The H19 locus acts in vivo as a tumor suppressor - PubMed (original) (raw)

The H19 locus acts in vivo as a tumor suppressor

Tomomi Yoshimizu et al. Proc Natl Acad Sci U S A. 2008.

Abstract

The H19 locus belongs to a cluster of imprinted genes that is linked to the human Beckwith-Wiedemann syndrome. The expression of H19 and its closely associated IGF2 gene is frequently deregulated in some human tumors, such as Wilms' tumors. In these cases, biallelic IGF2 expression and lack of expression of H19 are associated with hypermethylation of the imprinting center of this locus. These observations and others have suggested a potential tumor suppressor effect of the H19 locus. Some studies have also suggested that H19 is an oncogene, based on tissue culture systems. We show, using in vivo murine models of tumorigenesis, that the H19 locus controls the size of experimental teratocarcinomas, the number of polyps in the Apc murine model of colorectal cancer and the timing of appearance of SV40-induced hepatocarcinomas. The H19 locus thus clearly displays a tumor suppressor effect in mice.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.

Fig. 1.

Expression of the H19-Igf2 locus. (A) Maps of the H19-Igf2 locus. Wt, H19_Δ_3 and H19_Δ_enh mutants are represented with maternal (Mat) and paternal (Pat) alleles. Endodermal enhancers (E Enh) are indicated downstream from the H19 gene. Black arrows indicate full transcription and the gray arrow indicates weak transcription. R = EcoRI, S = SalI, X = XbaI, B = BamHI. (B) Igf2 expression analysis in H19_Δ_3 and wt mesoderm- and endoderm-derived organs. H19_Δ_3 and wt females were crossed with SD7 males. Semiquantitative RT-PCR was performed on 2 organ samples of 5-day-old mice, then digested with BsaA1 to detect the Mus spretus paternal polymorphism. Maternal Igf2 expression was only detected in mesoderm derived tissues, as shown by the Mat/Pat ratio under each slot.

Fig. 2.

Fig. 2.

Teratocarcinoma model. (A) Diagram of weights from wt and H_19_Δ_3_ derived tumors 35 days after grafting. Wt and H_19_Δ_3_ (indicated as _H19_−/−) are plotted in gray and black respectively, either on wt or _Igf2_−/− recipient background. (B) Histologic sections of embryo-derived teratocarcinomas. Tumor section after ectopic grafting to the kidney (Top). Differentiated tissues such as muscle (Middle) or bone (Bottom) in both wt and H19_Δ_3 derived tumors can be identified. (C) Expression analysis of Igf2 in tumors. Levels of Igf2 mRNA (orange) were determined by real-time qRT-PCR in wt and _H19_−/− derived tumors, produced on wt or _Igf2_−/− recipient background. H19 levels are shown in black (samples 1–4 are wt, others are _H19_−/−). In each set of samples, tumors are displayed by increasing weight as indicated in white. No significant difference in the levels of Igf2 was detected between the 2 sample sets (P = 0.39 on wt hosts and P = 0.45 on _Igf2_−/− hosts, respectively). (D) Methylation status of the ICR in tumors. Tumor DNA (wt in Left and H19_Δ_3 in Right) was digested with SacI (S) and HhaI (H) and analyzed with the CTCF3 probe (thick black line). The resulting 3.8 kb SacI fragment corresponds to the fully methylated paternal allele and the 0.3 kb HhaI fragment to the unmethylated maternal allele. Methylation index (MI) is indicated under each sample.

Fig. 3.

Fig. 3.

Colorectal cancer model. (A) Effect of H19 deletion on Apc_Δ_14/+ intestinal polyps at 180 days. Top shows the number of polyps in H19+/+Apc_Δ_14/+ (gray) and H19_Δ_3/+Apc_Δ_14/+ (black) mice on C57BL/6 background (P = 0.05). Bottom shows the number of polyps smaller and larger than 2 mm for both genotypes, with a significantly higher number of smaller polyps in the H19_Δ_3/+Apc_Δ_14/+mice. (B) Histologic analysis. Top shows H&E staining of adenomas in the colon and small intestine of H19_Δ_3/+Apc_Δ_14/+ mouse. Bottom shows H&E staining of the small intestine crypts from H19+/+Apc_Δ_14/+ and H19_Δ_3/+Apc_Δ_14/+ mice. Crypt length was measured (black vertical bar) and showed no significant difference between the two genotypes. (C) Expression analysis of Igf2 transcripts. Detection of Igf2 transcript level by quantitative RT-PCR in polyps from H19+/+Apc_Δ_14/+ and H19_Δ_3/+Apc_Δ_14/+ mice. Gapdh was used as control. (D) Methylation status of the ICR in intestine (I), colon (C), colon polyps (CP) and intestine polyps (IP) from H19_Δ_3/+Apc_Δ_14/+, H19+/+Apc_Δ_14/+, H19_Δ_3/+ and wt mice. The 3.8-kb SacI fragment corresponds to the fully methylated paternal allele and the 0.3 kb HhaI fragment to the unmethylated maternal allele as in Fig. 2. Methylation indexes (MI) are indicated.

Fig. 4.

Fig. 4.

Experimental liver carcinogenesis model. (A) Expression analysis of Igf2 and H19 transcripts. Northern analysis of 15 wt or Mat_Δ_Enh liver tumors. The blot was hybridized sequentially with the H19, Igf2 and ribosomal 28S probes. (B) Igf2 and H19 expression in Mat_Δ_Enh mice. Serial frozen sections of liver nodules of 148-day-old male Mat_Δ_Enh and wt mice were hybridized to S35-labeled Igf2 (Left) or H19 (Right) probes. (C) Latency of liver tumor appearance in the Mat_Δ_Enh mutants. Mice were killed between 120 and 134 days of age. The histogram shows the fraction of animals with liver tumors larger than 3 mm. The number of mice analyzed in each class is indicated above the columns.

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