RNase HIII. Remarkably, pervasive RNA:DNA hybrid formation at head-on genes completely stops replication and inhibits gene expression in a replication-dependent manner. Accordingly, we find that resolution of head-on conflicts by RNase HIII is crucial for survival upon exposure to various stresses, as many stress response genes are encoded head-on to replication. We conclude that R-loops, RNA:DNA hybrids formed outside of the transcription bubble, exacerbate head-on replication-transcription conflicts, thereby threatening life, especially upon exposure to environmental stresses.

molecular biology↗

bioRxiv2017

mRNA processing in mutant zebrafish lines generated by chemical and CRISPR-mediated mutagenesis produces potentially functional transcripts

As model organism-based research shifts from forward to reverse genetics approaches, largely due to the ease of genome editing technology, allow frequency of abnormal phenotypes is being observed in lines with mutations predicted to lead to deleterious effects on the encoded protein. In zebrafish, this low frequency is in part explained by compensation by genes of redundant or similar function, often resulting from the additional round of teleost-specific whole genome duplication within vertebrates. Here we offer additional explanations for the low frequency of mutant phenotypes. We analyzed mRNA processing in seven zebrafish lines with mutations expected to disrupt gene function, generated by CRISPR/Cas9 or ENU mutagenesis methods. Five of the seven lines showed evidence of genomic compensation by means of altered mRNA processing: one through a skipped exon that did not lead to a frame shift, one through nonsense-associated splicing that did not lead to a frame shift, and three through the use of cryptic splice sites. These results highlight the need for a methodical analysis of the mRNA produced in mutant lines before making conclusions or embarking on studies that assume loss of function as a result of a given genomic change. Furthermore, recognition of the types of genomic adaptations that can occur may inform the strategies of mutant generation.\n\nAuthor summaryThe recent rise of reverse genetic, gene targeting methods has allowed researchers to readily generate mutations in any gene of interest with relative ease. Should these mutations have the predicted effect on the mRNA and encoded protein, we would expect many more abnormal phenotypes than are typically being seen in reverse genetic screens. Here we set out to explore some of the reasons for this discrepancy by studying seven separate mutations in zebrafish. We present evidence that thorough cDNA sequence analysis is a key step in assessing the likelihood that a given mutation will produce hypomorphic or null alleles. This study reveals that alternative mRNA processing in the mutant background often produces transcripts that escape nonsense-mediated decay, thereby potentially preserving gene function. By understanding the ways that cells avoid the deleterious consequences of mutations, researchers can better design reverse genetic strategies to increase the likelihood of gene disruption.

molecular biology↗

bioRxiv2017

Nuclease escape elements protect messenger RNA against cleavage by multiple viral endonucleases

During lytic Kaposis sarcoma-associated herpesvirus (KSHV) infection, the viral endonu-clease SOX promotes widespread degradation of cytoplasmic messenger RNA (mRNA). However, select mRNAs, including the transcript encoding interleukin-6 (IL-6), escape SOX-induced cleavage. IL-6 escape is mediated through a 3 UTR RNA regulatory element that overrides the SOX targeting mechanism. Here, we reveal that this protective RNA element functions to broadly restrict cleavage by a range of homologous and non-homologous viral endonucleases. However, it does not impede cleavage by cellular endonucleases. The IL-6 protective sequence may be representative of a larger class of nuclease escape elements, as we identified a similar protective element in the GADD45B mRNA. The IL-6 and GADD45B-derived elements display similarities in their sequence, putative structure, and several associated RNA binding proteins. However, the overall composition of their ribonucleoprotein complexes appears distinct, leading to differences in the breadth of nucleases restricted. These findings highlight how RNA elements can selectively control transcript abundance in the background of widespread virus-induced mRNA degradation.\n\nAUTHOR SUMMARYThe ability of viruses to control the host gene expression environment is crucial to promote viral infection. Many viruses express factors that reduce host gene expression through widespread mRNA decay. However, some mRNAs escape this fate, like the transcript encoding the immunoregulatory cytokine IL-6 during KSHV infection. IL-6 escape relies on an RNA regulatory element located in its 3UTR and involves the recruitment of a protective protein complex. Here, we show that this escape extends beyond KSHV to a variety of related and unrelated viral endonucleases. However, the IL-6 element does not protect against cellular endonucleases, revealing for the first time a virus-specific nuclease escape element. We identified a related escape element in the GADD45B mRNA, which displays several similarities with the IL-6 element. However, these elements assemble a largely distinct complex of proteins, leading to differences in the breadth of their protective capacity. Collectively, these findings reveal how a putative new class of RNA elements function to control RNA fate in the background of widespread mRNA degradation by viral endonucleases.

molecular biology↗

bioRxiv2017

HyperTRIBE identifies many in vivo targets of a RNA-binding protein

We previously developed TRIBE, a method for the identification of cell-specific RNA binding protein targets. TRIBE expresses an RBP of interest fused to the catalytic domain (cd) of the RNA editing enzyme ADAR and performs Adenosine-to-Inosine editing on RNA targets of the RBP. However, target identification is limited by the low editing efficiency of the ADARcd. Here we describe HyperTRIBE, which carries a previously characterized hyperactive mutation (E488Q) of the ADARcd. HyperTRIBE identifies dramatically more editing sites, many of which are also edited by TRIBE but at a much lower editing frequency. HyperTRIBE therefore more faithfully recapitulates the known binding specificity of its RBP than TRIBE. In addition, separating RNA binding from the enhanced editing activity of the HyperTRIBE ADAR catalytic domain sheds light on the mechanism of ADARcd editing as well as the enhanced activity of the HyperADARcd.

molecular biology↗

bioRxiv2017

The mechanism of transcription start site selection

During transcription initiation, RNA polymerase (RNAP) binds to promoter DNA, unwinds promoter DNA to form an RNAP-promoter open complex (RPo) containing a single-stranded \"transcription bubble,\" and selects a transcription start site (TSS). TSS selection occurs at different positions within the promoter region, depending on promoter sequence and initiating-substrate concentration. Variability in TSS selection has been proposed to involve DNA \"scrunching\" and \"antiscrunching,\" the hallmarks of which are: (i) forward and reverse movement of the RNAP leading edge, but not trailing edge, relative to DNA, and (ii) expansion and contraction of the transcription bubble. Here, using in vitro and in vivo protein-DNA photocrosslinking and single-molecule nanomanipulation, we show bacterial TSS selection exhibits both hallmarks of scrunching and anti-scrunching, and we define energetics of scrunching and anti-scrunching. The results establish the mechanism of TSS selection by bacterial RNAP and suggest a general mechanism for TSS selection by bacterial, archaeal, and eukaryotic RNAP.

molecular biology↗

bioRxiv2017

Proteomic Architecture of Human Coronary and Aortic Atherosclerosis

The inability to detect premature atherosclerosis significantly hinders implementation of personalized therapy to prevent coronary heart disease. A comprehensive understanding of arterial protein networks and how they change in early atherosclerosis could identify new biomarkers for disease detection and improved therapeutic targets. Here we describe the human arterial proteome and the proteomic features strongly associated with early atherosclerosis based on mass-spectrometry analysis of coronary artery and aortic specimens from 100 autopsied young adults (200 arterial specimens). Convex analysis of mixtures, differential dependent network modeling and bioinformatic analyses defined the composition, network re-wiring and likely regulatory features of the protein networks associated with early atherosclerosis. Among other things the results reveal major differences in mitochondrial protein mass between the coronary artery and distal aorta in both normal and atherosclerotic samples - highlighting the importance of anatomic specificity and dynamic network structures in in the study of arterial proteomics. The publicly available data resource and the description of the analysis pipeline establish a new foundation for understanding the proteomic architecture of atherosclerosis and provide a template for similar investigations of other chronic diseases characterized by multi-cellular tissue phenotypes.\n\nHighlightsO_LILC MS/MS analysis performed on 200 human aortic or coronary artery samples\nC_LIO_LINumerous proteins, networks, and regulatory pathways associated with early atherosclerosis\nC_LIO_LIMitochondrial proteins mass and selected metabolic regulatory pathways vary dramatically by disease status and anatomic location\nC_LIO_LIPublically available data resource and analytic pipeline are provided or described in detail\nC_LI

molecular biology↗

bioRxiv2017

Widely used commercial ELISA for human Zonulin reacts with Complement C3 rather than pre-Haptoglobin 2

BACKGROUNDThere is increasing evidence for the role of impaired intestinal permeability in obesity and associated metabolic diseases. Zonulin is an established serum marker for intestinal permeability and identical to pre-haptoglobin2. Here, we aimed to investigate the relationship between circulating zonulin and metabolic traits related to obesity.\n\nMETHODSSerum zonulin was measured by using a widely used commercial ELISA kit in 376 subjects from the metabolically well-characterized cohort of Sorbs from Germany. In addition, haptoglobin genotype was determined in DNA samples from all study subjects.\n\nRESULTSAs zonulin concentrations did not correlate to the haptoglobin genotypes, we investigated the specificity of the zonulin ELISA assay using antibody capture experiments, mass spectrometry and Western blot analysis. Using serum samples that gave the highest or lowest ELISA signals, we detected several proteins that are likely to be captured by the antibody in the present kit. However, none of these proteins corresponds to pre-haptoglobin2. We used increasing concentrations of recombinant pre-haptoglobin 2 and complement C3 as one of the representative captured proteins and the ELISA kit did not detect either. Western blot analysis using both the polyclonal antibodies used in this kit and monoclonal antibodies rose against zonulin showed a similar protein recognition pattern but with different intensity of detection. The protein(s) measured using the ELISA kit was (were) significantly increased in patients with diabetes and obesity and correlated strongly with markers of the lipid and glucose metabolism. Combining mass spectrometry and Western blot analysis using the polyclonal antibodies used in the ELISA kit, we identified properdin as another member of the zonulin family.\n\nCONCLUSIONSOur study suggests that the zonulin ELISA does not recognize pre-haptoglobin 2, rather structural (and possibly functional) analogue proteins belonging to the mannose-associated serine protease family, with properdin being the most likely possible candidate.

molecular biology↗