Upstream binding factor association induces large-scale chromatin decondensation

Abstract

The function of upstream binding factor (UBF), an essential component of the RNA polymerase (pol) I preinitiation complex, is unclear. Recently, UBF was found distributed throughout ribosomal gene repeats rather than being restricted to promoter regions. This observation has led to the speculation that one role of UBF binding may be to induce chromatin remodeling. To directly evaluate the impact of UBF on chromatin structure, we used an in vivo assay in which UBF is targeted via a lac repressor fusion protein to a heterochromatic, amplified chromosome region containing lac operator repeats. We show that the association of UBF with this locus induces large-scale chromatin decondensation. This process does not appear to involve common remodeling complexes, including SWI/SNF and histone acetyltransferases, and is independent of histone H3 lysine 9 acetylation. However, UBF recruits the pol I-specific, TATA box-binding protein containing complex SL1 and pol I subunits. Our results suggest a working hypothesis in which the dynamic association of UBF with ribosomal DNA clusters recruits the pol I transcription machinery and maintains these loci in a transcriptionally competent configuration. These studies also provide an in vivo model simulating ribosomal DNA transactivation outside the nucleolus, allowing temporal and spatial analyses of chromatin remodeling and assembly of the pol I transcription machinery.

Fig. 1.

GFP- or YFP-repressor-UBF behaves similarly to its endogenous counterparts. (A) A diagram of the expression constructs for fusion proteins that contain GFP or YFP, lac repressor, and UBF. (B) The fusion protein GFP-repressor-UBF behaves similarly to endogenous UBF with regards to its subnucleolar localization pattern in HeLa cells (Upper) and its dimerization with cotransfected HcRed-UBF in A03_1 cells (Lower). (Scale bar, 10 μm.)

Fig. 2.

Targeting YFP-repressor-UBF to the lac operator array induces chromatin decondensation. (A) The decondensation induced by UBF (A Bottom) is often to a lesser extent than the decondensation induced by VP-16 (A Middle). A03_1 cells were transfected with YFP-repressor (Top), YFP-repressor-VP16 (Middle), and YFP-repressor-UBF (Bottom). (Left) The localization of the lac operator array visualized by its binding to YFP-repressor and fusion proteins. (Center) The same cells stained with DAPI. (Right) Overlay images. (B) The lac operator array loses characteristic HP1α binding of heterochromatin upon UBF binding. (Left) The expression of GFP or YFP-repressor and their fusion proteins. (Center) Immunolabeling of the same cells with either anti-HP1α antibody. (Right) The overlay images. (Scale bar, 10 μm.)

Fig. 3.

The decondensed chromatin induced by UBF association does not recruit components of SWI/SNF complex (Brg1) (A) and does not show massive histone acetylations. (B)(Left) The expression of GFP or YFP-repressor and their fusion proteins. (Center) Immunolabeling of the same cells with either anti-Brg1 antibody (A) or anti-K9 actylated histone H3 (B). (Right) Overlay images. (C) Double labeling BrUrd incorporation for 5 min and UBF at anaphase–telophase tranisition (Upper) and double labeling of acetylated K9 of histone H3 and UBF at the same stage of mitosis (Lower, arrowheads). (Scale bar, 10 μm.)

Fig. 4.

The association of UBF with the lac operator array recruits the pol I-specific TBP-containing complex, SL1, and pol I subunit RPA39. This finding is in contrast to the association of VP-16 with the lac operator array, which indiscriminately recruits all TBP-associated complexes and does not recruit RPA39. Arrowheads indicate the labeling of RPA39 in the endogenous nucleolus. Specifics are labeled in the images. (Scale bar, 10 μm.)