Unit: Nematode Genetics
Director: LAKOWSKI, Bernard
Mutations in the human presenilin genes can cause Familial Alzheimer's disease. Using the Nematode Caenorhabditis elegans we are characterizing several suppressor of presenilin' (spr) genes that regulate presenilin transcription and identifying additional genes. The human homologues of these genes regulate the expression of many neuronal genes and play an important role in neuronal determination and differentiation. We are also characterising dog-1, a C. elegans DNA helicase involved in genomic stability, in order to use it better as a tool for genetics and to understand the role of its homolog BACH1 in breast cancer.
1) Presenilin suppressors
The Nematode Caenorhabditis elegans is a powerful model system to study neurobiology and development. We are using C. elegans to study the genetics of presenilin genes. Presenilins are polytopic proteins that are found as part of a high molecular weight complex that cleaves certain types of type I transmembrane proteins in the middle of the transmembrane domain. Mutations in the human presenilin genes lead to Familial Alzheimer's Disease by affecting the processing of the Amyloid Precursor Protein (APP). In all animals, presenilin genes are essential for normal development as they are necessary for the activation of Notch receptors.
To try to understand more about this important class of proteins, we have been using genetic approaches in C. elegans to identify factors that can modify the effect of mutations in presenilin genes. Loss of sel-12 presenilin activity in C. elegans results in a strong egg-laying defect. In screens for mutations that suppress this defect, five spr genes (for suppressor of presenilin) were identified by us and others. These genes encode components of a putative transcriptional repressor complex that normally represses the transcription of a second presenilin gene, hop-1, and presumably other targets. The SPR proteins are similar to components of the human REST-CoREST complex, which is an important regulator of the final step of neuronal determination and differentiation.
In two new screens we have recovered over 50 new spr mutations and we are in the process of characterizing these mutations. As expected, we have recovered new alleles of all previously identified spr genes including several mutations that affect conserved domains of these proteins, shedding additional light on the structure and function of these genes. We have also identified several mutations in the gene sel-10, a component of an ubiquitin ligase complex known to regulate the stability of presenilin and Notch receptor proteins. In addition, we have recovered several mutations that define at least three additional spr genes which may encode additional components of the putative SPR complex or other types of genes that can suppress sel-12.
A CoREST-like complex has recently been shown to regulate neuronal gene expression in Drosophila, suggesting an ancient role for CoREST-like complexes in neuronal cell fate. Furthermore, the human homologues of the spr genes are implicated in several types of cancer. We are using the C. elegans spr genes to address the biological and biochemical function of CoREST-like complexes and their associated transcription factors in order to better understand the role of these complexes in neuronal cell fate, transcriptional repression and disease.
2) The mutator dog-1
Mutations in the human BACH1 gene (Brca1 associated helicase) are responsible for a small number of the cases of familial breast cancer. Although BACH1 binds to the breast cancer susceptibility gene Brca1, the role BACH1 plays in the development of breast cancer is unknown. The Caenorhabditis elegans gene dog-1 (for deletions of guanine rich DNA) encodes a DEAH box DNA helicase most similar to BACH1. It has been reported that loss of dog-1 function leads to genomic instability and causes a high frequency of small deletions at poly-guanine (poly-G) stretches longer than 17 nucleotides. Based on the spectrum of dog-1 induced mutations, it was suggested that DOG-1 may be required to resolve three-dimensional structures formed by poly-G stretches during lagging strand DNA synthesis. Studies on dog-1 may help to clarify the role of BACH1 and related genes in genomic instability and cancer. We are also trying to use the extremely unusual and restricted spectrum of dog-1 induced mutations to advance forward and reverse genetics in C. elegans.
To better characterise the spectrum of germline mutations induced by dog-1, we have carried out several screens. So far we have isolated dog-1 induced mutations in 13 genes, including spr-3 and spr-4, and determined the sequence of 10 of these mutations. We have cloned the gene dpy-1, which appears to encode a novel structural component of the worm's cuticle. We have also identified four new genes which we are in the process of cloning and characterizing. Our preliminary results confirm that dog-1 does induce a high frequency of deletions at poly-G stretches but that it is not totally specific for such sites. We also find that the deletions are often larger than previously reported and that DOG-1 may have additional functions to those proposed. However, the restricted spectrum of dog-1 induced mutations can indeed aid in cloning genes identified by mutation (forward genetics) and could be used to target deletions in selected gene sequences (reverse genetics).
Keywords: Genetics, Caenorhabditis elegans, Alzheimer’s disease, epigenetics, mutator