A mitotic transcriptional switch in polycystic kidney disease

Abstract

Hepatocyte nuclear factor-1beta (HNF-1beta) is a transcription factor required for the expression of several renal cystic genes and whose prenatal deletion leads to polycystic kidney disease (PKD). We show here that inactivation of Hnf1b from postnatal day 10 onward does not elicit cystic dilations in tubules after their proliferative morphogenetic elongation is over. Cystogenic resistance is intrinsically linked to the quiescent state of cells. In fact, when Hnf1b deficient quiescent cells are forced to proliferate by an ischemia-reperfusion injury, they give rise to cysts, owing to loss of oriented cell division. Remarkably, in quiescent cells, the transcription of crucial cystogenic target genes is maintained even in the absence of HNF-1beta. However, their expression is lost as soon as cells proliferate and the chromatin of target genes acquires heterochromatin marks. These results unveil a previously undescribed aspect of gene regulation. It is well established that transcription is shut off during the mitotic condensation of chromatin. We propose that transcription factors such as HNF-1beta might be involved in reprogramming gene expression after transcriptional silencing is induced by mitotic chromatin condensation. Notably, HNF-1beta remains associated with the mitotically condensed chromosomal barrels. This association suggests that HNF-1beta is a bookmarking factor that is necessary for reopening the chromatin of target genes after mitotic silencing.

Figure 1

Time-restricted cystogenic competence. (a) Morphology of kidneys from mice where Hnf1b was inactivated at either P1 or P10 and killed 30 d later. X-gal staining reveals the extent of Cre recombination (ROSA26R). Scale bar, 1 mm. (b) Percentage of Hnf1b deletion in mice injected at birth (perinatal) or in adulthood (adult), determined by qRT-PCR on genomic DNA. Data are means ± s.e.m.; n = 7 per group. **P < 0.01. (c) HNF-1β protein immunodetection in kidneys from 30-day old mice where Cre was activated at P10. Cx, cortex; Md, medulla. Scale bar, 100 μm. (d) Diagram showing the time-dependent cystogenic competence of Hnf1b deletion. Each circle represents one mouse.

Figure 2

Tubular regeneration elicits distorted cell division and dilations in Hnf1b deficiency. (a) Three-dimensional reconstruction of typical examples of longitudinally (left) or perpendicularly (right) oriented mitoses. Tubules are in green; mitoses are in red. (b) Comparison of the distribution of the mitotic angles in regenerating tubules from wild-type (n = 35, from six mice) or Hnf1b-deficient mice (n = 35, from four mice). The Mann-Whitney U test shows a highly statistically significant different distribution of the mitotic angle between mutant and control mice (P < 0.005). (c) Morphology (periodic acid–Schiff staining) of nonischemic control kidneys (top) and kidneys after ischemia (bottom) from wild-type or Hnf1b-inactivated mice, 2 weeks after surgery. Normal-shaped Hnf1b-inactivated tubules underwent dilations (arrows in bottom right image) when forced to reenter the cell cycle. Scale bar, 50 μm.

Figure 3

Effect of proliferation on HNF-1β target gene expression and chromatin modifications. (a) Renal expression of Umod, Pkhd1, Pkd2, Kif12, Crb3, Tcfap2b, Tmem27 and Bicc1 mRNA transcripts in wild-type or in mutant adult kidneys 3–4 weeks after the inactivation that was induced in proliferative P0 pups or in quiescent kidneys at P30, respectively. Data are means ± s.e.m.; n = 4–8 for each experimental group. *P < 0.05; **P < 0.01. Ribosomal protein L13 (Rpl13) was used to normalize values. (b) BrdU and Pkd2 (top) or Kif12 (bottom) staining in kidneys of wild-type and mutant mice 2 weeks after ischemia. Colocalization analysis shows that, in wild-type tubules after ischemia, Pkd2 and Kif12 expression (brown cytoplasm) is maintained in cells that have proliferated (blue nuclei). In contrast, in Hnf1b-deficient tubules, Pkd2 and Kif12 expression is selectively turned off in cells that have proliferated. Scale bars, 100 μm. (c) Chromatin modifications in the mIMCD3 cell line expressing a HNF-1β dominant-negative mutant. Chromatin immunoprecipitation of activating (H3K9ac) or inhibiting (H3K9me3 or H3K27me3) histone H3 modifications of the Pkhd1 gene at various locations with respect to the transcriptional start site (indicated on the x axis) in control cells or in cells expressing a dominant-negative mutant of HNF-1β. The results are representative of a set of three independent experiments. Error bars represent s.e.m. *P < 0.05. (d) Chromatin immunoprecipitation of activating modifications (H3K9ac or H3K4me3) of histone H3 on the Pkhd1 gene at various locations with respect to the transcriptional start site (indicated on the x axis) in kidneys from wild-type or Hnf1b-deleted mice inactivated shortly after birth. Immunoprecipitation of total histone H3 is used as a control. Enrichments are calculated with respect to the Rpl13 gene for activating modifications and for the Acta2 gene (encoding smooth muscle α-actin) for repressive modifications. The results are representative of a set of three independent experiments. *P < 0.05.

Figure 4

HNF-1β behaves as a bookmarking factor. (a) Time-lapse microscopy of mIMCD3 cells expressing either HNF1β–GFP or HNF4α–GFP fusion proteins. For each fusion protein, phase contrast (top) and green fluorescence (bottom) are shown at crucial time points. The time (in min) is indicated in the bottom right corner of each frame. The white arrows indicate the sudden appearance of cytoplasmic GFP fusion protein upon nuclear envelope breakdown. The red arrows indicate the persistence of the HNF-1β–GFP fusion protein on mitotically condensed chromosomes. Scale bars, 20 μm. (b) A mitotic transcriptional switch in polycystic kidney disease. HNF-1β activates gene expression via two distinct mechanisms: by recruiting the transcriptional machinery to the promoters of target genes and/or by inducing the reopening of chromatin after a silencing mitotic chromatin condensation. Class 1 genes are silenced whenever HNF-1β is inactivated, independent of the proliferative state of cells. Class 2 gene expression is transiently maintained in quiescence and is lost as soon as cells have progressed through a mitotic chromatin condensation in the cell cycle.