Journal: bioRxiv

Article Title: Natural variation modifies centromere-proximal meiotic crossover frequency and segregation distortion in Arabidopsis thaliana

doi: 10.1101/2025.01.04.631303

Figure Lengend Snippet: For each chromosome in the Col-CEN assembly , we plotted the position of the FTL red-encoding (red triangles) and green-encoding (green circles) T-DNAs. Beneath we show density plots for (i) CEN178 satellite repeats (green), CENH3 ChIP-seq enrichment (purple), and (ii) DNA methylation in CG (dark red), CHG (orange) and CHH (yellow) sequence contexts [ , ].

Article Snippet: The chromatin was incubated overnight at 4°C with 50 µl Protein A magnetic beads (Dynabeads, Thermo Fisher) pre-bound with either 5 µl α-CENH3 raised to peptide acetyl-RTK HRV TRS QPR NQT DAC-amide (Eurogentec).

Techniques: ChIP-sequencing, DNA Methylation Assay, Sequencing

Journal: bioRxiv

Article Title: Natural variation modifies centromere-proximal meiotic crossover frequency and segregation distortion in Arabidopsis thaliana

doi: 10.1101/2025.01.04.631303

Figure Lengend Snippet: A. Chromosome ideogram showing the location of FTL T-DNAs used in this study, together with the position of the CEN178 satellite repeat arrays (dark grey), against the Col-CEN genome assembly . Red and green triangles indicate the location of FTL T-DNAs expressing RFP and GFP in seed from the napA promoter, respectively [ , ]. B. Crossover frequency (centiMorgan) values of individual replicates of F 1 hybrids between the accession listed on the x-axis, and the CEN1 FTL reporters in a Col-0 background. Mean values are shown in orange diamonds. The inbred Col-0/Col-0-FTL control is highlighted in green, via the horizontal dashed line. Stars at the top of the plot indicate hybrids with significantly different crossover frequencies to the inbred strain based on Chi-square tests. Beneath, the same data are analysed for the ratios of green (circles, short dashed line) and red fluorescent (triangles, long dashed line) seed to non-fluorescent seed, and presented in the same way. C. Sequence similarity dot plots comparing the CEN178 arrays from a subset of the accessions analyzed in B. Red and blue shading indicate sequence similarity using a 161 base pair window. Beneath the dotplot, the cM value for the corresponding CEN1 hybrid from B. is shown, which is proportionally shaded. Additionally, beneath the dot plot are text indicators showing which accessions showed segregation distortion in B. D. For the Col-0 and Ler-0 Eurasian, and Cvi-0 and Tanz-1 relict accessions, we show the CENH3 ChIP-seq enrichment (upper, purple), and DNA methylation (lower) in CG (dark pink), CHG (pink) and CHH (light pink) contexts in the CEN1 region. The position of CEN178 arrays are indicated by ticks on the x-axis on the forward (red) or reverse (blue) strand.

Article Snippet: The chromatin was incubated overnight at 4°C with 50 µl Protein A magnetic beads (Dynabeads, Thermo Fisher) pre-bound with either 5 µl α-CENH3 raised to peptide acetyl-RTK HRV TRS QPR NQT DAC-amide (Eurogentec).

Techniques: Expressing, Control, Sequencing, ChIP-sequencing, DNA Methylation Assay

Journal: bioRxiv

Article Title: Natural variation modifies centromere-proximal meiotic crossover frequency and segregation distortion in Arabidopsis thaliana

doi: 10.1101/2025.01.04.631303

Figure Lengend Snippet: A. Chromosome ideogram showing the location of FTL T-DNAs used in this study, together with the position of the CEN178 satellite repeat arrays (dark grey), against the Col-CEN genome assembly . Red and green triangles indicate the location of FTL T-DNAs expressing RFP and GFP in seed from the napA promoter, respectively [ , ]. B. Crossover frequency (centiMorgan) values of individual replicates of F 1 hybrids between the accession listed on the x-axis, and the CEN2 FTL reporters in a Col-0 background. Mean values are shown in orange diamonds. The inbred Col-0/Col-0-FTL control is highlighted in green, via the horizontal dashed line. Stars at the top of the plot indicate hybrids with significantly different crossover frequencies to the inbred strain based on Chi-square tests. Beneath, the same data are analysed for the ratios of green (circles, short dashed line) and red (triangles, long dashed line) fluorescent seed to non-fluorescent seed, and presented in the same way. C. Sequence similarity dot plots comparing the CEN178 arrays from a subset of the accessions analyzed in B. Red and blue shading indicate sequence similarity using a 161 base pair window. Beneath the dotplot, the cM value for the corresponding CEN2 hybrid from B. is shown, which is proportionally shaded. Additionally, beneath the dot plot are text indicators showing which accessions showed segregation distortion in B. D. For the Col-0 and Ler-0 Eurasian, and Cvi-0 and Tanz-1 relict accessions, we show the CENH3 ChIP-seq enrichment (upper, purple), and DNA methylation (lower) in CG (dark pink), CHG (pink) and CHH (light pink) contexts in the CEN2 region. The position of CEN178 arrays are indicated by ticks on the x-axis on the forward (red) or reverse (blue) strand.

Article Snippet: The chromatin was incubated overnight at 4°C with 50 µl Protein A magnetic beads (Dynabeads, Thermo Fisher) pre-bound with either 5 µl α-CENH3 raised to peptide acetyl-RTK HRV TRS QPR NQT DAC-amide (Eurogentec).

Techniques: Expressing, Control, Sequencing, ChIP-sequencing, DNA Methylation Assay

Journal: bioRxiv

Article Title: Natural variation modifies centromere-proximal meiotic crossover frequency and segregation distortion in Arabidopsis thaliana

doi: 10.1101/2025.01.04.631303

Figure Lengend Snippet: A. Chromosome ideogram showing the location of FTL T-DNAs used in this study, together with the position of the CEN178 satellite repeat arrays (dark grey), against the Col-CEN genome assembly . Red and green triangles indicate the location of FTL T-DNAs expressing RFP and GFP in seed from the napA promoter, respectively [ , ]. B. Crossover frequency (centiMorgan) values of individual replicates of F 1 hybrids between the accession listed on the x-axis, and the CEN3 FTL reporters in a Col-0 background. Mean values are shown in orange diamonds. The inbred Col-0/Col-0-FTL control is highlighted in green, via the horizontal dashed line. Stars at the top of the plot indicate hybrids with significantly different crossover frequencies to the inbred strain based on Chi-square tests. Beneath, the same data are analysed for the ratios of green (circles, short dashed line) and red (triangles, long dashed line) fluorescent seed to non-fluorescent seed, and presented in the same way. C. Sequence similarity dot plots comparing the CEN178 arrays from a subset of the accessions analyzed in B. Red and blue shading indicate sequence similarity using a 161 base pair window. Beneath the dotplot, the cM value for the corresponding CEN3 hybrid from B. is shown, which is proportionally shaded. Additionally, beneath the dot plot are text indicators showing which accessions showed segregation distortion in B. D. For the Col-0 and Ler-0 Eurasian, and Cvi-0 and Tanz-1 relict accessions, we show the CENH3 ChIP-seq enrichment (upper, purple), and DNA methylation (lower) in CG (dark pink), CHG (pink) and CHH (light pink) contexts in the CEN3 region. The position of CEN178 arrays are indicated by ticks on the x-axis on the forward (red) or reverse (blue) strand.

Article Snippet: The chromatin was incubated overnight at 4°C with 50 µl Protein A magnetic beads (Dynabeads, Thermo Fisher) pre-bound with either 5 µl α-CENH3 raised to peptide acetyl-RTK HRV TRS QPR NQT DAC-amide (Eurogentec).

Techniques: Expressing, Control, Sequencing, ChIP-sequencing, DNA Methylation Assay

Journal: bioRxiv

Article Title: Natural variation modifies centromere-proximal meiotic crossover frequency and segregation distortion in Arabidopsis thaliana

doi: 10.1101/2025.01.04.631303

Figure Lengend Snippet: A. Chromosome ideogram showing the location of FTL T-DNAs used in this study, together with the position of the CEN178 satellite repeat arrays (dark grey), against the Col-CEN genome assembly . Red and green triangles indicate the location of FTL T-DNAs expressing RFP and GFP in seed from the napA promoter, respectively [ , ]. B. Crossover frequency (centiMorgan) values of individual replicates of F 1 hybrids between the accession listed on the x-axis, and the CEN4 FTL reporters in a Col-0 background. Mean values are shown in orange diamonds. The inbred Col-0/Col-0-FTL control is highlighted in green, via the horizontal dashed line. Stars at the top of the plot indicate hybrids with significantly different crossover frequencies to the inbred strain based on Chi-square tests. Beneath, the same data are analysed for the ratios of green (circles, short dashed line) and red (triangles, long dashed line) fluorescent seed to non-fluorescent seed, and presented in the same way. C. Sequence similarity dot plots comparing the CEN178 arrays from a subset of the accessions analyzed in B. Red and blue shading indicate sequence similarity using a 161 base pair window. Beneath the dotplot, the cM value for the corresponding CEN4 hybrid from B. is shown, which is proportionally shaded. Additionally, beneath the dot plot are text indicators showing which accessions showed segregation distortion in B. D. For the Col-0 and Ler-0 Eurasian, and Cvi-0 and Tanz-1 relict accessions, we show the CENH3 ChIP-seq enrichment (upper, purple), and DNA methylation (lower) in CG (dark pink), CHG (pink) and CHH (light pink) contexts in the CEN4 region. The position of CEN178 arrays are indicated by ticks on the x-axis on the forward (red) or reverse (blue) strand.

Article Snippet: The chromatin was incubated overnight at 4°C with 50 µl Protein A magnetic beads (Dynabeads, Thermo Fisher) pre-bound with either 5 µl α-CENH3 raised to peptide acetyl-RTK HRV TRS QPR NQT DAC-amide (Eurogentec).

Techniques: Expressing, Control, Sequencing, ChIP-sequencing, DNA Methylation Assay

Journal: bioRxiv

Article Title: Natural variation modifies centromere-proximal meiotic crossover frequency and segregation distortion in Arabidopsis thaliana

doi: 10.1101/2025.01.04.631303

Figure Lengend Snippet: A. Chromosome ideogram showing the location of FTL T-DNAs used in this study, together with the position of the CEN178 satellite repeat arrays (dark grey), against the Col-CEN genome assembly . Red and green triangles indicate the location of FTL T-DNAs expressing RFP and GFP in seed from the napA promoter, respectively [ , ]. B. Crossover frequency (centiMorgan) values of individual replicates of F 1 hybrids between the accession listed on the x-axis, and the CEN5 FTL reporters in a Col-0 background. Mean values are shown in orange diamonds. The inbred Col-0/Col-0-FTL control is highlighted in green, via the horizontal dashed line. Stars at the top of the plot indicate hybrids with significantly different crossover frequencies to the inbred strain based on Chi-square tests. Beneath, the same data are analysed for the ratios of green (circles, short dashed line) and red (triangles, long dashed line) fluorescent seed to non-fluorescent seed, and presented in the same way. C. Sequence similarity dot plots comparing the CEN178 arrays from a subset of the accessions analyzed in B. Red and blue shading indicate sequence similarity using a 161 base pair window. Beneath the dotplot, the cM value for the corresponding CEN5 hybrid from B. is shown, which is proportionally shaded. Additionally, beneath the dot plot are text indicators showing which accessions showed segregation distortion in B. D. For the Col-0 and Ler-0 Eurasian, and Cvi-0 and Tanz-1 relict accessions, we show the CENH3 ChIP-seq enrichment (upper, purple), and DNA methylation (lower) in CG (dark pink), CHG (pink) and CHH (light pink) contexts in the CEN5 region. The position of CEN178 arrays are indicated by ticks on the x-axis on the forward (red) or reverse (blue) strand.

Article Snippet: The chromatin was incubated overnight at 4°C with 50 µl Protein A magnetic beads (Dynabeads, Thermo Fisher) pre-bound with either 5 µl α-CENH3 raised to peptide acetyl-RTK HRV TRS QPR NQT DAC-amide (Eurogentec).

Techniques: Expressing, Control, Sequencing, ChIP-sequencing, DNA Methylation Assay

Journal: bioRxiv

Article Title: Natural variation modifies centromere-proximal meiotic crossover frequency and segregation distortion in Arabidopsis thaliana

doi: 10.1101/2025.01.04.631303

Figure Lengend Snippet: Protein sequences of the CENH3 coding region were extracted from the 13 studied Arabidopsis accessions, and A. lyrata (MN47), which contains two CENH3 paralogues (Al1G11082 and Al1G59290) . MAFFT ( https://github.com/GSLBiotech/mafft ) was used to align the protein sequences and snipit ( https://github.com/aineniamh/snipit ) was used for visualisation. Tick marks across the protein sequence represent polymorphisms compared to the reference Col-0 CENH3 sequence, and above is shown the reference amino acid. The position of the polymorphism and the amino acid change is shown at the top of the graph.

Article Snippet: The chromatin was incubated overnight at 4°C with 50 µl Protein A magnetic beads (Dynabeads, Thermo Fisher) pre-bound with either 5 µl α-CENH3 raised to peptide acetyl-RTK HRV TRS QPR NQT DAC-amide (Eurogentec).

Techniques: Sequencing

Journal: bioRxiv

Article Title: Natural variation modifies centromere-proximal meiotic crossover frequency and segregation distortion in Arabidopsis thaliana

doi: 10.1101/2025.01.04.631303

Figure Lengend Snippet: A. Diagram representing the developing female ovule in Arabidopsis. A sub-epidermal cell becomes specified to enter meiosis, and undergoes two cell divisions to produce a tetrad of haploid spores. Three of the megaspores undergo programmed cell death, and the surviving cell tends to be the innermost spore [ , ]. The surviving megaspore then divides mitotically and differentiates into the female gametophyte, which contains the egg cell [ , ]. B. In early prophase-I in the sub-epidermal cell that is specified to enter meiosis, the homologous chromosomes (red and blue) replicate and are held together by cohesin and the meiotic axis. During prophase I, the replicated chromosomes experience DNA double strand breaks (DSBs, stars) that initiate meiotic recombination. As cell division progresses, the replicated homologs pair to form a four-strand bivalent structure, and a subset of the DSBs are repaired using a homologous chromatid to form crossovers (black crosses). At this stage we propose there is an asymmetry in the spindle microtubules, either in number or arrangement (green). Due to centromere structural heterozygosity, we propose that the synapsed homologs form a buckle where the sequence is hemizygous. Therefore, the relative positioning of the CENH3 chromatin (ovals) is offset between the homologs in the bivalent. In this case, this synapse-buckle and CENH3-offset cause a biased association of the purple centromeres with the lower spindle. The chromosomes then segregate through meiosis-I and meiosis-II, and because of the initial bias, the blue centromere has a higher chance of segregating into the surviving female spore that escapes programmed cell death and differentiates into the female gametophyte and egg cell.

Article Snippet: The chromatin was incubated overnight at 4°C with 50 µl Protein A magnetic beads (Dynabeads, Thermo Fisher) pre-bound with either 5 µl α-CENH3 raised to peptide acetyl-RTK HRV TRS QPR NQT DAC-amide (Eurogentec).

Techniques: Sequencing

Journal: Breast Cancer Research : BCR

Article Title: Characterization of novel small molecule inhibitors of estrogen receptor-activation function 2 (ER-AF2)

doi: 10.1186/s13058-024-01926-2

Figure Lengend Snippet: Activity profile of VPC-260724. ( A ) Binding pose of VPC-260724 (cyan) in the AF2 site, predicted by computational docking. The compound occupies the hydrophobic cavity with the pyridine and the imidazopyridine moieties mimicking L694 and L690 residues (in bold italic) of the AF2-binding coactivator and interacting with hydrophobic and aromatic residues lining the AF2 binding site. Green = hydrophobic surfaces, purple = polar surfaces, red = solvent exposed. ( B ) A salt bridge is established between the imidazopyridine group and the side chain of Glu542 of the AF2 site. Proximity ligation assay to determine the effect of lead compound VPC-260724 on the interaction between ER and coactivator SRC3 in MCF7 ( C ) and TamR3 ( E ) cells. Interactions between ER and SRC3 are represented by the red PLA signals. 10 µM treatment of VPC-260724 for 48 h significantly reduced the number of PLA signals per nuclei in both MCF7 ( D ) and TamR3 ( F ) cells following starvation for 4 days and stimulation with 1 nM E2. ( G ) Dose response effect of lead compound VPC-260724 on area of MCF7 and TamR3 spheroids. Cells were starved for 3 days, and spheroids were formed in a round-bottom low-binding 96-well plate for 24 h in starvation media before treatment with a serial 1:2 dilution of VPC-260724 starting at 50 µM. ( H ) Quantification of the dose-dependent inhibitory effect of VPC-260724 on the size of spheroids. ( I ) qPCR showing reduction of ER-target genes mRNA levels. Treatment of MCF7 and TamR3 cells by 10 µM VPC-260724 for 72 h reduced mRNA levels of ER targets: CyclinD1, CDC2, E2F, PR and pS2. P values are indicated by stars: ns ≥ 0.05, * 0.01 to 0.05, ** 0.001 to 0.01, *** 0.0001 to 0.001, **** <0.0001

Article Snippet: We performed a TR-FRET assay to assess whether compounds displace the AF2 peptide from the ER-AF2 pocket using the LanthaScreen TR-FRET ER alpha Coactivator Assay Kit (Thermo Fisher, A15885).

Techniques: Activity Assay, Binding Assay, Solvent, Proximity Ligation Assay

Journal: Breast Cancer Research : BCR

Article Title: Characterization of novel small molecule inhibitors of estrogen receptor-activation function 2 (ER-AF2)

doi: 10.1186/s13058-024-01926-2

Figure Lengend Snippet: Profiling the activity of VPC-260724. ( A ) Proximity ligation assay to determine the effect of lead compound VPC-260724 on the interaction between ER and coactivator SRC3 in TamR3 cells. The cells were starved in CSS media without Tamoxifen for 4 days before treated with either 1 µM Tamoxifen alone or with 10 µM VPC-260724 and 1 µM Tamoxifen simultaneously. Interactions between ER and SRC3 are represented by the red PLA signals. ( B ) 10µM treatment of VPC-260724 significantly reduced the number of PLA signals per nuclei. ( C ) qPCR showing reduction of ER-target genes cyclinD1, CDC2, E2F, PR and pS2 mRNA levels after 72 h of 10 µM treatment of VPC-260724 in the presence of 1 µM Tamoxifen. ( D ) VPC-260724 is specific for ER, tested at indicated concentrations in luciferase assay transiently transfecting ER, AR, GR and PR with a luciferase reporter plasmid. P values are indicated by stars: ns ≥ 0.05, * 0.01 to 0.05, ** 0.001 to 0.01, *** 0.0001 to 0.001, **** <0.0001

Article Snippet: We performed a TR-FRET assay to assess whether compounds displace the AF2 peptide from the ER-AF2 pocket using the LanthaScreen TR-FRET ER alpha Coactivator Assay Kit (Thermo Fisher, A15885).

Techniques: Activity Assay, Proximity Ligation Assay, Luciferase, Plasmid Preparation