Zinc-finger-based transcriptional repression of rhodopsin in a model of dominant retinitis pigmentosa

Despite the recent success of gene-based complementation approaches for genetic recessive traits, the development of therapeutic strategies for gain-of-function mutations poses great challenges. General therapeutic principles to correct these genetic defects mostly rely on post-transcriptional gene regulation (RNA silencing). Engineered zinc-finger (ZF) protein-based repression of transcription may represent a novel approach for treating gain-of-function mutations, although proof-of-concept of this use is still lacking. Here, we generated a series of transcriptional repressors to silence human rhodopsin (hRHO), the gene most abundantly expressed in retinal photoreceptors. The strategy was designed to suppress both the mutated and the wild-type hRHO allele in a mutational-independent fashion, to overcome mutational heterogeneity of autosomal dominant retinitis pigmentosa due to hRHO mutations. Here we demonstrate that ZF proteins promote a robust transcriptional repression of hRHO in a transgenic mouse model of autosomal dominant retinitis pigmentosa. Furthermore, we show that specifically decreasing the mutated human RHO transcript in conjunction with unaltered expression of the endogenous murine Rho gene results in amelioration of disease progression, as demonstrated by significant improvements in retinal morphology and function. This zinc-finger-based mutation-independent approach paves the way towards a ‘repression–replacement’ strategy, which is expected to facilitate widespread applications in the development of novel therapeutics for a variety of disorders that are due to gain-of-function mutations

Mouse Embryonic Retina Delivers Information Controlling Cortical Neurogenesis

The relative contribution of extrinsic and intrinsic mechanisms to cortical development is an intensely debated issue and an outstanding question in neurobiology. Currently, the emerging view is that interplay between intrinsic genetic mechanisms and extrinsic information shape different stages of cortical development [1]. Yet, whereas the intrinsic program of early neocortical developmental events has been at least in part decoded [2], the exact nature and impact of extrinsic signaling are still elusive and controversial. We found that in the mouse developing visual system, acute pharmacological inhibition of spontaneous retinal activity (retinal waves-RWs) during embryonic stages increase the rate of corticogenesis (cell cycle withdrawal). Furthermore, early perturbation of retinal spontaneous activity leads to changes of cortical layer structure at a later time point. These data suggest that mouse embryonic retina delivers long-distance information capable of modulating cell genesis in the developing visual cortex and that spontaneous activity is the candidate long-distance acting extrinsic cue mediating this process. In addition, these data may support spontaneous activity to be a general signal coordinating neurogenesis in other developing sensory pathways or areas of the central nervous system

An allelic series of miR-17 ∼ 92-mutant mice uncovers functional specialization and cooperation among members of a microRNA polycistron.

Polycistronic microRNA (miRNA) clusters are a common feature of vertebrate genomes. The coordinated expression of miRNAs belonging to different seed families from a single transcriptional unit suggests functional cooperation, but this hypothesis has not been experimentally tested. Here we report the characterization of an allelic series of genetically engineered mice harboring selective targeted deletions of individual components of the miR-17 ∼ 92 cluster. Our results demonstrate the coexistence of functional cooperation and specialization among members of this cluster, identify a previously undescribed function for the miR-17 seed family in controlling axial patterning in vertebrates and show that loss of miR-19 selectively impairs Myc-driven tumorigenesis in two models of human cancer. By integrating phenotypic analysis and gene expression profiling, we provide a genome-wide view of how the components of a polycistronic miRNA cluster affect gene expression in vivo. The reagents and data sets reported here will accelerate exploration of the complex biological functions of this important miRNA cluster

Intact p53-Dependent Responses in miR-34–Deficient Mice

<div><p>MicroRNAs belonging to the miR-34 family have been proposed as critical modulators of the p53 pathway and potential tumor suppressors in human cancers. To formally test these hypotheses, we have generated mice carrying targeted deletion of all three members of this microRNA family. We show that complete inactivation of miR-34 function is compatible with normal development in mice. Surprisingly, p53 function appears to be intact in miR-34–deficient cells and tissues. Although loss of miR-34 expression leads to a slight increase in cellular proliferation <em>in vitro</em>, it does not impair p53-induced cell cycle arrest or apoptosis. Furthermore, in contrast to p53-deficient mice, miR-34–deficient animals do not display increased susceptibility to spontaneous, irradiation-induced, or c-Myc–initiated tumorigenesis. We also show that expression of members of the miR-34 family is particularly high in the testes, lungs, and brains of mice and that it is largely p53-independent in these tissues. These findings indicate that miR-34 plays a redundant function in the p53 pathway and suggest additional p53-independent functions for this family of miRNAs.</p> </div

Targeted deletion of miR-34a and miR-34b∼c.

<p>(A) Targeting and screening strategy for the generation of constitutive and conditional miR-34a KO alleles. The restriction sites used for the Southern blot screening are indicated (S = SphI, E = EcoRI). The gray bar with an asterisk represents a genomic region absent in the 129SvJae strain but present in the C57BL/6 strain, which results in two distinct sizes in digestions. (B) Targeting and screening strategy for the generation of miR-34b∼c KO allele (H = HindIII, S = SpeI). (C) Genotyping by tail genomic PCR showing germline transmission of the miR-34a deleted and floxed alleles (upper panel), and the miR-34b∼c deleted allele (lower panel). (D) Northern blotting (upper panel) on total RNA extracted from the testes of mice with the indicated genotypes. Probes specific for miR-34a and miR-34c were used. Complete loss of miR-34a and miR-34c expression was further confirmed in MEFs by qPCR (lower panel). Representative pictures of miR-34a^−/− (E), miR-34b∼c^−/− (F), and miR-34^TKO/TKO (G) males at 4 weeks of age. The table below each picture summarizes the expected and observed frequencies of mice of each genotype as obtained from heterozygous inter-crosses. For the miR-34^TKO allele (G), double heterozygous mice were inter-crossed.</p

MiR-34 expression in wild-type and p53^−/− mouse tissues.

<p>(A) Sequence alignment of mouse miR-34a, miR-34b and miR-34c. Differing nucleotides are colored in blue. The seed sequences are in bold. (B–D) MiR-34a and miR-34c expression as detected by qPCR (B,C) and by Northern blotting (D) in tissues of wild-type and p53^−/− mice.</p

Response to p53 activation in miR-34^TKO/TKO mouse embryonic fibroblasts (MEFs).

<p>(A) MiR-34a and miR-34c expression in serially-passaged wild-type MEFs, as measured by qPCR. Error bars indicate 1 standard deviation (SD). (B) Cumulative population doublings of wild-type, miR-34^TKO/TKO and p53^−/− MEFs. Error bars indicate 1 SD. (C) Growth curves of wild-type and miR-34^TKO/TKO MEFs. Error bars indicate 1 SD. (D) Immunoblots of p53, p21 and Mdm2 in wild-type (W) and miR-34^TKO/TKO (K) MEFs treated with 0.2 µg/ml doxorubicin for the indicated time. (E) Expression of selected p53 targets in total RNA from doxorubicin-treated MEFs. Cells were treated with 0.2 µg/ml doxorubicin for 12 hours (Dox) or left untreated (U). Expression of the indicated genes was determined by qPCR. Error bars represent 1 SD. (F) Immunoblots showing p53 activation in three wild-type and three miR-34^TKO/TKO MEF lines. Cells were left untreated or treated with 0.2 µg/ml doxorubicin for 12 hours. (G) Time course of miR-34a and miR-34c expression in wild-type and p53^−/− cells treated with 0.2 µg/ml doxorubicin. MicroRNA expression was determined by qPCR. Error bars indicate 1 SD. (H, I) Cell cycle distribution of wild-type and miR-34^TKO/TKO MEFs. Asynchronously growing MEFs of the indicated genotype were treated with increasing doses of doxorubicin for 16 hours (H), or with 0.2 µg/ml doxorubicin for increasing time (I). Error bars indicate 1 SD. (J) Upper panel: cell cycle distribution of wild-type, miR-34^TKO/TKO, and p53^−/− MEFs after 72 hours in starvation medium (gray histogram). Starved cells were released in complete medium containing colcemid and mock-treated (light blue histogram) or exposed to 20 Gy irradiation (red histogram). Cells were analyzed by 7-AAD staining at the indicated time after release in complete medium. Lower panel: percentages of irradiated and untreated cells in G1 and G2-M phases after 24 hours in complete medium. Experiments were performed on three independent wild-type and three independent miR-34^TKO/TKO MEF lines. (K) Immunoblot detection of predicted miR-34 targets on three independent wild-type and three independent miR-34^TKO/TKO MEF lines.</p