Isolation of the human peroxisome proliferator activated receptor gamma cDNA: expression in hematopoietic cells and chromosomal mapping

Abstract

The nuclear receptor superfamily of transcription factors, which includes the retinoic acid receptors and v-erb A, play important roles in the molecular control of hematopoiesis. To identify nuclear receptors expressed in hematopoietic cells, we screened a human bone marrow cDNA library using a degenerate oligonucleotide and isolated a 1.85-kb full-length cDNA encoding a new human member of this superfamily, the peroxisome proliferator activated receptor gamma (hPPAR gamma). Two different hPPAR gamma transcripts were expressed in hematopoietic cells: a 1.85-kb transcript, which corresponds to the full-length mRNA (PPAR gamma 1), and a 0.65-kb transcript (PPAR gamma 2), which cannot encode all of the nuclear receptor functional domains. Normal neutrophils and peripheral blood lymphocytes, as well as circulating leukemic cells from patients with AML, ALL, and CML, express only PPAR gamma 2 on Northern blot analysis. In contrast, only the PPAR gamma 1 transcript was detected in a variety of human leukemia cell lines and in cultured normal primary bone marrow stromal cells. Both transcripts were detected in various fetal and adult nonhematopoietic tissues. We mapped the location of the hPPAR gamma gene to human chromosome 3p25 by somatic cell hybridization and linkage analysis. PPARs have been shown to be activated by peroxisome proliferating agents, long-chain fatty acids and arachidonic acid. Human PPAR gamma, although homologous to the PPAR gamma s of other species, has unique sequence and amino acid differences. Identification of hPPAR gamma will allow further understanding of its role in human cellular leukotriene, prostaglandin, and peroxide degradative or synthetic pathways, as well as its role in lipid metabolism and regulation of adipocyte differentiation.

Figures

FIG. 1

DNA sequence of the full-length human PPARγ cDNA (clone 14). The 1434-bp open reading frame is predicted to begin at bp 173 and end at bp 1607, producing a 478 aa nuclear receptor protein with a predicted mol. wt. of approximately 54 kDa.

FIG. 2A

Comparison of the amino acid sequence of human PPARγ with its nearest relatives in the nuclear receptor superfamily, Xenopus PPARα, β, γ (Dreyer et al., 1992), murine PPARα (Issemann and Green, 1990), and human NUC1 (Schmidt et al., 1992), PPARα (Sher et al., 1993), and RARα (Giguere et al., 1987; Petkovich et al., 1987). The nuclear receptor superfamily structural regions are indicated by the shaded blocks as per Dreyer et al. (1992). The putative conserved tau 1 silencing domain (T1) (Forman and Samuels, 1990) is indicated. The location of a partially conserved activating motif (CAF) (Danielian et al., 1992) is within the last 20 amino acids of this family of receptors. Bold letters indicate amino acids identical to hPPARγ.

FIG. 2B

Comparative amino acid identity between hPPARγ, other members of the PPAR family, and hRARα. The schematic amino acid alignment was prepared according to Fig. 2A. The percentage of amino acid identity among the various receptors and hPPARγ is indicated for each domain. The E domain is further subdivided to indicate the location of a highly conserved (tau 1 putative silencing) domain (Forman and Samuels, 1990). h = human, m = murine, x = Xenopus.

FIG. 3

Northern blot analysis of hPPARγ mRNA expression in hematopoietic cell lines and primary peripheral blood cells as indicated. Mo = monocytic cell line, T = T-cell line, My = myeloid cell line, AML = acute myelogenous leukemia cell line, E/M = erythroid or myeloid cell line, B = B-cell line, PMN’s = polymorphonuclear leukocytes. Each lane contains 5 μg poly(A)-selected RNA. Probe shown is hPPARγ 1.6-kb (full-length) cDNA, hybridizing to hPPARγ transcripts either 1.8 kb or 0.65 kb in size. Control probe (β-actin, 1.85 kb) is shown below.

FIG. 4A

Northern blot analysis of hPPARγ in eight fresh acute lymphoid leukemia (ALL) cells, using the 1.6-kb (full-length) hPPARγ cDNA as probe. Each lane contains 10 μg total RNA isolated from the peripheral blood mononuclear cell fraction. Morphologic ALL sub-classification (L1-L2) is listed according to the French American British (FAB) classification (Sun, 1983). A Tdt (terminal deoxyribonucleotide transferase) negative, natural killer (NK) cell leukemia sample is included. The amount and integrity of the RNA in each lane of Figs. 4 and 5 were confirmed by ethidium bromide staining and by hybridizations with other probes (data not shown). Darker exposures failed to reveal hybridization in the 1.85-kb region for any of these blots (Figs. 4 and 5).

FIG. 4B

Northern blot analysis of hPPARγ RNA expression in the peripheral blood mononuclear fraction isolated from patients with chronic myelogenous leukemia (CML); 10 or 20 μg of total RNA was loaded per lane (as indicated).

FIG. 4C

Northern blot analysis of hPPARγ RNA expression in peripheral blood mononuclear fractions from patients diagnosed with acute myeloid leukemias (listed according to the FAB classification). Lanes contain 10 or 20 μg total RNA as indicated. RAEB (lane 9) is from a patient with refractory anemia with excess blasts (Sun, 1983). Lane 8 has an artifact above the true signal.

FIG. 5A

Northern blot analysis of hPPARγ mRNA expression in adult human organs. Each lane contain 5 μg poly(A)-selected RNA. The position of the two transcripts, 1.85 kb and 0.65 kb, is indicated. Hybridization using β actin cDNA is shown below.

FIG. 5B

Northern blot analysis of hPPARγ expression in human fetal organs. Lanes contain either 10 μg or 2 μg of poly(A)-selected RNA as indicated. Cortex refers to the cerebral cortex.

FIG. 6

Multipoint linkage analysis of loci in the telomeric region of chromosome 3p. The result of the analysis of these values indicate that the most likely order of these loci is as shown. Note that based on all information available PPARG and RAF1 cannot be ordered. The values in parentheses indicate the odds against reversing the order of the adjacent loci. The most likely sex-specific recombination fraction (Q) between each locus is shown. Sex average values represent most likely recombinationfractions assuming no sex difference. PPARG = hPPARγ gene; RAF1 = RAF1 gene; THRB = thyroid hormone receptor, beta gene; D3S### = genetic marker, single copy DNA fragments, human chromosome 3.