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The leukocyte nuclear envelope proteome varies with cell activation and contains novel transmembrane proteins that affect genome architecture.

Korfali N, Wilkie GS, Swanson SK, Srsen V, Batrakou DG, Fairley EA, Malik P, Zuleger N, Goncharevich A, de Las Heras J, Kelly DA, Kerr AR, Florens L, Schirmer EC - Mol. Cell Proteomics (2010)

Bottom Line: Several known proteins identified in both data sets have functions in chromatin organization and gene regulation.To test whether the novel NETs identified might include those that also regulate chromatin, nine were run through two screens for different chromatin effects.The variation in the protein milieu with pharmacological activation of the same cell population and consequences for gene regulation suggest that the nuclear envelope is a complex regulatory system with significant influences on genome organization.

View Article: PubMed Central - PubMed

Affiliation: Wellcome Trust Centre for Cell Biology, University of Edinburgh, Edinburgh EH93JR, United Kingdom.

ABSTRACT
A favored hypothesis to explain the pathology underlying nuclear envelopathies is that mutations in nuclear envelope proteins alter genome/chromatin organization and thus gene expression. To identify nuclear envelope proteins that play roles in genome organization, we analyzed nuclear envelopes from resting and phytohemagglutinin-activated leukocytes because leukocytes have a particularly high density of peripheral chromatin that undergoes significant reorganization upon such activation. Thus, nuclear envelopes were isolated from leukocytes in the two states and analyzed by multidimensional protein identification technology using an approach that used expected contaminating membranes as subtractive fractions. A total of 3351 proteins were identified between both nuclear envelope data sets among which were 87 putative nuclear envelope transmembrane proteins (NETs) that were not identified in a previous proteomics analysis of liver nuclear envelopes. Nuclear envelope localization was confirmed for 11 new NETs using tagged fusion proteins and antibodies on spleen cryosections. 27% of the new proteins identified were unique to one or the other of the two leukocyte states. Differences in expression between activated and resting leukocytes were confirmed for some NETs by RT-PCR, and most of these proteins appear to only be expressed in certain types of blood cells. Several known proteins identified in both data sets have functions in chromatin organization and gene regulation. To test whether the novel NETs identified might include those that also regulate chromatin, nine were run through two screens for different chromatin effects. One screen found two NETs that can recruit a specific gene locus to the nuclear periphery, and the second found a different NET that promotes chromatin condensation. The variation in the protein milieu with pharmacological activation of the same cell population and consequences for gene regulation suggest that the nuclear envelope is a complex regulatory system with significant influences on genome organization.

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Cell-based screen for PBMC NETs that promote recruitment of chromosome loci to nuclear periphery. A, schematic representation of a screen to determine whether overexpression of a particular NET can recruit a specific chromatin locus to the NE. NETs were transfected into cells containing a lacO repeat integration that is typically in the interior. The lacO position was visualized with GFP-lacI (green) and measured using an algorithm that partitions the nucleus based on DAPI staining (blue) into five concentric circles of roughly equal area. B, example of NET-transfected cells. The position of the lacO locus is highlighted by the white arrows. The lacO locus position is unaffected by C20orf3 expression but moves to the periphery with TAPBPL expression. DAPI staining added to the merged image confirms that the movement of the locus is not an artifact of generalized chromatin condensation at the periphery. Deconvolved images are shown. C, the ring containing the locus was recorded in roughly 100 transfected cells. p values were calculated for NETs that increased the locus at the periphery in comparison with untransfected (UT) or mRFP-transfected control cells using a χ2 test.
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Figure 7: Cell-based screen for PBMC NETs that promote recruitment of chromosome loci to nuclear periphery. A, schematic representation of a screen to determine whether overexpression of a particular NET can recruit a specific chromatin locus to the NE. NETs were transfected into cells containing a lacO repeat integration that is typically in the interior. The lacO position was visualized with GFP-lacI (green) and measured using an algorithm that partitions the nucleus based on DAPI staining (blue) into five concentric circles of roughly equal area. B, example of NET-transfected cells. The position of the lacO locus is highlighted by the white arrows. The lacO locus position is unaffected by C20orf3 expression but moves to the periphery with TAPBPL expression. DAPI staining added to the merged image confirms that the movement of the locus is not an artifact of generalized chromatin condensation at the periphery. Deconvolved images are shown. C, the ring containing the locus was recorded in roughly 100 transfected cells. p values were calculated for NETs that increased the locus at the periphery in comparison with untransfected (UT) or mRFP-transfected control cells using a χ2 test.

Mentions: The first assay utilized an HT1080-derived cell line carrying a lacO repeat insertion in chromosome 5, the position of which is visualized with a lac repressor-GFP fusion that binds to the array. This lacO array tends to be in the nuclear interior. These cells were transfected with expression constructs for NET-mRFP fusions and assayed for a change in the distribution of the lacO array position with respect to the nuclear periphery. If a NET altered the position of the lacO array by pulling it to the nuclear periphery it could be considered to function in genome organization (Fig. 7A, left). To determine lacO positioning, a previously published algorithm was utilized that erodes the nucleus based on DAPI staining for DNA into five concentric rings of roughly equal area (55). The ring containing the lacO array was marked in each cell and recorded (Fig. 7A, right). The NET C20orf3 had no effect on the position of the lacO array; however, TAPBPL expression increased the occurrence of the array at the periphery (Fig. 7B). The ring containing the lacO array was marked in ∼100 cells, and the distribution was plotted (Fig. 7C). The lacO array tended to be in the nuclear interior, occurring in the peripheral ring only ∼20% of the time. Most blood NETs tested yielded no significant differences, but in cells expressing STT3A and TAPBPL, the occurrence at the periphery roughly doubled in multiple separate experiments with p values <0.0015 and 0.0001, respectively (Fig. 7C).


The leukocyte nuclear envelope proteome varies with cell activation and contains novel transmembrane proteins that affect genome architecture.

Korfali N, Wilkie GS, Swanson SK, Srsen V, Batrakou DG, Fairley EA, Malik P, Zuleger N, Goncharevich A, de Las Heras J, Kelly DA, Kerr AR, Florens L, Schirmer EC - Mol. Cell Proteomics (2010)

Cell-based screen for PBMC NETs that promote recruitment of chromosome loci to nuclear periphery. A, schematic representation of a screen to determine whether overexpression of a particular NET can recruit a specific chromatin locus to the NE. NETs were transfected into cells containing a lacO repeat integration that is typically in the interior. The lacO position was visualized with GFP-lacI (green) and measured using an algorithm that partitions the nucleus based on DAPI staining (blue) into five concentric circles of roughly equal area. B, example of NET-transfected cells. The position of the lacO locus is highlighted by the white arrows. The lacO locus position is unaffected by C20orf3 expression but moves to the periphery with TAPBPL expression. DAPI staining added to the merged image confirms that the movement of the locus is not an artifact of generalized chromatin condensation at the periphery. Deconvolved images are shown. C, the ring containing the locus was recorded in roughly 100 transfected cells. p values were calculated for NETs that increased the locus at the periphery in comparison with untransfected (UT) or mRFP-transfected control cells using a χ2 test.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Figure 7: Cell-based screen for PBMC NETs that promote recruitment of chromosome loci to nuclear periphery. A, schematic representation of a screen to determine whether overexpression of a particular NET can recruit a specific chromatin locus to the NE. NETs were transfected into cells containing a lacO repeat integration that is typically in the interior. The lacO position was visualized with GFP-lacI (green) and measured using an algorithm that partitions the nucleus based on DAPI staining (blue) into five concentric circles of roughly equal area. B, example of NET-transfected cells. The position of the lacO locus is highlighted by the white arrows. The lacO locus position is unaffected by C20orf3 expression but moves to the periphery with TAPBPL expression. DAPI staining added to the merged image confirms that the movement of the locus is not an artifact of generalized chromatin condensation at the periphery. Deconvolved images are shown. C, the ring containing the locus was recorded in roughly 100 transfected cells. p values were calculated for NETs that increased the locus at the periphery in comparison with untransfected (UT) or mRFP-transfected control cells using a χ2 test.
Mentions: The first assay utilized an HT1080-derived cell line carrying a lacO repeat insertion in chromosome 5, the position of which is visualized with a lac repressor-GFP fusion that binds to the array. This lacO array tends to be in the nuclear interior. These cells were transfected with expression constructs for NET-mRFP fusions and assayed for a change in the distribution of the lacO array position with respect to the nuclear periphery. If a NET altered the position of the lacO array by pulling it to the nuclear periphery it could be considered to function in genome organization (Fig. 7A, left). To determine lacO positioning, a previously published algorithm was utilized that erodes the nucleus based on DAPI staining for DNA into five concentric rings of roughly equal area (55). The ring containing the lacO array was marked in each cell and recorded (Fig. 7A, right). The NET C20orf3 had no effect on the position of the lacO array; however, TAPBPL expression increased the occurrence of the array at the periphery (Fig. 7B). The ring containing the lacO array was marked in ∼100 cells, and the distribution was plotted (Fig. 7C). The lacO array tended to be in the nuclear interior, occurring in the peripheral ring only ∼20% of the time. Most blood NETs tested yielded no significant differences, but in cells expressing STT3A and TAPBPL, the occurrence at the periphery roughly doubled in multiple separate experiments with p values <0.0015 and 0.0001, respectively (Fig. 7C).

Bottom Line: Several known proteins identified in both data sets have functions in chromatin organization and gene regulation.To test whether the novel NETs identified might include those that also regulate chromatin, nine were run through two screens for different chromatin effects.The variation in the protein milieu with pharmacological activation of the same cell population and consequences for gene regulation suggest that the nuclear envelope is a complex regulatory system with significant influences on genome organization.

View Article: PubMed Central - PubMed

Affiliation: Wellcome Trust Centre for Cell Biology, University of Edinburgh, Edinburgh EH93JR, United Kingdom.

ABSTRACT
A favored hypothesis to explain the pathology underlying nuclear envelopathies is that mutations in nuclear envelope proteins alter genome/chromatin organization and thus gene expression. To identify nuclear envelope proteins that play roles in genome organization, we analyzed nuclear envelopes from resting and phytohemagglutinin-activated leukocytes because leukocytes have a particularly high density of peripheral chromatin that undergoes significant reorganization upon such activation. Thus, nuclear envelopes were isolated from leukocytes in the two states and analyzed by multidimensional protein identification technology using an approach that used expected contaminating membranes as subtractive fractions. A total of 3351 proteins were identified between both nuclear envelope data sets among which were 87 putative nuclear envelope transmembrane proteins (NETs) that were not identified in a previous proteomics analysis of liver nuclear envelopes. Nuclear envelope localization was confirmed for 11 new NETs using tagged fusion proteins and antibodies on spleen cryosections. 27% of the new proteins identified were unique to one or the other of the two leukocyte states. Differences in expression between activated and resting leukocytes were confirmed for some NETs by RT-PCR, and most of these proteins appear to only be expressed in certain types of blood cells. Several known proteins identified in both data sets have functions in chromatin organization and gene regulation. To test whether the novel NETs identified might include those that also regulate chromatin, nine were run through two screens for different chromatin effects. One screen found two NETs that can recruit a specific gene locus to the nuclear periphery, and the second found a different NET that promotes chromatin condensation. The variation in the protein milieu with pharmacological activation of the same cell population and consequences for gene regulation suggest that the nuclear envelope is a complex regulatory system with significant influences on genome organization.

Show MeSH
Related in: MedlinePlus