Transient knock-out of HP1.1 by RNA interference

3.5.1 Injection of HP1.1-dsRNA resulted in suppression of HP1.1 expression in an hp1.1::gfp strain

The dsRNA of full length HP1.1-cDNA was microinjected into the gonads of young hermaphrodites of a strain carrying hp1.1::gfp constructs integrated into the genome. Embryos of the F1 progeny were screened for GFP expression to find out whether dsRNA could abolish the whole GFP expression.

The results of the injection of the HP1.1 dsRNA are given in Table I-12.

Interestingly, 95% of the F1 progeny of the injected animals did not exhibit any visible GFP signal from the nuclei (Fig. 3-16). Furthermore, there were 12.3%

(94 out of 766) dead embryos observed in the F1 progeny. The results indicate that injection of dsHP1.1RNA did inhibit HP1.1 expression in F1 embryos.

Table I-12 Percentage of dead embryos and of GFP fluorescence loss after injection of HP1.1 dsRNA. The numbers in parentheses give the total number of animals screened in each experiment; n, the number of injected hermaphrodites; m, the number of dead embryos, *, integrated array.

RNAi Strain %Dead embryos % loss of GFP signal from nuclei

Fig. 3-16 Suppression of HP1.1::GFP expression by HP1.1 dsRNA. dsRNA corresponding to the total length of in hp1.1 cDNA was injected into a C. elegans strain, that carries hp1.1::gfp in its genome, and yielded a total loss of the GFP fluorescence (A). B:

Nomarski illumination.

3.5.2 Injection of HP1.1-dsRNA resulted in mutant phenotypes

The dsRNA of full-length cDNA of HP1.1 was microinjected into the gonads of young hermaphrodite worms of the wild type strain N2, and the embryos of the F1 offspring and their phenotypes were screened. In other experiments, dsRNA corresponding to the full-length cDNA of HP1.2 was injected separately or in combination with HP1.1 dsRNA.

Table I-13 Percentage of dead embryos in F1 after injection of dsRNA of hp1 genes. The numbers in parentheses give the total number of animals screened in each experiment; n, the number of injected hermaphrodites.

RNAi Strain %Dead embryos

HP1.1 N2 13.52(1769, n=116)

HP1.2 N2 10.05 (209, n=26)

HP1.1 & HP1.2 N2 11.84 (380, n=56)

Control injection Strain % Dead embryos

M9 N2 0.92 (1200, n=97)

M9 N2 0.9 (782, n=35)

A number of dead embryos was observed in the F1 progeny. 13.5% dead embryos were observed in the injection series with HP1.1 dsRNA, 10% with HP1.2 dsRNA, and 11.8% when both types of dsRNA had been injected (Table I-13). Approximately 5% of the F1 animals screened after injection of HP1.1 dsRNA alone or in combination with HP1.2 dsRNA (for the total numbers of animals screened see Table I-13) showed abnormal development. These embryos were smaller than those of the N2 wild type strain and had a different morphology. When they grew up, their larvae had a different body shape in comparison to wild-type larvae. Mostly they had abnormal tail regions. Fig. 3-17 shows a larva with this phenotype. These animals have a lumpy, dumbbell or twisted body, did not grow normally, and were arrested in their development.

They can be compared with vab mutants, specifically vab-6 (variable abnormal morphology) mutants. Usually the larvae could not move freely around on the NGM plates as N2 animals do but kept within a very small area. Other larvae grew and looked like dumpy mutants. Of the dumpy-like mutants, some had progeny whereas others were arrested in their development.

Fig. 3-17 Abnormal phenotype of an C. elegans larva due to transient knock-out of hp1.1. HP1.1 dsRNA was microinjected into young hermaphrodites of the N2 wild type strain, and the F1 was screened for embryonic lethality and for new phenotypes. The micrograph (Nomarski-DIC) shows severe defects in the tail region. Image recorded with a confocal laser scanning microscope (Zeiss 510).

3.5.3 dsRNA-mediated interference with HP1.1 expression did not show interaction with gene silencing in the germline of C. elegans

A germline specific reporter system (Kelly and Fire, 1998) was used in order to check whether HP1.1 is involved in chromatin silencing in the germline of C. elegans. In this system, a GFP-tagged gene (let-858) is established as a repetitive extrachromosomal transgenic array in a genetic background (pha-1, Granato et al., 1994), which allows positive selection for the transgene at the temperature of 25°C. The LET-858::GFP reporter was established as a

replicating transgene. The endogenous let-858 promoter is active in the germline, whereas LET-858::GFP expression as consequence is lost after a number of animal generation. The loss of expression of the reporter gene in the germline was used to indicate the status of gene silencing. HP1.1 dsRNA was injected into the gonads of healthy young hermaphrodite worms, that carry the reporter transgene as extrachromosomal arrays, and the gonads of adult F1 animals were screened for LET-858::GFP fluorescence.

No desilencing effect of the HP1.1 dsRNA injections was observed in F1 hermaphrodites and in male progeny (not shown). If HP1.1 played a role in gene silencing, one would expect to see the GFP fluorescence signal in the germline of the F1 progeny (adult hermaphrodites) of microinjected young hermaphrodites, because of the suppression of HP1.1. However, no indication of any fluorescence signal in any of the animals was observed that might have indicated germline desilencing.

In additional experiments, the SS222 (mes-3(bn21) I) strain was crossed with a strain that carried the hp1.1::gfp construct as integrated arrays, and the F1 progeny was screened for HP1.1::GFP expression in the gonad arms. The hermaphrodite gonads in mes-3 animals are deprived of mitotic nuclei as well as of differentiated germ cells. It was checked whether HP1.1 is involved in chromatin silencing in the germline using the mes-3 strain. Only the abnormally enlarged germ nuclei with prominent nucleoli were observed, that are a trait of the mes-3 strain. It must be concluded that the HP1.1::GFP expression pattern did not cause any noticeable changes in the mes-3 strain comparable to let-858 (Kelly and Fire, 1998).

Taken together, the experiments performed did not indicate that HP1.1 is involved in the remarkable chromatin silencing occurring in the germline of C.

elegans.