title: The leukocyte nuclear envelope proteome varies with cell activation and contains novel transmembrane proteins that affect genome architecture.
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.
authors.affiliation: Wellcome Trust Centre for Cell Biology, University of Edinburgh, Edinburgh EH93JR, United Kingdom.
authors.forename: Nadia
authors.initials: N
authors.lastname: Korfali
authors.forename: Gavin S
authors.initials: GS
authors.lastname: Wilkie
authors.forename: Selene K
authors.initials: SK
authors.lastname: Swanson
authors.forename: Vlastimil
authors.initials: V
authors.lastname: Srsen
authors.forename: Dzmitry G
authors.initials: DG
authors.lastname: Batrakou
authors.forename: Elizabeth A L
authors.initials: EA
authors.lastname: Fairley
authors.forename: Poonam
authors.initials: P
authors.lastname: Malik
authors.forename: Nikolaj
authors.initials: N
authors.lastname: Zuleger
authors.forename: Alexander
authors.initials: A
authors.lastname: Goncharevich
authors.forename: Jose
authors.initials: J
authors.lastname: de Las Heras
authors.forename: David A
authors.initials: DA
authors.lastname: Kelly
authors.forename: Alastair R W
authors.initials: AR
authors.lastname: Kerr
authors.forename: Laurence
authors.initials: L
authors.lastname: Florens
authors.forename: Eric C
authors.initials: EC
authors.lastname: Schirmer
mesh_terms: D000818:Animals; D015153:Blotting, Western; D002460:Cell Line; D015894:Genome, Human; D006801:Humans; D007962:Leukocytes / Q000378:metabolism*; D008565:Membrane Proteins / Q000378:metabolism*; D008856:Microscopy, Fluorescence; D009685:Nuclear Envelope / Q000378:metabolism*; D020543:Proteome; D051381:Rats
chemical_list: D008565:Membrane Proteins; D020543:Proteome