In vivo interactions of the Acanthamoeba TBP gene promoter

Abstract

Transcription of the TATA box binding protein (TBP) gene in Acanthamoeba castellanii is regulated by TATA box binding protein promoter binding factor (TPBF), which binds to an upstream TBP promoter element to stimulate transcription, and to a TATA proximal element, where it represses transcription. In order to extend these observations to the in vivo chromatin context, the TBP gene was examined by in situ footprinting and chromatin immunoprecipitation (ChIP). Acanthamoeba DNA is nucleosomal with a repeat of approximately 160 bp, and an intranucleosomal DNA periodicity of 10.5 bp. The TBP gene comprises a 220 bp micrococcal nuclease hypersensitive site corresponding to the promoter regulatory elements previously identified, flanked by protected regions of a size consistent with the presence of nucleosomes. ChIP data indicated that TPBF is associated with the TBP, TPBF and MIL gene promoters, but not to the CSP21, MIIHC, 5SrRNA or 39SrRNA promoters, or to the MIL gene C-terminal region. Binding by TPBF to the TPBF and MIL gene promoters was confirmed by in vitro assays. These results validate the in vitro model for TBP gene regulation and further suggest that TPBF may be autoregulated and may participate in the regulation of the MIL gene.

Figure 1

Acanthamoeba DNA is packaged as nucleosomes. (A) Naked DNA or nuclei were digested with MNase as described in Materials and Methods and fractionated on a 1% agarose gel. Each band represents a preferential cut by MNase between nucleosomes. (B) DNA samples prepared from Acanthamoeba nuclei digested with DNase I were end-labeled with [γ-³²P]ATP and fractionated on a 6% denaturing polyacrylamide gel. The 10 bp intervals between bands (shown as triangles) reflect the average DNA helical turn within a nucleosome. The sizes of DNA ladders are indicated on the side.

Figure 2

In situ MNase footprinting of the TBP gene. The MNase digested DNA samples were fractionated on a 1.2% agarose gel (left panels), and analyzed by Southern blotting (center and right panels). The sizes of the DNA markers and the nucleosomal ladders are indicated on the right side of the left panels. The open box represents the hypersensitive site, and the dashed ellipses I–V indicate possible binding by nucleosomes. The nuclease-sensitive site between nucleosomes II and III is marked by asterisks in the center panels. Dots denote bands within nucleosome II that are less sensitive to nuclease digestion. (A) DNA digested with PstI, and hybridized with probe 1 (center panel) and probe 3 (right panel). (B) DNA digested with XhoI, and hybridized with probe 2 (center panel) and probe 4 (right panel). (C) Diagram showing the upstream and 5′ end of the TBP gene. The four probes used in Southern blotting are shown on the top of the diagram, with the lengths drawn to scale. The hypersensitive site and the possible nucleosome positions deduced from Figure 4 are shown, respectively, as an open box and ellipses. The numbers denote the nucleotide positions relative to the transcription start site.

Figure 3

ChIP assays of several A.castellanii genes. (A) Diagrams of genes tested in the ChIP assays. The primer pairs used for each gene are shown as arrows and the sizes of each PCR product are indicated. (B) ChIP by anti-TPBF rabbit serum; –FA, cells without formaldehyde crosslinking; +FA, cells with formaldehyde crosslinking; WCE, whole cell extract; MILP, MIL promoter amplified by primers MILP5P and MILP3P; MILC, MIL gene C-terminus amplified by primers MILC5P and MILC3P.

Figure 4

TPBF is recruited to its own promoter. (A) DNA sequence of the TPBF gene promoter. The TATA box is indicated as bold letters, the transcription start site is denoted as bold underlined characters, the translational start codon is illustrated as italic bold letters. Underlined sequences represent TPBF binding sites. (B) In vitro footprinting experiment demonstrating stable binding of TPBF to its own promoter. Lane 1, no TPBF protein; lanes 2–6, 100 ng, 200 ng, 500 ng, 1 µg, 2 µg recombinant TPBF, respectively; lane 7, 2 µg protein and 2 µg TPE oligonucleotide as competitor; lane 8, no DNase I digestion. Protected regions are denoted by solid lines. (C) In vitro transcription from various TPBF gene promoter constructs. Arrows point to transcription signals.

Figure 5

Electrophoretic mobility shift assay of myoTPE and TPE. (A) Sequence comparison of the myoTPE from the MIL gene and the TPE from the TBP gene. myoTPE contains two TPEs which partially overlap each other. (B) Approximately 5 ng of γ-³²P-end-labeled double-stranded DNA probes were incubated with the indicated amounts of TPBF. Solid arrows point to the complex formed by the TPBF tetramer and the open arrow represents the binding of two TPBF tetramers.