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Search indexed bioRxiv preprints in genomics, neuroscience, cell biology and bioinformatics. Read source abstracts and check manuscript versions; preprints are not peer reviewed.

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bioRxiv2017

Impact of hypoxia induced VEGF and its signaling during caudal fin regeneration in Zebrafish

ABSTRACTHypoxia is known to play important role during various cellular process, including regeneration. Regeneration is a complex process involving wound healing and tissue repair. We propose that hypoxia might mediate regeneration through angiogenesis involving angiogenic factors such as VEGF, VEGF-R2, NRP1a during the wound healing process. We have chosen Zebrafish model to study the role of hypoxia induced regeneration. Unlike mammals Zebrafish has the ability to regenerate. Hypoxic condition was mimicked using inorganic salt cobalt chloride to study caudal fin regeneration in adult Zebrafish. Intense blood vessel formation, with increased tail fin length experimented at various time points have been observed when adult zebrafish caudal fin partially amputated were exposed to 1% CoCl2. Regeneration is enhanced under hypoxia, with increased VEGF expression. To study the significance of VEGF signaling during wound healing and tissue regeneration, sunitinib well known inhibitor of VEGF receptor is used against CoCl2-induced caudal fin regeneration. Diminished fin length, lowering of blood vessel formation was documented using angioquant software, reduction in mRNA level of hypoxia inducible factors, VEGF and other pro-angiogenic genes such as VEGF, VEGF-R2, NRP1A, FGFR2, ANGPT1 were observed, while reduction in VEGF protein was demonstrated using western blot analysis. Genistein inhibitor of HIF-1 completely arrested regeneration, with suppression of VEGF highlighting the significance of hypoxia induced VEGF signaling during fin regeneration. Our results suggest that hypoxia through HIF-1 might lead to angiogenesis involving VEGF signaling during wound healing and this might throw light on therapeutic efficacy of cobalt chloride during regeneration.

molecular biology↗

bioRxiv2017

Histone H3 threonine 11 phosphorylation is catalyzed directly by the meiosis-specific kinase Mek1 and provides a molecular readout of Mek1 activity in vivo

Saccharomyces cerevisiae Mek1 is a CHK2/Rad53-family kinase that regulates meiotic recombination and progression upon its activation in response to DNA double-strand breaks (DSBs). The full catalog of direct Mek1 phosphorylation targets remains unknown. Here, we show that phosphorylation of histone H3 on threonine 11 (H3 T11ph) is induced by meiotic DSBs in S. cerevisiae and Schizosaccharomyces pombe. Molecular genetic experiments in S. cerevisiae confirmed that Mek1 is required for H3 T11ph and revealed that phosphorylation is rapidly reversed when Mek1 kinase is no longer active. Reconstituting histone phosphorylation in vitro with recombinant proteins demonstrated that Mek1 directly catalyzes H3 T11 phosphorylation. Mutating H3 T11 to nonphosphorylatable residues conferred no detectable defects in otherwise unperturbed meiosis, although the mutations modestly reduced spore viability in certain strains where Rad51 is used for strand exchange in place of Dmc1. H3 T11ph is therefore mostly dispensable for Mek1 function. However, H3 T11ph provides an excellent marker of ongoing Mek1 kinase activity in vivo. Anti-H3 T11ph chromatin immunoprecipitation followed by deep sequencing demonstrated that H3 T11ph was highly enriched at presumed sites of attachment of chromatin to chromosome axes, gave a more modest signal along chromatin loops, and was present at still lower levels immediately adjacent to DSB hotspots. These localization patterns closely tracked the distribution of Red1 and Hop1, axis proteins required for Mek1 activation. These findings provide insight into the spatial disposition of Mek1 kinase activity and the higher order organization of recombining meiotic chromosomes.\n\nbioRxiv version 2 (June 2017)One major experimental change was incorporated into the revised manuscript: We repeated the anti-H3 T11ph ChIP-seq experiment on larger scale, including two meiotic time points from each of two wild type cultures and one time point from a spo11-Y135F mutant culture. To facilitate comparison of different samples, we used meiotic S. pombe cells as a spike-in control for all samples for both anti-H3 and anti-H3 T11ph ChIP-seq. Most conclusions described in the first bioRxiv submission were confirmed, but the improved datasets allowed us to derive more detailed information in particular about H3 T11ph patterns around DSB sites.\n\nbioRxiv version 3 (October 2017)The following experimental changes were incorporated, along with more minor changes in response to reviewer comments: O_LIWe added previously unpublished ChIP-seq data for Red1 protein, generated by Masaru Ito and Kunihiro Ohta, who have been added as coauthors.\nC_LIO_LIWe repeated key experiments with the H3-T11V single point mutant. No conclusions were changed relative to prior experiments with the H3-S10, T11V mutant.\nC_LIO_LIWe repeated the analysis of spore viability in a dmc1 rad54-T132A background using a more appropriate isogenic control, and recapitulated the original conclusion that the H3-T11V mutation modestly decreases spore viability in this sensitized background.\nC_LI

molecular biology↗

bioRxiv2017

A simple centrifugation protocol leads to a 55-fold mitochondrial DNA enrichment and paves the way for future mitogenomic research

DNA (meta)barcoding is used to study biodiversity and is available for standardised assessments. However, it suffers from PCR bias, which can lead to the loss of specific taxa. PCR-free techniques such as shotgun metagenomics are therefore thought to be more suited for biodiversity assessments, but are currently limited by incomplete reference libraries.\n\nThe technique of mitogenome-skimming or mitogenomics, in which complete mitochondrial genomes are sequenced, is ideal to bridge the techniques of (meta)barcoding and metagenomics. However, without the enrichment of mitochondria, roughly 99 % of all sequencing reads are of non-mitochondrial origin and mostly useless for common applications, e.g. species identification.\n\nHere, we present a simple centrifugation protocol that leads to an average 140-fold enrichment of mitochondrial DNA. By sequencing six mock- communities - comprising the freshwater taxa Corbicula fluminea, Gammarus roeselii and Hydropsyche exocellata each - we recovered whole mitochondrial genomes of these species and the acanthocephalan endoparasite Pomphorhynchus laevis.\n\nThe presented protocol will greatly speed up building reference libraries for whole mitochondrial genomes, as dozens of species could be sequenced on a single MiSeq run. Subsequently, it will also allow biodiversity assessments using mitogenomics at greatly reduced costs in comparison to mitogenomic approaches without prior enrichment for mitochondria.

molecular biology↗

bioRxiv2017

Antibacterial nucleoside-analog inhibitor of bacterial RNA polymerase: pseudouridimycin

There is an urgent need for new antibacterial drugs effective against bacterial pathogens resistant to current drugs1-2. Nucleoside-analog inhibitors (NAIs) of viral nucleotide polymerases have had transformative impact in treatment of HIV3 and HCV4. NAIs of bacterial RNA polymerase (RNAP) potentially could have major impact on treatment of bacterial infection, particularly because functional constraints on substitution of RNAP nucleoside triphosphate (NTP) binding sites4-5 could limit resistance emergence4-5. Here we report the discovery, from microbial extract screening, of an NAI that inhibits bacterial RNAP and exhibits antibacterial activity against a broad spectrum of drug-sensitive and drug-resistant bacterial pathogens: pseudouridimycin (PUM). PUM is a novel microbial natural product consisting of a formamidinylated, N-hydroxylated Gly-Gln dipeptide conjugated to 6'-amino-pseudouridine. PUM potently and selectively inhibits bacterial RNAP in vitro, potently and selectively inhibits bacterial growth in culture, and potently clears infection in a mouse model of Streptococcus pyogenes peritonitis. PUM inhibits RNAP through a binding site on RNAP (the \"i+1\" NTP binding site) and mechanism (competition with UTP for occupancy of the \"i+1\" NTP binding site) that differ from those of the RNAP inhibitor and current antibacterial drug rifampin (Rif). PUM exhibits additive antibacterial activity when co-administered with Rif, exhibits no cross-resistance with Rif, and exhibits a spontaneous resistance rate an order-of-magnitude lower than that of Rif. The results provide the first example of a selective NAI of bacterial RNAP, provide an advanced lead compound for antibacterial drug development, and provide structural information and synthetic routes that enable lead optimization for antibacterial drug development.

molecular biology↗

bioRxiv2017

Taenia solium TAF6 and TAF9 binds to a putative Downstream Promoter Element present in TsTBP1 gene core promoter

We have cloned and characterized the gene encoding to Taenia solium TATA binding protein 1 (TsTBP1). It spans 1481 bp and its coding region is interrupted by four introns that possess the consensus donor/acceptor sequences. It produces a protein of 238 amino acids residues, which presented all the classical motives of the TBP1. On the core promoter region we identified putative binding sites for NF1, AP-1, YY1, TAF1/TAF2 and TAF6/TAF9, and the TSS that corresponds to an A+1. Southern and Northern blot analysis showed that TsTBP1 is encoded by a single gene, which produce a messenger of about 1.1 kbp with a higher differential expression in adult than the larval stage. Moreover, two putative TATA boxes were identified at -97 and -69 bp (relative to the TSS), likewise a Downstream Promoter Element (DPE) was located at +27 to +31 bp. EMSA experiments did not show any component of cysticerci nuclear extracts bound to the putative TATA-box elements; in contrast TAF6 and TAF9 bind to the DPE, showing that TsTBP1 is a TATA-less gen. On the other hand, TAF6 and TAF9 were localized on the nucleus from cells of T. crassiceps cysticerci bladder walls. By using the amino acid sequences of T. solium TAF6 (TsTAF6) and TAF9 (TsTAF9), we constructed a molecular model showing interaction between DPE-TsTAF6 and TsTAF6-TsTAF9. Finally, an in silico analysis of the core promoters of Taeniidae family genes showed that TATA-box and DPE are homologous to the mammalian elements, but not so the Inr. The low identity between TAF9 from cestodes and mammals open the possibility to use it as target to interrupt or modulate the transcription of TATA-box less genes in cestodes as a novel therapeutic strategy.\n\nAuthor summaryNeurocysticercosis still being a health problem in developing countries. Mexico reports a prevalence of 2.5% in the total patients from National Institute of Neurology and Neurosurgery. Several efforts have been made in different fields to study Taenia solium, and although the Genome Project has been published and released the genomic sequence of this parasite; the transcriptional mechanisms this organism remains unexplored. We isolated and characterized Taenia solium TATA-Binding Protein 1 (TsTBP1) gene and TBP-associated factor 6 and 9 cDNAs (TsTAF6 and TsTAF9, respectively). Our principal findings are: 1.- Identification of cis and trans elements in the core promoter of TsTBP1 gene. 2.- The TsTBP1 gene is a TATA-less promoter. 3.- Cloning of cDNAs to TsTAF6 and TsTAF9, 4.- Identification of a DPE in the core promoter of TsTBP1 gene, which interacts with TAF6 and TAF9, 5.- Construction of a molecular model that shows the interaction between DPE, TAF6, and TAF9; and 6.- A proposal of consensus sequences for TATA-box, Inr and DPE presented on Taeniidae family core promoters.

molecular biology↗

bioRxiv2017

Inhibition of DNA2 nuclease as a therapeutic strategy targeting replication stress in cancer cells.

Replication stress is a characteristic feature of cancer cells, which is resulted from sustained proliferative signaling induced by activation of oncogenes or loss of tumor suppressors. In cancer cells, oncogene-induced replication stress manifests as replication-associated lesions, predominantly double-strand DNA breaks (DSBs). An essential mechanism utilized by cells to repair replication-associated DSBs is homologous recombination (HR). In order to overcome replication stress and survive, cancer cells often require enhanced HR repair capacity. Therefore, the key link between HR repair and cellular tolerance to replication-associated DSBs provides us with a mechanistic rationale for exploiting synthetic lethality between HR repair inhibition and replication stress. Our studies showed that DNA2 nuclease is an evolutionarily conserved essential component of HR repair machinery. Here we demonstrate that DNA2 is indeed overexpressed in pancreatic cancers, one of the deadliest and more aggressive forms of human cancers, where mutations in the KRAS are present in 90%-95% of cases. In addition, depletion of DNA2 significantly reduces pancreatic cancer cell survival and xenograft tumor growth, suggesting the therapeutic potential of DNA2 inhibition. Finally, we develop a robust high-throughput biochemistry assay to screen for inhibitors of the DNA2 nuclease activity. The top inhibitors were shown to be efficacious against both yeast Dna2 and human DNA2. Treatment of cancer cells with DNA2 inhibitors recapitulates phenotypes observed upon DNA2 depletion, including decreased DNA end resection and attenuation of HR repair. Similar to genetic ablation of DNA2, chemical inhibition of DNA2 selectively attenuates the growth of various cancer cells with oncogene-induced replication stress. Taken together, our findings open a new avenue to develop a new class of anti-cancer drugs by targeting druggable nuclease DNA2. We 4, 16. In propose DNA2 inhibition as new strategy in cancer therapy by targeting replication stress, a molecular property of cancer cells that is acquired as a result of oncogene activation instead of targeting undruggable oncoprotein itself such as KRAS.

molecular biology↗

bioRxiv2017

Inferring intracellular signal transduction circuitry from molecular perturbation experiments

The development of network inference methodologies that accurately predict connectivity in dysregulated pathways may enable the rational selection of patient therapies. Accurately inferring an intracellular network from data remains a very challenging problem in molecular systems biology. Living cells integrate extremely robust circuits that exhibit significant heterogeneity, but still respond to external stimuli in predictable ways. This phenomenon allows us to introduce a network inference methodology that integrates measurements of protein activation from perturbation experiments. The methodology relies on logic-based networks to provide a predictive approximation of the transfer of signals in a network. The approach presented was validated in silico with a set of test networks and applied to investigate the epidermal growth factor receptor signaling of a breast epithelial cell line, MFC10A. In our analysis, we predict the potential signaling circuitry most likely responsible for the experimental readouts of several proteins in the mitogen activated protein kinase and phosphatidylinositol-3 kinase pathways. The approach can also be used to identify additional necessary perturbation experiments to distinguish between a set of possible candidate networks.

systems biology↗

bioRxiv2017

The regulation of tDNA transcription during the directed differentiation of stem cells.

The human genome consists of 625 tDNA copies that encode 51 distinct isoacceptor families. Recent studies demonstrated that changes in chromatin structure during cellular differentiation can alter the expression of these tDNA. However, the mechanism by which tDNA can be differentially regulated remains unclear. Here we used the directed differentiation of pluripotent human embryonic stem cells (hESCs) towards the endoderm lineage as a model system to study the developmental regulation of individual tDNA. We demonstrated a significant change in the Pol III occupancy at 49 tDNA (22 reduced and 27 increased). The regulation of tDNA did not correlate with changes in TFIIIB or TFIIIC occupancy, H3K4me3, or H3K27me3 levels. However, tDNA that had an increase in Pol III binding were preferentially found within strong CTCF-COHESIN chromatin loops. The knockdown of either Ctcf or Rad21 in mouse tail tip fibroblasts had similar effects on changes in tRNA levels. We identified 7 isoacceptors that were differentially expressed during the directed differentiation of hESCs. The open reading frames of the ribosomal protein genes, which are translationally repressed during hESC differentiation, are enriched for codons that are decoded by these downregulated isoacceptors. Thus, translation efficiency during cellular differentiation may be affected by changes in tDNA regulation.

molecular biology↗

bioRxiv2017

Poly(A)-ClickSeq: click-chemistry for next-generation 3´-end sequencing without RNA enrichment or fragmentation

The recent emergence of alternative polyadenylation (APA) as an engine driving transcriptomic diversity has stimulated the development of sequencing methodologies designed to assess genome-wide polyadenylation events. The goal of these approaches is to enrich, partition, capture, and ultimately sequence poly(A) site junctions. However, these methods often require poly(A) enrichment, 3{acute} linker ligation steps, and RNA fragmentation, which can necessitate higher levels of starting RNA, increase experimental error, and potentially introduce bias. We recently reported a click-chemistry based method for generating RNAseq libraries called \"ClickSeq\". Here, we adapt this method to direct the cDNA synthesis specifically toward the 3{acute} UTR/poly(A) tail junction of cellular RNA. With this novel approach, we demonstrate sensitive and specific enrichment for poly(A) site junctions without the need for complex sample preparation, fragmentation or purification. Poly(A)-ClickSeq (PAC-seq) is therefore a simple procedure that generates high-quality RNA-seq poly(A) libraries. As a proof-of-principle, we utilized PAC-seq to explore the poly(A) landscape of both human and Drosophila cells in culture and observed outstanding overlap with existing poly(A) databases and also identified previously unannotated poly(A) sites. Moreover, we utilize PAC-seq to quantify and analyze APA events regulated by CFIm25 illustrating how this technology can be harnessed to identify alternatively polyadenylated RNA.

molecular biology↗

bioRxiv2017

Gal4 activation domain 9aaTAD could be inactivated by adjacent mini-inhibitory domain and reactivated by distal re-activation domain

The characterisation of the activation domains started three decades ago with Gcn4 and Gal4 activators. The amorphous character of the activation domains strongly hindered their definition. Moreover, during the attempts to localise the Gal4 activation domain, the artificial peptides, an unintended consequence of cloning, were responsible for artificial transcriptional activity of the several Gal4 constructs. These artefacts produced enormous experimental bias and misconception. The presence of inhibitory domains in some Gal4 constructs made the misperception even worse. Previously, we reported that the nine amino acid transactivation domain, 9aaTAD, is the exclusive activation domain in the Gal4 protein. The activation domain 9aaTAD could be identified in Gal4 paralogs Oaf1, Pip2, Pdr1, Pdr3 and other activators p53, E2A and MLL. Surprisingly, the activation domain 9aaTAD was reported as misconception for Gal4 activator. Here we demonstrated that small region of 10 amino acids adjacent to the Gal4 activation domain 9aaTAD is an inhibitory domain, which the authors included in their constructs. Moreover, we identified Gal4 region, which was able to the reverse the inhibitory effect. The 9aaTAD re-activation domain was localized to the 13 amino acid long region. In this report we clarified the numerous confusions and rebutted supposed 9aaTAD misconception.\n\nSummaryThe activation domain 9aaTAD has decisive function in Gal4 activation. Gal4 activation domain 9aaTAD could be inhibited by adjacent region of 10 amino acids. The inhibited Gal4 activation domain 9aaTAD could be reactivated by 13 amino acid long Gal4 region. The activation domains 9aaTAD could be identified by our 9aaTAD prediction algorithm, especially in the Gal4 family.

molecular biology↗

bioRxiv2017

Observation of histone nuclear import in living cells: implications in the processing of newly synthesised H3.1 & H4

Highlights- Small-molecule-gated tether-and-release system for rapid pulse-chase of nuclear proteins\n- Tracking nuclear import of histone H3.1 and H4 and their incorporation at sites of active replication\n- Tethered H3.1 and H4 are monomeric and do not associate with ASF1, NASP, RbAp46 or HAT1 in the cytosol\n- Importin-{beta} proteins as cytosolic binders of monomeric histones\n\n\nSummaryWe present here a cytosolic tether-and-release system to study the import and dynamics of newly synthesised nuclear proteins. Release is gated by rapamycin-induced recruitment and activation of a viral protease, with cleavage of a peptide linker releasing the tethered cargo. We use this system to investigate nucleo-cytoplasmic divisions in the histone H3.1 & H4 deposition pathway, revealing that, contrary to previous analyses, H3.1 and H4 are predominantly monomeric in the cytosol, and only associate with the core histone chaperoning machinery after translocation to the nucleus. Whilst we do not detect interaction with known H3-H4 chaperones in the cytosol we do detect interaction with a number of importin-{beta} proteins, that may serve a dual import and chaperoning function, preventing aggregation of histones until they are handed-off to the core histone chaperoning machinery in the nucleus.

molecular biology↗

bioRxiv2017

Subdomains of the Pol V largest subunit CTD mediate locus-specific RNA-directed DNA methylation

Plant multisubunit RNA Polymerase V transcription recruits Argonaute siRNA complexes that specify sites of RNA-directed DNA methylation (RdDM) for gene silencing. Pol V's largest subunit, NRPE1, evolved from the largest subunit of Pol II but has a distinctive carboxyl-terminal domain (CTD). We show that the Pol V CTD is dispensable for catalytic activity in vitro, yet essential in vivo. One CTD subdomain (DeCL), is required for Pol V function at virtually all loci. other CTD subdomains have locusspecific effects. In a yeast two-hybrid screen, the 3'->5' exoribonuclease, RRP6L1 was identified as an interactor with the DeCL subdomain and DeCL and glutamine-serine-rich (QS) subdomains, located downstream from an Argonaute-binding repeat subdomain. Experimental evidence indicates that RRP6L1 trims the 3 ends of Pol V transcripts sliced by ARGONAUTE 4 (AGO4), suggesting a model whereby the CTD enables the spatial and temporal coordination of AGO4 and RRP6L1 RNA processing activities.

molecular biology↗

bioRxiv2017

Random Sequences Rapidly Evolve Into De Novo Promoters

How do new promoters evolve? To follow evolution of de novo promoters, we put various random sequences upstream to the lac operon in Escherichia coli and evolved the cells in the presence of lactose. We found that a typical random sequence of ~100 bases requires only one mutation in order to enable growth on lactose by increasing resemblance to the canonical promoter motifs. We further found that ~10% of random sequences could serve as active promoters even without any period of evolutionary adaptation. Such a short mutational distance from a random sequence to an active promoter may improve evolvability yet may also lead to undesirable accidental expression. We found that across the E. coli genome accidental expression is minimized by avoiding codon combinations that resemble promoter motifs. Our results suggest that the promoter recognition machinery has been tuned to allow high accessibility to new promoters, and similar findings might also be observed in higher organisms or in other motif recognition machineries, like transcription factor binding sites or protein-protein interactions.

molecular biology↗

bioRxiv2017

The mechanism of sphingolipid processing revealed by a GALC-SapA complex structure

Sphingolipids are essential components of cellular membranes and defects in their synthesis or degradation cause severe human diseases. The efficient degradation of sphingolipids in the lysosome requires lipid-binding saposin proteins and hydrolytic enzymes. The glycosphingolipid galactocerebroside is the primary lipid component of the myelin sheath and is degraded by the hydrolase {beta}-galactocerebrosidase (GALC). This enzyme requires the saposin SapA for lipid processing and defects in either of these proteins causes a severe neurodegenerative disorder, Krabbe disease. Here we present the structure of a glycosphingolipid-processing complex, revealing how SapA and GALC form a heterotetramer with an open channel connecting the enzyme active site to the SapA hydrophobic cavity. This structure defines how a soluble hydrolase can cleave the polar glycosyl headgroups of these essential lipids from their hydrophobic ceramide tails. Furthermore, the molecular details of this interaction reveal how specificity of saposin binding to hydrolases is encoded.

molecular biology↗

bioRxiv2017

BRD4 inhibitors block telomere elongation

Cancer cells maintain telomere length equilibrium to avoid senescence and apoptosis induced by short telomeres, which are triggered by the DNA damage response. Limiting the potential for telomere maintenance in cancer cells has been long been proposed as a therapeutic target. Using an unbiased shRNA screen targeting known kinases, we identified bromodomain 4 (BRD4) as a telomere length regulator. Four independent BRD4 inhibitors blocked telomere elongation, in a dose dependent manner, in mouse cells overexpressing telomerase. Long-term treatment with BRD4 inhibitors caused telomere shortening in both mouse and human cells, suggesting BRD4 plays a role in telomere maintenance in vivo. Telomerase enzymatic activity was not directly affected by BRD4 inhibition. BRD4 is in clinical trials for a number of cancers, but its effects on telomere maintenance have not been previously reported investigated.

molecular biology↗

bioRxiv2017

Evolution of ribosomal internal transcribed spacers in Deuterostomia

Sequences of ribosomal internal transcribed spacers (ITSs) are of great importance to molecular phylogenetics and DNA barcoding, but remain unstudied in some large taxa of Deuterostomia. We have analyzed complete ITS1 and ITS2 sequences in 62 species from 16 Deuterostomia classes, with ITSs sequences in 24 species from 11 classes initially obtained using unannotated contigs and raw read sequences. A general tendency for both ITS length and GC-content increase from interior to superior Deuterostomia taxa, a uniform GC-content in both ITSs within the same species, thymine content decrease in sense DNA sequences of both ITSs are shown. A possible role of GC-based gene conversion in Deuterostomia ITSs evolutionary changes is hypothesized. The first example of non-LTR retrotransposon insertion into ITS sequence in Deuterostomia is described in turtle Geochelone nigra. The roles of mobile genetic element insertions in the evolution of ITS sequences in some Sauropsida taxa are discussed as well.

molecular biology↗

bioRxiv2017

Histone gene replacement reveals a post-transcriptional role for H3K36 in maintaining metazoan transcriptome fidelity

Histone H3 lysine 36 methylation (H3K36me) is thought to participate in a host of co-transcriptional regulatory events. To study the function of this residue independent from the enzymes that modify it, we used a \"histone replacement\" system in Drosophila to generate a non-modifiable H3K36 lysine-to-arginine (H3K36R) mutant. We observed global dysregulation of mRNA levels in H3K36R animals that correlates with the incidence of H3K36me3. Similar to previous studies, we found that mutation of H3K36 also resulted in H4 hyperacetylation. However, neither cryptic transcription initiation, nor alternative pre-mRNA splicing, contributed to the observed changes in expression, in contrast with previously reported roles for H3K36me. Interestingly, knockdown of the RNA surveillance nuclease, Xrn1, and members of the CCR4-Not deadenylase complex, restored mRNA levels for a class of downregulated, H3K36me3-rich genes. We propose a posttranscriptional role for modification of replication-dependent H3K36 in the control of metazoan gene expression.\n\nImpact StatementPost-translational modification of histone H3K36 is neither required to suppress cryptic transcription initiation nor to include alternative exons in Drosophila; instead it promotes expression of active genes by stimulating polyadenylation.

molecular biology↗

bioRxiv2017

DNA sequence homology induces cytosine-to-thymine mutation by a heterochromatin-related pathway

In the genomes of many eukaryotes, including mammals, highly repetitive DNA is normally associated with histone H3 lysine-9 di-/trimethylation (H3K9me2/3) and C5-cytosine methylation (5mC) in the context of heterochromatin. In the fungus Neurospora crassa, H3K9me3 and 5mC are catalyzed, respectively, by a conserved SUV39 histone methylase DIM-5 and a DNMT1-like cytosine methylase DIM-2. Here we show that DIM-2 can also mediate cytosine-to-thymine mutation of repetitive DNA during the pre-meiotic process known as Repeat-Induced Point mutation (RIP) in N. crassa. We further show that DIM-2-dependent RIP requires DIM-5, HP1, and other heterochromatin factors, implying the involvement of a repeat-induced heterochromatin-related process. Our previous findings suggest that the mechanism of homologous repeat recognition for RIP involves direct pairwise interactions between co-aligned double-stranded (ds) DNA segments. Our current findings therefore raise the possibility that such pairing interactions may occur not only in pre-meiotic but also in vegetative cells, where they may direct heterochromatin assembly on repetitive DNA. In accord with this possibility, we find that, in vegetative cells of N. crassa, our model repeat array comprising only four 674-bp sequence copies can trigger a low level of DIM-5-dependent 5meC. We thus propose that homologous dsDNA/dsDNA interactions between a small number of repeat copies can nucleate a transient state of heterochromatin and that, on longer repeat arrays, such interactions lead to the formation of stable heterochromatin. Since the number of possible pairwise dsDNA/dsDNA interactions will scale non-linearly with the number of repeats, this mechanism provides an attractive way of creating the extended domains of constitutive heterochromatin found in pericentromeric and subtelomeric regions.

molecular biology↗