Terminally differentiated human neurons repair transcribed genes but display attenuated global DNA repair and modulation of repair gene expression

Abstract

Repair of UV-induced DNA lesions in terminally differentiated human hNT neurons was compared to that in their repair-proficient precursor NT2 cells. Global genome repair of (6-4)pyrimidine-pyrimidone photoproducts was significantly slower in hNT neurons than in the precursor cells, and repair of cyclobutane pyrimidine dimers (CPDs) was not detected in the hNT neurons. This deficiency in global genome repair did not appear to be due to denser chromatin structure in hNT neurons. By contrast, CPDs were removed efficiently from both strands of transcribed genes in hNT neurons, with the nontranscribed strand being repaired unexpectedly well. Correlated with these changes in repair during neuronal differentiation were modifications in the expression of several repair genes, in particular an up-regulation of the two structure-specific nucleases XPG and XPF/ERCC1. These results have implications for neuronal dysfunction and aging.

FIG. 1

Global genome repair of CPDs in NT2 precursor cells and hNT neurons. NT2 cells and hNT neurons were either not irradiated (open symbols) or irradiated with 254-nm UV light at 10 J/m² and harvested immediately (solid symbols) or 24 h after irradiation (crossed symbols). Total DNA was digested with T4 endo V, fractionated on alkaline sucrose gradients, slot blotted, and detected with a ³²P-labeled genomic probe. The arrowheads indicate the positions of the molecular size markers: bacteriophage T2 (166 kbp), bacteriophage λ (48 kbp), and the largest fragment of HindIII-digested phage λ (23 kbp).

FIG. 2

Global genome repair of CPDs and (6-4)PPs in NT2 and hNT cells. DNA from NT2 cells (circles) and hNT neurons (diamonds) that were irradiated (solid symbols) or not (open symbols) was slot blotted on a nylon membrane and assayed with monoclonal antibodies specific for CPDs or (6-4)PPs. The results are the means of two to four experiments; the error bars indicate standard error of the mean.

FIG. 3

TCR in NT2 and hNT cells. DNA from UV-irradiated NT2 cells and hNT neurons was prepared at various times after irradiation, digested with restriction enzymes and then with T4 endo V, and subjected to a Southern analysis with riboprobes specific for the transcribed or nontranscribed strand of the gene of interest. Quantification of the fragment resistant to T4 endo V is an indication of the number of CPDs present at that time, from which the percentage of repair can be calculated. DNA from nonirradiated cells is included as a control; the corresponding points are reported on the y axis.

FIG. 4

Search for transcription products from either strand of the DHFR and CK18 genes. Total RNA from NT2 or hNT cells was reverse transcribed with primers specific for the DHFR or CK18 mRNA or for putative antisense RNA generated by transcription of the nontranscribed strand of these genes. As a control, RT reactions were performed with no primer. The resulting cDNA was then measured by semiquantitative PCR.

FIG. 5

Detection of CPDs by T4 endo V in NT2 and hNT cells. NT2 cells and hNT neurons were either not irradiated (open symbols) or irradiated (solid symbols) with 254-nm UV light at 10 J/m², permeabilized, treated with T4 endo V, and lysed at the top of alkaline sucrose gradients. The DNA in gradient fractions was blotted and assayed with a ³²P-labeled genomic probe. In some cases (crossed symbols), chromatin was disrupted by treating the cells with high salt prior to T4 endo V digestion.

FIG. 6

Effect of trichostatin A on the repair of CPDs and (6-4)PPs. DNA from NT2 cells (circles) and hNT neurons (diamonds) harvested at various times after UV irradiation was blotted onto a nylon membrane and assayed with monoclonal antibodies specific for CPDs or (6-4)PPs. For 6 h prior to irradiation, the cells were treated (solid symbols) or not treated (open symbols) with trichostatin A at 25 μg/ml for (6-4)PPs and 50 μg/ml for CPDs. Results are taken from two experiments performed in triplicate; the error bars indicate standard deviation.

FIG. 7

Effect of differentiation on gene expression. Total RNA from NT2 precursor cells and hNT neurons was reverse transcribed, and quantitative PCRs were performed for repair genes (top panel) or nonrepair genes (bottom panel) (note the change in scale). Normalization was achieved by coamplifying a reference gene, either the GAPDH or β-actin gene. Bars show the means of one to six measurements, depending on the gene; the error bars indicate standard error of the mean. Data are expressed as the ratio of expression in hNT neurons to that in NT2 cells.