Search Results (10)

Proteins are localized and concentrated at cellular and genomic locations for specific and efficient functions. Efforts to understand protein accumulation in eukaryotic organisms have primarily focused on multivalent interactions between intrinsically disordered regions (IDRs) as mediators of protein condensation. We previously showed that α-crystalline domain (ACD) proteins 15 (ACD15) and 21 (ACD21) were required for multimerization and the accumulation of gene-silencing methyl-CpG-binding domain protein 6 (MBD6) at chromocenters in Arabidopsis thaliana. Here, we demonstrate that ACDs and IDRs can act as parallel mechanisms, facilitating higher-order MBD6 assemblies. Using human IDRs known to be important for protein accumulation, we replicated and enhanced the accumulation of MBD6 at chromocenters. In addition, IDRs fused to MBD6 could substitute for ACD function and partially reconstitute the MBD6 gene-silencing function. However, the accumulation of MBD6 by IDRs still required ACD15 and ACD21 for full effect. These results establish that ACD-mediated protein accumulation is a mechanism that can function similarly to and together with IDR-mediated mechanisms. Full article

Arabidopsis ASYMMETRIC LEAVES2 (AS2) plays a key role in the formation of flat symmetric leaves. AS2 represses the expression of the abaxial gene ETTIN/AUXIN RESPONSE FACTOR3 (ETT/ARF3). AS2 interacts in vitro with the CGCCGC sequence in ETT/ARF3 exon 1. In cells of leaf primordia, AS2 localizes at peripheral regions of the nucleolus as two AS2 bodies, which are partially overlapped with chromocenters that contain condensed 45S ribosomal DNA repeats. AS2 contains the AS2/LOB domain, which consists of three sequences conserved in the AS2/LOB family: the zinc finger (ZF) motif, the ICG sequence including the conserved glycine residue, and the LZL motif. AS2 and the genes NUCLEOLIN1 (NUC1), RNA HELICASE10 (RH10), and ROOT INITIATION DEFECTIVE2 (RID2) that encode nucleolar proteins coordinately act as repressors against the expression of ETT/ARF3. Here, we examined the formation and patterning of AS2 bodies made from as2 mutants with amino acid substitutions in the ZF motif and the ICG sequence in cells of cotyledons and leaf primordia. Our results showed that the amino acid residues next to the cysteine residues in the ZF motif were essential for both the formation of AS2 bodies and the interaction with ETT/ARF3 DNA. The conserved glycine residue in the ICG sequence was required for the formation of AS2 bodies, but not for the DNA interaction. We also examined the effects of nuc1, rh10, and rid2 mutations, which alter the metabolism of rRNA intermediates and the morphology of the nucleolus, and showed that more than two AS2 bodies were observed in the nucleolus and at its periphery. These results suggested that the patterning of AS2 bodies is tightly linked to the morphology and functions of the nucleolus and the development of flat symmetric leaves in plants. Full article

Triatoma infestans (Klug) is an insect recognized as not only an important vector of South American trypanosomiasis (Chagas disease) but also a model of specific cellular morphofunctional organization and epigenetic characteristics. The purpose of the present review is to highlight certain cellular processes that are particularly unveiled in T. infestans, such as the following: (1) somatic polyploidy involving nuclear and cell fusions that generate giant nuclei; (2) diversification of nuclear phenotypes in the Malpighian tubules during insect development; (3) heterochromatin compartmentalization into large bodies with specific spatial distribution and presumed mobility in the cell nuclei; (4) chromatin remodeling and co-occurrence of necrosis and apoptosis in the Malpighian tubules under stress conditions; (5) epigenetic markers; and (6) response of heterochromatin to valproic acid, an epidrug that inhibits histone deacetylases and induces DNA demethylation in other cell systems. These cellular processes and epigenetic characteristics emphasize the role of T. infestans as an attractive model for cellular research. A limitation of these studies is the availability of insect supply by accredited insectaries. For studies that require the injection of drugs, the operator’s dexterity to perform insect manipulation is necessary, especially if young nymphs are used. For studies involving in vitro cultivation of insect organs, the culture medium should be carefully selected to avoid inconsistent results. Full article

The combination of ever-increasing microscopy resolution with cytogenetical tools allows for detailed analyses of nuclear functional partitioning. However, the need for reliable qualitative and quantitative methodologies to detect and interpret chromatin sub-nuclear organization dynamics is crucial to decipher the underlying molecular processes. Having access to properly automated tools for accurate and fast recognition of complex nuclear structures remains an important issue. Cognitive biases associated with human-based curation or decisions for object segmentation tend to introduce variability and noise into image analysis. Here, we report the development of two complementary segmentation methods, one semi-automated (iCRAQ) and one based on deep learning (Nucl.Eye.D), and their evaluation using a collection of A. thaliana nuclei with contrasted or poorly defined chromatin compartmentalization. Both methods allow for fast, robust and sensitive detection as well as for quantification of subtle nucleus features. Based on these developments, we highlight advantages of semi-automated and deep learning-based analyses applied to plant cytogenetics. Full article

One of the most popular tools for species discovery and resolution is the DNA barcode, typically based on the cytochrome c oxidase I (COI) gene. However, other non-genic barcodes are available for Diptera. The banding sequence of polytene chromosomes in some dipteran cells, particularly of the larval silk glands, can provide a unique species barcode. We used the sequence of bands to reveal a new species of black fly in the Simulium (Boreosimulium) annulus species group from California, USA. To further characterize the species and provide more integrated taxonomy, we morphologically described all life stages above the egg, formally named the species Simulium ustulatum n. sp., and provided a conventional COI barcode. The COI barcode confirmed the chromosomal and morphological evidence that the species is a new member of the S. annulus group, and enabled identification of the larva and female, which are structurally similar to those of other species. The chromosomal barcode shows that this species has the most rearranged complement, compared with the eight other North American members of its species group, with up to 12 times the number of fixed rearrangements. Up to six chromosomal rearrangements, including autosomal polymorphisms and sex-linked phenomena, are shared with other members of the group. The most unique and conspicuous chromosomal feature of this new species is a large, pale-staining chromocenter from which the six chromosomal arms radiate. The distribution of this univoltine species in lowland rivers of California’s Central Valley could make it vulnerable, given climate change and increasing land development. Full article

Although the pericentromeric regions of chromosomes that are enriched in tandemly repeated satellite DNA represent a significant part of eukaryotic genomes, they remain understudied, which is mainly due to interdisciplinary knowledge gaps. Recent studies suggest their important role in genome regulation, karyotype stability, and evolution. Thus, the idea of satellite DNA as a junk part of the genome has been refuted. The integration of data regarding molecular composition, chromosome behaviour, and the details of the in situ organization of pericentromeric regions is of great interest. The objective of this work was a cytogenetic analysis of the interactions between pericentromeric regions from non-homologous chromosomes in mouse spermatocytes using immuno-FISH. We analysed two events: the associations between centromeric regions of the X chromosome and autosomes and the associations between the centromeric regions of the autosomal bivalents that form chromocenters. We concluded that the X chromosome forms temporary synaptic associations with different autosomes in early meiotic prophase I, which can normally be found until the pachytene–diplotene, without signs of pachytene arrest. These associations are formed between the satellite-DNA-rich centromeric regions of the X chromosome and different autosomes but do not involve the satellite-DNA-poor centromeric region of the Y chromosome. We suggest the hypothetical model of X chromosome competitive replacement from such associations during synaptic correction. We showed that the centromeric region of the X chromosome in association remains free of γH2Ax-dependent chromatin inactivation, while the Y chromosome is completely inactivated. This finding highlights the predominant role of associations between satellite DNA-rich regions of different chromosomes, including the X chromosome. We suppose that X-autosomal transient associations are a manifestation of an additional synaptic disorder checkpoint. These associations are normally corrected before the late diplotene stage. We revealed that the intense spreading conditions that were applied to the spermatocyte I nuclei did not lead to the destruction of stretched chromatin fibers of elongated chromocenters enriched in satellite DNA. The tight associations that we revealed between the pericentromeric regions of different autosomal bivalents and the X chromosome may represent the basis for a mechanism for maintaining the repeats stability in the autosomes and in the X chromosome. The consequences of our findings are discussed. Full article

Robertsonian (centric-fusion) translocation is the form of chromosomal translocation in which two long arms of acrocentric chromosomes are fused to form one metacentric. These translocations reduce the number of chromosomes while preserving existing genes and are considered to contribute to speciation. We asked whether hypomorphic mutations in genes that disrupt the formation of pericentromeric regions could lead to centric fusion. TBP-related factor 2 (Trf2) encodes an alternative general transcription factor. A decrease of TRF2 expression disrupts the structure of the pericentromeric regions and prevents their association into chromocenter. We revealed several centric fusions in two lines of Drosophila melanogaster with weak Trf2 alleles in genetic experiments. We performed an RNAi-mediated knock-down of Trf2 in Drosophila and S2 cells and demonstrated that Trf2 upregulates expression of D1—one of the major genes responsible for chromocenter formation and nuclear integrity in Drosophila. Our data, for the first time, indicate that Trf2 may be involved in transcription program responsible for structuring of pericentromeric regions and may contribute to new karyotypes formation in particular by promoting centric fusion. Insight into the molecular mechanisms of Trf2 function and its new targets in different tissues will contribute to our understanding of its phenomenon. Full article

Leaves of Arabidopsis develop from a shoot apical meristem grow along three (proximal–distal, adaxial–abaxial, and medial–lateral) axes and form a flat symmetric architecture. ASYMMETRIC LEAVES2 (AS2), a key regulator for leaf adaxial–abaxial partitioning, encodes a plant-specific nuclear protein and directly represses the abaxial-determining gene ETTIN/AUXIN RESPONSE FACTOR3 (ETT/ARF3). How AS2 could act as a critical regulator, however, has yet to be demonstrated, although it might play an epigenetic role. Here, we summarize the current understandings of the genetic, molecular, and cellular functions of AS2. A characteristic genetic feature of AS2 is the presence of a number of (about 60) modifier genes, mutations of which enhance the leaf abnormalities of as2. Although genes for proteins that are involved in diverse cellular processes are known as modifiers, it has recently become clear that many modifier proteins, such as NUCLEOLIN1 (NUC1) and RNA HELICASE10 (RH10), are localized in the nucleolus. Some modifiers including ribosomal proteins are also members of the small subunit processome (SSUP). In addition, AS2 forms perinucleolar bodies partially colocalizing with chromocenters that include the condensed inactive 45S ribosomal RNA genes. AS2 participates in maintaining CpG methylation in specific exons of ETT/ARF3. NUC1 and RH10 genes are also involved in maintaining the CpG methylation levels and repressing ETT/ARF3 transcript levels. AS2 and nucleolus-localizing modifiers might cooperatively repress ETT/ARF3 to develop symmetric flat leaves. These results raise the possibility of a nucleolus-related epigenetic repression system operating for developmental genes unique to plants and predict that AS2 could be a molecule with novel functions that cannot be explained by the conventional concept of transcription factors. Full article

Methyl-CpG binding protein 2 (MeCP2) is a multi-function factor involved in locus-specific transcriptional modulation and the regulation of genome architecture, e.g., pericentric heterochromatin (PCH) organization. MECP2 mutations are responsible for Rett syndrome (RTT), a devastating postnatal neurodevelopmental disorder, the pathogenetic mechanisms of which are still unknown. MeCP2, together with Alpha-thalassemia/mental retardation syndrome X-linked protein (ATRX), accumulates at chromocenters, which are repressive PCH domains. As with MECP2, mutations in ATRX cause ATR-X syndrome which is associated with severe intellectual disability. We exploited two murine embryonic stem cell lines, in which the expression of MeCP2 or ATRX is abolished. Through immunostaining, chromatin immunoprecipitation and western blot, we show that MeCP2 and ATRX are reciprocally dependent both for their expression and targeting to chromocenters. Moreover, ATRX plays a role in the accumulation of members of the heterochromatin protein 1 (HP1) family at PCH and, as MeCP2, modulates their expression. Furthermore, ATRX and HP1 targeting to chromocenters depends on an RNA component. 3D-DNA fluorescence in situ hybridization (FISH) highlighted, for the first time, a contribution of ATRX in MeCP2-mediated chromocenter clustering during neural differentiation. Overall, we provide a detailed dissection of the functional interplay between MeCP2 and ATRX in higher-order PCH organization in neurons. Our findings suggest molecular defects common to RTT and ATR-X syndrome, including an alteration in PCH. Full article

Background: Genomes are non-randomly organized within the interphase nucleus; and spermatozoa are proposed to have a unique hairpin-loop configuration, which has been hypothesized to be critical for the ordered exodus of the paternal genome following fertilization. Recent studies suggest that the hairpin-loop model of sperm chromatin organization is more segmentally organized. The purpose of this study is to examine the 3D organization and hairpin-loop configurations of chromosomes in human spermatozoa. Methods: Three-color sperm-fluorescence in-situ hybridization was utilized against the centromeres, and chromosome p- and q-arms of eight chromosomes from five normozoospermic donors. Wide-field fluorescence microscopy and 3D modelling established the radial organization and hairpin-loop chromosome configurations in spermatozoa. Results: All chromosomes possessed reproducible non-random radial organization (p < 0.05) and formed discrete hairpin-loop configurations. However, chromosomes preferentially formed narrow or wide hairpin-loops. We did not find evidence to support the existence of a centralized chromocenter(s) with centromeres being more peripherally localized than one or both of their respective chromosome arms. Conclusion: This provides further evidence to support a more segmental organization of chromatin in the human sperm nucleus. This may be of significance for fertilization and early embryogenesis as specific genomic regions are likely to be exposed, remodeled, and activated first, following fertilization. Full article