William Lawrence Tower's Beetles: Experimental Evolution and the Manipulation of Inheritance.

William Lawrence Tower's work on the evolution of the Colorado Potato Beetle (Leptinotarsa decemlineata), documenting the environmental induction of mutation and speciation, made him a leading figure in experimental genetics during the first decade of the 20th century. His research program served as a model for other experimental evolution studies seeking to demonstrate the environmental modification of inheritance. Tower enjoyed the support of influential figures in the field, despite well-known problems that plagued Tower's earlier academic career. The validity of his genetic work, and other findings reported by Tower, were later challenged. The Tower affair illustrates how questionable and possibly fraudulent scientific practices can be tolerated to explore certain experimental directions and theoretical frameworks, particularly at the frontier of expanding disciplines. When needed, those explorations can be forestalled or extinguished by exploiting conspicuous vulnerabilities of rogue practitioners. In Tower's case, both unrefuted allegations of scientific misconduct and personal problems dissolved his institutional support, leading to a swift ouster from academic science. Tower's downfall discredited soft inheritance and neo-Lamarckian conceptions in the field of experimental genetics, facilitating the discipline's embrace of a hard inheritance model that featured a hereditary material resistant to environmental modification.

The nuclear lamina is required for proper development and nuclear shape distortion in tomato.

The nuclear lamina in plant cells is composed of plant-specific proteins, including nuclear matrix constituent proteins (NMCPs), which have been postulated to be functional analogs of lamin proteins that provide structural integrity to the organelle and help stabilize the three-dimensional organization of the genome. Using genomic editing, we generated alleles for the three genes encoding NMCPs in cultivated tomato (Solanum lycopersicum) to determine if the consequences of perturbing the nuclear lamina in this crop species were similar to or distinct from those observed in the model Arabidopsis thaliana. Loss of the sole NMCP2-class protein was lethal in tomato but is tolerated in Arabidopsis. Moreover, depletion of NMCP1-type nuclear lamina proteins leads to distinct developmental phenotypes in tomato, including leaf morphology defects and reduced root growth rate (in nmcp1b mutants), compared with cognate mutants in Arabidopsis. These findings suggest that the nuclear lamina interfaces with different developmental and signaling pathways in tomato compared with Arabidopsis. At the subcellular level, however, tomato nmcp mutants resembled their Arabidopsis counterparts in displaying smaller and more spherical nuclei in differentiated cells. This result argues that the plant nuclear lamina facilitates nuclear shape distortion in response to forces exerted on the organelle within the cell.

Seminars in cell and development biology on histone variants remodelers of H2A variants associated with heterochromatin.

Variants of the histone H2A occupy distinct locations in the genome. There is relatively little known about the mechanisms responsible for deposition of specific H2A variants. Notable exceptions are chromatin remodelers that control the dynamics of H2A.Z at promoters. Here we review the steps that identified the role of a specific class of chromatin remodelers, including LSH and DDM1 that deposit the variants macroH2A in mammals and H2A.W in plants, respectively. The function of these remodelers in heterochromatin is discussed together with their multiple roles in genome stability.

The edge of the nucleus: Variations on a theme.

The plant nuclear lamina utilizes distinct and highly divergent proteins to mediate chromatin interactions at the nuclear edge. In this issue of Developmental Cell, Tang et al. show that members of PNET2, a family of inner nuclear membrane proteins in Arabidopsis, are capable of binding histones and are involved in large-scale genome organization.

Coordination of NMCP1- and NMCP2-class proteins within the plant nucleoskeleton.

Plants lack lamin proteins but contain a class of coiled-coil proteins that serve as analogues to form a laminal structure at the nuclear periphery. These nuclear matrix constituent proteins (NMCPs) play important roles in regulating nuclear morphology and are partitioned into two distinct groups. We investigated Arabidopsis NMCPs (called CRWNs) to study the interrelationship between the three NMCP1-type paralogues (CRWN1, 2, and 3) and the lone NMCP2-type paralogue, CRWN4. An examination of crwn mutants using protein immunoblots demonstrated that CRWN4 abundance depends on the presence of the NMCP1-type proteins, particularly CRWN1. The possibility that CRWN4 is coimported into the nucleus with nuclear localization signal (NLS)-bearing paralogues in the NMCP1-clade was discounted based on recovery of a crwn4-2 missense allele that disrupts a predicted NLS and lowers the abundance of CRWN4 in the nucleus. Further, a screen for mutations that suppress the effects of the crwn4-2 mutation led to the discovery of a missense allele, impa-1G146E, in one of the nine importin-α genes in the Arabidopsis genome. Our results indicate that the CRWN4 carries a functional NLS that interacts with canonic nuclear import machinery. Once imported, the level of CRWN4 within the nucleus is modulated by the abundance of NMCP1 proteins.

The role of CRWN nuclear proteins in chromatin-based regulation of stress response genes.

The periphery in animal nuclei is generally considered to be a transcriptionally repressive environment. Recent studies indicate that chromatin-based mechanisms establish a similar situation in plant nuclei. We demonstrated recently that the loss of CRWN nuclear lamina proteins in Arabidopsis leads to the misregulation of a group of genes involved in plant defense. How this defense response is triggered is largely unknown. Here, we briefly review recent findings that identify several layers of chromatin-based regulation responsible for this response. Further, we introduce new data suggesting that histone H3 lysine 27 tri-methylation levels are reduced in the absence of CRWNs near genes encoding transcription factors regulating SA biosynthesis, providing an explanation for SA induction. These discoveries begin to uncover the interplay between nuclear architecture and stress response in plants.

Loss of CRWN Nuclear Proteins Induces Cell Death and Salicylic Acid Defense Signaling.

Defects in the nuclear lamina of animal cell nuclei have dramatic effects on nuclear structure and gene expression as well as diverse physiological manifestations. We report that deficiencies in CROWDED NUCLEI (CRWN), which are candidate nuclear lamina proteins in Arabidopsis (Arabidopsis thaliana), trigger widespread changes in transcript levels and whole-plant phenotypes, including dwarfing and spontaneous cell death lesions. These phenotypes are caused in part by ectopic induction of plant defense responses via the salicylic acid pathway. Loss of CRWN proteins induces the expression of the salicylic acid biosynthetic gene ISOCHORISMATE SYNTHASE1, which leads to spontaneous defense responses in crwn1 crwn2 and crwn1 crwn4 mutants, which are deficient in two of the four CRWN paralogs. The symptoms of ectopic defense response, including pathogenesis marker gene expression and cell death, increase in older crwn double mutants. These age-dependent effects are postulated to reflect an increase in nuclear dysfunction or damage over time, a phenomenon reminiscent of aging effects seen in animal nuclei and in some human laminopathy patients.

Cell Biology of the Plant Nucleus.

The eukaryotic nucleus is enclosed by the nuclear envelope, which is perforated by the nuclear pores, the gateways of macromolecular exchange between the nucleoplasm and cytoplasm. The nucleoplasm is organized in a complex three-dimensional fashion that changes over time and in response to stimuli. Within the cell, the nucleus must be viewed as an organelle (albeit a gigantic one) that is a recipient of cytoplasmic forces and capable of morphological and positional dynamics. The most dramatic reorganization of this organelle occurs during mitosis and meiosis. Although many of these aspects are less well understood for the nuclei of plants than for those of animals or fungi, several recent discoveries have begun to place our understanding of plant nuclei firmly into this broader cell-biological context.

Arabidopsis VIM proteins regulate epigenetic silencing by modulating DNA methylation and histone modification in cooperation with MET1.

Methylcytosine-binding proteins containing SRA (SET- and RING-Associated) domain are required for the establishment and/or maintenance of DNA methylation in both plants and animals. We previously proposed that Arabidopsis VIM/ORTH proteins with an SRA domain maintain DNA methylation and epigenetic gene silencing in heterochromatic regions. However, their endogenous targets of epigenetic gene silencing have not been analyzed globally and the mechanisms by which VIM proteins coordinate DNA methylation and epigenetic silencing are largely unknown. In this study, a genome-wide transcript profiling analysis revealed 544 derepressed genes in a vim1/2/3 triple mutant, including 133 known genes. VIM1 bound to promoter and transcribed regions of the up-regulated genes in vim1/2/3 and VIM deficiency caused severe DNA hypomethylation in all sequence contexts at direct VIM1 targets. We found a drastic loss of H3K9me2 at heterochromatic chromocenters in vim1/2/3 nuclei. Furthermore, aberrant changes in transcriptionally active and repressive histone modifications were observed at VIM1 targets in vim1/2/3. VIM1-binding capacity to target genes was significantly reduced in the met1 background, indicating that VIM1 primarily recognizes CG methylation deposited by MET1. Overall, our data indicate that VIM proteins regulate genome-wide epigenetic gene silencing through coordinated modulation of DNA methylation and histone modification status in collaboration with MET1.

VIM proteins regulate transcription exclusively through the MET1 cytosine methylation pathway.

In Arabidopsis, variant in methylation (VIM) proteins are required for the maintenance of DNA methylation in the CpG dinucleotide context. VIM1 acts as a cofactor of DNA methyltransferase 1 (MET1), although the mechanism for this co-regulation remains unclear. In this study, we used RNA-seq analysis to profile the transcriptomes of vim1, vim1 vim2 vim3, and met1 null mutants. Consistent with previous studies indicating functional redundancy between these VIM proteins, we found no transcripts that were significantly misregulated in vim1 mutants. However, we identified a large set of VIM protein regulatory targets through analysis of vim1 vim2 vim3 mutants, and we observed that this set is essentially identical to that regulated by MET1. Log 2 fold changes in gene expression relative to wild type are strongly correlated between vim1 vim2 vim3 and met1 mutants. While the largest subset of these transcripts is upregulated and enriched with transposable elements, we also found small subsets of downregulated genes in each mutant, which are enriched with protein-coding genes. Together, these results expand on previous studies that profiled cytosine methylation in the vim1 vim2 vim3 mutant, and show that VIM proteins function in transcriptional regulation via their roles in the MET1 DNA methylation pathway.

Arabidopsis CROWDED NUCLEI (CRWN) proteins are required for nuclear size control and heterochromatin organization.

BACKGROUND: Plant nuclei superficially resemble animal and fungal nuclei, but the machinery and processes that underlie nuclear organization in these eukaryotic lineages appear to be evolutionarily distinct. Among the candidates for nuclear architectural elements in plants are coiled-coil proteins in the NMCP (Nuclear Matrix Constituent Protein) family. Using genetic and cytological approaches, we dissect the function of the four NMCP family proteins in Arabidopsis encoded by the CRWN genes, which were originally named LINC (LITTLE NUCLEI). RESULTS: CRWN proteins are essential for viability as evidenced by the inability to recover mutants that have disruptions in all four CRWN genes. Mutants deficient in different combinations of the four CRWN paralogs exhibit altered nuclear organization, including reduced nuclear size, aberrant nuclear shape and abnormal spatial organization of constitutive heterochromatin. Our results demonstrate functional diversification among CRWN paralogs; CRWN1 plays the predominant role in control of nuclear size and shape followed by CRWN4. Proper chromocenter organization is most sensitive to the deficiency of CRWN4. The reduction in nuclear volume in crwn mutants in the absence of a commensurate reduction in endoreduplication levels leads to an increase in average nuclear DNA density. CONCLUSIONS: Our findings indicate that CRWN proteins are important architectural components of plant nuclei that play diverse roles in both heterochromatin organization and the control of nuclear morphology.

Natural epigenetic variation in plant species: a view from the field.

Researchers are beginning to use wild plant populations to survey and assess cytosine methylation polymorphisms in a population and ecological genetic framework. These studies support the plausibility of adaptive epigenetic alleles, but uncertainty remains due to the difficulty in untangling genetic and epigenetic variation in wild populations. The increasing emphasis on stress-induced epigenetic alterations and transgenerational phenomena among researchers focused on epigenetic mechanisms should push practitioners of this subfield to consider the questions and tools of colleagues grappling with epigenetics from ecological and evolutionary perspectives.

The structure, organization and radiation of Sadhu non-long terminal repeat retroelements in Arabidopsis species.

BACKGROUND: Sadhu elements are non-autonomous retroposons first recognized in Arabidopsis thaliana. There is a wide degree of divergence among different elements, suggesting that these sequences are ancient in origin. Here we report the results of several lines of investigation into the genomic organization and evolutionary history of this element family. RESULTS: We present a classification scheme for Sadhu elements in A. thaliana, describing derivative elements related to the full-length elements we reported previously. We characterized Sadhu5 elements in a set of A. thaliana strains in order to trace the history of radiation in this subfamily. Sequences surrounding the target sites of different Sadhu insertions are consistent with mobilization by LINE retroelements. Finally, we identified Sadhu elements grouping into distinct subfamilies in two related species, Arabidopsis arenosa and Arabidopsis lyrata. CONCLUSIONS: Our analyses suggest that the Sadhu retroelement family has undergone target primed reverse transcription-driven retrotransposition during the divergence of different A. thaliana strains. In addition, Sadhu elements can be found at moderate copy number in three distinct Arabidopsis species, indicating that the evolutionary history of these sequences can be traced back at least several millions of years.

Gene duplication and hypermutation of the pathogen Resistance gene SNC1 in the Arabidopsis bal variant.

The bal defect in the Arabidopsis thaliana Columbia strain was spontaneously generated in an inbred ddm1 (decrease in DNA methylation 1) mutant background in which various genetic and epigenetic alterations accumulate. The bal variant displays short stature and curled leaves due to the constitutive activation of defense signaling. These bal phenotypes are metastable and phenotypic suppression is evident in more than one-third of ethyl methanesulfonate (EMS)-treated bal M(1) plants. The semidominant bal allele maps to the RPP5 (recognition of Peronospora parasitica 5) locus, which includes a cluster of disease Resistance (R) genes, many of which show an increase in steady-state expression levels in the bal variant. Here, we report that activation of RPP5 locus R genes and dwarfing in the bal variant are caused by a 55-kb duplication within the RPP5 locus. Although many RPP5 locus R genes are duplicated in the bal variant, the duplication of SNC1 alone is necessary and sufficient for the phenotypic changes in the bal variant. Missense mutations in the SNC1 gene were identified in all three phenotypically suppressed EMS-treated bal lines investigated, indicating that the high-frequency phenotypic instability induced by EMS treatment is caused by a genetic mechanism. We propose that the high degree of variation in SNC1-related sequences among Arabidopsis natural accessions follows the two-step mechanism observed in the bal variant: gene duplication followed by hypermutation.

Quantitative epigenetics: DNA sequence variation need not apply.

Two recent reports, including one by Reinders and colleagues (pp. 939-950) in the April 15, 2009, issue of Genes & Development, describe the construction of Arabidopsis recombinant inbred populations that maximize epigenetic rather than genetic variation. The distribution and behavior of phenotypic variation in these populations suggest that stable epialleles can control complex quantitative traits. However, stochastic epimutation and transposon movement in these populations present some unexpected technical hurdles to implementing quantitative epigenetic analysis.

Natural variation in DNA methylation in ribosomal RNA genes of Arabidopsis thaliana.

BACKGROUND: DNA methylation is an important biochemical mark that silences repetitive sequences, such as transposons, and reinforces epigenetic gene expression states. An important class of repetitive genes under epigenetic control in eukaryotic genomes encodes ribosomal RNA (rRNA) transcripts. The ribosomal genes coding for the 45S rRNA precursor of the three largest eukaryotic ribosomal RNAs (18S, 5.8S, and 25-28S) are found in nucleolus organizer regions (NORs), comprised of hundreds to thousands of repeats, only some of which are expressed in any given cell. An epigenetic switch, mediated by DNA methylation and histone modification, turns rRNA genes on and off. However, little is known about the mechanisms that specify and maintain the patterns of NOR DNA methylation. RESULTS: Here, we explored the extent of naturally-occurring variation in NOR DNA methylation among accessions of the flowering plant Arabidopsis thaliana. DNA methylation in coding regions of rRNA genes was positively correlated with copy number of 45S rRNA gene and DNA methylation in the intergenic spacer regions. We investigated the inheritance of NOR DNA methylation patterns in natural accessions with hypomethylated NORs in inter-strain crosses and defined three different categories of inheritance in F1 hybrids. In addition, subsequent analysis of F2 segregation for NOR DNA methylation patterns uncovered different patterns of inheritance. We also revealed that NOR DNA methylation in the Arabidopsis accession Bor-4 is influenced by the vim1-1 (variant in methylation 1-1) mutation, but the primary effect is specified by the NORs themselves. CONCLUSION: Our results indicate that the NORs themselves are the most significant determinants of natural variation in NOR DNA methylation. However, the inheritance of NOR DNA methylation suggests the operation of a diverse set of mechanisms, including inheritance of parental methylation patterns, reconfiguration of parental NOR DNA methylation, and the involvement of trans-acting modifiers.

Three SRA-domain methylcytosine-binding proteins cooperate to maintain global CpG methylation and epigenetic silencing in Arabidopsis.

Methylcytosine-binding proteins decipher the epigenetic information encoded by DNA methylation and provide a link between DNA methylation, modification of chromatin structure, and gene silencing. VARIANT IN METHYLATION 1 (VIM1) encodes an SRA (SET- and RING-associated) domain methylcytosine-binding protein in Arabidopsis thaliana, and loss of VIM1 function causes centromere DNA hypomethylation and centromeric heterochromatin decondensation in interphase. In the Arabidopsis genome, there are five VIM genes that share very high sequence similarity and encode proteins containing a PHD domain, two RING domains, and an SRA domain. To gain further insight into the function and potential redundancy among the VIM proteins, we investigated strains combining different vim mutations and transgenic vim knock-down lines that down-regulate multiple VIM family genes. The vim1 vim3 double mutant and the transgenic vim knock-down lines showed decreased DNA methylation primarily at CpG sites in genic regions, as well as repeated sequences in heterochromatic regions. In addition, transcriptional silencing was released in these plants at most heterochromatin regions examined. Interestingly, the vim1 vim3 mutant and vim knock-down lines gained ectopic CpHpH methylation in the 5S rRNA genes against a background of CpG hypomethylation. The vim1 vim2 vim3 triple mutant displayed abnormal morphological phenotypes including late flowering, which is associated with DNA hypomethylation of the 5' region of FWA and release of FWA gene silencing. Our findings demonstrate that VIM1, VIM2, and VIM3 have overlapping functions in maintenance of global CpG methylation and epigenetic transcriptional silencing.

Signaling silence--breaking ground and spreading out.

Among the persistent mysteries in epigenetics are the criteria by which specific regions of the genome are chosen for deposition of distinguishing chromatin marks. Once a particular region is modified, will the newly acquired epigenetic state spill onto neighboring regions of the genome or be confined to tidy patches? The report by Henderson and Jacobsen in the the previous issue of Genes & Development (1597-1606) addresses these questions while providing insight into the utility of DNA methylation for a eukaryotic genome.

Phenotypic instability of Arabidopsis alleles affecting a disease Resistance gene cluster.

BACKGROUND: Three mutations in Arabidopsis thaliana strain Columbia - cpr1, snc1, and bal - map to the RPP5 locus, which contains a cluster of disease Resistance genes. The similar phenotypes, gene expression patterns, and genetic interactions observed in these mutants are related to constitutive activation of pathogen defense signaling. However, these mutant alleles respond differently to various conditions. Exposure to mutagens, such as ethyl methanesulfonate (EMS) and gamma-irradiation, induce high frequency phenotypic instability of the bal allele. In addition, a fraction of the bal and cpr1 alleles segregated from bal x cpr1 F1 hybrids also show signs of phenotypic instability. To gain more insight into the mechanism of phenotypic instability of the bal and cpr1 mutations, we systematically compared the behavior of these unusual alleles with that of the missense gain-of-function snc1 allele in response to DNA damage or passage through F1 hybrids. RESULTS: We found that the cpr1 allele is similar to the bal allele in its unstable behavior after EMS mutagenesis. For both the bal and cpr1 mutants, destabilization of phenotypes was observed in more than 10% of EMS-treated plants in the M1 generation. In addition, exceptions to simple Mendelian inheritance were identified in the M2 generation. Like cpr1 x bal F1 hybrids, cpr1 x snc1 F1 hybrids and bal x snc1 F1 hybrids exhibited dwarf morphology. While only dwarf F2 plants were produced from bal x snc1 F1 hybrids, about 10% wild-type F2 progeny were produced from cpr1 x snc1 F1 hybrids, as well as from cpr1 x bal hybrids. Segregation analysis suggested that the cpr1 allele in cpr1 x snc1 crosses was destabilized during the late F1 generation to early F2 generation. CONCLUSION: With exposure to EMS or different F1 hybrid contexts, phenotypic instability is induced for the bal and cpr1 alleles, but not for the snc1 allele. Our results suggest that the RPP5 locus can adopt different metastable genetic or epigenetic states, the stability of which is highly susceptible to mutagenesis and pairing of different alleles.

Population epigenetics.

Our knowledge of the mechanisms that specify and propagate epigenetic states of gene expression is expanding rapidly; however, the significance of variation in epigenetic states at the population level remains largely unexplored. Population epigenetics, emerging as an active subfield at the interface of molecular genetics, genomics, and population biology, addresses questions concerning the prevalence and importance of epigenetic variation in the natural world.