C/EBPβ Induces Chromatin Opening at a Cell-Type-Specific Enhancer

Abstract

We have used the chicken mim-1 gene as a model to study the mechanisms by which transcription factors gain initial access to their target sites in compacted chromatin. The expression of mim-1 is restricted to the myelomonocytic lineage of the hematopoietic system where it is regulated synergistically by the Myb and CCAAT/enhancer binding protein (C/EBP) factors. Myb and C/EBP␤ cooperate at two distinct cis elements of mim-1, the promoter and a cell-type-specific enhancer, both of which are associated with DNase I hypersensitive sites in myelomonocytic cells but not in mim-1-nonexpressing cells. Previous work has shown that ectopic expression of Myb and C/EBP␤ activates the endogenous mim-1 gene in a nonhematopoietic cell type (fibroblasts), where the gene is normally completely silent. Here, we investigated the molecular details of this finding and show that the activation of mim-1 occurs by two independent mechanisms. In the absence of Myb, C/EBP␤ triggers the initial steps of chromatin opening at the mim-1 enhancer without inducing transcription of the gene. mim-1 transcription occurs only in the presence of Myb and is associated with chromatin opening at the promoter. Our work identifies a novel function for C/EBP␤ in the initial steps of a localized chromatin opening at a specific, physiologically relevant target region.The significant changes in gene expression that occur during processes such as differentiation, proliferation, and apoptosis are often controlled at the transcriptional level. In eukaryotic organisms, the transcriptional activation of genes occurs in the chromatin context. The basic unit of chromatin is the nucleosome, which consists of an octameric complex of the core histones H2A, H2B, H3, and H4 and 146 base pairs of DNA wrapped around the protein core and connected by linker DNA. Further folding of nucleosomes through the binding of histone H1 leads to compact higher-order structures in which the DNA is not easily accessible for transcription factors or the transcriptional machinery. Access by the transcription machinery to DNA is facilitated by various chromatin remodeling enzymes that cause localized changes of the chromatin structure. These remodeling complexes are themselves recruited by transcription factors to specific cis-acting sequences (for reviews see references 4, 23, 26, 41, 47, and 48). Although we have a rather detailed picture of the chromatin remodeling events that occur after transcription factors have associated with their binding sites, the mechanisms by which these proteins gain initial access to their binding sites in nucleosomally organized and tightly packed DNA are less well understood. Cirillo et al. (14) have used the term "pioneer factor" to describe a class of transcription factors that are capable of entering into silent chromatin and initiating chromatin opening.

Pdcd4 directly binds the coding region of c-myb mRNA and suppresses its translation

“…In the resulting plasmid, the HCV translational start codon is linked via a 123 base-pair sequence in-frame to the c-Myb start codon. pCDNA4-CCR encodes full-length chicken C/EBPb (Plachetka et al, 2008). pCMVluc was obtained by inserting the CMV promoter into pGL3-basic (Promega, Mannheim, Germany).…”

Myb-induced Chromatin Remodeling at a Dual Enhancer/Promoter Element Involves Non-coding RNA Transcription and Is Disrupted by Oncogenic Mutations of v-myb

“…In Vivo Footprinting and Chromatin ImmunoprecipitationIn vivo dimethylsulfate footprinting and chromatin immunoprecipitation were done as described (25).…”

“…Furthermore, we have demonstrated that Myb-induced nucleosomal remodeling is accompanied by increased occupancy at functionally relevant C/EBP and Ets binding sites. We have recently shown that C/EBP␤ induces the initial opening of the chromatin at the mim-1 enhancer in a step that occurs prior to the actual transcription of the gene and is independent of Myb (25). Taken together with the work reported here this suggests a stepwise mechanism for activation of the enhancer in which C/EBP␤ is responsible for the initial opening of the chromatin structure and Myb induces subsequent remodeling events which lead to additional binding of transcription factors and, finally, to the transcription of the mim-1 gene.…”

“…Additional insight into mim-1 gene activation was provided by the identification of a Myb-responsive enhancer located 2-kb upstream of the mim-1 promoter (24). We have recently shown that the transcription factor C/EBP␤ induces opening of the compact chromatin structure at the enhancer in a step preceding the transcription of the gene (25). These observations suggested that the mim-1 enhancer plays an important role during the activation of the gene and have made mim-1 an interesting model to study the stepwise activation of a gene by Myb.…”

Inhibition of Myb-dependent gene expression by the sesquiterpene lactone mexicanin-I

“…[11][12][13] One of the most strongly activated Myb target genes is the chicken myb-inducible myeloid-specific gene-1 (mim-1), originally identified by Ness et al 14 Recent work has shown that mim-1 expression is regulated by Myb in a complex manner, involving several cis-acting DNA-elements, as well as several cooperating proteins. [15][16][17][18][19] Although myb was originally identified as the oncogene of the avian myeloblastosis virus, it was suspected that c-myb is also involved in the development of human cancer. Recent work has indeed provided strong evidence that deregulated c-myb expression has a role in the development of certain leukemias, and of breast and colon tumors.…”