No abstract

Neutrophil-specific granule deficiency (SGD) is a rare congenital disorder marked by recurrent bacterial infections. Neutrophils from SGD patients lack secondary and tertiary granules and their content proteins and lack normal neutrophil functions. Gene-inactivating mutations in the C/EBPepsilon gene have been identified in 2 SGD patients. Our studies on a third SGD patient revealed a heterozygous mutation in the C/EBPepsilon gene. However, we demonstrate elevated levels of C/EBPepsilon and PU.1 proteins in the patient's peripheral blood neutrophils. The expression of the transcription factor growth factor independence-1 (Gfi-1), however, was found to be markedly reduced in our SGD patient despite the absence of an obvious mutation in this gene. This may explain the elevated levels of both C/EBPepsilon and PU.1, which are targets of Gfi-1 transcriptional repression. We have generated a growth factor-dependent EML cell line from the bone marrow of Gfi-1(+/-) and Gfi-1(+/+) mice as a model for Gfi-1-deficient SGD, and demonstrate that lower levels of Gfi-1 expression in the Gfi-1(+/-) EML cells is associated with reduced levels of secondary granule protein (SGP) gene expression. Furthermore, we demonstrate a positive role for Gfi-1 in SGP expression, in that Gfi-1 binds to and up-regulates the promoter of neutrophil collagenase (an SGP gene), in cooperation with wild-type but not with mutant C/EBPepsilon. We hypothesize that decreased Gfi-1 levels in our SGD patient, together with the mutant C/EBPepsilon, block SGP expression, thereby contributing to the underlying etiology of the disease in our patient.

IntroductionAlthough a number of transcription factors have been implicated in myelopoiesis, 1-4 several members of the CCAAT enhancer binding protein (C/EBP) family of transcription factors have been shown to be indispensable for normal development of the myeloid lineage. The C/EBP family of transcription factors is a class of basic region/leucine zipper (bZIP) factors that recognize the consensus DNA-binding sequence, 5Ј-ATTGCGCAAT-3Ј, as obligate dimers. Currently, 6 members of this family of transcription factors have been described, including C/EBP ␣, ␤, ␥, ␦, ⑀, and CHOP-10/ GADD 153, all of which contain highly homologous dimerization (leucine-zipper) domains and DNA-binding (basic-region) motifs. These proteins have been implicated in regulating gene expression in a variety of cell types, including adipocytes (reviewed in Darlington et al 5 ), constitutive and acute phase response genes in the liver (reviewed in Diehl 6 ), and myelomonocytic cells (reviewed in Lekstrom-Himes and Xanthopoulos 7 ).Severe hematopoietic abnormalities have been reported for mice nullizygous for C/EBP␣ and ⑀. Although C/EBP␣ Ϫ/Ϫ mice die perinatally due to defects in gluconeogenesis, 8,9 they also demonstrate an early block in the differentiation of granulocytes. C/EBP␣ has been postulated to be a master regulator of the granulopoietic developmental program. Although C/EBP␣ is expressed in several tissues, its expression in the hematopoietic compartment is restricted to the granulocytic lineage, where it is expressed in the most primitive precursors. Recently, mutations within the C/EBP␣ gene have been demonstrated in patients with acute myeloid leukemia (AML) (French-American-British [FAB] classification M1 and M2). 10 Additionally, the AML1/ETO fusion protein that results from the t(8;21) translocation in patients with M2 AML has been shown to down-regulate C/EBP␣ expression, 11 suggesting that loss of C/EBP␣ function in AML patients may contribute to the observed early block in the granulocytic maturation pathway.C/EBP⑀ is expressed relatively late in the granulocytic maturation pathway and has been shown to play a role in late myeloid gene expression. [12][13][14] Absence of C/EBP⑀ in mutant mice causes a late block in the differentiation of granulocytes. C/EBP⑀ Ϫ/Ϫ mice produce hyposegmented granulocytes that are functionally defective. Mutant mice usually survive 2 to 5 months but eventually succumb to opportunistic bacterial infections. 15 It has been demonstrated that C/EBP⑀ Ϫ/Ϫ mice express absent or low levels of RNA for several genes, including the secondary granule protein (SGP) genes (murine lactoferrin [mLF], murine neutrophil gelatinase [mNG], and murine neutrophil collagenase [mNC]). Studies from our laboratory have demonstrated that expression of mLF and mNC in the developing neutrophil is dependent on intact C/EBP binding sites within their gene promoters (Khanna-Gupta et al 16 ; and A.K.-G. and N.B., manuscript in preparation, 2003). Mutations within the C/EBP⑀ gene have been described in patients with spec...

Neutrophils from CCAAT enhancer binding protein epsilon (C͞ EBP) knockout mice have morphological and biochemical features similar to those observed in patients with an extremely rare congenital disorder called neutrophil-specific secondary granule deficiency (SGD). SGD is characterized by frequent bacterial infections attributed, in part, to the lack of neutrophil secondary granule proteins (SGP). A mutation that results in loss of functional C͞EBP activity has recently been described in an SGD patient, and has been postulated to be the cause of the disease in this patient. We have previously demonstrated that overexpression of CCAAT displacement protein (CDP͞cut), a highly conserved transcriptional repressor of developmentally regulated genes, suppresses expression of SGP genes in 32Dcl3 cells. This phenotype resembles that observed in both C͞EBP؊͞؊ mice and in SGD patients. Based on these observations we investigated potential interactions between C͞EBP and CDP͞cut during neutrophil maturation. In this study, we demonstrate that inducible expression of C͞EBP in 32Dcl3͞tet cells results in granulocytic differentiation. Furthermore, Northern blot analysis of G-CSF-induced CDP͞cut overexpressing 32Dcl3 cells revealed absence of C͞EBP mRNA. We therefore hypothesize that C͞EBP positively regulates SGP gene expression, and that C͞EBP is itself negatively regulated by CDP͞cut during neutrophil maturation. We further demonstrate that the C͞EBP promoter is regulated by CDP͞cut during myeloid differentiation.

We have performed transient-expression experiments with CV1 monkey kidney cells to investigate the role of the bovine papillomavirus type 1 (BPV1) E2 gene in the regulation of the E5 transforming gene. Direct analysis of the 7-kilodalton open reading frame (ORF) E5 protein and measurements of the expression of an E5-chloramphenicol acetyltransferase fusion protein indicate that the efficient expression of ORF E5 requires the full-length E2 gene, which can be supplied in trans. The viral long control region is required in cis for this response to ORF E2, and it acts in a positionand orientation-independent fashion characteristic of a transcriptional enhancer. Deletion analysis suggests that the P2443 promoter is required for efficient expression of the E5 gene. The E2 repressor activity encoded in the 3' end of the E2 gene inhibits the expression of ORF E5. These effects define a major BPV1 regulatory circuit and appear to explain the transformation behavior of a variety of BPV1 mutants.