Clara Wong

Skeletal Muscle Proteolysis in Rats With Acute Streptozocin-Induced Diabetes

Skeletal muscle proteolysis was studied in rats 1 day after induction of diabetes with 65 mg/kg streptozocin. An evisceration procedure, including functional hepatectomy-nephrectomy, was performed, and the rate of proteolysis in the remaining tissues, primarily skeletal muscles, was evaluated over 2 h. With cycloheximide to block protein synthesis, total protein breakdown was measured from the rate of rise in plasma tyrosine concentration. The rate of degradation of contractile (myofibrillar) protein was estimated from the rate of rise in plasma concentration of 3-methylhistidine released from the breakdown of actomyosin. Compared with nondiabetic control preparations, the total protein degradation rate was increased 30% by diabetes (P < .001), and myofibrillar catabolism was accelerated by 60% (P < .005). In diabetes, the increase in proteolysis was accompanied by reductions in circulating insulin to 25–50% of normal level, whereas food intake did not differ from control. Treatment of diabetic rats with exogenous insulin, including acute infusions postoperatively, completely reversed the proteolytic effects of diabetes. The findings demonstrate that the hypoinsulinemia of acute diabetes increases the catabolism of skeletal muscle protein and that the inhibitory effect of normal levels of insulin includes a specific action to restrain myofibrillar proteolysis.

Chromatin Architecture and Transcription Factor Binding Regulate Expression of Erythrocyte Membrane Protein Genes

ABSTRACT Erythrocyte membrane protein genes serve as excellent models of complex gene locus structure and function, but their study has been complicated by both their large size and their complexity. To begin to understand the intricate interplay of transcription, dynamic chromatin architecture, transcription factor binding, and genomic organization in regulation of erythrocyte membrane protein genes, we performed chromatin immunoprecipitation (ChIP) coupled with microarray analysis and ChIP coupled with massively parallel DNA sequencing in both erythroid and nonerythroid cells. Unexpectedly, most regions of GATA-1 and NF-E2 binding were remote from gene promoters and transcriptional start sites, located primarily in introns. Cooccupancy with FOG-1, SCL, and MTA-2 was found at all regions of GATA-1 binding, with cooccupancy of SCL and MTA-2 also found at regions of NF-E2 binding. Cooccupancy of GATA-1 and NF-E2 was found frequently. A common signature of histone H3 trimethylation at lysine 4, GATA-1, NF-E2, FOG-1, SCL, and MTA-2 binding and consensus GATA-1-E-box binding motifs located 34 to 90 bp away from NF-E2 binding motifs was found frequently in erythroid cell-expressed genes. These results provide insights into our understanding of membrane protein gene regulation in erythropoiesis and the regulation of complex genetic loci in erythroid and nonerythroid cells and identify numerous candidate regions for mutations associated with membrane-linked hemolytic anemia.

Teleost Growth Factor Independence (Gfi) Genes Differentially Regulate Successive Waves of Hematopoiesis

Growth Factor Independence (Gfi) transcription factors play essential roles in hematopoiesis, differentially activating and repressing transcriptional programs required for hematopoietic stem/progenitor cell (HSPC) development and lineage specification. In mammals, Gfi1a regulates hematopoietic stem cells (HSC), myeloid and lymphoid populations, while its paralog, Gfi1b, regulates HSC, megakaryocyte and erythroid development. In zebrafish, gfi1aa is essential for primitive hematopoiesis; however, little is known about the role of gfi1aa in definitive hematopoiesis or about additional gfi factors in zebrafish. Here, we report the isolation and characterization of an additional hematopoietic gfi factor, gfi1b. We show that gfi1aa and gfi1b are expressed in the primitive and definitive sites of hematopoiesis in zebrafish. Our functional analyses demonstrate that gfi1aa and gfi1b have distinct roles in regulating primitive and definitive hematopoietic progenitors, respectively. Loss of gfi1aa silences markers of early primitive progenitors, scl and gata1. Conversely, loss of gfi1b silences runx-1, c-myb, ikaros and cd41, indicating that gfi1b is required for definitive hematopoiesis. We determine the epistatic relationships between the gfi factors and key hematopoietic transcription factors, demonstrating that gfi1aa and gfi1b join lmo2, scl, runx-1 and c-myb as critical regulators of teleost HSPC. Our studies establish a comparative paradigm for the regulation of hematopoietic lineages by gfi transcription factors.

Influence of Glucocorticoids on Skeletal Muscle Proteolysis in Normal and Diabetic-Adrenalectomized Eviscerated Rats

The effect on skeletal muscle proteolysis of acute (20-hour) glucocorticoid treatment (dexamethasone 1.5 mg/kg, subcutaneously [SC]) was tested using the eviscerated rat preparation. According to this method, the peripheral tissues (primarily the skeletal muscles) are isolated by functional hepatectomy-nephrectomy. Total proteolysis is estimated from the rate of rise of plasma tyrosine concentration in the presence of cycloheximide to block protein synthesis. Myofibrillar proteolysis is measured from the rate of release into the plasma of the nonreutilized, nonmetabolized amino acid 3-methylhistidine (3MH), in the absence of cycloheximide. In normal rats, dexamethasone increased total proteolysis by 20% and myofibrillar proteolysis by 75% (both P less than .025 v saline controls). In diabetic-adrenalectomized rats prepared 2 weeks earlier (65 mg/kg streptozocin [STZ] followed by adrenalectomy), dexamethasone caused much greater increments in rates of total proteolysis (94%) and myofibrillar proteolysis (240%) (both P less than .001 v saline controls). Because diabetic animals are extremely sensitive to glucocorticoid-induced proteolysis, we also examined whether the acute proteolytic effect of diabetes itself might be mediated by adrenal cortical hormones. Previously adrenalectomized rats studied 20 hours after STZ showed a 40% augmentation of total proteolysis (P less than .01), an effect similar to that produced by acute diabetes in rats with intact adrenals. We conclude that glucocortical hormones cause a catabolic effect on total and myofibrillar skeletal muscle protein which is exaggerated when the counteracting action of insulin is reduced, but that the excess proteolysis of acute insulin deficiency is independent of the endogenous glucocorticoids secretion.

Patterns of Histone H3 Lysine 27 Monomethylation and Erythroid Cell Type-specific Gene Expression

Post-translational histone modifications, acting alone or in a context-dependent manner, influence numerous cellular processes via their regulation of gene expression. Monomethylation of histone H3 lysine 27 (K27me1) is a poorly understood histone modification. Some reports describe depletion of K27Me1 at promoters and transcription start sites (TSS), implying its depletion at TSS is necessary for active transcription, while others have associated enrichment of H3K27me1 at TSS with increased levels of mRNA expression. Tissue- and gene-specific patterns of H3K27me1 enrichment and their correlation with gene expression were determined via chromatin immunoprecipitation on chip microarray (ChIP-chip) and human mRNA expression array analyses. Results from erythroid cells were compared with those in neural and muscle cells. H3K27me1 enrichment varied depending on levels of cell-type specific gene expression, with highest enrichment over transcriptionally active genes. Over individual genes, the highest levels of H3K27me1 enrichment were found over the gene bodies of highly expressed genes. In contrast to H3K4me3, which was highly enriched at the TSS of actively transcribing genes, H3K27me1 was selectively depleted at the TSS of actively transcribed genes. There was markedly decreased to no H3K27me1 enrichment in genes with low expression. At some locations, H3K27 monomethylation was also found to be associated with chromatin signatures of gene enhancers.

Genetic basis of the polymorphisms of the αI domain of spectrin

Defects of α spectrin have been identified in many cases of hereditary elliptocytosis (HE) and hereditary pyropoikilocytosis (HPP). To aid in the genetic analysis of families with these disorders, the locations of three α‐spectrin gene polymorphisms were mapped, the genetic basis of these polymorphisms identified, and PCR‐based assays designed for their identification. The frequencies of these polymorphisms were determined in two populations and in patients with αI/50a HE and HPP. These studies identified two distinct haplotypes and provided evidence that two HE/HPP mutations associated with the αI/50a protein phenotype, L207P and L260P, arose on separate chromosomal backgrounds. Am. J. Hematol. 56:107–111, 1997.

Functional analysis of a novel cis-acting regulatory region within the human ankyrin gene (ANK-1) promoter.

ABSTRACT The characterization of atypical mutations in loci associated with diseases is a powerful tool to discover novel regulatory elements. We previously identified a dinucleotide deletion in the human ankyrin-1 gene (ANK-1) promoter that underlies ankyrin-deficient hereditary spherocytosis. The presence of the deletion was associated with a decrease in promoter function both in vitro and in vivo establishing it as a causative hereditary spherocytosis mutation. The dinucleotide deletion is located in the 5′ untranslated region of the ANK-1 gene and disrupts the binding of TATA binding protein and TFIID, components of the preinitiation complex. We hypothesized that the nucleotides surrounding the mutation define an uncharacterized regulatory sequence. To test this hypothesis, we generated a library of more than 16,000 ANK-1 promoters with degenerate sequence around the mutation and cloned the functional promoter sequences after cell-free transcription. We identified the wild type and three additional sequences, from which we derived a consensus. The sequences were shown to be functional in cell-free transcription, transient-transfection, and transgenic mouse assays. One sequence increased ANK-1 promoter function 5-fold, while randomly chosen sequences decreased ANK-1 promoter function. Our results demonstrate a novel functional motif in the ANK-1 promoter.