SIM1

Abstract: Studies of mice and humans have revealed a number of genes that when mutated result in severe obesity. We have studied a unique girl with early-onset obesity and a de novo balanced translocation between chromosomes 1p22.1 and 6q16.2. Her weight gain is most likely due to excessive food intake, since measured energy expenditure was normal. We cloned and sequenced both translocation breakpoints. The translocation does not appear to affect any transcription unit on 1p, but it disrupts the SIM1 gene on 6q. SIM1 encodes a human homolog of Drosophila Sim (Single-minded), a transcription factor involved in midline neurogenesis, and is a prototypical member of the bHLH-PAS (basic helix-loop-helix + period, aryl hydrocarbon receptor, Single-minded) gene family. Our subject's trans- location separates the 5' promoter region and bHLH domain from the 3' PAS and putative transcriptional regulation domains. The transcriptional targets of SIM1 are not known. Mouse Sim1 is expressed in the developing kidney and central nervous system, and is essential for formation of the supraoptic and paraventricular (PVN) nuclei of the hypothalamus. Previous neuroanatomical and pharmacological studies have implicated the PVN in the regulation of body weight: PVN neurons express the melanocortin 4 receptor and appear to be physiological targets of alpha-melanocyte-stimulating hormone, which inhibits food intake. We hypothesize that haploinsufficiency of SIM1, possibly acting upstream or downstream of the melanocortin 4 receptor in the PVN, is responsible for severe obesity in our subject.

Abstract: The bHLH-PAS transcription factor SIM1 is required for the development of the paraventricular nucleus (PVN) of the hypothalamus. Mice homozygous for a null allele of Sim1 (Sim1 -/- ) lack a PVN and die perinatally. In contrast, we show here that Sim1 heterozygous mice are viable but develop early-onset obesity, with increased linear growth, hyperinsulinemia and hyperleptinemia. Sim1 +/- mice are hyperphagic but their energy expenditure is not decreased, distinguishing them from other mouse models of early-onset obesity such as deficiencies in leptin and melanocortin receptor 4. Quantitative histological comparison with normal littermates showed that the PVN of Sim1 +/- mice contains on average 24% fewer cells without a selective loss of any identifiable major cell type. Since acquired lesions in the PVN also induce increased appetite without a decrease in energy expenditure, we propose that abnormalities of PVN development cause the obesity of Sim1 +/- mice. Severe obesity was described recently in a patient with a balanced translocation disrupting SIM1. Pathways controlling the development of the PVN thus have the potential to cause obesity in both mice and humans.

Abstract: INTRODUCTION Loss-of-function mutations in one gene copy can lead to reduced amounts of protein and, consequently, human disease, a condition termed haploinsufficiency. It is currently estimated that more than 660 genes cause human disease as a result of haploinsufficiency. The delivery of extra copies of the gene by way of gene therapy is a promising therapeutic strategy to increase gene dosage in such conditions. Recombinant adeno-associated virus (rAAV) provides a promising tool for delivery of transgenes in an efficient and safe way for gene therapy. However, it has some limitations, including an optimal DNA packaging constraint of 4700 base pairs and ectopic expression. RATIONALE Increasing the expression levels of the normal gene copy by directly targeting the endogenous gene regulatory elements that control it could potentially correct haploinsufficiency. CRISPR-mediated activation (CRISPRa), whereby a nuclease-deficient Cas9 (dCas9) is used to target a transcriptional activator to the gene’s regulatory element (promoter or enhancer), could be used for this purpose. Such an approach could overcome the ectopic expression and DNA packaging limitations of rAAV. Using obesity as a model, we tested in mice whether CRISPR-mediated activation of the existing normal copy of two different genes, Sim1 or Mc4r, where loss-of-function mutations that lead to haploinsufficiency are a major cause of human obesity, can rescue their obesity phenotype. RESULTS We first generated a transgenic CRISPRa system using dCas9 fused to a transcriptional activator, VP64, to test whether it can rescue the obesity phenotype in a Sim1 haploinsufficient mouse model. CRISPRa targeting of the Sim1 promoter or its hypothalamus-specific enhancer, which is 270 kilobases away from the gene, in Sim1 haploinsufficient mice increased the expression of the normal copy of Sim1. This up-regulation was sufficient to rescue the obesity phenotype of Sim1 heterozygous mice and led to significantly reduced food intake and body fat content in these mice. We assessed the off-targeting effects of CRISPRa using both RNA sequencing (RNA-seq) and Cas9 chromatin immunoprecipitation sequencing (ChIP-seq) analyses. We found CRISPRa targeting to be highly specific and without any overt changes in the expression of other genes. We also observed that Sim1 up-regulation occurred only in tissues where the regulatory element (promoter or enhancer) that was being targeted was active. Although promoter-CRISPRa–targeted mice up-regulated Sim1 in all the tissues where it is expressed, the enhancer-CRISPRa–targeted mice showed Sim1 up-regulation only in the hypothalamus. We then delivered CRISPRa packaged into rAAV targeting the Sim1 promoter or its hypothalamus-specific enhancer using either Streptococcus pyogenes or the shorter Staphylococcus aureus CRISPRa system. We show that postnatal injection of CRISPRa-rAAV into the hypothalamus can up-regulate Sim1 expression and rescue the obesity phenotype in Sim1 haploinsufficient mice in a long-lasting manner. To further highlight the therapeutic potential of this approach to rescue other haploinsufficient genes, we targeted Mc4r, where haploinsufficiency leads to severe obesity in mice and humans. CRISPRa-rAAV targeting of the Mc4r promoter rescued the obesity phenotype of Mc4r heterozygous mice. CONCLUSION These findings show that the CRISPRa system can rescue a haploinsufficient phenotypein vivo. This CRISPR-mediated activation strategy is different from a conventional gene therapy strategy, as it uses the endogenous regulatory elements to up-regulate the existing functional gene copy. As such, it can overcome the problem of ectopic gene expression. In addition, it could be used for genes that are not amenable to conventional gene therapy because their coding sequences are longer than the rAAV packaging limit. Our results provide a framework to further develop CRISPRa as a potential tool to treat gene dosage–sensitive diseases.

Abstract: We generated mice carrying a loss-of-function mutation in Brn-2, a gene encoding a nervous system specific POU transcription factor, by gene targeting in embryonic stem cells. In homozygous mutant embryos, migratory precursor cells for neurons of the paraventricular nuclei (PVN) and the supraoptic nuclei (SO) of the hypothalamus die at approximately E12.5. All homozygous mutants suffered mortality within 10 days after birth, possibly because of a complete deficiency of these neurons in the hypothalamus. Although neither developmental nor histological abnormalities were observed in heterozygous mice, the levels of expression of vasopressin and oxytocin in the hypothalamus of these animals were half these of wild-type mice. These results strongly suggest that Brn-2 plays an essential role in the determination and development of the PVN and SO neuronal lineages in the hypothalamus.