Abstract
Upstream Binding Factor (UBF) is a unique multi-HMGB-box protein first identified as a co-factor in RNA polymerase I (RPI/PolI) transcription. However, its poor DNA sequence selectivity and its ability to generate nucleosome-like nucleoprotein complexes suggest a more generalized role in chromatin structure. We previously showed that extensive depletion of UBF reduced the number of actively transcribed ribosomal RNA (rRNA) genes, but had little effect on rRNA synthesis rates or cell proliferation, leaving open the question of its requirement for RPI transcription. Using gene deletion in mouse, we now show that UBF is essential for embryo development beyond morula. Conditional deletion in cell cultures reveals that UBF is also essential for transcription of the rRNA genes and that it defines the active chromatin conformation of both gene and enhancer sequences. Loss of UBF prevents formation of the SL1/TIF1B pre-initiation complex and recruitment of the RPI-Rrn3/TIF1A complex. It is also accompanied by recruitment of H3K9me3, canonical histone H1 and HP1α, but not by de novo DNA methylation. Further, genes retain penta-acetyl H4 and H2A.Z, suggesting that even in the absence of UBF the rRNA genes can maintain a potentially active state. In contrast to canonical histone H1, binding of H1.4 is dependent on UBF, strongly suggesting that it plays a positive role in gene activity. Unexpectedly, arrest of rRNA synthesis does not suppress transcription of the 5S, tRNA or snRNA genes, nor expression of the several hundred mRNA genes implicated in ribosome biogenesis. Thus, rRNA gene activity does not coordinate global gene expression for ribosome biogenesis. Loss of UBF also unexpectedly induced the formation in cells of a large sub-nuclear structure resembling the nucleolar precursor body (NPB) of oocytes and early embryos. These somatic NPBs contain rRNA synthesis and processing factors but do not associate with the rRNA gene loci (NORs).
Highlights
The nucleolus is the largest visible structure in the mammalian cell nucleus and the site of ribosome biogenesis
Upstream Binding Factor (UBF) is essential for mouse development To establish the in vivo requirements for UBF, we generated mouse embryonic stem (ES) cells carrying a potentially conditional ‘‘flox-neo’’ Ubf allele in which Lox recombination sites were placed in intron 2 and intron 5, and a neo selective marker gene flanked by FRT sites was inserted within intron 5 (Figure S1A & B)
UBF has been termed an ‘‘architectural’’ protein due to its unique ability to induce a complex similar in size and proteinDNA content to the nucleosome of histone chromatin [54]. The discovery of this structure, its modulation by ERK phosphorylation, and the finding that UBF is broadly distributed across the rDNA repeat [8,11,24,25], have provided a potential explanation for growth factor regulation of RNA Polymerase I (RPI) elongation rates in vivo [3]
Summary
The nucleolus is the largest visible structure in the mammalian cell nucleus and the site of ribosome biogenesis. The nucleolus is the site of assembly of ribonucleoprotein (RNP) complexes ranging from spliceosomes to telomerase, and is of key importance in mounting cellular responses to oncogenic stress [2]. The formation of the nucleolus is the result of transcription of the genes for the major ribosomal RNAs (rRNAs), the 18S, 5.8S and 28S rRNAs, which are encoded as part of the 47S precursor RNA. Transcription of the rRNA genes is highly responsive to nutrient availability and growth factors [3] as well as the actions of oncogenes such as Myc [4] and tumour suppressors such as ARF [5]. Knowledge of how the activity of these genes is determined and controlled is of fundamental importance to an understanding of cell growth, oncogenic transformation and tumour suppression
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