Characterization of the components of the putative mammalian sister chromatid cohesion complex

Abstract

Establishing and maintaining proper sister chromatid cohesion throughout the cell cycle are essential for maintaining genome integrity. To understand how sister chromatid cohesion occurs in mammals, we have cloned and characterized mouse orthologs of proteins known to be involved in sister chromatid cohesion in other organisms. The cDNAs for the mouse orthologs of SMC1S.c. and SMC3S.c. , mSMCB and mSMCD respectively, were cloned, and the corresponding transcripts and proteins were characterized. mSMCB and mSMCD are transcribed at similar levels in adult mouse tissues except in testis, which has an excess of mSMCD transcripts. The mSMCB and mSMCD proteins, as well as the PW29 protein, a mouse homolog of Mcd1pS.c./Rad21S.p., form a complex similar to cohesin in X. laevis. mSMCB, mSMCD and PW29 protein levels show no significant cell-cycle dependence. The bulk of the mSMCB, mSMCD and PW29 proteins undergo redistribution from the chromosome vicinity to the cytoplasm during prometaphase and back to the chromatin in telophase. This pattern of intracellular localization suggests a complex role for this group of SMC proteins in chromosome dynamics. The PW29 protein and PCNA, which have both been implicated in sister chromatid cohesion, do not colocalize, indicating that these proteins may not function in the same cohesion pathway. Overexpression of a PW29-GFP fusion protein in mouse fibroblasts leads to inhibition of proliferation, implicating this protein and its complex with SMC proteins in the control of mitotic cycle progression.

Fig. 1

mSMCB and mSMCD protein sequences. (A) Protein sequence inferred from the mSMCB cDNA. GeneBank Accession No. AF047600. (B) Protein sequence encoded by mSMCD cDNA. GeneBank Accession No. AF047601. The amino-terminal ATP-binding region and carboxy-terminal DA-box are underlined.

Fig. 2

mSMCB and mSMCD protein sequence comparison to yeast SMC proteins. A dot matrix analysis was performed with a window of 23 and a stringency of 7.

Fig. 3

Characterization of mSMCB and mSMCD mRNA. (A) The mouse multiple tissue Multiple-tissue Northern blot (Clontech) was probed with internal probes derived from mSMCB and mSMCD cDNA. Two micrograms of poly-A RNA were loaded per lane. h, heart; b, brain; sp, spleen; ln, lung; lv, liver; sm, skeletal muscle; k, kidney; t, testis. A Northern hybridization using actin as a loading-control probe revealed a uniform loading in all lanes, except the skeletal muscle lane (data not shown).

Fig. 4

Coimmunoprecipitation of mSMCD and PW29 proteins with mSMCB. Immunoprecipitates from mouse embryonic extract (see Section 2) were separated on a NUPAGE Bis-Tris gel (Novex) and probed with affinity-purified anti-mSMCB (#2966, 1:500 dilution), affinity-purified anti-mSMCD (#3009, 1:250 dilution) and crude anti-PW29 (1:10 000 dilution) antibodies. Mock immunoprecipitations were performed under identical conditions, with precipitating antibody omitted.

Fig. 5

Cell-cycle expression pattern for mSMCB, mSMCD and PW29 proteins. (A) Characterization of the cell-cycle arrest in NIH3T3 cells by FACS analysis. Arresting factors: G1, lovastatin; G0, 0.5% serum starvation; S, aphidicolin; G2/M, nocodazole. (B) Comparison of intracellular levels of mSMCB, mSMCD and PW29 proteins at the arresting conditions corresponding to (A). Fifteen micrograms of protein were loaded per lane. GAP3DH levels are shown as a control for equal loading.

Fig. 6

Localization of mouse mSMCB, mSMCD and PW29 proteins in asynchronously growing NIH3T3 cells. (A–D) Cells at different stages of the cell cycle, stained for PW29 with crude specific antiserum (1:10 000). Cells were also stained for DNA with DAPI and tubulin (not shown) with YOL1/34 antibody. (E) Interphase and (F) metaphase NIH3T3 cells stained with affinity-purified anti-mSMCB antibodies (1:2000) and DAPI. (G) Interphase and (H) anaphase NIH3T3 cells stained with affinity-purified anti-mSMCD antibodies (1:1000) and DAPI. Bars: 5 μm.

Fig. 7

Localization of mouse PW29 protein and PCNA in asynchronously growing NIH3T3 cells. Two cells in the same field are at different stages of S-phase, based on staining for PCNA marker. PW29 localization (1:10 000 staining with crude serum) does not change with changed PCNA localization. Cells were also stained for DNA with DAPI and tubulin (not shown) with YOL1/34 antibody. Bar: 5 μm.