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 stem cell fate and neuronal determination. 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/BRIP1 in Fanconi anemia and 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 neuronal gene expression, especially during neurogenesis.
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 many 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 mutations that define at least three additional spr genes that may encode additional components of the putative SPR complex or other types of genes that can suppress sel-12. We have cloned spr-6 and its characterisation is ongoing. We have found that spr-6 encodes a novel protein with no domains similar to those in the PFAM database. However, SPR-6 is clearly conserved in other nematodes species and multiple alignments of SPR-6 homologous point to the existence of at least three conserved domains that may bind zinc and mediate protein-protein interactions. This suggests that SPR-6 may be an additional component of the SPR complex. As the SPR proteins are predicted to modify histones, we have begun to characterise the nature of chromatin around the hop-1 locus by chromatin immunoprecipitation (ChIP) both in the wild type and in spr mutants strains.
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/BRIP1 gene are responsible for a small number of the cases of familial breast cancer and Fanconi anemia. Although BRIP1 binds to the breast cancer susceptibility gene BRCA1, the role BRIP1 plays in the development of disease and cancer is unknown. The Caenorhabditis elegans gene dog-1 (for deletions of guanine rich DNA) encodes a DEAH box DNA helicase homologous to BRIP1. It had 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 BRIP1 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 two types of genetic screens: one for simple visible phenotypes and the other for spr genes. So far we have isolated dog-1 induced mutations in 13 genes and determined the exact nature of the mutations for 23 alleles. Most dog-1 induced mutations are indeed small deletions occurring at Poly-G and other G-rich sequences. However we have also identified three other classes of mutations that indicate that dog-1 can cause more complex types of genomic rearrangements. Many dog-1 induced mutations have small regions of micro-homology at their breakpoints implicating Non-Homologous End Joining in the repair of dog-1 induced genomic instability.
We have also found that the restricted spectrum of dog-1 induced mutations can aid in cloning genes identified by mutation (forward genetics) and can be used to target deletions in selected gene sequences (reverse genetics). dog-1 induced mutations are also often very useful genetic tools and we have recovered both null and partial loss of function alleles of various genes. For example, we have used dog-1 to generate putative null alleles of spr-3 and a strong loss of function alleles of spr-4. Furthermore, for two genes, we have recovered small deletions in the promoter regions that point to the presence of important domains regulating gene expression. The presence of poly-G stretches in the proximity of candidate genes has also allowed us to clone four new genes: W08G11.4, ngn-1, nhr-67 and dpy-1. W08G11.4, ngn-1 and nhr-67 all have clear homologs in other species and their phenotypes indicate that these genes play important roles in nervous system development and function. dpy-1 appears to encode a novel structural component of the worm's cuticle. To better understand the function of dpy-1 we are trying to determine the structure of dpy-1 homologues in other nematode species. In collaboration with the group of Ralf Sommer at the Max Planck Institute for Developmental biology in Tuebingen, we have determined the structure of pdl-1, the Pristionchus pacificus homolog of dpy-1, and shown that mutations in pdl-1 have very similar consequences to mutations in dpy-1. Comparison of dpy-1 and pdl-1 has helped to define conserved domains in dpy-1 and will help us to understand its biological function.
Keywords: Genetics, Caenorhabditis elegans, Alzheimer’s disease, epigenetics, mutator