Chromatin Remodeler Recruitment during Macrophage Differentiation Facilitates Transcription Factor Binding to Enhancers in Mature Cells.

We show how enhancers of macrophage-specific genes are rendered accessible in differentiating macrophages to allow their induction in mature cells in response to an appropriate stimulus. Using a lentiviral knockdown approach in primary differentiating macrophages from mouse bone marrow, we demonstrate that enhancers of Il12b and Il1a are kept relatively lowly occupied by nucleosomes and accessible through recruitment of the nucleosome remodeler BAF/PBAF. Our results using an inducible cell line that expresses an estrogen receptor fusion of the macrophage-specific transcription factor PU.1 (PUER) show that BAF/PBAF recruitment to these enhancers is a consequence of translocation of PUER to the nucleus in the presence of tamoxifen, and we speculate that remodeler recruitment may be directly mediated by PU.1. In the absence of BAF/PBAF recruitment, nucleosome occupancy at the enhancer of Il12b (and to a lesser extent at Il1a) reaches high levels in bone marrow-derived macrophages (BMDMs), and the enhancers are not fully cleared of nucleosomes upon LPS induction, resulting in impaired gene expression. Analysis of Il12b expression in single cells suggests that recruitment of the remodeler is necessary for high levels of transcription from the same promoter, and we propose that remodelers function by increasing nucleosome turnover to facilitate transcription factor over nucleosome binding in a process we have termed "remodeler-assisted competition."

The Lineage-Specific Transcription Factor PU.1 Prevents Polycomb-Mediated Heterochromatin Formation at Macrophage-Specific Genes.

Lineage-specific transcription factors (TFs) are important determinants of cellular identity, but their exact mode of action has remained unclear. Here we show using a macrophage differentiation system that the lineage-specific TF PU.1 keeps macrophage-specific genes accessible during differentiation by preventing Polycomb repressive complex 2 (PRC2) binding to transcriptional regulatory elements. We demonstrate that the distal enhancer of a gene becomes bound by PRC2 as cells differentiate in the absence of PU.1 binding and that the gene is wrapped into heterochromatin, which is characterized by increased nucleosome occupancy and H3K27 trimethylation. This renders the gene inaccessible to the transcriptional machinery and prevents induction of the gene in response to an external signal in mature cells. In contrast, if PU.1 is bound at the transcriptional regulatory region of a gene during differentiation, PRC2 is not recruited, nucleosome occupancy is kept low, and the gene can be induced in mature macrophages. Similar results were obtained at the enhancers of other macrophage-specific genes that fail to bind PU.1 as an estrogen receptor fusion (PUER) in this system. These results show that one role of PU.1 is to exclude PRC2 and to prevent heterochromatin formation at macrophage-specific genes.

Nucleosomes are stably evicted from enhancers but not promoters upon induction of certain pro-inflammatory genes in mouse macrophages.

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.

RNA polymerase III repression by the retinoblastoma tumor suppressor protein.

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. This article is part of a Special Issue entitled: Transcription by Odd Pols.

Regulation of human RNA polymerase III transcription by DNMT1 and DNMT3a DNA methyltransferases.

The human small nuclear RNA (snRNA) and small cytoplasmic RNA (scRNA) gene families encode diverse non-coding RNAs that influence cellular growth and division. Many snRNA and scRNA genes are related via their compact and yet powerful promoters that support RNA polymerase III transcription. We have utilized the human U6 snRNA gene family to examine the mechanism for regulated transcription of these potent transcription units. Analysis of nine U6 family members showed enriched CpG density within the promoters of actively transcribed loci relative to inert genes, implying a relationship between gene potency and DNA methylation. Indeed, both pharmacological inhibition of DNA methyltransferase (DNMT) activity and the forced diminution of DNMT-1, DNMT-3a, and DNMT-3b by siRNA targeting resulted in increased U6 levels in asynchronously growing MCF7 adenocarcinoma cells. In vitro transcription assays further showed that template methylation impedes U6 transcription by RNA polymerase III. Both DNMT-1 and DNMT-3a were detected at the U6-1 locus by chromatin immunoprecipitation directly linking these factors to RNA polymerase III regulation. Despite this association, the endogenous U6-1 locus was not substantially methylated in actively growing cells. However, both DNMT occupancy and low frequency methylation were correlated with increased Retinoblastoma tumor suppressor (RB) expression, suggesting that the RB status can influence specific epigenetic marks.

The perinucleolar compartment is directly associated with DNA.

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 anti-cancer 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.