Reduced RNA polymerase II transcription in intact and permeabilized Cockayne syndrome group B cells - PubMed (original) (raw)
Reduced RNA polymerase II transcription in intact and permeabilized Cockayne syndrome group B cells
A S Balajee et al. Proc Natl Acad Sci U S A. 1997.
Abstract
Cockayne syndrome (CS) is characterized by increased photosensitivity, growth retardation, and neurological and skeletal abnormalities. The recovery of RNA synthesis is abnormally delayed in CS cells after exposure to UV radiation. Gene-specific repair studies have shown a defect in the transcription-coupled repair (TCR) of active genes in CS cells from genetic complementation groups A and B (CS-A and CS-B). We have analyzed transcription in vivo in intact and permeabilized CS-B cells. Uridine pulse labeling in intact CS-B fibroblasts and lymphoblasts shows a reduction of approximately 50% compared with various normal cells and with cells from a patient with xeroderma pigmentosum (XP) group A. In permeabilized CS-B cells transcription in chromatin isolated under physiological conditions is reduced to about 50% of that in normal chromatin and there is a marked reduction in fluorescence intensity in transcription sites in interphase nuclei. Transcription in CS-B cells is sensitive to alpha-amanitin, suggesting that it is RNA polymerase II-dependent. The reduced transcription in CS-B cells is complemented in chromatin by the addition of normal cell extract, and in intact cells by transfection with the CSB gene. CS-B may be a primary transcription deficiency.
Figures
Figure 1
(A) RNA synthesis in intact normal (▪) and CS-B (•) lymphoblastoid cells. [14C]Thymidine-prelabeled cells were pulse labeled with [3H]uridine for 30 and 90 min. Radioactivity incorporated into acid-insoluble material was measured by scintillation counting. The absolute transcription rate was expressed as [3H]uridine incorporation per 106 cells based on the specific activity of [14C]thymidine per cell. (B) RNA synthesis in intact normal (▪) and three different CS-B fibroblast cell lines (•, ▴, ▾). [14C]Prelabeled cells were given a pulse with [3H]uridine for 30 and 90 min and the radioactivity incorporated into acid-insoluble material was measured. The transcription rate was expressed as [3H]uridine incorporation per 106 cells based on the specific activity of [14C]thymidine per cell. (C and D). Visualization of transcription sites in permeabilized normal (C) and CS-B (D) fibroblast cells. Cells were encapsulated in 0.5% agarose beads, lysed with 0.05% Triton X-100. The transcription reaction was carried out for 30 min using triphosphates and 0.1 mM Br-UTP at 37°C. After the reaction, the beads were washed in PB and the nuclei were permeabilized with 0.5% Triton X-100 for 10 min. Transcription sites were indirectly immunolabeled using a primary antibody against bromodeoxyuridine-BSA conjugate, which reacts only with single-stranded Br-RNA (Anti-BrdU mouse monoclonal IgG). After washing, the beads were incubated with Texas red conjugated secondary antibody (Sheep anti-mouse IgG). (E) Transcription in normal and CS-B chromatin in permeabilized cells. [3H]Thymidine-labeled normal (▪) and CS-B (•) lymphoblastoid cells were encapsulated in 0.5% agarose beads and lysed with 0.05% Triton X-100. The transcription was carried out as described. The beads were incubated at 37°C and 100-μl aliquots were collected at 0, 15, and 30 min to determine the rate of incorporation. The beads were washed thrice in ice-cold PB and lysed with 0.1% SDS for 2 hr at 37°C. The radioactivity incorporated into acid-insoluble material was measured and the transcription was expressed as pmol UMP incorporated per 106 cells. (F) In vitro transcription using different concentrations of Triton X-100. [3H]Thymidine-labeled normal (GM1712A, ▪, ▴) and CSB (•, ▾) lymphoblastoid cells were encapsulated and lysed with 0.05% and 0.5% Triton X-100. The beads were incubated at 37°C in transcription mixture, 100-μl aliquots were collected, and the incorporated radioactivity was determined by scintillation counting. The radioactivity incorporated into acid-insoluble material was measured and the transcription was expressed as pmol UMP incorporated per 106 cells.
Figure 1
(A) RNA synthesis in intact normal (▪) and CS-B (•) lymphoblastoid cells. [14C]Thymidine-prelabeled cells were pulse labeled with [3H]uridine for 30 and 90 min. Radioactivity incorporated into acid-insoluble material was measured by scintillation counting. The absolute transcription rate was expressed as [3H]uridine incorporation per 106 cells based on the specific activity of [14C]thymidine per cell. (B) RNA synthesis in intact normal (▪) and three different CS-B fibroblast cell lines (•, ▴, ▾). [14C]Prelabeled cells were given a pulse with [3H]uridine for 30 and 90 min and the radioactivity incorporated into acid-insoluble material was measured. The transcription rate was expressed as [3H]uridine incorporation per 106 cells based on the specific activity of [14C]thymidine per cell. (C and D). Visualization of transcription sites in permeabilized normal (C) and CS-B (D) fibroblast cells. Cells were encapsulated in 0.5% agarose beads, lysed with 0.05% Triton X-100. The transcription reaction was carried out for 30 min using triphosphates and 0.1 mM Br-UTP at 37°C. After the reaction, the beads were washed in PB and the nuclei were permeabilized with 0.5% Triton X-100 for 10 min. Transcription sites were indirectly immunolabeled using a primary antibody against bromodeoxyuridine-BSA conjugate, which reacts only with single-stranded Br-RNA (Anti-BrdU mouse monoclonal IgG). After washing, the beads were incubated with Texas red conjugated secondary antibody (Sheep anti-mouse IgG). (E) Transcription in normal and CS-B chromatin in permeabilized cells. [3H]Thymidine-labeled normal (▪) and CS-B (•) lymphoblastoid cells were encapsulated in 0.5% agarose beads and lysed with 0.05% Triton X-100. The transcription was carried out as described. The beads were incubated at 37°C and 100-μl aliquots were collected at 0, 15, and 30 min to determine the rate of incorporation. The beads were washed thrice in ice-cold PB and lysed with 0.1% SDS for 2 hr at 37°C. The radioactivity incorporated into acid-insoluble material was measured and the transcription was expressed as pmol UMP incorporated per 106 cells. (F) In vitro transcription using different concentrations of Triton X-100. [3H]Thymidine-labeled normal (GM1712A, ▪, ▴) and CSB (•, ▾) lymphoblastoid cells were encapsulated and lysed with 0.05% and 0.5% Triton X-100. The beads were incubated at 37°C in transcription mixture, 100-μl aliquots were collected, and the incorporated radioactivity was determined by scintillation counting. The radioactivity incorporated into acid-insoluble material was measured and the transcription was expressed as pmol UMP incorporated per 106 cells.
Figure 1
(A) RNA synthesis in intact normal (▪) and CS-B (•) lymphoblastoid cells. [14C]Thymidine-prelabeled cells were pulse labeled with [3H]uridine for 30 and 90 min. Radioactivity incorporated into acid-insoluble material was measured by scintillation counting. The absolute transcription rate was expressed as [3H]uridine incorporation per 106 cells based on the specific activity of [14C]thymidine per cell. (B) RNA synthesis in intact normal (▪) and three different CS-B fibroblast cell lines (•, ▴, ▾). [14C]Prelabeled cells were given a pulse with [3H]uridine for 30 and 90 min and the radioactivity incorporated into acid-insoluble material was measured. The transcription rate was expressed as [3H]uridine incorporation per 106 cells based on the specific activity of [14C]thymidine per cell. (C and D). Visualization of transcription sites in permeabilized normal (C) and CS-B (D) fibroblast cells. Cells were encapsulated in 0.5% agarose beads, lysed with 0.05% Triton X-100. The transcription reaction was carried out for 30 min using triphosphates and 0.1 mM Br-UTP at 37°C. After the reaction, the beads were washed in PB and the nuclei were permeabilized with 0.5% Triton X-100 for 10 min. Transcription sites were indirectly immunolabeled using a primary antibody against bromodeoxyuridine-BSA conjugate, which reacts only with single-stranded Br-RNA (Anti-BrdU mouse monoclonal IgG). After washing, the beads were incubated with Texas red conjugated secondary antibody (Sheep anti-mouse IgG). (E) Transcription in normal and CS-B chromatin in permeabilized cells. [3H]Thymidine-labeled normal (▪) and CS-B (•) lymphoblastoid cells were encapsulated in 0.5% agarose beads and lysed with 0.05% Triton X-100. The transcription was carried out as described. The beads were incubated at 37°C and 100-μl aliquots were collected at 0, 15, and 30 min to determine the rate of incorporation. The beads were washed thrice in ice-cold PB and lysed with 0.1% SDS for 2 hr at 37°C. The radioactivity incorporated into acid-insoluble material was measured and the transcription was expressed as pmol UMP incorporated per 106 cells. (F) In vitro transcription using different concentrations of Triton X-100. [3H]Thymidine-labeled normal (GM1712A, ▪, ▴) and CSB (•, ▾) lymphoblastoid cells were encapsulated and lysed with 0.05% and 0.5% Triton X-100. The beads were incubated at 37°C in transcription mixture, 100-μl aliquots were collected, and the incorporated radioactivity was determined by scintillation counting. The radioactivity incorporated into acid-insoluble material was measured and the transcription was expressed as pmol UMP incorporated per 106 cells.
Figure 2
(A) Effect of α-amanitin and actinomycin D on transcription in intact normal and CS-B cells. 14C-labeled cells were pretreated with α-amanitin (2 μg/ml) alone or α-amanitin and actinomycin D (0.2 μg/ml) for 16 hr. The cells were then labeled with [3H]uridine for 30 and 90 min. The absolute transcription rate was expressed as [3H]uridine incorporation per 106 cells based on the specific activity of [14C]thymidine per cell. Normal lymphoblastoid cells (▪, •, ▾), CS-B (▾, ♦, +). (B) Effect of α-amanitin on in vitro transcription in normal and CS-B chromatin. 14C-prelabeled cells were encapsulated in 0.5% agarose beads and lysed with 0.05% Triton X-100. Prior to the transcription reaction, a fraction of the beads were incubated with the inhibitor of RNA pol II, α-amanitin (5 μg/ml, 30 min on ice), and the transcription was carried out with triphosphates and [32P]UTP using the conditions described in Fig. 3. Aliquot of beads (100 μl) was collected at 0, 5, and 30 min. After washing the beads in PB to remove the unincorporated nucleotides, the beads were lysed in 0.2% SDS for 2 hr and the acid insoluble radioactivity was measured by scintillation counting. The transcription rate was expressed as pmol UMP incorporated per 106 cells. Normal (▴, untreated; ▾, α-amanitin treated) and CS-B (▪, untreated; •, α-amanitin treated).
Figure 3
(A) Complementation of transcription in CS-B chromatin by WCEs. [14C]Thymidine-prelabeled lymphoblastoid cells were encapsulated and lysed in 0.5% Triton X-100 in PB. Before the transcription reaction, the beads containing the protein depleted chromatin were incubated either with 100 μg of whole cell CS-B (•) or normal (▴) extract for 30 min on ice. The chromatin not supplemented with either of the WCE (▪) served as control. The transcription reaction was carried out using triphosphates and [3H]UTP and the radioactivity incorporated into acid-insoluble material was measured. The transcription rate was expressed as pmol UMP incorporated per 106 cells. (B and C) Transcription in complemented cell lines. Transcription in intact (B) SV40-transformed normal fibroblasts (GM00637D) (▪) and in CS-B cells transfected with the CSB c-DNA (CS1AN/CSB/pDR2) (•). CS-B cells transfected with the plasmid alone (CS1AN/pDR2) (▴) were used for comparison. Transcription measured in chromatin (C) SV40-transformed normal fibroblasts (▴), CS-B cells transfected with CSB cDNA (•), and CS-B transfected with plasmid alone (▪).
Figure 4
In vitro NER by normal and CS-B cell free extract. Reactions contained 0.3 μg of each of _N_-acetoxy-2-acetylaminofluorene damaged and undamaged plasmid supplemented with 100 μg protein of either normal (lane 1) or CS-B (lane 2) cell free extract. Following incubation at 30°C for 1 hr, plasmid DNA was linearized and electrophoresed on 1% agarose gel. (A) Ethidium bromide-stained agarose gel showing equal loading of both damaged and undamaged plasmids. (B) Autoradiograph of the same gel showing repair incorporation.
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