Interactions of Chromatin Context,Binding Site Sequence Content,and Sequence Evolution in Stress-Induced p53 Occupancy and Transactivation

Cellular stresses activate the tumor suppressor p53 protein leading to selective binding to DNA response elements (REs) and gene transactivation from a large pool of potential p53 REs (p53REs). To elucidate how p53RE sequences and local chromatin context interact to affect p53 binding and gene transactivation, we mapped genome-wide binding localizations of p53 and H3K4me3 in untreated and doxorubicin (DXR)-treated human lymphoblastoid cells. We examined the relationships among p53 occupancy, gene expression, H3K4me3, chromatin accessibility (DNase 1 hypersensitivity, DHS), ENCODE chromatin states, p53RE sequence, and evolutionary conservation. We observed that the inducible expression of p53-regulated genes was associated with the steady-state chromatin status of the cell. Most highly inducible p53-regulated genes were suppressed at baseline and marked by repressive histone modifications or displayed CTCF binding. Comparison of p53RE sequences residing in different chromatin contexts demonstrated that weaker p53REs resided in open promoters, while stronger p53REs were located within enhancers and repressed chromatin. p53 occupancy was strongly correlated with similarity of the target DNA sequences to the p53RE consensus, but surprisingly, inversely correlated with pre-existing nucleosome accessibility (DHS) and evolutionary conservation at the p53RE. Occupancy by p53 of REs that overlapped transposable element (TE) repeats was significantly higher (p<10⁻⁷) and correlated with stronger p53RE sequences (p<10⁻¹¹⁰) relative to nonTE-associated p53REs, particularly for MLT1H, LTR10B, and Mer61 TEs. However, binding at these elements was generally not associated with transactivation of adjacent genes. Occupied p53REs located in L2-like TEs were unique in displaying highly negative PhyloP scores (predicted fast-evolving) and being associated with altered H3K4me3 and DHS levels. These results underscore the systematic interaction between chromatin status and p53RE context in the induced transactivation response. This p53 regulated response appears to have been tuned via evolutionary processes that may have led to repression and/or utilization of p53REs originating from primate-specific transposon elements.     

Loss of TDP-43 in male germ cells causes meiotic failure and impairs fertility in mice

Meiotic arrest is a common cause of human male infertility, but the causes of this arrest are poorly understood. Transactive response DNA-binding protein of 43 kDa (TDP-43) is highly expressed in spermatocytes in the preleptotene and pachytene stages of meiosis. TDP-43 is linked to several human neurodegenerative disorders wherein its nuclear clearance accompanied by cytoplasmic aggregates underlies neurodegeneration. Exploring the functional requirement for TDP-43 for spermatogenesis for the first time, we show here that conditional KO (cKO) of the Tardbp gene (encoding TDP-43) in male germ cells of mice leads to reduced testis size, depletion of germ cells, vacuole formation within the seminiferous epithelium, and reduced sperm production. Fertility trials also indicated severe subfertility. Spermatocytes of cKO mice showed failure to complete prophase I of meiosis with arrest at the midpachytene stage. Staining of synaptonemal complex protein 3 and γH2AX, markers of the meiotic synaptonemal complex and DNA damage, respectively, and super illumination microscopy revealed nonhomologous pairing and synapsis defects. Quantitative RT–PCR showed reduction in the expression of genes critical for prophase I of meiosis, including Spo11 (initiator of meiotic double-stranded breaks), Rec8 (meiotic recombination protein), and Rad21L (RAD21-like, cohesin complex component), as well as those involved in the retinoic acid pathway critical for entry into meiosis. RNA-Seq showed 1036 upregulated and 1638 downregulated genes (false discovery rate <0.05) in the Tardbp cKO testis, impacting meiosis pathways. Our work reveals a crucial role for TDP-43 in male meiosis and suggests that some forms of meiotic arrest seen in infertile men may result from the loss of function of TDP-43.     

Comparative genomic analysis reveals species-dependent complexities that explain difficulties with microsatellite marker development in molluscs

Reliable population DNA molecular markers are difficult to develop for molluscs, the reasons for which are largely unknown. Identical protocols for microsatellite marker development were implemented in three gastropods. Success rates were lower for Gibbula cineraria compared to Littorina littorea and L. saxatilis. Comparative genomic analysis of 47.2 kb of microsatellite containing sequences (MCS) revealed a high incidence of cryptic repetitive DNA in their flanking regions. The majority of these were novel, and could be grouped into DNA families based upon sequence similarities. Significant inter-specific variation in abundance of cryptic repetitive DNA and DNA families was observed. Repbase scans show that a large proportion of cryptic repetitive DNA was identified as transposable elements (TEs). We argue that a large number of TEs and their transpositional activity may be linked to differential rates of DNA multiplication and recombination. This is likely to be an important factor explaining inter-specific variation in genome stability and hence microsatellite marker development success rates. Gastropods also differed significantly in the type of TEs classes (autonomous vs non-autonomous) observed. We propose that dissimilar transpositional mechanisms differentiate the TE classes in terms of their propensity for transposition, fixation and/or silencing. Consequently, the phylogenetic conservation of non-autonomous TEs, such as CvA, suggests that dispersal of these elements may have behaved as microsatellite-inducing elements. Results seem to indicate that, compared to autonomous, non-autonomous TEs maybe have a more active role in genome rearrangement processes. The implications of the findings for genomic rearrangement, stability and marker development are discussed.     

Methylation-Sensitive Expression of a DNA Demethylase Gene Serves As an Epigenetic Rheostat

Genomes must balance active suppression of transposable elements (TEs) with the need to maintain gene expression. In Arabidopsis, euchromatic TEs are targeted by RNA-directed DNA methylation (RdDM). Conversely, active DNA demethylation prevents accumulation of methylation at genes proximal to these TEs. It is unknown how a cellular balance between methylation and demethylation activities is achieved. Here we show that both RdDM and DNA demethylation are highly active at a TE proximal to the major DNA demethylase gene ROS1. Unexpectedly, and in contrast to most other genomic targets, expression of ROS1 is promoted by DNA methylation and antagonized by DNA demethylation. We demonstrate that inducing methylation in the ROS1 proximal region is sufficient to restore ROS1 expression in an RdDM mutant. Additionally, methylation-sensitive expression of ROS1 is conserved in other species, suggesting it is adaptive. We propose that the ROS1 locus functions as an epigenetic rheostat, tuning the level of demethylase activity in response to methylation alterations, thus ensuring epigenomic stability.     

Cytoplasmic mislocalization and mitochondrial colocalization of TDP-43 are common features between normal aged and young mice

Transactive response DNA binding protein 43 (TDP-43) pathologies have been well recognized in various neurodegenerative disorders including frontotemporal lobar degeneration (FTLD), amyotrophic lateral sclerosis (ALS), and Alzheimer’s disease (AD). However, there have been limited studies on whether there are any TDP-43 alterations in normal aging. We investigated TDP-43 distribution in different brain regions in normal aged (n = 3 for 26- or 36-month-old) compared to young (n = 3 for 6- or 12-month-old) mice. In both normal aged and young mice, TDP-43 and phosphorylated TDP-43 (pTDP-43) demonstrated a unique pattern of distribution in neurons in some specific brain regions including the pontine nuclei, thalamus, CA3 region of the hippocampus, and orbital cortex. This pattern was demonstrated on higher magnification of high-resolution double fluorescence images and confocal microscopy as mislocalization of TDP-43 and pTDP-43, characterized by neuronal nuclear depletion and cytoplasmic accumulation in these brain regions, as well as colocalization between TDP-43 or pTDP-43 and mitochondria, similar to what has been described previously in neurodegenerative disorders. All these findings were identical in both normal aged and young mice. In summary, TDP-43 and pTDP-43 mislocalization from nucleus to cytoplasm and their colocalization with mitochondria in the specific brain regions are present not only in aging, but also in young healthy states. Our findings provide a new insight for the role of TDP-43 proteinopathy in health and diseases, and that aging may not be a critical factor for the development of TDP-43 proteinopathy in subpopulations of neurons.Impact statementDespite increasing evidence implicating the important role of TDP-43 in the pathogenesis of a wide range of age-related neurodegenerative diseases, there is limited study of TDP-43 proteinopathy and its association with mitochondria during normal aging. Our findings of cytoplasmic accumulation of TDP-43 that is highly colocalized with mitochondria in neurons in selective brain regions in young animals in the absence of neuronal loss provide a novel insight into the development of TDP-43 proteinopathy and its contribution to neuronal loss.     

Transgenic Rat Model of Neurodegeneration Caused by Mutation in the TDP Gene

TDP-43 proteinopathies have been observed in a wide range of neurodegenerative diseases. Mutations in the gene encoding TDP-43 (i.e., TDP) have been identified in amyotrophic lateral sclerosis (ALS) and in frontotemporal lobe degeneration associated with motor neuron disease. To study the consequences of TDP mutation in an intact system, we created transgenic rats expressing normal human TDP or a mutant form of human TDP with a M337V substitution. Overexpression of mutant, but not normal, TDP caused widespread neurodegeneration that predominantly affected the motor system. TDP mutation reproduced ALS phenotypes in transgenic rats, as seen by progressive degeneration of motor neurons and denervation atrophy of skeletal muscles. This robust rat model also recapitulated features of TDP-43 proteinopathies including the formation of TDP-43 inclusions, cytoplasmic localization of phosphorylated TDP-43, and fragmentation of TDP-43 protein. TDP transgenic rats will be useful for deciphering the mechanisms underlying TDP-43–related neurodegenerative diseases.     

Transposable Element Regulation in Rice and Arabidopsis: Diverse Patterns of Active Expression and siRNA-mediated Silencing

Transposable elements (TEs) are DNA segments that can mediate or cause movement within genomes. We performed a comprehensive, whole-genome analysis of annotated TEs in rice (Oryza sativa L.) and Arabidopsis thaliana, focusing on their expression (mRNA data) and silencing (small RNA data), and we compared these data with annotated genes that are not annotated as transposons. TEs demonstrated higher levels of antisense mRNA expression in comparison to non-TE genes. The majority of the TEs were silenced, as demonstrated by higher levels of small RNAs and a lack of mRNA MPSS data. When TEs were expressed, their activity was usually limited to just one or a few of the mRNA libraries. When we examined TE expression at the whole-genome level and across the complete mRNA dataset, we observed that most activity was contributed by a few highly expressed transposable elements. These TEs were characterized by their low copy number and few matching small RNAs. Our results help define the relationship between gene expression and gene silencing for TEs, and indicate that TE silencing can impact neighboring genes, perhaps via a mechanism of heterochromatin formation and spreading. These data may be used to define active TEs and families of transposable elements that continue to shape plant genomes.     

Minocycline as a potential therapeutic agent in neurodegenerative disorders characterised by protein misfolding

Many neurodegenerative disorders share common features including the accumulation of aggregated misfolded proteins, neuroinflammation and the induction of apoptosis. While the contributions of each of these individual elements to neuronal death remain unclear, a commonly used antibiotic, minocycline, has been shown to reduce the progression and severity of disease in several models of neurodegeneration by variously downregulating these molecular pathways. Here we discuss the evidence for the potential of minocycline as a broad-specificity therapeutic agent for those neurodegenerative diseases that are characterized by the presence of misfolded proteins.Key words: minocycline, protein aggregation, neuroinflammation, neurodegeneration, protein misfolding     

Macrotransposition and other complex chromosomal restructuring in maize by closely linked transposons in direct orientation

Several observations indicate that compatible ends of separate, yet closely linked, transposable elements (TEs) can interact in alternative transposition reactions. First, pairs of TEs cause chromosome breaks with frequencies inversely related to the intertransposon distance. Second, some combinations of two TEs produce complex rearrangements that often include DNA adjacent to one or both elements. In pairs of TEs in direct orientation, alternative reactions involving the external ends of the two TEs should lead to the transposition of a macrotransposon consisting of both elements plus the intervening chromosomal segment. Such macrotransposons have been hypothesized previously based on deletions, but no macrotransposon insertions have been recovered. To detect macrotransposition, we have analyzed heritable chromosomal rearrangements produced by a chromosome-breaking pair of Ac and Ds elements situated 6.5 kb apart in direct orientation in a part of the maize (Zea mays) genome dispensable for viability. Here, we show that the postulated macrotransposon can excise and reinsert elsewhere in the genome. In addition, this transposon pair produces other complex rearrangements, including deletions, inversions, and reshuffling of the intertransposon segment. Thus, closely linked TE pairs, a common transposition outcome in some superfamilies, are adept at restructuring chromosomes and may have been instrumental in reshaping plant genomes.     

Potentiated Hsp104 variants suppress toxicity of diverse neurodegenerative disease-linked proteins

Protein misfolding is implicated in numerous lethal neurodegenerative disorders, including amyotrophic lateral sclerosis (ALS) and Parkinson disease (PD). There are no therapies that reverse these protein-misfolding events. We aim to apply Hsp104, a hexameric AAA+ protein from yeast, to target misfolded conformers for reactivation. Hsp104 solubilizes disordered aggregates and amyloid, but has limited activity against human neurodegenerative disease proteins. Thus, we have previously engineered potentiated Hsp104 variants that suppress aggregation, proteotoxicity and restore proper protein localization of ALS and PD proteins in Saccharomyces cerevisiae, and mitigate neurodegeneration in an animal PD model. Here, we establish that potentiated Hsp104 variants possess broad substrate specificity and, in yeast, suppress toxicity and aggregation induced by wild-type TDP-43, FUS and α-synuclein, as well as missense mutant versions of these proteins that cause neurodegenerative disease. Potentiated Hsp104 variants also rescue toxicity and aggregation of TAF15 but not EWSR1, two RNA-binding proteins with a prion-like domain that are connected with the development of ALS and frontotemporal dementia. Thus, potentiated Hsp104 variants are not entirely non-specific. Indeed, they do not unfold just any natively folded protein. Rather, potentiated Hsp104 variants are finely tuned to unfold proteins bearing short unstructured tracts that are not recognized by wild-type Hsp104. Our studies establish the broad utility of potentiated Hsp104 variants.KEY WORDS: FUS, Hsp104, TDP-43, α-synuclein, Disaggregase, Neurodegeneration     

Rotenone-Dependent Changes of Anterograde Motor Protein Expression and Mitochondrial Mobility in Brain Areas Related to Neurodegenerative Diseases

The presence of protein aggregates is common in neurodegenerative disorders; however, the real cause and effect of these aggregates during neurodegeneration is still a matter of investigation. We hypothesize that impairment of intracellular traffic may appear in the absence of protein inclusions and might trigger protein aggregation. In the present study, we aimed to evaluate mitochondria mobility as well as protein and messenger RNA expression of KIF1B and KIF5 that are molecular motors for neuronal anterograde traffic, in hippocampus, substantia nigra, and locus coeruleus of 10-month-old Lewis rats and cultured cells, from these same areas, following exposure to low doses of rotenone that do not lead to protein inclusions. The present study showed alteration in KIF1B and KIF5 expression, as well as in mitochondria mobility prior to protein aggregation involved in neurodegenerative disorders. These findings suggest that change in intracellular trafficking might be critical and one of the primary events for impairment of cell physiology during neurodegeneration associated with protein inclusions.     

Commentary: ZFYVE26/SPASTIZIN: A close link between complicated hereditary spastic paraparesis and autophagy

Defective autophagy is associated with neurodegenerative disorders including Alzheimer, Parkinson and Huntington diseases, amyotrophic lateral sclerosis and SCA (spinocerebellar ataxias). Autophagy defects were detected also in SPG49, a complicated form of hereditary spastic paraparesis (cHSP) associated with mutations in the TECPR2 gene, suggesting a role of autophagy also in this heterogeneous group of neurodegenerative diseases. We recently found defective autophagy in SPG15, another HSP subtype associated with mutations in the ZFYVE26/SPG15 gene. Patient-derived cells (fibroblasts/lymphoblasts) carrying different ZFYVE26 mutations show accumulation of immature autophagosomes and increased MAP1LC3B-II and SQSTM1/p62 levels. These findings indicate that ZFYVE26 is a key determinant of autophagosome maturation, which is impaired when the protein is defective or absent. Replication of these findings in primary neurons supports the relevance of defective autophagy in SPG15-related neurodegeneration.     

TDP‐43 loss of function increases TFEB activity and blocks autophagosome–lysosome fusion

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease that is characterized by selective loss of motor neurons in brain and spinal cord. TAR DNA‐binding protein 43 (TDP‐43) was identified as a major component of disease pathogenesis in ALS, frontotemporal lobar degeneration (FTLD), and other neurodegenerative disease. Despite the fact that TDP‐43 is a multi‐functional protein involved in RNA processing and a large number of TDP‐43 RNA targets have been discovered, the initial toxic effect and the pathogenic mechanism underlying TDP‐43‐linked neurodegeneration remain elusive. In this study, we found that loss of TDP‐43 strongly induced a nuclear translocation of TFEB, the master regulator of lysosomal biogenesis and autophagy, through targeting the mTORC1 key component raptor. This regulation in turn enhanced global gene expressions in the autophagy–lysosome pathway (ALP) and increased autophagosomal and lysosomal biogenesis. However, loss of TDP‐43 also impaired the fusion of autophagosomes with lysosomes through dynactin 1 downregulation, leading to accumulation of immature autophagic vesicles and overwhelmed ALP function. Importantly, inhibition of mTORC1 signaling by rapamycin treatment aggravated the neurodegenerative phenotype in a TDP‐43‐depleted Drosophila model, whereas activation of mTORC1 signaling by PA treatment ameliorated the neurodegenerative phenotype. Taken together, our data indicate that impaired mTORC1 signaling and influenced ALP may contribute to TDP‐43‐mediated neurodegeneration.     

Three Groups of Transposable Elements with Contrasting Copy Number Dynamics and Host Responses in the Maize (Zea mays ssp. mays) Genome

Most angiosperm nuclear DNA is repetitive and derived from silenced transposable elements (TEs). TE silencing requires substantial resources from the plant host, including the production of small interfering RNAs (siRNAs). Thus, the interaction between TEs and siRNAs is a critical aspect of both the function and the evolution of plant genomes. Yet the co-evolutionary dynamics between these two entities remain poorly characterized. Here we studied the organization of TEs within the maize (Zea mays ssp mays) genome, documenting that TEs fall within three groups based on the class and copy numbers. These groups included DNA elements, low copy RNA elements and higher copy RNA elements. The three groups varied statistically in characteristics that included length, location, age, siRNA expression and 24∶22 nucleotide (nt) siRNA targeting ratios. In addition, the low copy retroelements encompassed a set of TEs that had previously been shown to decrease expression within a 24 nt siRNA biogenesis mutant (mop1). To investigate the evolutionary dynamics of the three groups, we estimated their abundance in two landraces, one with a genome similar in size to that of the maize reference and the other with a 30% larger genome. For all three accessions, we assessed TE abundance as well as 22 nt and 24 nt siRNA content within leaves. The high copy number retroelements are under targeted similarly by siRNAs among accessions, appear to be born of a rapid bust of activity, and may be currently transpositionally dead or limited. In contrast, the lower copy number group of retrolements are targeted more dynamically and have had a long and ongoing history of transposition in the maize genome.     

Mobility and Generation of Mosaic Non-Autonomous Transposons by Tn3-Derived Inverted-Repeat Miniature Elements (TIMEs)

Functional transposable elements (TEs) of several Pseudomonas spp. strains isolated from black shale ore of Lubin mine and from post-flotation tailings of Zelazny Most in Poland, were identified using a positive selection trap plasmid strategy. This approach led to the capture and characterization of (i) 13 insertion sequences from 5 IS families (IS3, IS5, ISL3, IS30 and IS1380), (ii) isoforms of two Tn3-family transposons – Tn5563a and Tn4662a (the latter contains a toxin-antitoxin system), as well as (iii) non-autonomous TEs of diverse structure, ranging in size from 262 to 3892 bp. The non-autonomous elements transposed into AT-rich DNA regions and generated 5- or 6-bp sequence duplications at the target site of transposition. Although these TEs lack a transposase gene, they contain homologous 38-bp-long terminal inverted repeat sequences (IRs), highly conserved in Tn5563a and many other Tn3-family transposons. The simplest elements of this type, designated TIMEs (Tn3 family-derived Inverted-repeat Miniature Elements) (262 bp), were identified within two natural plasmids (pZM1P1 and pLM8P2) of Pseudomonas spp. It was demonstrated that TIMEs are able to mobilize segments of plasmid DNA for transposition, which results in the generation of more complex non-autonomous elements, resembling IS-driven composite transposons in structure. Such transposon-like elements may contain different functional genetic modules in their core regions, including plasmid replication systems. Another non-autonomous element “captured” with a trap plasmid was a TIME derivative containing a predicted resolvase gene and a res site typical for many Tn3-family transposons. The identification of a portable site-specific recombination system is another intriguing example confirming the important role of non-autonomous TEs of the TIME family in shuffling genetic information in bacterial genomes. Transposition of such mosaic elements may have a significant impact on diversity and evolution, not only of transposons and plasmids, but also of other types of mobile genetic elements.