Gretchen J. Darlington

Impaired energy homeostasis in C/EBP alpha knockout mice

Mice homozygous for the targeted deletion of the c/ebp alpha gene, which expresses the CCAAT/enhancer-binding protein alpha (C/EBP alpha), did not store hepatic glycogen and died from hypoglycemia within 8 hours after birth. In these mutant mice, the amounts of glycogen synthase messenger RNA were 50 to 70 percent of normal and the transcriptional induction of the genes for two gluconeogenic enzymes, phosphoenolpyruvate carboxykinase and glucose-6-phosphatase, was delayed. The hepatocytes and adipocytes of the mutant mice failed to accumulate lipid and the expression of the gene for uncoupling protein, the defining marker of brown adipose tissue, was reduced. This study demonstrates that C/EBP alpha is critical for the establishment and maintenance of energy homeostasis in neonates.

Cross-Regulation of C/EBPα and PPARγ Controls the Transcriptional Pathway of Adipogenesis and Insulin Sensitivity

Abstract Mice deficient in C/EBPα have defective development of adipose tissue, but the precise role of C/EBPα has not been defined. Fibroblasts from C/EBPα(−/−) mice undergo adipose differentiation through expression and activation of PPARγ, though several clear defects are apparent. C/EBPα-deficient adipocytes accumulate less lipid, and they do not induce endogenous PPARγ, indicating that cross-regulation between C/EBPα and PPARγ is important in maintaining the differentiated state. The cells also show a complete absence of insulin-stimulated glucose transport, secondary to reduced gene expression and tyrosine phosphorylation for the insulin receptor and IRS-1. These results define multiple roles for C/EBPα in adipogenesis and show that cross-regulation between PPARγ and C/EBPα is a key component of the transcriptional control of this cell lineage.

The Role of C/EBP Genes in Adipocyte Differentiation

One of the central problems facing higher animals is that cells require a continuous source of energy; however, it is impractical for organisms to meet this need by supplying a constant external source of calories. Two specialized tissues, brown and white adipose tissues, have evolved to meet the ongoing requirement for energy. White adipose tissue is able to store excess calories in the form of triacylglycerol. When cells require energy, such as during periods of fasting, these needs are largely met by fatty acids and glycerol formed from lipolysis of stored triacylglycerol. Brown adipose tissues use stored triacylglycerols to maintain body temperature. In particular, these cells convert energy from fatty acid metabolism to heat through the action of uncoupling protein 1 (UCP1), a mitochondrial protein found only in brown adipose tissue. Brown adipocytes contain less triacylglycerol and many more mitochondria than white adipocytes, resulting in their characteristic color. Humans and rats develop brown adipose tissue depots prenatally, and although these depots largely disappear in humans during childhood, some brown adipocytes likely remain interspersed in white adipose tissue throughout adulthood (1). In view of the prevalence of obesity and obesity-related diseases, such as type II diabetes, it is important to understand how white and brown adipose tissues develop and how the activities of these tissues are regulated. Many factors are important for normal adipocyte development and function. In this minireview, we explore the role of one family of transcription factors, the CCAAT/enhancer-binding proteins (C/EBPs), in inducing preadipocyte differentiation and in modulating gene expression in the fully differentiated adipocyte. Analyses of cultured cell lines, and more recently, genetically altered mice have contributed significantly to our understanding of the way in which adipose-specific gene expression is directed by C/EBPs. These models have also elucidated some of the molecular mechanisms that regulate the expression of the C/EBP genes themselves.

Dnmt3a is essential for hematopoietic stem cell differentiation

Loss of the de novo DNA methyltransferases Dnmt3a and Dnmt3b in embryonic stem cells obstructs differentiation; however, the role of these enzymes in somatic stem cells is largely unknown. Using conditional ablation, we show that Dnmt3a loss progressively impairs hematopoietic stem cell (HSC) differentiation over serial transplantation, while simultaneously expanding HSC numbers in the bone marrow. Dnmt3a-null HSCs show both increased and decreased methylation at distinct loci, including substantial CpG island hypermethylation. Dnmt3a-null HSCs upregulate HSC multipotency genes and downregulate differentiation factors, and their progeny exhibit global hypomethylation and incomplete repression of HSC-specific genes. These data establish Dnmt3a as a critical participant in the epigenetic silencing of HSC regulatory genes, thereby enabling efficient differentiation.

A combination of prebiotic short- and long-chain inulin-type fructans enhances calcium absorption and bone mineralization in young adolescents

BACKGROUND Short-term studies in adolescents have generally shown an enhancement of calcium absorption by inulin-type fructans (prebiotics). Results have been inconsistent; however, and no studies have been conducted to determine whether this effect persists with long-term use. OBJECTIVE The objective was to assess the effects on calcium absorption and bone mineral accretion after 8 wk and 1 y of supplementation with an inulin-type fructan. DESIGN Pubertal adolescents were randomly assigned to receive 8 g/d of a mixed short and long degree of polymerization inulin-type fructan product (fructan group) or maltodextrin placebo (control group). Bone mineral content and bone mineral density were measured before randomization and after 1 y. Calcium absorption was measured with the use of stable isotopes at baseline and 8 wk and 1 y after supplementation. Polymorphisms of the Fok1 vitamin D receptor gene were determined. RESULTS Calcium absorption was significantly greater in the fructan group than in the control group at 8 wk (difference: 8.5 +/- 1.6%; P < 0.001) and at 1 y (difference: 5.9 +/- 2.8%; P = 0.04). An interaction with Fok1 genotype was present such that subjects with an ff genotype had the least initial response to fructan. After 1 y, the fructan group had a greater increment in both whole-body bone mineral content (difference: 35 +/- 16 g; P = 0.03) and whole-body bone mineral density (difference: 0.015 +/- 0.004 g/cm(2); P = 0.01) than did the control group. CONCLUSION Daily consumption of a combination of prebiotic short- and long-chain inulin-type fructans significantly increases calcium absorption and enhances bone mineralization during pubertal growth. Effects of dietary factors on calcium absorption may be modulated by genetic factors, including specific vitamin D receptor gene polymorphisms.

CCAAT/enhancer binding protein alpha regulates p21 protein and hepatocyte proliferation in newborn mice.

CCAAT/enhancer binding protein alpha (C/EBP alpha) is expressed at high levels in quiescent hepatocytes and in differentiated adipocytes. In cultured cells, C/EBP alpha inhibits cell proliferation in part via stabilization of the p21 protein. The role of C/EBP alpha in regulating hepatocyte proliferation in vivo is presented herein. In C/EBP alpha knockout newborn mice, p21 protein levels are reduced in the liver, and the fraction of hepatocytes synthesizing DNA is increased. Greater than 30% of the hepatocytes in C/EBP alpha knockout animals continue to proliferate at day 17 of postnatal life when cell division in wild-type littermates is low (3%). p21 protein levels are relatively high in wild-type neonates but undetectable in C/EBP alpha knockout mice. The reduction of p21 protein in the highly proliferating livers that lack C/EBP alpha suggests that p21 is responsible for C/EBP alpha-mediated control of liver proliferation in newborn mice. During rat liver regeneration, the amounts of both C/EBP alpha and p21 proteins are decreased before DNA synthesis (6 to 12 h) and then return to presurgery levels at 48 h. Although C/EBP alpha controls p21 protein levels, p21 mRNA is not influenced by C/EBP alpha in liver. Using coimmunoprecipitation and a mammalian two-hybrid assay system, we have shown the interaction of C/EBP alpha and p21 proteins. Study of p21 stability in liver nuclear extracts showed that C/EBP alpha blocks proteolytic degradation of p21. Our data demonstrate that C/EBP alpha regulates hepatocyte proliferation in newborn mice and that in liver, the level of p21 protein is under posttranscriptional control, consistent with the hypothesis that protein-protein interaction with C/EBP alpha determines p21 levels.

C/EBPα is required for differentiation of white, but not brown, adipose tissue

The transcription factor CCAAT enhancer binding protein α (C/EBPα) is expressed at high levels in liver and adipose tissue. Cell culture studies show that C/EBPα is sufficient to trigger differentiation of preadipocytes into mature adipocytes, suggesting a central role for C/EBPα in the development of adipose tissue. C/EBPα knockout mice die within 7–12 h after birth. Defective gluconeogenesis of the liver and subsequent hypoglycemia contribute to the early death of these animals. This short life span impairs investigation of the development of adipose tissue in these mice. To improve the survival of C/EBPα−/− animals, we generated a transgenic line that expresses C/EBPα under the control of the albumin enhancer/promoter. This line was bred into the knockout strain to generate animals that express C/EBPα in the liver but in no other tissue. The presence of the transgene improved survival of C/EBPα−/− animals almost 3-fold. Transgenic C/EBPα−/− animals at 7 days of age show an absence of s.c., perirenal, and epididymal white fat despite excess lipid substrate in the serum, whereas brown adipose tissue is somewhat hypertrophied and shows minimal biochemical alterations. Interestingly, mammary gland fat tissue is present and exhibits normal morphology. The absence of white adipose tissue in many depots in the presence of high serum lipid levels shows that C/EBPα is required for the in vivo development of this tissue. In contrast, brown adipose tissue differentiation is independent of C/EBPα expression. The presence of lipid in brown adipose tissue serves as an internal nutritional control, indicating that neither nutritional intake nor lipoprotein composition is likely responsible for the absence of white fat.

Epigenomic profiling of young and aged HSCs reveals concerted changes during aging that reinforce self-renewal

To investigate the cell-intrinsic aging mechanisms that erode the function of somatic stem cells during aging, we have conducted a comprehensive integrated genomic analysis of young and aged cells. We profiled the transcriptome, DNA methylome, and histone modifications of young and old murine hematopoietic stem cells (HSCs). Transcriptome analysis indicated reduced TGF-β signaling and perturbation of genes involved in HSC proliferation and differentiation. Aged HSCs exhibited broader H3K4me3 peaks across HSC identity and self-renewal genes and showed increased DNA methylation at transcription factor binding sites associated with differentiation-promoting genes combined with a reduction at genes associated with HSC maintenance. Altogether, these changes reinforce HSC self-renewal and diminish differentiation, paralleling phenotypic HSC aging behavior. Ribosomal biogenesis emerged as a particular target of aging with increased transcription of ribosomal protein and RNA genes and hypomethylation of rRNA genes. This data set will serve as a reference for future epigenomic analysis of stem cell aging.

Large conserved domains of low DNA methylation maintained by Dnmt3a

Gains and losses in DNA methylation are prominent features of mammalian cell types. To gain insight into the mechanisms that promote shifts in DNA methylation and contribute to changes in cell fate, including malignant transformation, we performed genome-wide mapping of 5-methylcytosine and 5-hydroxymethylcytosine in purified mouse hematopoietic stem cells. We discovered extended regions of low methylation (canyons) that span conserved domains frequently containing transcription factors and are distinct from CpG islands and shores. About half of the genes in these methylation canyons are coated with repressive histone marks, whereas the remainder are covered by activating histone marks and are highly expressed in hematopoietic stem cells (HSCs). Canyon borders are demarked by 5-hydroxymethylcytosine and become eroded in the absence of DNA methyltransferase 3a (Dnmt3a). Genes dysregulated in human leukemias are enriched for canyon-associated genes. The new epigenetic landscape we describe may provide a mechanism for the regulation of hematopoiesis and may contribute to leukemia development.

Autoregulation of the human C/EBP alpha gene by stimulation of upstream stimulatory factor binding.

The human C/EBP alpha gene promoter shares significant sequence homology with that of the mouse but has a different mechanism of autoregulation. Activation of the murine promoter by direct binding of C/EBP alpha to a site within 200 bp of the transcriptional start was shown to elevate activity by approximately threefold (R. J. Christy, K. H. Kaestner, D. E. Geiman, and M. D. Lane, Proc. Natl. Acad. Sci. USA 88:2593-2597, 1991; K. Legraverend, P. Antonson, P. Flodby, and K. G. Xanthapoulos, Nucleic Acids Res. 21:1735-1742, 1993). Unlike its murine counterpart, the human C/EBP alpha gene promoter does not contain a cis element that binds the C/EBP alpha protein. Neither C/EBP alpha nor C/EBP beta (NF-Il-6) binds the human C/EBP alpha promoter within 437 bp. However, cotransfection studies show that C/EBP alpha stimulates transcription of a reporter gene driven by 437 bp of the C/EBP alpha promoter. Our studies show that the human C/EBP alpha protein stimulates USF to bind to a USF consensus element within C/EBP alpha promoter and activates it by two- to threefold. We propose that the human gene employs the ubiquitously expressed DNA-binding protein factor USF to carry out autoregulation. Autoregulation of the human C/EBP alpha promoter was abolished by deletion of the USF binding site, CACGTG. Expression of human C/EBP beta following transfection did not stimulate USF binding. These studies suggest a mechanism whereby tissue-specific autoregulation can be achieved via a trans-acting factor that is expressed in all cell types. Thus, direct binding of the C/EBP alpha protein to the promoter of the C/EBP alpha gene is not required for autoregulation.