Promoter Regulatory Elements and DNase I-hypersensitive Sites Involved in Serglycin Proteoglycan Gene Expression in Human Erythroleukemia, CHRF 288-11, and HL-60 Cells

Abstract

We have compared regulation of the serglycin gene in human erythroleukemia (HEL) and CHRF 288-11 cells, which have megakaryocytic characteristics, with promyelocytic HL-60 cells. Deletion constructs were prepared from the region ؊1123/؉42 to ؊20/؉42, and putative regulatory sites were mutated. In all three cell lines, the two major regulatory elements for constitutive expression were the (؊80)ets site and the cyclic AMP response element (CRE) half-site at ؊70. A protein from HEL and CHRF, but not HL60, nuclear extracts bound to the (-80)ets site. Another protein from all three cell lines bound to the (-70)CRE. Phorbol 12-myristate 13-acetate (PMA) and dibutyryl cyclic AMP (dbcAMP) increased expression of the reporter in HEL cells 2.5-3-and 4.5-fold, respectively, from all constructs except those with (-70)CRE mutations. PMA virtually eliminated expression of serglycin mRNA and promoter constructs, but dbcAMP increased expression in HL-60 cells. The effects of PMA and dbcAMP on promoter expression correlated with mRNA expression. The strengths of two DNase Ihypersensitive sites in the 5-flanking region and the first intron in all three cells correlated with relative endogenous serglycin mRNA expression. An additional DNase I-hypersensitive site in HL60 DNA in the first intron may be related to the high serglycin expression in HL60 relative to HEL or CHRF cells.The serglycin proteoglycan is synthesized by a number of hematopoietic cells, including platelets (1) and their parent megakaryocytes (2), granulocytes, macrophages, and lymphocytes (3-6), mast cells (4), and a large number of hematopoietic tumor cell lines (3, 7-10). Mature erythrocytes do not contain proteoglycans. We have recently found that serglycin is a major component of endothelial (11), murine uterine mesometrial decidual (12), and murine embryonic stem cell proteoglycans. 1Serglycin is thought to be critical for packaging and storage of various proteins in and secretion from hematopoietic cell granules and for modulating their activity. Serglycin binds to and stabilizes the chymases in the protease-containing secretory granules in mast cells (4, 13) and binds to granule-associated cytokines or chemokines such as MIP-1␣ and platelet factor 4 (14). Serglycin can bind to a number of matrix proteins such as fibronectin and collagen (9,15,16) and thus can influence cell/matrix interactions (16). We have reported that the serglycin proteoglycan of platelets of Wistar-Furth rats, which have a severe defect in ␣ granule structure and protein content, has abnormally shortened glycyosaminoglycan chains (15). Mice with a gene deletion of an enzyme required for heparin biosynthesis have severe defects in mast cell granules because of the defect in the heparin serglycin glycyosaminoglycan chains (17,18). Serglycins from other hematopoietic cells, endothelial cells, and decidua contain primarily chondroitin sulfate (2,4,8,9,19). The overall structure of the intact proteoglycan, rather than just the nature of the glycyosaminoglycan chains, appears to govern the ...

DNase I Hypersensitivity Patterns of the Serglycin Proteoglycan Gene in Resting and Phorbol 12-Myristate 13-Acetate-stimulated Human Erythroleukemia (HEL), CHRF 288-11, and HL-60 Cells Compared with Neutrophils and Human Umbilical Vein Endothelial Cells

“…In the present report, we have extended our studies of the serglycin gene to mapping the serglycin DHSS in resting and PMA-stimulated HEL, CHRF 288-11, and HL-60 cells and have compared these DHSS with those of normal primary blood neutrophils and HUVEC. We also correlated the pattern of hypersensitivity with the previous studies on the endogenous levels of serglycin expression in both resting and PMA-stimulated hematopoietic tumor cells (11,12,14,26,31). Our data suggest that cell-and differentiation-specific alterations in chromatin structure may potentially control serglycin expression.…”

“…Immortal human skin keratinocytes, HaCaT (35), were used as a control because serglycin expression was not detected by reverse transcriptase-PCR of HaCaT RNA (26).…”

“…Schick et al (26) showed the presence of DNase I-hypersensitive sites (DHSS) in the serglycin gene in resting hematopoietic tumor cells. DHSS, in their most general form, represent regions of less compact chromatin that is more susceptible to nuclease digestion and gaps in the nucleosome array of the chromatin fiber that reveal the location of regulatory motifs (27).…”

Serglycin Is Essential for Maturation of Mast Cell Secretory Granule

“…Based on the corresponding SG cDNA sequence, exons 1-3 were identified in the genomic DNA sequence. The SG gene is spanning over ϳ15 kb including regulatory sequences (48). We decided to make a classical knockout by replacing exon 1 and the regulatory sequences with a neomycin-resistant cassette.…”

Serglycin: A Structural and Functional Chameleon with Wide Impact on Immune Cells

“…For example, megakaryocytic differentiation is associated with increased serglycin expression (42,43), whereas treatment of leukemic cell lines with PMA decreased the expression of serglycin mRNA (44). During myeloblast differentiation, increased serglycin expression was shown to coincide with granule biogenesis (45), whereas, in contrast, serglycin expression is downregulated during promyelocyte differentiation into mature neutrophils (23).…”