UBF levels determine the number of active ribosomal RNA genes in mammals

Research Division, Peter MacCallum Cancer Centre, East Melbourne, Victoria 3002, Australia.
The Journal of Cell Biology (Impact Factor: 9.83). 01/2009; 183(7):1259-74. DOI: 10.1083/jcb.200805146
Source: PubMed


In mammals, the mechanisms regulating the number of active copies of the approximately 200 ribosomal RNA (rRNA) genes transcribed by RNA polymerase I are unclear. We demonstrate that depletion of the transcription factor upstream binding factor (UBF) leads to the stable and reversible methylation-independent silencing of rRNA genes by promoting histone H1-induced assembly of transcriptionally inactive chromatin. Chromatin remodeling is abrogated by the mutation of an extracellular signal-regulated kinase site within the high mobility group box 1 domain of UBF1, which is required for its ability to bend and loop DNA in vitro. Surprisingly, rRNA gene silencing does not reduce net rRNA synthesis as transcription from remaining active genes is increased. We also show that the active rRNA gene pool is not static but decreases during differentiation, correlating with diminished UBF expression. Thus, UBF1 levels regulate active rRNA gene chromatin during growth and differentiation.

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    • "Antihyperacetylated H4 (06-946) and anti H3K9ac (07-352) antibodies were from Upstate (Millipore). Antibodies targeting UBF1/2 and the largest subunit of the Pol I complex (POLR1A/RPA194) were raised inhouse and were used as reported in Sanij et al.[3]. "
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    ABSTRACT: The upstream binding transcription factor (UBTF, also called UBF) is thought to function exclusively in RNA polymerase I (Pol I)-specific transcription of the ribosomal genes. We recently reported in Sanij et al.(2014) [1] that the two isoforms of UBF (UBF1/2) are enriched at Pol II-transcribed genes throughout the mouse and human genomes. By using chromatin immunoprecipitation coupled with deep sequencing (ChIP-seq) of UBF1/2, Pol I, Pol II, H3K9me3, H3K4me4, H3K9ac and H4 hyperacetylation, we reported a correlation of UBF1/2 binding with enrichments in Pol II and markers of active chromatin. In addition, we examined a functional role for UBF1/2 in mediating Pol II transcription by performing expression array analysis in control and UBF1/2 depleted NIH3T3 cells. Our data demonstrate that UBF1/2 bind highly active Pol II-transcribed genes and mediate their expression without recruiting Pol I. Furthermore, we reported ChIP-sequencing analysis of UBF1/2 in immortalized human epithelial cells and their isogenically matched transformed counterparts. Here we report the experimental design and the description of the ChIP-sequencing and microarray expression datasets uploaded to NCBI Sequence Research Archive (SRA) and Gene Expression Omnibus (GEO).
    Full-text · Article · Jan 2015 · Genomics Data
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    • "UBF plays a particularly critical role at the rRNA gene promoter, where it serves as a scaffold for the binding of RNA Pol I transcription factors and other processing proteins (Mais et al., 2005; Prieto and McStay, 2007). As this facilitates RNA Pol I promoter escape (Panov et al., 2006), UBF therefore orchestrates the level of rRNA transcription at active rRNA genes (O'Mahony and Rothblum, 1991; O'Sullivan et al., 2002; Chen et al., 2004; Sanij et al., 2008). UBF activity is itself tightly regulated (Sanij and Hannan, 2009), including by post-translational modifications, which control how UBF reactivates rRNA transcription after it has temporarily ceased during mitosis (Voit et al., 1999; Meraner et al., 2006). "

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    • "Chromatin immunoprecipitation (ChIP) was performed as described previously [55]. For each ChIP assay, we incubated 25 µl of anti-FLAG M2 affinity beads (Sigma-Aldrich) overnight with cross-linked chromatin fragments from 1.5×107 BE cells stably expressing pBABE-FLAG-FRA1 or empty vector (negative control). "
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