Tumor necrosis factor alpha (TNF-␣) is a potent cytokine which regulates inflammation via the induction of adhesion molecules and chemokine expression. Its expression is known to be regulated in a complex manner with transcription, message turnover, message splicing, translation, and protein cleavage from the cell surface all being independently regulated. This study examined both cell lines and primary cells to understand the developmental regulation of epigenetic changes at the TNF-␣ locus. We demonstrate that epigenetic modifications of the TNF-␣ locus occur both developmentally and in response to acute stimulation and, importantly, that they actively regulate expression. DNA demethylates early in development, beginning with the hematopoietic stem cell. The TNF-␣ locus migrates from heterochromatin to euchromatin in a progressive fashion, reaching euchromatin slightly later in differentiation. Finally, histone modifications characteristic of a transcriptionally competent gene occur with myeloid differentiation and progress with differentiation. Additional histone modifications characteristic of active gene expression are acquired with stimulation. In each case, manipulation of these epigenetic variables altered the ability of the cell to express TNF-␣. These studies demonstrate the importance of epigenetic regulation in the control of TNF-␣ expression. These findings may have relevance for inflammatory disorders in which TNF-␣ is overproduced.

Tumor necrosis factor alpha (TNF-␣) is an important mediator of inflammation, apoptosis, and the development of secondary lymphoid structures. Multiple polymorphic microsatellites have been identified in and around the gene, and there are also multiple single-base pair biallelic polymorphisms in the introns and promoter. The TNF-␣ ؊308 promoter polymorphism is a G-to-A transition which has been statistically associated with various autoimmune disorders. Some studies have found that it may directly mediate the increased transcription of TNF-␣ in some circumstances. This study characterizes proteins interacting at the polymorphic promoter site. Affinity purification of binding proteins and confirmatory chromatin immunoprecipitation assays were used to identify the proteins. Electrophoretic mobility shift analyses and surface plasmon resonance were used to define binding characteristics. Proteins interacting at this site include GCF2/LRRFIP1 and Ets-1. GCF2/LRRFIP1 appears to act as a repressor and occupies the ؊308 site in cells that do not make TNF-␣. Cells competent to produce TNF-␣ have Ets-1 bound to the ؊308 promoter site. Active transcription is accompanied by NF-B and c-Jun binding to the proximal promoter. Thus, dynamic changes on the TNF-␣ promoter, particularly at the ؊308 site, accompany the transition from repressed to active transcription. GCF2/LRRFIP1 is the first TNF-␣ repressor identified.Tumor necrosis factor (TNF-␣) is a proinflammatory cytokine which plays a role in humoral immunity, chemokine expression, the regulation of adhesion molecule expression, and apoptosis. The expression of TNF-␣ is regulated at many levels, including transcription, message turnover, protein production, and protein release (15,21,25,36). The transcription of TNF-␣ is complex, with tissue-specific expression and stimulusinducible expression, which in turn can be tissue specific. Some of the TNF-␣ promoter motifs which have been implicated in transcription include binding sites for NF-B, NFAT, ATF-2, Ets-1, C/EBP␤, cis-acting replication element, AP-1, and AP-2 (13, 14, 24, 26-28, 35, 37, 39, 40, 48, 49, 58, 59). Several promoter polymorphisms have been identified, and a polymorphism at Ϫ308 has been implicated in the regulation of TNF-␣ transcription (24,29,52,56). (This work uses the traditional numbering system for the promoter polymorphisms [55].)The TNF-␣ promoter polymorphism at Ϫ308 involves a biallelic single-base pair transition from G to A. The polymorphic sequence, designated as TNF-␣ Ϫ308A, has gene frequencies of approximately 0.12 to 0.17 in Caucasians and 0.08 in African Americans (9,10,42,45,4). Studies have found associations of TNF-␣ Ϫ308A with systemic lupus erythematosus, sarcoidosis, alopecia areata, rheumatoid arthritis, and dermatitis herpetiformis (9,17,18,34,42,43,45,54). Other studies have found no association of TNF-␣ Ϫ308A with rheumatoid arthritis, ankylosing spondylitis, and Felty's syndrome (5, 51, 53). Certain infectious diseases, such as cerebral malaria and mucocutaneous leishmania, als...