Spann TP et al. (2002), Alteration of nuclear lamin organization inhibi...

XB-ART-7614

J Cell Biol 2002 Feb 18;1564:603-8. doi: 10.1083/jcb.200112047.

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Alteration of nuclear lamin organization inhibits RNA polymerase II-dependent transcription.

Spann TP , Goldman AE , Wang C , Huang S , Goldman RD .

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Regulation of gene activity is mediated by alterations in chromatin organization. In addition, chromatin organization may be governed in part by interactions with structural components of the nucleus. The nuclear lamins comprise the lamina and a variety of nucleoplasmic assemblies that together are major structural components of the nucleus. Furthermore, lamins and lamin-associated proteins have been reported to bind chromatin. These observations suggest that the nuclear lamins may be involved in the regulation of gene activity. In this report, we test this possibility by disrupting the normal organization of nuclear lamins with a dominant negative lamin mutant lacking the NH2-terminal domain. We find that this disruption inhibits RNA polymerase II activity in both mammalian cells and transcriptionally active embryonic nuclei from Xenopus laevis. The inhibition appears to be specific for polymerase II as disruption of lamin organization does not detectably inhibit RNA polymerases I and III. Furthermore, immunofluorescence observations indicate that this selective inhibition of polymerase II-dependent transcription involves the TATA binding protein, a component of the basal transcription factor TFIID.

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Species referenced: Xenopus laevis
Genes referenced: lmnb3 mt-tr sp1 taf6 tbp trna

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	Figure 1. Disruption of lamin organization alters the distribution of splicing factors. The organization of A- and B-type lamins in untreated BHK 21 cells (A and B) and cells microinjected with ΔNLA (C and D) were examined by immunofluorescence with antibodies specific to A- or B-type lamins. Microinjection of ΔNLA disrupted the organization of both B- and A-type lamins (C and D). Immunofluorescence showing the distribution of LA and B′′, a U2-specific binding protein (E–G). In the control cell on the left, splicing factor B′′ is distributed in a characteristic pattern of speckles and interconnecting material within the nucleoplasm. In the ΔNLA-injected cell (upper right), the interconnecting material is absent and the number of B′′ speckles is greatly reduced (E and F). Note that the lamin aggregates and the B′′ speckles do not co-align (G). Bars, 5 μm.
	Figure 2. Disruption of lamin organization results in reduced incorporation of BrUTP. After microinjection of ΔNLA (arrows in A and B), cells were assayed for transcriptional activity by in situ incorporation of BrUTP. Cells were fixed and stained by immunofluorescence using antibodies directed against LA and BrU. At the bottom is an uninjected cell (A and B). The nucleolus of the uninjected cell is marked with an arrowhead. Lamin (C) and BrU (E) staining of another cell after microinjection of ΔNLA reveals that BrU incorporation is confined to nucleolar regions (D). Bars, 10 μm.
	Figure 3. Disruption of lamin organization in embryonic nuclei inhibits the incorporation of BrU. Buffer (A–D) or ΔNLA (E–J) was added to embryonic extracts. After 60 min, BrUTP was added, and 15 min later, the cells were fixed. Chromatin was stained with TOTO3 (A, C, E, and H). Immunofluorescence using antibodies against BrU (D and J), LB3 (B and G), and human LA (F and I) was used to monitor the incorporation of BrU and the distribution of lamins. Images are confocal sections through the center of the nuclei. Bars, 5 μm.
	Figure 4. Disruption of lamin organization inhibits the synthesis of mRNA-sized products in embryonic nuclei. The size of transcription products synthesized in the embryonic extract was determined by the addition of [α32P]UTP. After 15 min, total RNA was prepared from each sample and resolved by denaturing gel electrophoresis, and the dried gel was used for autoradiography. The addition of α-amanitin (10 μg/ml) blocked the synthesis of upper molecular weight products (bracket, compare A and B). Alternatively, buffer (control) or ΔNLA (D) was added to embryonic extracts 1 h before the addition of [α32P]UTP. The incorporation of [32P]UTP into upper molecular weight products was inhibited by disruption of LB3 organization, whereas the synthesis of products the size of tRNA (arrow) was not affected.
	Figure 5. Disruption of lamin organization does not alter the distribution of Sp1. BHK 21 cells were microinjected with ΔNLA. Cells were stained with rabbit anti–human LA (A) and a monoclonal anti–human Sp1 (B). The cell on the left in each panel was not injected with ΔNLA and displays normal lamin and Sp1 staining. The cell on the right was injected with ΔNLA, and although lamin organization is disrupted, it also displays a normal distribution of Sp1. Bar, 5 μm.
	Figure 6. Disruption of lamin organization alters the distribution of TBP. BHK 21 cells (A–D) and embryonic nuclei (E–H) stained with a rabbit anti-TBP (B, D, F, and H), rat anti–human LA (A and C), and a monoclonal anti–Xenopus LB3 (E and G). An uninjected BHK 21 cell is shown (A and B). An embryonic nucleus in extract treated with buffer is shown (E and F). Both display normal distributions of lamin (A and E) and TBP (B and F). In BHK 21 cells (C and D) and embryonic nuclei (G and H), ΔNLA treatment resulted in TBP (D and H) and lamins (C and G) colocalizing in nucleoplasmic aggregates. Arrows point to the location of nucleolar regions as seen by phase contrast (unpublished data). Images are confocal sections through the center of the nuclei. Bars, 5 μm.

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Albright, TAFs revisited: more data reveal new twists and confirm old ideas. 2000, Pubmed