Journal: Nucleic Acids Research

Article Title: Phosphorylation of Barrier-to-Autointegration Factor (BAF) regulates anchoring of centromeric heterochromatin to the nuclear envelope (NE)

doi: 10.1093/nar/gkag021

Figure Lengend Snippet: Impaired BAF phosphorylation reinforces heterochromatin anchoring. ( A ) Immunostaining with αLamin antibodies (in cyan) in stable S2 cells co-expressing mCherry::HP1a (in red) and either N-BAF, C-BAF, or N-BAF T4A/S5A . mCherry::HP1a signal is direct fluorescence. Scale bars are 2 μm. ( B ) Quantification of the results shown in panel (A). The proportion of HP1a foci in the peripheral, intermediate, and central nuclear regions is shown for cells expressing N-BAF, C-BAF, or N-BAF T4A/S5A (see “Materials and methods” section for details). As a control, a similar quantification was performed in S2 cells after co-immunostaining with αLamin and αHP1a antibodies. Results are the sum of two independent experiments. ( N = 91 for N-BAF, 93 for C-BAF, and 111 for N-BAF T4A/S5A ; P -values with respect to control: ns > 0.05, ***** < 0.00001; two-tailed paired Student’s t -test). ( C ) Quantification of the results shown in panel (A). The number of HP1a foci per nucleus is shown for cells expressing N-BAF, C-BAF, or N-BAF T4A/S5A . Results are the sum of two independent experiments. Error bars are SD. ( N = 35 for N-BAF, 34 for C-BAF, and 33 for N-BAF T4A/S5A ; P -values with respect to control: ***** < .00001; two-tailed paired Student’s t -test). ( D ) ChIP-seq genomic profiling of endogenous BAF, N-BAF, C-BAF, and N-BAF T4A/S5A . For endogenous BAF, ChIP was performed with αBAF antibodies in Drosophila S2 cells. For N-BAF, C-BAF, and N-BAF T4A/S5A , ChIPs were performed with αGFP antibodies in stable S2 cells expressing the corresponding BAF forms. The IP/Input coverage ratio is presented for two representative genomic regions and compared to Lamin (GEO GSM509086 ) . LADs are indicated in gray. The Spearman correlation coefficients respect to Lamin for the whole genome are also indicated.

Article Snippet: The rest of antibodies used are commercially available: mouse monoclonal αHP1a (DSHB C1A9), mouse monoclonal αLaminDm0 (DSHB ADL67.10), rabbit polyclonal αGFP (Invitrogen A11122), mouse polyclonal αGFP (Roche 118144600), rabbit polyclonal αFlag (Merck Life Science F7425), rabbit polyclonal αH3 (Cell Signaling 9715), rabbit polyclonal αH3K9me3 (Millipore 67–442), rabbit polyclonal αH3PS10 (Cell Signaling 9701), and mouse monoclonal αTubulin (MAB3408 Millipore).

Techniques: Phospho-proteomics, Immunostaining, Expressing, Fluorescence, Control, Two Tailed Test, ChIP-sequencing

Journal: Nucleic Acids Research

Article Title: Phosphorylation of Barrier-to-Autointegration Factor (BAF) regulates anchoring of centromeric heterochromatin to the nuclear envelope (NE)

doi: 10.1093/nar/gkag021

Figure Lengend Snippet: Non-phosphorylatable BAF forms compromise NE integrity. ( A ) Images of metaphase cells expressing N-BAF, C-BAF, or N-BAF T4A/S5A . N-BAF, C-BAF, and N-BAF T4A/S5A signals are direct fluorescence (in green). DNA was stained with DAPI (in gray). Scale bars are 2 μm. ( B ) In vivo time-lapse recordings of a stable S2 cell co-expressing N-BAF (in green) and mCherry::N-BAF T4A/S5A (in red) that is undergoing mitosis. Time is in minutes. Scale bars are 5 μm. ( C ) Immunostaining with αLamin antibodies (in cyan) of stable S2 cells co-expressing mCherry::HP1a (in red) and either N-BAF (left), C-BAF (center), or N-BAF T4A/S5A (right). mCherry::HP1a signal is direct fluorescence. Scale bars are 2 μm. ( D ) Quantification of the results shown in panel (C). The proportion of cells showing NE defects is presented for cells expressing the indicated BAF constructs. ( N = 323 for N-BAF, 360 for C-BAF, and 341 for N-BAF T4A/S5A ; P -values with respect to N-BAF expressing cells: ***** < .00 001; Fisher’s exact test). ( E ) Immunostaining with αLamin antibodies (in red) of stable S2 cells expressing N-BAF (top), C-BAF (center), or N-BAF T4A/S5A (bottom). Representative images of NE regions showing foci of the corresponding BAF forms are presented. N-BAF, C-BAF, and N-BAF T4A/S5A signals (in green) are direct fluorescence. Scale bars are 0.5 μm. ( F ) Quantification of the results shown in panel (E). Box plots showing median αLamin intensity at NE regions showing N-BAF, C-BAF, or N-BAF T4A/S5A foci relative to the median αLamin intensity across the entire NE (see “Materials and methods” section for details). Boxes represent the median and IQR, whiskers are 1.5 × IQR. ( N = 168 for N-BAF, 119 for C-BAF, and 149 for N-BAF T4A/S5A ; P -values: ***** < .00 001; two-tailed paired Student’s t -test).

Article Snippet: The rest of antibodies used are commercially available: mouse monoclonal αHP1a (DSHB C1A9), mouse monoclonal αLaminDm0 (DSHB ADL67.10), rabbit polyclonal αGFP (Invitrogen A11122), mouse polyclonal αGFP (Roche 118144600), rabbit polyclonal αFlag (Merck Life Science F7425), rabbit polyclonal αH3 (Cell Signaling 9715), rabbit polyclonal αH3K9me3 (Millipore 67–442), rabbit polyclonal αH3PS10 (Cell Signaling 9701), and mouse monoclonal αTubulin (MAB3408 Millipore).

Techniques: Expressing, Fluorescence, Staining, In Vivo, Immunostaining, Construct, Two Tailed Test

Journal: Nucleic Acids Research

Article Title: Phosphorylation of Barrier-to-Autointegration Factor (BAF) regulates anchoring of centromeric heterochromatin to the nuclear envelope (NE)

doi: 10.1093/nar/gkag021

Figure Lengend Snippet: BAF associates with centromeric heterochromatin. ( A ) Immuno-PLA with αHP1a antibodies and either αBAF (top), αH3K9me3 (center), or αGFP (bottom) antibodies in S2 cells. The PLA signal is shown in red. Immunostaining with αHP1a antibodies is shown in cyan. DNA was stained with DAPI (in gray). In the top and center panels, enlarged images of representative nuclei show the overlap of PLA and αHP1a signals. Scale bars are 10 μm (in the top, center, and bottom panels) and 2 μm (in the enlarged images). ( B ) Quantification of the results shown in panel (C). The percentages of PLA-positive cells are presented for assays performed with αHP1a antibodies and either αGFP, αH3K9me3, or αBAF antibodies. Results are the sum of two independent experiments. ( N = 224 for αGFP, 228 for αH3K9me3, and 230 for αBAF; P -values: ns > 0.05; *** < 0.001; Fisher’s exact test).

Article Snippet: The rest of antibodies used are commercially available: mouse monoclonal αHP1a (DSHB C1A9), mouse monoclonal αLaminDm0 (DSHB ADL67.10), rabbit polyclonal αGFP (Invitrogen A11122), mouse polyclonal αGFP (Roche 118144600), rabbit polyclonal αFlag (Merck Life Science F7425), rabbit polyclonal αH3 (Cell Signaling 9715), rabbit polyclonal αH3K9me3 (Millipore 67–442), rabbit polyclonal αH3PS10 (Cell Signaling 9701), and mouse monoclonal αTubulin (MAB3408 Millipore).

Techniques: Immunostaining, Staining

Journal: Nucleic Acids Research

Article Title: Phosphorylation of Barrier-to-Autointegration Factor (BAF) regulates anchoring of centromeric heterochromatin to the nuclear envelope (NE)

doi: 10.1093/nar/gkag021

Figure Lengend Snippet: The localization patterns of N-BAF and C-BAF. ( A ) The pattern of localization of N-BAF is determined by direct fluorescence in stable S2 cells co-expressing N-BAF (in green) and mCherry::HP1a (in red). Scale bars are 2 μm. ( B ) As in panel (A), but for C-BAF in stable S2 cells co-expressing C-BAF (in green) and mCherry::HP1a (in red). Scale bars are 2 μm. ( C ) Quantification of the results shown in panels (A) and (B). Box plots of the fluorescence intensity at the perinuclear ring relative to centromeric heterochromatin are presented for N-BAF and C-BAF expressing cells. Boxes represent the median and interquartile range (IQR), whiskers are 1.5 × IQR. Results are the sum of three independent experiments. ( N = 42 for N-BAF and C-BAF; P -value: **** < .0001; unpaired Student’s t -test). ( D ) Immuno-PLA with αHP1a antibodies and αGFP antibodies in stable S2 cells expressing N-BAF (top) or C-BAF (bottom). The PLA signal is shown in red. Immunostaining with αHP1a antibodies is shown in cyan. DNA was stained with DAPI (in gray). Scale bars are 10 μm. ( E ) Quantification of the results shown in panel (D). The percentages of PLA-positive cells are presented for assays performed with αHP1a antibodies and αGFP in stable S2 cells expressing N-BAF (green) or C-BAF (gray). The percentage of αHP1a/αGFP PLA-positive cells shown in Fig. is included for comparison. Results are the sum of two independent experiments. ( N = 379 for N-BAF and 266 for C-BAF; P -value: *** < .001; Fisher’s exact test).

Article Snippet: The rest of antibodies used are commercially available: mouse monoclonal αHP1a (DSHB C1A9), mouse monoclonal αLaminDm0 (DSHB ADL67.10), rabbit polyclonal αGFP (Invitrogen A11122), mouse polyclonal αGFP (Roche 118144600), rabbit polyclonal αFlag (Merck Life Science F7425), rabbit polyclonal αH3 (Cell Signaling 9715), rabbit polyclonal αH3K9me3 (Millipore 67–442), rabbit polyclonal αH3PS10 (Cell Signaling 9701), and mouse monoclonal αTubulin (MAB3408 Millipore).

Techniques: Fluorescence, Expressing, Immunostaining, Staining, Comparison

Journal: Nucleic Acids Research

Article Title: Phosphorylation of Barrier-to-Autointegration Factor (BAF) regulates anchoring of centromeric heterochromatin to the nuclear envelope (NE)

doi: 10.1093/nar/gkag021

Figure Lengend Snippet: The phospho-dead N-BAF T4A/S5A mutant mimics C-BAF. ( A ) The pattern of localization of N-BAF T4A/S5A is determined by direct fluorescence in stable S2 cells co-expressing N-BAF T4A/S5A (in green) and mCherry::HP1a (in red). Scale bars are 2 μm. ( B ) Box plots showing the fluorescence intensity at the perinuclear ring relative to centromeric heterochromatin for cells expressing N-BAF T4A/S5A (in red) or C-BAF T4E/S5E (in yellow). Results are the sum of 3 and 2 independent experiments, respectively. Results in Fig. for N-BAF (in green) and C-BAF (in gray) expressing cells are included for comparison. Boxes represent the median and IQR, whiskers are 1.5 × IQR. ( N = 40 for N-BAF T4A/S5A and 31 for C-BAF T4E/S5E ; P -value: **** < .0001; unpaired Student’s t -test). ( C ) The patterns of localization of N-BAF and N-BAF T4A/S5A are determined by direct fluorescence in stable S2 cells co-expressing N-BAF (in green) and N-BAF T4A/S5A (in red). Scale bars are 2 μm. ( D ) Immuno-PLA with αHP1a antibodies and αGFP antibodies in stable S2 cells expressing N-BAF T4A/S5A . The PLA signal is shown in red. Immunostaining with αHP1a antibodies is shown in cyan. DNA was stained with DAPI (in gray). Scale bars are 10 μm. ( E ) Quantification of the results shown in panel (D). The percentage of PLA positive cells is presented for assays performed with αHP1a antibodies and αGFP in stable S2 cells expressing N-BAF T4A/S5A (red). For comparison, the percentages of αHP1a/αGFP PLA positive cells shown in Fig. for N-BAF (green) and C-BAF (gray) expressing cells are included. Results are the sum of two independent experiments. ( N = 173; P -values: * < .05, *** < .001; Fisher’s exact test). ( F ) In the top, recordings of FRAP experiments in stable S2 cells expressing N-BAF T4A/S5A . Squares indicate the bleached regions. In the bottom, quantification of the results showing FRAP for N-BAF T4A/S5A . The t -half and proportion of mobile fraction are indicated. Results are expressed as mean ± SD. Quantification of the results obtained for N-BAF and C-BAF shown in Fig. are included for comparison. ( N = 33 for N-BAF T4A/S5A ). (see “Materials and methods” section for details). ( G ) The proportion of cells expressing the indicated BAF constructs that show perinuclear (in green) or centromeric heterochromatin (in red) localization of the corresponding BAF protein. Results are the sum of two independent experiments ( N > 180; P -values of the N-BAF/C-BAF mutated forms respect to the corresponding wild-type form: ns > 0.5; **** < .0001; Fisher’s exact test).

Article Snippet: The rest of antibodies used are commercially available: mouse monoclonal αHP1a (DSHB C1A9), mouse monoclonal αLaminDm0 (DSHB ADL67.10), rabbit polyclonal αGFP (Invitrogen A11122), mouse polyclonal αGFP (Roche 118144600), rabbit polyclonal αFlag (Merck Life Science F7425), rabbit polyclonal αH3 (Cell Signaling 9715), rabbit polyclonal αH3K9me3 (Millipore 67–442), rabbit polyclonal αH3PS10 (Cell Signaling 9701), and mouse monoclonal αTubulin (MAB3408 Millipore).

Techniques: Mutagenesis, Fluorescence, Expressing, Comparison, Immunostaining, Staining, Construct