Cheng-Han Huang

The Human Rh50 Glycoprotein Gene STRUCTURAL ORGANIZATION AND ASSOCIATED SPLICING DEFECT RESULTING IN Rhnull DISEASE

The Rh (Rhesus) protein family comprises Rh50 glycoprotein and Rh30 polypeptides, which form a complex essential for Rh antigen expression and erythrocyte membrane integrity. This article describes the structural organization of Rh50 gene and identification of its associated splicing defect causing Rhnulldisease. The Rh50 gene, which maps at chromosome 6p11–21.1, has an exon/intron structure nearly identical to Rh30 genes, which map at 1p34–36. Of the 10 exons assigned, conservation of size and sequence is confined mainly to the region from exons 2 to 9, suggesting thatRH50 and RH30 were formed as two separate genetic loci from a common ancestor via a transchromosomal insertion event. The available information on the structure of RH50facilitated search for candidate mutations underlying the Rh deficiency syndrome, an autosomal recessive disorder characterized by mild to moderate chronic hemolytic anemia and spherostomatocytosis. In one patient with the Rhnull disease of regulator type, a shortened Rh50 transcript lacking the sequence of exon 7 was detected, while no abnormality was found in transcripts encoding Rh30 polypeptides and Rh-related CD47 glycoprotein. Amplification and sequencing of the genomic region spanning exon 7 revealed a G → A transition in the invariant GT motif of the donor splice site in both Rh50 alleles. This splicing mutation caused not only a total skipping of exon 7 but also a frameshift and premature chain termination. Thus, the deduced translation product contained 351 instead of 409 amino acids, with an entirely different C-terminal sequence following Thr315. These results identify the donor splicing defect, for the first time, as a loss-of-function mutation at theRH50 locus and pinpoint the importance of the C-terminal region of Rh50 in Rh complex formation via protein-protein interactions.

Regulation of fat storage and reproduction by Kruppel-like transcription factor KLF3 and fat-associated genes in Caenorhabditis elegans.

Coordinated regulation of fat storage and utilization is essential for energy homeostasis, and its disruption is associated with metabolic syndrome and atherosclerosis in humans. Across species, Krüppel-like transcription factors (KLFs) have been identified as key components of adipogenesis. In humans, KLF14 acts as a master transregulator of adipose gene expression in type 2 diabetes and cis-acting expression quantitative trait locus associated with high-density lipoprotein cholesterol. Herein we report that, in Caenorhabditis elegans, mutants in klf-3 accumulate large fat droplets rich in neutral lipids in the intestine; this lipid accumulation is associated with an increase in triglyceride levels. The klf-3 mutants show normal pharyngeal pumping; however, they are sterile or semisterile. We explored important genetic interactions of klf-3 with the genes encoding enzymes involved in fatty acid (FA) β-oxidation in mitochondria or peroxisomes and FA synthesis in the cytosol, namely acyl-CoA synthetase (acs-1 and acs-2), acyl-CoA oxidase (F08A8.1 and F08A8.2), and stearoyl-CoA desaturase (fat-7). We show that mutations or RNA interference in these genes increases fat deposits in the intestine of acs-1, acs-2, F08A8.1, and F08A8 animals. We further show that acs-1 and F08A8.1 influence larval development and fertility, respectively. Thus, KLF3 may regulate FA utilization in the intestine and reproductive tissue. We demonstrate that depletion of F08A8.1 activity, but not of acs-1, acs-2, F08A8.2, or fat-7 activity, enhances the fat phenotype of the klf-3 mutant. Taken together, these results suggest that klf-3 regulates lipid metabolism, along with acs-1, acs-2, F08A8.1, and F08A8.2, by promoting FA β-oxidation and, in parallel, may contribute to normal reproductive behavior and fecundity in C. elegans.

A Krüppel-Like Factor in Caenorhabditis elegans with Essential Roles in Fat Regulation, Cell Death, and Phagocytosis

We demonstrate that a Caenorhabditis elegans Krüppel-like transcription factor is involved in fat regulation, cell death, and phagocytosis in C. elegans. Suppression of C. elegans klf-1 function by RNA interference (RNAi) results in increased fat storage in the intestine of the RNAi worm that directly or indirectly causes germ cells to die. These dead cells are not engulfed or phagocytosed in the RNAi worm. High-level expression of Ce-klf-1 during larval development, as well as its specific localization in the worms intestine, supports a direct role for Ce-klf-1 in fat regulation. The C. elegans klf-1 encodes a C(2)H(2) zinc finger protein that is known to act as transcriptional modulator of tissue-specific expression. Members of the Krüppel-like factor (KLF) family play a variety of important roles in vertebrate tissue differentiation. KLFs have recently been implicated in energy and glucose homeostasis through their expression in pancreas, adipose, liver, and muscle tissues. The extensive fat storage and increased cell death in the Ce-klf-1 RNAi worm is important in that it may explain the connection between Ce-klf-1 signaling, cell death, and fat storage. This is the first evidence involving Ce-KLF-1 protein in such functions. In future studies, a thorough analysis of cellular functions of other members of C. elegans Krüppel-like transcription factors together with their interactions and pathway activities with other molecular partners should yield significant insights into mammalian KLF proteins.