Nucleolin is one component of the B cell-specific transcription factor and switch region binding protein, LR1 - PubMed (original) (raw)

Nucleolin is one component of the B cell-specific transcription factor and switch region binding protein, LR1

L A Hanakahi et al. Proc Natl Acad Sci U S A. 1997.

Abstract

LR1 is a B cell-specific, sequence-specific DNA binding activity that regulates transcription in activated B cells. LR1 also binds Ig heavy chain switch region sequences and may function in class switch recombination. LR1 contains two polypeptides, of 106 kDa and 45 kDa, and here we report that the 106-kDa component of LR1 is nucleolin. This identification, initially made by microsequence analysis, was verified by showing that (i) LR1-DNA binding activity increased in B cells transfected with a nucleolin cDNA expression construct; (ii) LR1-DNA binding activity was recognized by antibodies raised against recombinant human nucleolin; and (iii) in B cells transfected with epitope-tagged nucleolin expression constructs, the LR1-DNA complex was recognized by the anti-tag antibody. Nucleolin is an abundant nucleolar protein which is believed to play a role in rDNA transcription or organization, or rRNA processing. Homology between nucleolin and histone H1 suggests that nucleolin may alter DNA organization in response to cell cycle controls, and the nucleolin component of LR1 may therefore function to organize switch regions before, during, or after switch recombination. The demonstration that nucleolin is a component of a B cell-specific complex that binds switch region sequences suggests that the G-rich switch regions may have evolved from rDNA.

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Figures

Figure 1

Figure 1

Nucleolin. (A) Predicted amino acid sequence of human nucleolin. Residues 624–626 were omitted from the original published report (GenBank file humnucleo, accession number J05584J05584; ref. 22). (B) Schematic of nucleolin, showing the histone H1-like N-terminal region, which includes long runs of acidic amino acids and sites for cdc2 kinase; the nuclear localization signal and N-glycosylation sites; the four RRMs; and the RGG motifs in the C terminus.

Figure 2

Figure 2

Specificity of antinucleolin antibodies. (A) In situ immunofluorescent staining of PD31 murine pre-B cells with antinucleolin antibodies. Arrows indicate two of the nucleoli evident by staining. (B) Immunoblot analysis of crude PD31 nuclear extract; the single band at 106 kDa is indicated.

Figure 3

Figure 3

Gel mobility-shift analysis of the effect of antinucleolin antibodies on LR1–DNA binding activity. (A) Nuclear extract from PD31 pre-B cells was treated with no antibodies (no Ab) or 2 μg of protein A-purified antibodies from pre-immune serum (pre) or from a rabbit immunized with recombinant human nucleolin (α-nuc). Arrows indicate bands corresponding to the LR1–DNA complex (LR1), the subshift, and free DNA duplex. (B) LR1 purified 12,000-fold from PD31 pre-B cells was treated with 0, 0.016, 0.08, 0.4 or 2 μg of protein A purified antibodies from pre-immune serum (pre) or from a rabbit immunized with recombinant human nucleolin (α-nuc). Control lanes on the right show that neither antibody preparation alone altered mobility of the DNA duplex. (C) Purified LR1 was treated with 2 μg rabbit polyclonal antinucleolin antibodies in the presence of antirabbit Ig (α-rIg) or antirabbit IgM antibodies (α-rIgM) at 1 or 5 μg/reaction.

Figure 4

Figure 4

Epitope-tagged nucleolin is found in the LR1–DNA binding complex. PD31 pre-B cells were mock-transfected (−) or transfected with the constructs indicated. (A) Gel mobility-shift analysis of nuclear extracts of PD31 pre-B cells transfected with increasing amounts (1, 2, 4, or 16 μg) of pNtag4, which expresses nucleolin cDNA carrying an N-terminal HA tag. (B) Gel mobility-shift analysis of nuclear extracts from cells transfected with 4 μg pNtag4, which expresses nucleolin cDNA carrying an N-terminal HA tag; or 4 μg pNfor4, which expresses untagged nucleolin cDNA. Reactions were treated with 0.3 or 1 μl of anti-tag mAb 12CA5, as indicated. Control lanes on the right show that the 12CA5 antibody preparation alone did not alter the mobility of the DNA duplex.

Figure 5

Figure 5

Gel mobility-shift analysis of LR1 binding to sites substituted with inosine (I) and 2-aminopurine (2-AP). Binding specificity of LR1 in PD31 nuclear extracts is compared in the absence (Left) and presence (Right) of antinucleolin antibodies.

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