Loss of Rearranged L-Myc Fusion (RLF) results in defects in heart development in the mouse.

Recently we reported that Rearranged L-Myc Fusion, RLF, acts as an epigenetic modifier maintaining low levels of DNA methylation at CpG island shores and enhancers across the genome. Here we focus on the phenotype of Rlf null mutant mice generated via an ENU mutagenesis screen, to identify genes required for epigenetic regulation. RLF is expressed in a range of fetal mouse tissues, including the fetal heart. Comprehensive timed-mating studies are consistent with our previously reported findings that Rlf homozygous mutant mice rarely survive to adulthood, with the majority dying shortly after birth. Histological analysis of two independent Rlf ENU mutant lines at E11.5-E14.5 showed heart defects resembling those present in humans with Left Ventricular Non-Compaction (LVNC). In situ hybridisation analysis localized expression of Rlf to the endocardium and epicardium of embryonic and postnatal hearts, and transiently to cardiomyocytes during heart looping and early chamber formation stages. RNA-seq analysis of Rlf mutant hearts highlighted defective NOTCH pathway signalling, recently describe as one cause of LVNC. This study provides the first evidence that RLF is required for normal heart development in the mouse. The heart morphological defects present at high penetrance in Rlf mutants are consistent with features of LVNC in humans, and molecular analysis identified attenuated JAGGED 1 expression and NOTCH signalling as likely contributors to these defects. Our study highlights the importance of RLF-dependent epigenetic modifications to DNA for maintaining correct gene regulatory network and intercellular signalling interactions during heart chamber and septal development. Further investigations are needed to define the biochemical role of RLF in the developing heart, and whether RLF mutations are a cause of heart defects in humans.

Biochemical characteristics and inhibitor selectivity of mouse indoleamine 2,3-dioxygenase-2.

The first step in the kynurenine pathway of tryptophan catabolism is the cleavage of the 2,3-double bond of the indole ring of tryptophan. In mammals, this reaction is performed independently by indoleamine 2,3-dioxygenase-1 (IDO1), tryptophan 2,3-dioxygenase (TDO) and the recently discovered indoleamine 2,3-dioxygenase-2 (IDO2). Here we describe characteristics of a purified recombinant mouse IDO2 enzyme, including its pH stability, thermal stability and structural features. An improved assay system for future studies of recombinant/isolated IDO2 has been developed using cytochrome b (5) as an electron donor. This, the first description of the interaction between IDO2 and cytochrome b (5), provides further evidence of the presence of a physiological electron carrier necessary for activity of enzymes in the "IDO family". Using this assay, the kinetic activity and substrate range of IDO2 were shown to be different to those of IDO1. 1-Methyl-D-tryptophan, a current lead IDO inhibitor used in clinical trials, was a poor inhibitor of both IDO1 and IDO2 activity. This suggests that its immunosuppressive effect may be independent of pharmacological inhibition of IDO enzymes, in the mouse at least. The different biochemical characteristics of the mouse IDO proteins suggest that they have evolved to have distinct biological roles.

The molecular mechanisms of congenital hypofibrinogenaemia.

Congenital hypofibrinogenaemia is characterized by abnormally low levels of fibrinogen and is usually caused by heterozygous mutations in the fibrinogen chain genes (alpha, beta and gamma). However, it does not usually result in a clinically significant condition unless inherited in a homozygous or compound heterozygous state, where it results in a severe bleeding disorder, afibrinogenaemia. Various protein and expression studies have improved our understanding of how mutations causing hypo- and afibrinogenaemia affect secretion of the mature fibrinogen molecule from the hepatocyte. Some mutations can perturb chain assembly as in the gamma153 Cys-->Arg case, while others such as the Bbeta Leu-->Arg and the Bbeta414 Gly-->Ser mutations allow intracellular hexamer assembly but inhibit protein secretion. An interesting group of mutations, such as gamma284 Gly-->Arg and gamma375 Arg-->Trp, not only cause hypofibrinogenaemia but are also associated with liver disease. The nonexpression of these variant chains in plasma fibrinogen is due to retention in the endoplasmic reticulum, which in turn leads to hypofibrinogenaemia.