GATA-1 Interferes with Monopoiesis by Blocking a Positive Regulatory Circuit Involving VDR and PU.1 - Results from a Retroviral Dominant Effector Screen.

In multipotent hematopoietic progenitors the co-expression of several lineage affiliated transcription factors occurs prior to commitment. The mechanism underlying the repression of alternative lineage pathways is poorly understood. We screened a complex retroviral fetal liver cDNA library for regulatory proteins that repress monopoiesis in a phenotype-based differentiation assay system. This functional genetic “dominant effector” strategy revealed a positive circuit regulating monopoiesis that is established by PU.1 and vitamin D receptor (VDR) as well as a novel repressor function of GATA-1. Increasing levels of PU.1 sensitize progenitors to VDR-dependent monopoiesis, and VDR/retinoic X receptor (RXR) signalling directly transactivates PU.1 gene expression. This positive regulatory loop is repressed by GATA-1 via the VDR. Specifically, we show that the GATA-1 N-terminal region, which is required for the inhibition of monocytic cell proliferation, represses and physically interacts with VDR. Additionally, PU.1 binding via the GATA-1 C-terminus is sufficient to inhibit monocyte differentiation without impairing myeloid cell proliferation. Thus, loss of GATA-1 N-terminal function allows for the expansion of immature myeloid progenitors. We propose that GATA-1 represses monopoiesis in multipotent myeloid progenitors or precursors by interacting with both VDR and PU.1, ultimately enabling alternative lineage programs.

Molecular cloning and expression analysis of a new gene for short-chain dehydrogenase/reductase 9.

We report here the cloning and characterization of a novel human short-chain dehydrogenases/reductase gene SCDR9, isolated from a human liver cDNA library, and mapped to 4q22.1 by browsing the UCSC genomic database. SCDR9 containing an ORF with a length of 900 bp, encoding a protein with a signal peptide sequence and an adh_short domain. GFP localization shows SCDR9 protein concentrated in some site of the cytoplasm, but not in the ER. Expression pattern in eighteen tissues revealed that SCDR9 is expressed highly in liver. Soluble recombinant protein was successfully purified from Escherichia coli using pET28A(+) expression vector. Our data provides important information for further study of the function of the SCDR9 gene and its products.

Isolation and functional expression of human COQ2, a gene encoding a polyprenyl transferase involved in the synthesis of CoQ

The COQ2 gene in Saccharomyces cerevisiae encodes a Coq2 (p-hydroxybenzoate:polyprenyl transferase), which is required in the biosynthetic pathway of CoQ (ubiquinone). This enzyme catalyses the prenylation of p-hydroxybenzoate with an all-trans polyprenyl group. We have isolated cDNA which we believe encodes the human homologue of COQ2 from a human muscle and liver cDNA library. The clone contained an open reading frame of length 1263 bp, which encodes a polypeptide that has sequence homology with the Coq2 homologues in yeast, bacteria and mammals. The human COQ2 gene, when expressed in yeast Coq2 null mutant cells, rescued the growth of this yeast strain in the absence of a non-fermentable carbon source and restored CoQ biosynthesis. However, the rate of CoQ biosynthesis in the rescued cells was lower when compared with that in cells rescued with the yeast COQ2 gene. CoQ formed when cells were incubated with labelled decaprenyl pyrophosphate and nonaprenyl pyrophosphate, showing that the human enzyme is active and that it participates in the biosynthesis of CoQ.

Identification, cloning and expression of the mouse N-acetylglutamate synthase gene

In ureotelic animals, N-acetylglutamate (NAG) is an essential allosteric activator of carbamylphosphate synthetase I (CPSI), the first enzyme in the urea cycle. NAG synthase (NAGS; EC 2.3.1.1) catalyses the formation of NAG from glutamate and acetyl-CoA in liver and intestinal mitochondria. This enzyme is supposed to regulate ureagenesis by producing variable amounts of NAG, thus modulating CPSI activity. Moreover, inherited deficiencies in NAGS have been associated with hyperammonaemia, probably due to the loss of CPSI activity. Although the existence of the NAGS protein in mammals has been known for decades, the gene has remained elusive. We identified the mouse (Mus musculus) and human NAGS genes using their similarity to the respective Neurospora crassa gene. NAGS was cloned from a mouse liver cDNA library and was found to encode a 2.3kb message, highly expressed in liver and small intestine with lower expression levels in kidney, spleen and testis. The deduced amino acid sequence contains a putative mitochondrial targeting signal at the N-terminus. The cDNA sequence complements an argA (NAGS)-deficient Escherichia coli strain, reversing its arginine auxotrophy. His-tagged versions of the pre-protein and two putative mature proteins were each overexpressed in E. coli, and purified to apparent homogeneity by using a nickel-affinity column. The pre-protein and the two putative mature proteins catalysed the NAGS reaction but one of the putative mature enzymes had significantly higher activity than the pre-protein. The addition of l-arginine increased the catalytic activity of the purified recombinant NAGS enzymes by approx. 2–6-fold.