Mouse centric and pericentric satellite repeats form distinct functional heterochromatin

Heterochromatin is thought to play a critical role for centromeric function. However, the respective contributions of the distinct repetitive sequences found in these regions, such as minor and major satellites in the mouse, have remained largely unsolved. We show that these centric and pericentric repeats on the chromosomes have distinct heterochromatic characteristics in the nucleus. Major satellites from different chromosomes form clusters associated with heterochromatin protein 1α, whereas minor satellites are individual entities associated with centromeric proteins. Both regions contain methylated histone H3 (Me-K9 H3) but show different micrococcal nuclease sensitivities. A dinucleosome repeating unit is found specifically associated with major satellites. These domains replicate asynchronously, and chromatid cohesion is sustained for a longer time in major satellites compared with minor satellites. Such prolonged cohesion in major satellites is lost in the absence of Suv39h histone methyltransferases. Thus, we define functionally independent centromeric subdomains, which spatio-temporal isolation is proposed to be important for centromeric cohesion and dissociation during chromosome segregation.

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Repetitive DNA landscape in essential A and supernumerary B chromosomes of Festuca pratensis Huds

Scientific Reports ◽

10.1038/s41598-019-56383-1 ◽

2019 ◽

Vol 9 (1) ◽

Author(s):

Rahman Ebrahimzadegan ◽

Andreas Houben ◽

Ghader Mirzaghaderi

Keyword(s):

Repetitive Sequences ◽

Nuclear Genome ◽

B Chromosome ◽

B Chromosomes ◽

Festuca Pratensis ◽

Ltr Retrotransposons ◽

Chromosome Identification ◽

Satellite Repeats ◽

Low Pass ◽

Illumina Sequence

AbstractHere, we characterized the basic properties of repetitive sequences in essential A and supernumerary B chromosomes of Festuca pratensis Huds. This was performed by comparative analysis of low-pass Illumina sequence reads of B chromosome lacking (−B) and B chromosome containing (+B) individuals of F. pratensis. 61% of the nuclear genome is composed of repetitive sequences. 43.1% of the genome are transposons of which DNA transposons and retrotransposons made up 2.3% and 40.8%, respectively. LTR retrotransposons are the most abundant mobile elements and contribute to 40.7% of the genome and divided into Ty3-gypsy and Ty1-copia super families with 32.97% and 7.78% of the genome, respectively. Eighteen different satellite repeats were identified making up 3.9% of the genome. Five satellite repeats were used as cytological markers for chromosome identification and genome analysis in the genus Festuca. Four satellite repeats were identified on B chromosomes among which Fp-Sat48 and Fp-Sat253 were specific to the B chromosome of F. pratensis.

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Cul4-Ddb1 ubiquitin ligases facilitate DNA replication-coupled sister chromatid cohesion through regulation of cohesin acetyltransferase Esco2

10.1101/414888 ◽

2018 ◽

Cited By ~ 1

Author(s):

Haitao Sun ◽

Jiaxin Zhang ◽

Jingjing Zhang ◽

Zhen Li ◽

Qinhong Cao ◽

...

Keyword(s):

Dna Replication ◽

Ubiquitin Ligase ◽

Sister Chromatid ◽

Critical Role ◽

E3 Ubiquitin Ligase ◽

Ubiquitin Ligases ◽

Sister Chromatid Cohesion ◽

Vital Role ◽

Chromatid Cohesion ◽

Evolutionarily Conserved

AbstractCohesin acetyltransferases Esco1 and Esco2 play a vital role in establishing sister chromatid cohesion. How Esco1 and Esco2 are controlled to achieve this in a DNA replication-coupled manner remains unclear in higher eukaryotes. Here we show that Cul4-RING ligases (CRL4s) play a critical role in sister chromatid cohesion in human cells. Depletion of Cul4A, Cul4B or Ddb1 subunits substantially reduces normal cohesion efficiency. We also show that Mms22L, a vertebrate ortholog of yeast Mms22, is one of Ddb1 and Cul4-associated factors (DCAFs) involved in cohesion. Several lines of evidence suggest a selective interaction of CRL4s with Esco2, but not Esco1. Depletion of either CRL4s or Esco2 causes a defect in Smc3 acetylation which can be rescued by HDAC8 inhibition. More importantly, both CRL4s and PCNA act as mediators for efficiently stabilizing Esco2 on chromatin and catalyzing Smc3 acetylation. Taken together, we propose an evolutionarily conserved mechanism in which CRL4s and PCNA regulate Esco2-dependent establishment of sister chromatid cohesion.Author summaryWe identified human Mms22L as a substrate specific adaptor of Cul4-Ddb1 E3 ubiquitin ligase. Downregulation of Cul4A, Cul4B or Ddb1 subunit causes reduction of acetylated Smc3, via interaction with Esco2 acetyltransferase, and then impairs sister chromatid cohesion in 293T cells. We found functional complementation between Cul4-Ddb1-Mms22L E3 ligase and Esco2 in Smc3 acetylation and sister chromatid cohesion. Interestingly, both Cul4-Ddb1 E3 ubiquitin ligase and PCNA contribute to Esco2 mediated Smc3 acetylation. To summarise, we demonstrated an evolutionarily conserved mechanism in which Cul4-Ddb1 E3 ubiquitin ligases and PCNA regulate Esco2-dependent establishment of sister chromatid cohesion.

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An efficient method to clone TAL effector genes from Xanthomonas oryzae using Gibson assembly

10.1101/723064 ◽

2019 ◽

Author(s):

Chenhao Li ◽

Chonghui Ji ◽

José C. Huguet-Tapia ◽

Frank F. White ◽

Hansong Dong ◽

...

Keyword(s):

Pathogenic Bacteria ◽

Critical Role ◽

Repetitive Sequences ◽

Xanthomonas Oryzae ◽

Transcription Activator ◽

Gibson Assembly ◽

Vector Systems ◽

Single Strand Annealing ◽

Strand Annealing

AbstractTALes (Transcription Activator-Like effectors) represent the largest family of type III effectors among pathogenic bacteria and play a critical role in the process of infection. Strains of Xanthomonas oryzae pv. oryzae (Xoo) and some strains of other Xanthomonas pathogens contain large numbers of TALe genes. Previous techniques to clone individual or a complement of TALe genes through conventional strategies are inefficient and time-consuming due to multiple genes (up to 29 copies) in a given genome and technically challenging due to the repetitive sequences (up to 33 nearly identical 102-nucleotide repeats) of individual TALe genes. Thus, only a limited number of TALe genes have been molecularly cloned and characterized, and the functions of most TALe genes remain unknown. Here, we present an easy and efficient cloning technique to clone TALe genes selectively through in vitro homologous recombination and single strand annealing and demonstrate the feasibility of this approach with four different Xoo strains. Based on the Gibson assembly strategy, two complementary vectors with scaffolds that can preferentially capture all TALe genes from a pool of genomic fragments were designed. Both vector systems enabled cloning of a full complement of TALe genes from each of four Xoo strains and functional analysis of individual TALes in rice in approximately one month compared to three months by previously used methods. The results demonstrate a robust tool to advance TALe biology and a potential for broad usage of this approach to clone multiple copies of highly competitive DNA elements in any genome of interest.

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S-Palmitoylation determines TMEM55B-dependent positioning of lysosomes

Journal of Cell Science ◽

10.1242/jcs.258566 ◽

2021 ◽

Author(s):

Sönke Rudnik ◽

Saskia Heybrock ◽

Paul Saftig ◽

Markus Damme

Keyword(s):

Molecular Motors ◽

Protein Complexes ◽

Critical Role ◽

Adaptor Protein ◽

Retrograde Transport ◽

Microtubule Motors ◽

Lysosomal Sorting ◽

Sorting Motif ◽

Spatio Temporal

The spatio-temporal cellular distribution of lysosomes depends on active transport mainly driven by microtubule-motors such as kinesins and dynein. Different protein complexes attach these molecular motors to their vesicular cargo: TMEM55B, as an integral lysosomal membrane protein, is a component of such a complex mediating the retrograde transport of lysosomes by establishing an interaction with the cytosolic scaffold protein JIP4 and dynein/dynactin. Here we show that TMEM55B and its paralog TMEM55A are S-palmitoylated proteins and lipidated at multiple cysteine-residues. Mutation of all cysteines in TMEM55B prevents S-palmitoylation and causes the retention of the mutated protein in the Golgi-apparatus. Consequently, non-palmitoylated TMEM55B is no longer able to modulate lysosomal positioning and the perinuclear clustering of lysosomes. Additional mutagenesis of the dileucine-based lysosomal sorting motif in non-palmitoylated TMEM55B leads to partial missorting to the plasma membrane instead of retention in the Golgi, implicating a direct effect of S-palmitoylation on the adaptor-protein-dependent sorting of TMEM55B. Our data suggest a critical role of S-palmitoylation on the trafficking of TMEM55B and TMEM55B-dependent lysosomal positioning.

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Mapping native R-loops genome-wide using a targeted nuclease approach

10.1101/457226 ◽

2018 ◽

Cited By ~ 1

Author(s):

Qingqing Yan ◽

Emily J. Shields ◽

Roberto Bonasio ◽

Kavitha Sarma

Keyword(s):

Affinity Purification ◽

Repetitive Sequences ◽

Mapping Method ◽

Rnase H ◽

Micrococcal Nuclease ◽

Genome Wide ◽

Input Material ◽

Prokaryotic Genomes ◽

High Level ◽

Nuclear Processes

AbstractR-loops are three-stranded DNA:RNA hybrids that are pervasive in the eukaryotic and prokaryotic genomes and have been implicated in a variety of nuclear processes, including transcription, replication, DNA repair, and chromosome segregation. While R-loops may have physiological roles, the formation of stable, aberrant R-loops has been observed in disease, particularly neurological disorders and cancer. Despite the importance of these structures, methods to assess their distribution in the genome invariably rely on affinity purification, which requires large amounts of input material, is plagued by high level of noise, and is poorly suited to capture dynamic and unstable R-loops. Here, we present a new method that leverages the affinity of RNase H for DNA:RNA hybrids to target micrococcal nuclease to genomic sites that contain R-loops, which are subsequently cleaved, released, and sequenced. Our R-loop mapping method, MapR, is as specific as existing techniques, less prone to recover non-specific repetitive sequences, and more sensitive, allowing for genome-wide coverage with low input material and read numbers, in a fraction of the time.

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Experimental and Bioinformatic upgrade for genome-wide mapping of nucleosomes in Trypanosoma cruzi

10.1101/2021.07.02.450927 ◽

2021 ◽

Author(s):

Paula Beati ◽

Milena Massimino Stepnicka ◽

Salome Vilchez Larrea ◽

Guillermo Daniel Alonso ◽

Josefina Ocampo

Keyword(s):

Trypanosoma Cruzi ◽

Cell Cycle Progression ◽

Repetitive Sequences ◽

Nucleosome Position ◽

Regulatory Elements ◽

Histone Variants ◽

Chromatin Organization ◽

Micrococcal Nuclease ◽

Cell Manipulation ◽

Genome Wide

In Trypanosoma cruzi, as in every eukaryotic cell, DNA is packaged into chromatin by octamers of histone proteins that constitute nucleosomes. Besides compacting DNA, nucleosomes control DNA dependent processes by modulating the access of DNA binding proteins to regulatory elements on the DNA; or by providing the platform for additional layers of regulation given by histone variants and histone post-translational modifications. In trypanosomes, protein coding genes are constitutively transcribed as polycistronic units. Therefore, gene expression is controlled mainly post transcriptionally. However, chromatin organization and the histone code influence transcription, cell cycle progression, replication and DNA repair. Hence, determining nucleosome position is of uppermost importance to understand the peculiarities of these processes in trypanosomes. Digestion of chromatin with micrococcal nuclease followed by deep sequencing has been widely applied for genome-wide mapping of nucleosomes in several organisms. Nonetheless, this parasite presents numerous singularities. On one hand, special growth conditions and cell manipulation are required. On the other hand, chromatin organization shows some uniqueness that demands a specially designed analytical approach. An additional entanglement is given by the nature of its genome harboring a large content of repetitive sequences and the poor quality of the genome assembly and annotation of many strains. Here, we adapted this broadly used method to the hybrid reference strain, CL Brener. Particularly, we developed an exhaustive and thorough computational workflow for data analysis, highlighting the relevance of using its whole genome as a reference instead of the commonly used Esmeraldo-like haplotype. Moreover, the performance of two aligners, Bowtie2 and HISAT2 was tested to find the most appropriate tool to map any genomic read to reference genomes bearing this complexity.

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Critical Role for Cold Shock Protein YB-1 in Cytokinesis

Cancers ◽

10.3390/cancers12092473 ◽

2020 ◽

Vol 12 (9) ◽

pp. 2473 ◽

Cited By ~ 1

Author(s):

Sunali Mehta ◽

Michael Algie ◽

Tariq Al-Jabry ◽

Cushla McKinney ◽

Srinivasaraghavan Kannan ◽

...

Keyword(s):

Cold Shock ◽

Cleavage Plane ◽

Critical Role ◽

Temporal Distribution ◽

Confocal Imaging ◽

Cold Shock Protein ◽

Atomistic Modelling ◽

Protein Partners ◽

Spatio Temporal ◽

Chromosome Passenger Complex

High levels of the cold shock protein Y-box-binding protein-1, YB-1, are tightly correlated with increased cell proliferation and progression. However, the precise mechanism by which YB-1 regulates proliferation is unknown. Here, we found that YB-1 depletion in several cancer cell lines and in immortalized fibroblasts resulted in cytokinesis failure and consequent multinucleation. Rescue experiments indicated that YB-1 was required for completion of cytokinesis. Using confocal imaging we found that YB-1 was essential for orchestrating the spatio-temporal distribution of the microtubules, β-actin and the chromosome passenger complex (CPC) to define the cleavage plane. We show that phosphorylation at six serine residues was essential for cytokinesis, of which novel sites were identified using mass spectrometry. Using atomistic modelling we show how phosphorylation at multiple sites alters YB-1 conformation, allowing it to interact with protein partners. Our results establish phosphorylated YB-1 as a critical regulator of cytokinesis, defining precisely how YB-1 regulates cell division.

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Mitotic centromeric targeting of HP1 and its binding to Sgo1 are dispensable for sister-chromatid cohesion in human cells

Molecular Biology of the Cell ◽

10.1091/mbc.e11-01-0009 ◽

2011 ◽

Vol 22 (8) ◽

pp. 1181-1190 ◽

Cited By ~ 56

Author(s):

Jungseog Kang ◽

Jaideep Chaudhary ◽

Hui Dong ◽

Soonjoung Kim ◽

Chad A. Brautigam ◽

...

Keyword(s):

Sister Chromatid ◽

Sister Chromatid Cohesion ◽

Human Cells ◽

Structural Studies ◽

Heterochromatin Protein 1 ◽

Heterochromatin Protein ◽

Chromatid Cohesion ◽

A Subunit ◽

Chromosome Passenger Complex ◽

Chromo Shadow Domain

Human Shugoshin 1 (Sgo1) protects centromeric sister-chromatid cohesion during prophase and prevents premature sister-chromatid separation. Heterochromatin protein 1 (HP1) has been proposed to protect centromeric sister-chromatid cohesion by directly targeting Sgo1 to centromeres in mitosis. Here we show that HP1α is targeted to mitotic centromeres by INCENP, a subunit of the chromosome passenger complex (CPC). Biochemical and structural studies show that both HP1–INCENP and HP1–Sgo1 interactions require the binding of the HP1 chromo shadow domain to PXVXL/I motifs in INCENP or Sgo1, suggesting that the INCENP-bound, centromeric HP1α is incapable of recruiting Sgo1. Consistently, a Sgo1 mutant deficient in HP1 binding is functional in centromeric cohesion protection and localizes normally to centromeres in mitosis. By contrast, INCENP or Sgo1 mutants deficient in HP1 binding fail to localize to centromeres in interphase. Therefore, our results suggest that HP1 binding by INCENP or Sgo1 is dispensable for centromeric cohesion protection during mitosis of human cells, but might regulate yet uncharacterized interphase functions of CPC or Sgo1 at the centromeres.

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The Large Isoform of Drosophila melanogaster Heterochromatin Protein 2 Plays a Critical Role in Gene Silencing and Chromosome Structure

Genetics ◽

10.1534/genetics.106.057604 ◽

2006 ◽

Vol 174 (3) ◽

pp. 1189-1204 ◽

Cited By ~ 14

Author(s):

Christopher D. Shaffer ◽

Giovanni Cenci ◽

Brandi Thompson ◽

Gena E. Stephens ◽

Elizabeth E. Slawson ◽

...

Keyword(s):

Drosophila Melanogaster ◽

Gene Silencing ◽

Chromosome Structure ◽

Critical Role ◽

Heterochromatin Protein

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Research on the Spatio-Temporal Impacts of Environmental Factors on the Fresh Agricultural Product Supply Chain and the Spatial Differentiation Issue—An Empirical Research on 31 Chinese Provinces

International Journal of Environmental Research and Public Health ◽

10.3390/ijerph182212141 ◽

2021 ◽

Vol 18 (22) ◽

pp. 12141

Author(s):

Xuemei Fan ◽

Ziyue Nan ◽

Yuanhang Ma ◽

Yingdan Zhang ◽

Fei Han

Keyword(s):

Supply Chain ◽

Environmental Factors ◽

Critical Role ◽

Generalized Method Of Moments ◽

Spatial Differentiation ◽

Weighted Regression ◽

Agricultural Product ◽

Entropy Weight ◽

Comprehensive Development ◽

Spatio Temporal

Environmental factors in time and space play a critical role in advancing the sustainable development of the fresh agricultural product supply chain. This paper, availing the panel data of 31 Chinese provinces from 2008 to 2019, constructs a system of indicators assessing the development of the fresh agricultural product supply chain, and obtains the comprehensive development level in the Entropy Weight Method (EWM). Furthermore, it establishes a comparison between optimal solutions generated by the Instrumental Variables Method (IVM) and the Generalized Method of Moments (GMM) over the endogeneity issue of variables, creates the comparison between the weighted regression methods of Geographically Weighted Regression (GWR) and Multi-scale Geographic Weighted Regression (MGWR), and obtains the relationship among the 14 environmental factors in their spatio-temporal impacts on the development of the fresh agricultural product supply chain. The results indicate that: (1) the environmental influencing factors in this paper have significant endogenous problems and various environmental factors impact on the fresh agricultural product supply chain in different trends and to different degrees. (2) With different bandwidths, the environmental factors could impact the fresh agricultural product supply chain to greatly varied degrees, demonstrating a strong attribute of regional correlation.

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