Histone H2B.8 compacts flowering plant sperm through chromatin phase separation.

Sperm chromatin is typically transformed by protamines into a compact and transcriptionally inactive state1,2. Sperm cells of flowering plants lack protamines, yet they have small, transcriptionally active nuclei with chromatin condensed through an unknown mechanism3,4. Here we show that a histone variant, H2B.8, mediates sperm chromatin and nuclear condensation in Arabidopsis thaliana. Loss of H2B.8 causes enlarged sperm nuclei with dispersed chromatin, whereas ectopic expression in somatic cells produces smaller nuclei with aggregated chromatin. This result demonstrates that H2B.8 is sufficient for chromatin condensation. H2B.8 aggregates transcriptionally inactive AT-rich chromatin into phase-separated condensates, which facilitates nuclear compaction without reducing transcription. Reciprocal crosses show that mutation of h2b.8 reduces male transmission, which suggests that H2B.8-mediated sperm compaction is important for fertility. Altogether, our results reveal a new mechanism of nuclear compaction through global aggregation of unexpressed chromatin. We propose that H2B.8 is an evolutionary innovation of flowering plants that achieves nuclear condensation compatible with active transcription.

Molecular basis of CENP-C association with the CENP-A nucleosome at yeast centromeres.

Histone CENP-A-containing nucleosomes play an important role in nucleating kinetochores at centromeres for chromosome segregation. However, the molecular mechanisms by which CENP-A nucleosomes engage with kinetochore proteins are not well understood. Here, we report the finding of a new function for the budding yeast Cse4/CENP-A histone-fold domain interacting with inner kinetochore protein Mif2/CENP-C. Strikingly, we also discovered that AT-rich centromere DNA has an important role for Mif2 recruitment. Mif2 contacts one side of the nucleosome dyad, engaging with both Cse4 residues and AT-rich nucleosomal DNA. Both interactions are directed by a contiguous DNA- and histone-binding domain (DHBD) harboring the conserved CENP-C motif, an AT hook, and RK clusters (clusters enriched for arginine-lysine residues). Human CENP-C has two related DHBDs that bind preferentially to DNA sequences of higher AT content. Our findings suggest that a DNA composition-based mechanism together with residues characteristic for the CENP-A histone variant contribute to the specification of centromere identity.

Understanding the paradoxical mechanical response of in-phase A-tracts at different force regimes.

A-tracts are A:T rich DNA sequences that exhibit unique structural and mechanical properties associated with several functions in vivo. The crystallographic structure of A-tracts has been well characterized. However, the mechanical properties of these sequences is controversial and their response to force remains unexplored. Here, we rationalize the mechanical properties of in-phase A-tracts present in the Caenorhabditis elegans genome over a wide range of external forces, using single-molecule experiments and theoretical polymer models. Atomic Force Microscopy imaging shows that A-tracts induce long-range (∼200 nm) bending, which originates from an intrinsically bent structure rather than from larger bending flexibility. These data are well described with a theoretical model based on the worm-like chain model that includes intrinsic bending. Magnetic tweezers experiments show that the mechanical response of A-tracts and arbitrary DNA sequences have a similar dependence with monovalent salt supporting that the observed A-tract bend is intrinsic to the sequence. Optical tweezers experiments reveal a high stretch modulus of the A-tract sequences in the enthalpic regime. Our work rationalizes the complex multiscale flexibility of A-tracts, providing a physical basis for the versatile character of these sequences inside the cell.

A non-canonical promoter element drives spurious transcription of horizontally acquired bacterial genes.

RNA polymerases initiate transcription at DNA sequences called promoters. In bacteria, the best conserved promoter feature is the AT-rich -10 element; a sequence essential for DNA unwinding. Further elements, and gene regulatory proteins, are needed to recruit RNA polymerase to the -10 sequence. Hence, -10 elements cannot function in isolation. Many horizontally acquired genes also have a high AT-content. Consequently, sequences that resemble the -10 element occur frequently. As a result, foreign genes are predisposed to spurious transcription. However, it is not clear how RNA polymerase initially recognizes such sequences. Here, we identify a non-canonical promoter element that plays a key role. The sequence, itself a short AT-tract, resides 5 base pairs upstream of otherwise cryptic -10 elements. The AT-tract alters DNA conformation and enhances contacts between the DNA backbone and RNA polymerase.

The fungal CCAAT-binding complex and HapX display highly variable but evolutionary conserved synergetic promoter-specific DNA recognition.

To sustain iron homeostasis, microorganisms have evolved fine-tuned mechanisms for uptake, storage and detoxification of the essential metal iron. In the human pathogen Aspergillus fumigatus, the fungal-specific bZIP-type transcription factor HapX coordinates adaption to both iron starvation and iron excess and is thereby crucial for virulence. Previous studies indicated that a HapX homodimer interacts with the CCAAT-binding complex (CBC) to cooperatively bind bipartite DNA motifs; however, the mode of HapX-DNA recognition had not been resolved. Here, combination of in vivo (genetics and ChIP-seq), in vitro (surface plasmon resonance) and phylogenetic analyses identified an astonishing plasticity of CBC:HapX:DNA interaction. DNA motifs recognized by the CBC:HapX protein complex comprise a bipartite DNA binding site 5'-CSAATN12RWT-3' and an additional 5'-TKAN-3' motif positioned 11-23 bp downstream of the CCAAT motif, i.e. occasionally overlapping the 3'-end of the bipartite binding site. Phylogenetic comparison taking advantage of 20 resolved Aspergillus species genomes revealed that DNA recognition by the CBC:HapX complex shows promoter-specific cross-species conservation rather than regulon-specific conservation. Moreover, we show that CBC:HapX interaction is absolutely required for all known functions of HapX. The plasticity of the CBC:HapX:DNA interaction permits fine tuning of CBC:HapX binding specificities that could support adaptation of pathogens to their host niches.

Dynamics of DNA replication in a eukaryotic cell.

Each genomic locus in a eukaryotic cell has a distinct average time of replication during S phase that depends on the spatial and temporal pattern of replication initiation events. Replication timing can affect genomic integrity because late replication is associated with an increased mutation rate. For most eukaryotes, the features of the genome that specify the location and timing of initiation events are unknown. To investigate these features for the fission yeast, Schizosaccharomyces pombe, we developed an integrative model to analyze large single-molecule and global genomic datasets. The model provides an accurate description of the complex dynamics of S. pombe DNA replication at high resolution. We present evidence that there are many more potential initiation sites in the S. pombe genome than previously identified and that the distribution of these sites is primarily determined by two factors: the sequence preferences of the origin recognition complex (ORC), and the interference of transcription with the assembly or stability of prereplication complexes (pre-RCs). We suggest that in addition to directly interfering with initiation, transcription has driven the evolution of the binding properties of ORC in S. pombe and other eukaryotic species to target pre-RC assembly to regions of the genome that are less likely to be transcribed.

GAPDH as a model non-canonical AU-rich RNA binding protein.

Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) plays a key role in glycolysis but is also known for its involvement in a myriad of extra-glycolytic functions. While GAPDH is not the only enzyme with established moonlighting roles, it shows great diversity in terms of its functions, cellular localizations, protein partners, and post-translational modifications. This review focuses on GAPDH's role as a non-canonical RNA binding protein to regulate the stability and translation of cellular mRNAs. Despite the clear involvement of GAPDH in gene expression regulation, how and where GAPDH binds to its RNA targets is still unknown. In addition, the mechanism by which GAPDH switches among its various cellular functions is also unknown. This review will summarize our current understanding of GAPDH-mediated regulation of RNA function.

Downstream sequence-dependent RNA cleavage and pausing by RNA polymerase I.

The sequence of the DNA template has long been thought to influence the rate of transcription by DNA-dependent RNA polymerases, but the influence of DNA sequence on transcription elongation properties of eukaryotic RNA polymerase I (Pol I) from Saccharomyces cerevisiae has not been defined. In this study, we observe changes in dinucleotide production, transcription elongation complex stability, and Pol I pausing in vitro in response to downstream DNA. In vitro studies demonstrate that AT-rich downstream DNA enhances pausing by Pol I and inhibits Pol I nucleolytic cleavage activity. Analysis of Pol I native elongating transcript sequencing data in Saccharomyces cerevisiae suggests that these downstream sequence elements influence Pol I in vivo Native elongating transcript sequencing studies reveal that Pol I occupancy increases as downstream AT content increases and decreases as downstream GC content increases. Collectively, these data demonstrate that the downstream DNA sequence directly impacts the kinetics of transcription elongation prior to the sequence entering the active site of Pol I both in vivo and in vitro.

The genomic architecture and molecular evolution of ant odorant receptors.

The massive expansions of odorant receptor (OR) genes in ant genomes are notable examples of rapid genome evolution and adaptive gene duplication. However, the molecular mechanisms leading to gene family expansion remain poorly understood, partly because available ant genomes are fragmentary. Here, we present a highly contiguous, chromosome-level assembly of the clonal raider ant genome, revealing the largest known OR repertoire in an insect. While most ant ORs originate via local tandem duplication, we also observe several cases of dispersed duplication followed by tandem duplication in the most rapidly evolving OR clades. We found that areas of unusually high transposable element density (TE islands) were depauperate in ORs in the clonal raider ant, and found no evidence for retrotransposition of ORs. However, OR loci were enriched for transposons relative to the genome as a whole, potentially facilitating tandem duplication by unequal crossing over. We also found that ant OR genes are highly AT-rich compared to other genes. In contrast, in flies, OR genes are dispersed and largely isolated within the genome, and we find that fly ORs are not AT-rich. The genomic architecture and composition of ant ORs thus show convergence with the unrelated vertebrate ORs rather than the related fly ORs. This might be related to the greater gene numbers and/or potential similarities in gene regulation between ants and vertebrates as compared to flies.

Plastomes in the holoparasitic family Balanophoraceae: Extremely high AT content, severe gene content reduction, and two independent genetic code changes.

The transition to a heterotrophic lifestyle in angiosperms is characterized by convergent evolutionary changes. Plastid genome remodeling includes dramatic functional and physical reductions with the highest degrees observed in fully heterotrophic plants. Genes related to photosynthesis are generally absent or pseudogenized, while a few genes related to other metabolic processes that take place within the plastid are almost invariably maintained. The family Balanophoraceae consists of root holoparasites that present reduced plastid genomes with an extraordinarily elevated AT content and the single genetic code change ever documented in land plant plastomes (the stop codon TAG now codes for tryptophan). Here, we studied the plastomes of Lophophytum leandri and Ombrophytum subterraneum (Balanophoraceae) that showed the remarkable absence of the gene trnE, a highly biased nucleotide composition, and an independent genetic code change (the standard stop codon TGA codes for tryptophan). This is the second genetic code change identified in land plant plastomes. Analysis of the transcriptome of Lophophytum indicated that the entire C5 pathway typical of plants is conserved despite the lack of trnE in its plastome. A hypothetical model of plastome evolution in the Balanophoraceae is presented.

Integrase-mediated spacer acquisition during CRISPR-Cas adaptive immunity.

Bacteria and archaea insert spacer sequences acquired from foreign DNAs into CRISPR loci to generate immunological memory. The Escherichia coli Cas1-Cas2 complex mediates spacer acquisition in vivo, but the molecular mechanism of this process is unknown. Here we show that the purified Cas1-Cas2 complex integrates oligonucleotide DNA substrates into acceptor DNA to yield products similar to those generated by retroviral integrases and transposases. Cas1 is the catalytic subunit and Cas2 substantially increases integration activity. Protospacer DNA with free 3'-OH ends and supercoiled target DNA are required, and integration occurs preferentially at the ends of CRISPR repeats and at sequences adjacent to cruciform structures abutting AT-rich regions, similar to the CRISPR leader sequence. Our results demonstrate the Cas1-Cas2 complex to be the minimal machinery that catalyses spacer DNA acquisition and explain the significance of CRISPR repeats in providing sequence and structural specificity for Cas1-Cas2-mediated adaptive immunity.

Repeat elements organise 3D genome structure and mediate transcription in the filamentous fungus Epichloë festucae.

Structural features of genomes, including the three-dimensional arrangement of DNA in the nucleus, are increasingly seen as key contributors to the regulation of gene expression. However, studies on how genome structure and nuclear organisation influence transcription have so far been limited to a handful of model species. This narrow focus limits our ability to draw general conclusions about the ways in which three-dimensional structures are encoded, and to integrate information from three-dimensional data to address a broader gamut of biological questions. Here, we generate a complete and gapless genome sequence for the filamentous fungus, Epichloë festucae. We use Hi-C data to examine the three-dimensional organisation of the genome, and RNA-seq data to investigate how Epichloë genome structure contributes to the suite of transcriptional changes needed to maintain symbiotic relationships with the grass host. Our results reveal a genome in which very repeat-rich blocks of DNA with discrete boundaries are interspersed by gene-rich sequences that are almost repeat-free. In contrast to other species reported to date, the three-dimensional structure of the genome is anchored by these repeat blocks, which act to isolate transcription in neighbouring gene-rich regions. Genes that are differentially expressed in planta are enriched near the boundaries of these repeat-rich blocks, suggesting that their three-dimensional orientation partly encodes and regulates the symbiotic relationship formed by this organism.

Evidence for convergent evolution of SINE-directed Staufen-mediated mRNA decay.

Primate-specific Alu short interspersed elements (SINEs) as well as rodent-specific B and ID (B/ID) SINEs can promote Staufen-mediated decay (SMD) when present in mRNA 3'-untranslated regions (3'-UTRs). The transposable nature of SINEs, their presence in long noncoding RNAs, their interactions with Staufen, and their rapid divergence in different evolutionary lineages suggest they could have generated substantial modification of posttranscriptional gene-control networks during mammalian evolution. Some of the variation in SMD regulation produced by SINE insertion might have had a similar regulatory effect in separate mammalian lineages, leading to parallel evolution of the Staufen network by independent expansion of lineage-specific SINEs. To explore this possibility, we searched for orthologous gene pairs, each carrying a species-specific 3'-UTR SINE and each regulated by SMD, by measuring changes in mRNA abundance after individual depletion of two SMD factors, Staufen1 (STAU1) and UPF1, in both human and mouse myoblasts. We identified and confirmed orthologous gene pairs with 3'-UTR SINEs that independently function in SMD control of myoblast metabolism. Expanding to other species, we demonstrated that SINE-directed SMD likely emerged in both primate and rodent lineages >20-25 million years ago. Our work reveals a mechanism for the convergent evolution of posttranscriptional gene regulatory networks in mammals by species-specific SINE transposition and SMD.

BLM prevents instability of structure-forming DNA sequences at common fragile sites.

Genome instability often arises at common fragile sites (CFSs) leading to cancer-associated chromosomal rearrangements. However, the underlying mechanisms of how CFS protection is achieved is not well understood. We demonstrate that BLM plays an important role in the maintenance of genome stability of structure-forming AT-rich sequences derived from CFSs (CFS-AT). BLM deficiency leads to increased DSB formation and hyper mitotic recombination at CFS-AT and induces instability of the plasmids containing CFS-AT. We further showed that BLM is required for suppression of CFS breakage upon oncogene expression. Both helicase activity and ATR-mediated phosphorylation of BLM are important for preventing genetic instability at CFS-AT sequences. Furthermore, the role of BLM in protecting CFS-AT is not epistatic to that of FANCM, a translocase that is involved in preserving CFS stability. Loss of BLM helicase activity leads to drastic decrease of cell viability in FANCM deficient cells. We propose that BLM and FANCM utilize different mechanisms to remove DNA secondary structures forming at CFS-AT on replication forks, thereby preventing DSB formation and maintaining CFS stability.

Complete mitochondrial genome of Spilosoma lubricipedum (Noctuoidea: Erebidae) and implications for phylogeny of noctuid insects.

Mitochondrial genomes (mitogenomes) have been widely used for studies on phylogenetic relationships and molecular evolutionary biology. Here, the complete mitogenome sequence of Spilosoma lubricipedum (Noctuoidea: Erebidae: Arctiinae) was determined (total length 15,375 bp) and phylogenetic analyses S. lubricipedum were inferred from available noctuid sequence data. The mitogenome of S. lubricipedum was found to be highly A + T-biased (81.39%) and exhibited negative AT- and GC-skews. All 13 protein-coding genes (PCGs) were initiated by ATN codons, except for cox1 with CGA. All tRNAs exhibited typical clover-leaf secondary structures, except for trnS1. The gene order of the S. lubricipedum mitogenome was trnM-trnI-trnQ-nad2. The A + T-rich region of S. lubricipedum contained several conservative features common to noctuid insects. Phylogenetic analysis within Noctuoidea was carried out based on mitochondrial data. Results showed that S. lubricipedum belonged to Erebidae and the Noctuoidea insects could be divided into five well-supported families (Notodontidae + (Erebidae + (Nolidae + (Euteliidae + Noctuidae)))).

The complete mitochondrial genome of Calappa bilineata: The first representative from the family Calappidae and its phylogenetic position within Brachyura.

In this study, we determined the complete mitogenome sequence of Calappa bilineata, which is the first mitogenome of Calappidae up to now. The total length is 15,606 bp and includes 13 protein-coding genes, 22 transfer RNAs, two ribosomal RNAs and one control region. The genome composition is highly A + T biased (68.7%), and exhibits a negative AT-skew (-0.010) and GC-skew (-0.267). As with other invertebrate mitogenomes, the PCGs start with the standard ATN and stop with the standard TAN codons or incomplete T. Phylogenetic analysis showed that C. bilineata was most closely related to Matuta planipes (Matutidae), and these two species formed a sister clade, constituting a Calappoidea group and forming a sister clade with part of Eriphioidea. The existence of the polyphyletic families raised doubts over the traditional classification system. These results will help to better understand the features of the C. bilineata mitogenome and lay foundation for further evolutionary relationships within Brachyura.

The Active Mechanism of Nucleosome Depletion by Poly(dA:dT) Tracts In Vivo.

Poly(dA:dT) tracts cause nucleosome depletion in many species, e.g., at promoters and replication origins. Their intrinsic biophysical sequence properties make them stiff and unfavorable for nucleosome assembly, as probed by in vitro nucleosome reconstitution. The mere correlation between nucleosome depletion over poly(dA:dT) tracts in in vitro reconstituted and in in vivo chromatin inspired an intrinsic nucleosome exclusion mechanism in vivo that is based only on DNA and histone properties. However, we compile here published and new evidence that this correlation does not reflect mechanistic causation. (1) Nucleosome depletion over poly(dA:dT) in vivo is not universal, e.g., very weak in S. pombe. (2) The energy penalty for incorporating poly(dA:dT) tracts into nucleosomes is modest (<10%) relative to ATP hydrolysis energy abundantly invested by chromatin remodelers. (3) Nucleosome depletion over poly(dA:dT) is much stronger in vivo than in vitro if monitored without MNase and (4) actively maintained in vivo. (5) S. cerevisiae promoters evolved a strand-biased poly(dA) versus poly(dT) distribution. (6) Nucleosome depletion over poly(dA) is directional in vivo. (7) The ATP dependent chromatin remodeler RSC preferentially and directionally displaces nucleosomes towards 5' of poly(dA). Especially distribution strand bias and displacement directionality would not be expected for an intrinsic mechanism. Together, this argues for an in vivo mechanism where active and species-specific read out of intrinsic sequence properties, e.g., by remodelers, shapes nucleosome organization.

Innate immune recognition of an AT-rich stem-loop DNA motif in the Plasmodium falciparum genome.

Although Toll-like receptor 9 (TLR9) has been implicated in cytokine and type I interferon (IFN) production during malaria in humans and mice, the high AT content of the Plasmodium falciparum genome prompted us to examine the possibility that malarial DNA triggered TLR9-independent pathways. Over 6000 ATTTTTAC ("AT-rich") motifs are present in the genome of P. falciparum, which we show here potently induce type I IFNs. Parasite DNA, parasitized erythrocytes and oligonucleotides containing the AT-rich motif induce type I IFNs via a pathway that did not involve the previously described sensors TLR9, DAI, RNA polymerase-III or IFI16/p204. Rather, AT-rich DNA sensing involved an unknown receptor that coupled to the STING, TBK1 and IRF3-IRF7 signaling pathway. Mice lacking IRF3, IRF7, the kinase TBK1 or the type I IFN receptor were resistant to otherwise lethal cerebral malaria. Collectively, these observations implicate AT-rich DNA sensing via STING, TBK1 and IRF3-IRF7 in P. falciparum malaria.

IGS sequences in Cestrum present AT- and GC-rich conserved domains, with strong regulatory potential for 5S rDNA.

The 35S and 5S ribosomal DNA (rDNA) organized in thousands of copies in genomes, have been widely used in numerous comparative cytogenetic studies. Nevertheless, several questions related to the diversity and organization of regulatory motifs in 5S rDNA remain to be addressed. The 5S rDNA unit is composed of a conserved 120 bp length coding region and an intergenic spacer (IGS) containing potential regulatory motifs (Poly-T, AT-rich and GC-rich) differing in number, redundancy and position along the IGS. The Cestrum species (Solanaceae) have large genomes (about 10 pg/1C) and conserved 2n = 16 karyotypes. Strikingly, these genomes show high diversity of heterochromatin distribution, variability in 35S rDNA loci and the occurrence of B chromosomes. However, the 5S rDNA loci are highly conserved in the proximal region of chromosome 8. Comparison of seventy-one IGS sequences in plants revealed several conserved motifs with potential regulatory function. The AT- and GC-rich domains appeared highly conserved in Cestrum chromosomes. The 5S genic and the GC-rich IGS probe produced FISH signals in both A (pair 8) and B chromosomes. The GC-rich domain presented a strong potential for regulation because it may be associated with CpG islands organization, as well as to hairpin and loop organization. Another interesting aspect was the ability of AT- and GC-rich motifs to produce non-heterochromatic CMA/DAPI signals. While the length of the 5S rDNA IGS region varied in size between the Cestrum species, the individual sequence motifs seem to be conserved suggesting their regulatory function. The most striking feature was the conserved GC-rich domain in Cestrum, which is recognized as a signature trait of the proximal region of chromosome pair 8.

High-resolution 3D models of Caulobacter crescentus chromosome reveal genome structural variability and organization.

High-resolution three-dimensional models of Caulobacter crescentus nucleoid structures were generated via a multi-scale modeling protocol. Models were built as a plectonemically supercoiled circular DNA and by incorporating chromosome conformation capture based data to generate an ensemble of base pair resolution models consistent with the experimental data. Significant structural variability was found with different degrees of bending and twisting but with overall similar topologies and shapes that are consistent with C. crescentus cell dimensions. The models allowed a direct mapping of the genomic sequence onto the three-dimensional nucleoid structures. Distinct spatial distributions were found for several genomic elements such as AT-rich sequence elements where nucleoid associated proteins (NAPs) are likely to bind, promoter sites, and some genes with common cellular functions. These findings shed light on the correlation between the spatial organization of the genome and biological functions.