The perinucleolar compartment (PNC) is a nuclear subdomain that is unique to tumor cells, and the percentage of cells in a population containing PNCs (PNC prevalence) indicates the level of malignancy of that population. Here, we utilize anticancer drugs and other exogenous stimuli to investigate the structure and function of the PNC. Screening of clinically used anti-cancer drugs revealed two types of drugs disassemble PNCs and do so through their specific molecular actions. Transcription inhibitors reduce PNC prevalence in parallel with RNA polymerase III transcription reduction, and a subset of DNA-damaging drugs and stimuli (UV radiation) disassemble the PNC. Inhibition of cellular DNA damage response demonstrated that the DNA damage itself, not the response or polymerase III inhibition, is responsible for PNC disassembly, suggesting that the maintenance of the PNC is dependent upon DNA integrity. Analyses of the types of DNA damage that cause PNC disassembly show that interstrand DNA base pairing, not strand continuity, is important for PNC integrity, indicating that the PNC components are directly interacting with the DNA. Complementary cell biology experiments demonstrated that the number of PNCs per cell increases with the rounds of endoreplication and that PNCs split into doublets during mid S phase, both of which are phenotypes that are typical of a replicating DNA loci. Together, these studies validate PNC disassembly as a screening marker to identify chemical probes and revealed that the PNC is directly nucleated on a DNA locus, suggesting a potential role for the PNC in gene expression regulation. The perinucleolar compartment (PNC)2 is a non-membrane-bound nuclear subdomain that is associated with, but structurally distinct from, the nucleolus. The PNC is a generally heritable trait, in which the number of PNCs per cell in daughter cells often mimics that of their mother cells. The PNC is heterogeneous in shape and ranges from 0.5 to 4 m in size (1), is stable through interphase, disassembles during mitosis, and reassembles in early G 1 (1). The PNC is concentrated with newly synthesized RNA polymerase III RNAs (MRP RNase RNA, RNase P H1 RNA, hY RNAs (hY1, -2, and -5), AluRNA, and SRP (7SL) RNA) and RNA-binding proteins (nucleolin, PTB, CUG repeat-binding protein, KSRP, Raver1, Raver2, and Rod1) (2-9).3 Continuous transcription by pol III is necessary for the structure integrity of the PNC, implicating involvement of PNCs in pol III RNA metabolism (9). However, the complete molecular composition and function of the PNC remain to be elucidated.Extensive in vitro studies showed that the PNC is unique to tumor cells and preferentially forms in tumor cells derived from solid tissues (1, 10). In vitro studies of cancer cell lines from various origins and malignant capacities have shown that PNC prevalence correlates with the malignancy of tumors and has the potential to be developed as a pan-cancer prognostic marker (10). In addition, in vivo investigations using human breast tissue samples demonstrated...

Background: Human small nuclear RNA genes exhibit powerful transcription potential. Results: The DNMT1 and DNMT3a DNA methyltransferases down-regulate snRNA transcription by RNA polymerase III. The RB tumor suppressor facilitates DNMT promoter recruitment. Conclusion: Human RNA polymerase III transcription is regulated by epigenetic modification. Significance: This study uncovers a novel relationship between DNA methyltransferases and RB for epigenetic regulation.

Chromatin is thought to act as a barrier for binding of cis-regulatory transcription factors (TFs) to their sites on DNA and recruitment of the transcriptional machinery. Here we have analyzed changes in nucleosome occupancy at the enhancers as well as at the promoters of three pro-inflammatory genes when they are induced by bacterial lipopolysaccharides (LPS) in primary mouse macrophages. We find that nucleosomes are removed from the distal enhancers of IL12B and IL1A, as well as from the distal and proximal enhancers of IFNB1, and that clearance of enhancers correlates with binding of various cis-regulatory TFs. We further show that for IFNB1 the degree of nucleosome removal correlates well with the level of induction of the gene under different conditions. Surprisingly, we find that nucleosome occupancy at the promoters of IL12B and IL1A does not change significantly when the genes are induced, and that a considerably fraction of the cells is occupied by nucleosomes at any given time. We hypothesize that competing nucleosomes at the promoters of IL12B and IL1A may play a role in limiting the size of transcriptional bursts in individual cells, which may be important for controlling cytokine production in a population of immune cells.

The Retinoblastoma (RB) tumor suppressor protein regulates multiple pathways that influence cell growth, and as a key regulatory node, its function is inactivated in most cancer cells. In addition to its canonical roles in cell cycle control, RB functions as a global repressor of RNA polymerase (Pol) III transcription. Indeed, Pol III transcripts accumulate in cancer cells and their heightened levels are implicated in accelerated growth associated with RB dysfunction. Herein we review the mechanisms of RB repression for the different types of Pol III genes. For type 1 and type 2 genes, RB represses transcription through direct contacts with the core transcription machinery, notably Brf1-TFIIIB, and inhibits preinitiation complex formation and Pol III recruitment. A contrasting model for type 3 gene repression indicates that RB regulation involves stable and simultaneous promoter association by RB, the general transcription machinery including SNAPc, and Pol III, suggesting that RB may impede Pol III promoter escape or elongation. Interestingly, analysis of published genomic association data for RB and Pol III revealed added regulatory complexity for Pol III genes both during active growth and during arrested growth associated with quiescence and senescence.