BCCIP Is Required For Lamin B1 Dependent Nuclear Integrity

The nuclear envelope is composed of two layers of lipid membranes that separate the cytoplasm from the nucleoplasm,making the nucleus the largest organelle in the cell.Beneath the inner nuclear membrane,there is mess structure,nuclear lamina,composed of lamin complexes.Nuclear lamina acts as an interface which interacts with many trans-membrane proteins bound to the inner nuclear membrane and chromatin proteins.Lamin proteins also form intermediate filament network providing nucleoskeleton.Along with nucleoskeleton,nuclear lamina provides rigid structure to support nuclear envelope and maintain nuclear structure,as well as regulates important nuclear activities.BCCIP is a BRCA2-and CDKN1A(p21)-interacting protein that has been implicated in the maintenance of many cellular processes,including cytokinesis,genomic stability.Down-regulation of BCCIP has also been found closely correlated to tumor progression and malignancy.In BCCIP transgenic mouse model,whole body BCCIP knockdown causes embryonic lethality,while specifically knockdown BCCIP in brain during early embryonic development results in neurodegeneration and microcephaly,suggesting an important role of BCCIP in mouse neural/embryo development.In lamin B1 knockout model,small forebrain and other neuronal abnormalities have been reported,demonstrating essential function of lamin B1 in brain development.Since there is overlap in the function of neuron development,we sought to test the correlation between BCCIP and lamin B1 proteins.In my thesis study.I used immuno-precipitation approach to identify the interaction between BCCIP and B-type lamin,lamin B1.Also,I purified recombinant BCCIP and lamin B1,and further the interaction using GST-pull down assay.The interaction domain of lamin B1 is mapped to common region of BCCIP,amino acid 168 to amino acid 258,and BCCIP mainly interacts with lamin B1 through its C-terminal Ig-like fold,amino acid 390 to amino acid 586.By stable transfecting pBS-U6-LoxPneo-BCCIP shRNA construct.I constructed BCCIP conditional knock-down cell line(HT-CKD)to investigate the biological function of the interaction between BCCIP and lamin B1.I also used mouse embryonic fibroblast(MEF)derived from BCCIP conditional knockout mouse(BCCIPFlox/Flox)to further confirm the results.Significantly decreased lamin B1 distribution along nuclear envelope is observed in HT-CKD cells with BCCIP knockdown and MEF cells after BCCIP knockout using confocal microscopy.The deficiency of BCCIP results in lamin B1 degradation,at least partially through ubiquitin-proteasome pathway,which may be attributed to the abrogation of protein interaction.Significantly more abnormal nuclear are detected with CellPorfiler software in BCCIP deficient cells than BCCIP wild type cells.Furthermore,the organization of nucleoli is distorted in BCCIP deficient cells,which is associated with decreased RNA polymerase II(RNA pol II)activity visualized by fluorescent immunostaining with antibody against phosphor-RNA pol II,implying impaired RNA transcription caused by BCCIP deficiency.As a consequence of BCCIP deficiency,significant growth inhibition is observed in both HT-CKD cells and BCCIP knockout MEF cells.The growth defect in HT-CKD cells can be partially rescued by exogenous expression of BCCIP in the cells with BCCIP knockdown.These data for the first time demonstrate the interaction between BCCIP and lamin B1,which is required for mediating the normal function of lamin B1,implying the essential role of BCCIP in maintaining nuclear structure and regulating intracellular processes.