Ute Christine Rogner

CONGENIC MICE: CUTTING TOOLS FOR COMPLEX IMMUNE DISORDERS

Autoimmune diseases are, in general, under complex genetic control and subject to strong interactions between genetics and the environment. Greater knowledge of the underlying genetics will provide immunologists with a framework for study of the immune dysregulation that occurs in such diseases. Ascertaining the number of genes that are involved and their characterization have, however, proven to be difficult. Improved methods of genetic analysis and the availability of a draft sequence of the complete mouse genome have markedly improved the outlook for such research, and they have emphasized the advantages of mice as a model system. In this review, we provide an overview of the genetic analysis of autoimmune diseases and of the crucial role of congenic and consomic mouse strains in such research.

Control of neurulation by the nucleosome assembly protein-1–like 2

Neurulation is a complex process of histogenesis involving the precise temporal and spatial organization of gene expression. Genes influencing neurulation include proneural genes determining primary cell fate, neurogenic genes involved in lateral inhibition pathways and genes controlling the frequency of mitotic events. This is reflected in the aetiology and genetics of human and mouse neural tube defects, which are of both multifactorial and multigenic origin. The X-linked gene Nap1l2, specifically expressed in neurons, encodes a protein that is highly similar to the nucleosome assembly (NAP) and SET proteins. We inactivated Nap1l2 in mice by gene targeting, leading to embryonic lethality from mid-gestation onwards. Surviving mutant chimaeric embryos showed extensive surface ectoderm defects as well as the presence of open neural tubes and exposed brains similar to those observed in human spina bifida and anencephaly. These defects correlated with an overproduction of neuronal precursor cells. Protein expression studies showed that the Nap1l2 protein binds to condensing chromatin during S phase and in apoptotic cells, but remained cytoplasmic during G1 phase. Nap1l2 therefore likely represents a class of tissue-specific factors interacting with chromatin to regulate neuronal cell proliferation.

Nap1l2 Promotes Histone Acetylation Activity during Neuronal Differentiation

ABSTRACT The deletion of the neuronal Nap1l2 (nucleosome assembly protein 1-like 2) gene in mice causes neural tube defects. We demonstrate here that this phenotype correlates with deficiencies in differentiation and increased maintenance of the neural stem cell stage. Nap1l2 associates with chromatin and interacts with histones H3 and H4. Loss of Nap1l2 results in decreased histone acetylation activity, leading to transcriptional changes in differentiating neurons, which include the marked downregulation of the Cdkn1c (cyclin-dependent kinase inhibitor 1c) gene. Cdkn1c expression normally increases during neuronal differentiation, and this correlates with the specific recruitment of the Nap1l2 protein and an increase in acetylated histone H3K9/14 at the site of Cdkn1c transcription. These results lead us to suggest that the Nap1l2 protein plays an important role in regulating transcription in developing neurons via the control of histone acetylation. Our data support the idea that neuronal nucleosome assembly proteins mediate cell-type-specific mechanisms of establishment/modification of a chromatin-permissive state that can affect neurogenesis and neuronal survival.

Mapping of X chromosome inversion breakpoints [inv(X)(q11q28)] associated with FG syndrome: A second FG locus [FGS2]?

FG syndrome is an X-linked condition comprising mental retardation, congenital hypotonia, macrocephaly, distinctive facial changes, and constipation or anal malformations. In a linkage analysis, we mapped a major FG syndrome locus [FGS1] to Xq13, between loci DXS135 and DXS1066. The same data, however, clearly demonstrated genetic heterogeneity. Recently, we studied a French family in which an inversion [inv(X)(q12q28)] segregates with clinical symptoms of FG syndrome. This suggests that one of the breakpoints corresponds to a second FG syndrome locus [FGS2]. We report the results of fluorescence in situ hybridization analysis performed in this family using YACs and cosmids encompassing the Xq11q12 and Xq28 regions. Two YACs, one positive for the DXS1 locus at Xq11.2 and one positive for the color vision pigment genes and G6PD loci at Xq28, were found to cross the breakpoints, respectively. We postulate that a gene might be disrupted by one of the breakpoints.

Interaction between Nucleosome Assembly Protein 1-like Family Members

Mammals possess five nucleosome assembly protein 1-like (NAP1L) proteins, with three of them being expressed exclusively in the nervous system. The biological importance of the neuron-specific NAP1L2 protein is demonstrated by the neural tube defects occurring during the embryonic development of Nap1l2 mutant mice, which are associated with an overproliferation of neural stem cells and decreased neuronal differentiation. NAP1L2 controls the expression of its target genes, such as the cell cycle regulator Cdkn1c, at least in part via an effect on histone acetylation. Using a two-hybrid analysis, we have identified several proteins interacting with NAP1L2, including the ubiquitously expressed members of the nucleosome assembly protein family, NAP1L1 and NAP1L4. Structural studies further predict that all five NAP1-like proteins are able to interact directly via their highly conserved α-helices. These elements, in conjunction with the coexpression of all the NAP1-like proteins in neurons and the finding that deletion of Nap1l2 affects the cytoplasmic-nuclear distribution patterns of both NAP1L1 and NAP1L4 and their recruitment to target genes, suggest that combinatorial variation within the NAP family may ensure adaptation to the specific requirements for neuronal differentiation such as intercellular repartition, chromatin modification, transcriptional regulation, or the recruitment of specific transcription factors.

Lrmp and Bcat1 are candidates for the type I diabetes susceptibility locus Idd6

Three type 1 diabetes associated regions on distal mouse chromosome 6 have recently been defined by the construction and analysis of a series of congenic strains, carrying C3H/HeJ genomic material on a NOD/Lt genetic background. Whilst NOD/Lt alleles at the most distal locus Idd6 confer susceptibility, C3H/HeJ alleles confer resistance to diabetes. Idd6 overlaps with a locus controlling low rates of proliferation in immature NOD-thymocytes, suggesting that Idd6 could be controlling diabetes development through an effect on T cell proliferation rates. Candidates for Idd6 therefore include genes, which are implicated in the immune system and/or in the control of cell proliferation rates, such as Lrmp (Jaw1), Bcat1 and Kras2 that map to the Idd6 candidate region. In the present study, we have undertaken an expression and mutational analysis of all three genes. A surprisingly large number of polymorphisms and amino acid changes were identified in both Lrmp and Bcat1 indicating that they are candidates for Idd6. The two genes are located within a genomic interval of about 3 Mb that contains a large number of single nucleotide polymorphisms (SNP) and which has possibly been derived from distinct ancestral haplotypes in the C3H/HeJ and NOD/Lt strains.

Mapping of the murine type 1 diabetes locus Idd20 by genetic interaction

In the nonobese diabetes mouse, the murine type 1 diabetes susceptibility locus Idd20 interacts genetically with the diabetes resistance locus Idd19. Both Idds are located on distal mouse Chromosome 6, and previous studies on NOD.C3H congenic strains have shown that C3H alleles at Idd20 can suppress the disease-promoting effects of C3H alleles at Idd19 in both spontaneous and cyclophosphamide-induced diabetes. In this article we present the construction of novel congenic strains which, while maintaining the C3H alleles at Idd19, have allowed the candidate interval of Idd20 to be reduced from 4 to 1.8 cM. The analysis of these strains shows that Idd20 controls the progression of insulitis. Idd20 also increases the suppressive but not the pathogenic activity of splenocytes in diabetes transfer experiments. Our results suggest that the two Chromosome 6 susceptibility loci, Idd6 and Idd20, interact with the resistance locus Idd19 by regulating the activity of suppressor cells in the peripheral immune system.

Differential aiolos expression in human hematopoietic subpopulations

The Aiolos transcription factor plays a crucial role in the control of lymphocyte differentiation and proliferation. The expression of Aiolos isoform has been studied in lymphoid pathologies but nothing is known about its expression in unaffected human hematopoietic subpopulations. In this manuscript we show for the first time the differential Aiolos expression at the RNA and protein level in hematopoietic cell subpopulations. B cells express higher levels of Aiolos than NK and T cells while monocytes express almost undetectable levels of Aiolos. Moreover, human CD34 (+) progenitors do not express Aiolos. We did not observe significant difference when comparing naive to memory T and B cells, but we observed an important difference between Bright and Dim NK cells. Furthermore, we show that, in addition to hematopoietic cells, non hematopoietic cell lines such as MCF-7, SW480, HEK, PC3 and HeLa also express Aiolos.

Inhibition of type 1 diabetes by upregulation of the circadian rhythm-related aryl hydrocarbon receptor nuclear translocator-like 2

The genetic locus Idd6 is involved in type 1 diabetes development in the non-obese diabetic (NOD) mouse through its effect on the immune system and in particular, on T cell activities. Analysis of congenic strains for Idd6 has established the Aryl hydrocarbon receptor nuclear translocator-like 2 (Arntl2) as a likely candidate gene. In this study we investigate the role of Arntl2 in the autoimmune disease and T cell activation. An Arntl2 expressing plasmid was transfected into CD4+ T cells by nucleofection. Expression levels of cytokines and CD4+ T cell activation markers, cell death, apoptosis, and cell proliferation rates were characterized in ex vivo experiments whilst in vivo the transfected cells were transferred into NOD.SCID mice to monitor diabetes development. The results demonstrate that Arntl2 overexpression leads to inhibition of CD4+ T cell proliferation and decreases in their diabetogenic activity without influence on the expression levels of cytokines, CD4+ T cell activation markers, cell death, and apoptosis. Our findings suggest that Arntl2 at the Idd6 locus may act via the inhibition of CD4+ T cell proliferation and the reduction in the diabetogenic activity of CD4+ T cells to protect against autoimmune type 1 diabetes in the NOD mice.

Downregulation of the circadian rhythm related gene Arntl2 suppresses diabetes protection in Idd6 NOD.C3H congenic mice.

1. Our previous studies of the murine genetic locus Idd6 revealed the aryl hydrocarbon receptor nuclear translocator‐like protein 2 (Arntl2) as a candidate gene for type 1 diabetes; and in Idd6 NOD.C3H congenic mice, Arntl2 upregulation is linked to decreased diabetes development.