Communication between NF-kappa B and Sp1 controls histone acetylation within the proximal promoter of the monocyte chemoattractant protein 1 gene

The induction of the monocyte chemoattractant protein 1 gene (MCP-1) by TNF occurs through an NF-kappaB-dependent distal regulatory region and an Sp1-dependent proximal regulatory region that are separated by 2.2 kb of sequence. To investigate how these regions coordinate activation of MCP-1 in response to TNF, experiments were performed to examine the role of coactivators, changes in local chromatin structure, and the acetylation of histones at the MCP-1 regulatory regions. An E1a-sensitive coactivator was found to be required for expression. In vivo nuclease sensitivity assays identified changes in response to TNF at both the proximal and distal regions that were dependent on the p65 subunit of NF-kappaB and Sp1. Chromatin immunoprecipitations used to analyze factor assembly and histone acetylation at the distal and proximal regions showed that Sp1 binding to and histone acetylation of the proximal region was dependent on NF-kappaB p65. Conversely, Sp1 assembly at the proximal region was required for p65 binding to and acetylation of the distal region, suggesting communication between the two regions during gene activation. These data and the NF-kappaB p65-dependent histone acetylation of a middle region sequence suggest a potential order for the assembly, acetylation and accessibility of the MCP-1 regulatory regions in response to TNF.     

Structural and functional characterizations of the 5'-flanking region of the mouse glucagon receptor gene: comparison with the rat gene

A putative proximal promoter was defined previously for the mouse glucagon receptor (GR) gene. In the present study, a distal promoter was characterized upstream from a novel non-coding exon revealed by the 5'-rapid amplification of cDNA ends from mouse liver tissue. The 5'-flanking region of the mouse GR gene was cloned up to 6 kb and the structural organization was compared to the 5' untranslated region of the rat gene cloned up to 7 kb. The novel exon, separated by an intron of 3.8 kb from the first coding exon, displayed a high homology (80%) with the most distal of the two untranslated exons found in the 5' region of the rat GR gene. The mouse distal promoter region, extending up to -1 kb from the novel exon, displayed 85% identity with the rat promoter. Both contain a highly GC-rich sequence with five putative binding sites for Sp1, but no consensus TATA or CAAT elements. To evaluate basal promoter activities, 5'-flanking sequences of mouse or rat GR genes were fused to a luciferase reporter gene and transiently expressed in a mouse and in a rat cell line, respectively or in rat hepatocytes. Both mouse and rat distal promoter regions directed a high level of reporter gene activity. Deletion of the Sp1 binding sites region or mutation of the second proximal Sp1 sequence markedly reduced the distal promoter activity of the reporter gene. The mouse proximal promoter activity was 2- to 3-fold less than the distal promoter, for which no functional counterpart was observed in the similar region of the rat gene.