Experimental Autoimmune Encephalomyelitis Ameliorated by Passive Transfer of Polymerase 1-Silenced MOG35-55 Lymphatic Node Cells: Verification of a Novel Therapeutic Approach in Multiple Sclerosis.

In the current study, we present an innovative concept based on the knowledge that enhancing naturally occurring biological mechanisms is effective in preventing neuronal damage and maintaining low disease activity in about 15% of multiple sclerosis (MS) patients presenting the benign type of MS. Recently, we have demonstrated that low disease activity in benign MS is associated with suppression of RNA polymerase 1 (POL1) pathway; therefore, targeting POL1 transcription machinery as a strategy for suppressing active forms of MS is suggested. To further establish our approach, we aimed to suppress POL1 pathway by silencing of the POL1-related RRN3, POLR1D and LRPPRC genes in specific MOG35-55-activated lymphocytes and assess their capacity to induce experimental autoimmune encephalomyelitis (EAE) by passive transfer. We have demonstrated that silencing of specific POL1 pathway-related genes significantly decreased viability and increased the proportion of CD4+/AnnexinV+/PI+ apoptotic cells in MOG35-55-primed lymphocytes. POL1-gene silencing significantly decreased the proportion of CD4+IL17+ and increased proportion of CD4+IL10+ and CD4+TNFa+ lymphocytes that occurred simultaneously with over-presentation of Treg CD4+CD25+FoxP3+ cells. Passive transfer of MOG35-55-primed lymphocytes after POL1-gene silencing suppressed EAE development in mice as demonstrated by delayed onset and peak of disease accompanied by significantly lower maximal and cumulative EAE scores. Our study supports a basis for direct targeting of POL1 transcription pathway as a strategy for selective induction of apoptosis and suppression of inflammation in EAE and consequently paves the way for innovative and targeted MS therapeutic strategy that is based on naturally existing biological mechanism.

Cellular sensitivity to UV-irradiation is mediated by RNA polymerase I transcription.

The nucleolus has long been considered to be a pure ribosome factory. However, over the last two decades it became clear that the nucleolus is involved in numerous other functions besides ribosome biogenesis. Our experiments indicate that the activity of RNA polymerase I (Pol I) transcription monitors the integrity of the DNA and influences the response to nucleolar stress as well as the rate of survival. Cells with a repressed ribosomal DNA (rDNA) transcription activity showed an increased and prolonged p53 stabilisation after UVC-irradiation. Furthermore, p53 stabilisation after inhibition and especially after UVC-irradiation might be due to abrogation of the HDM2-p53 degradation pathway by ribosomal proteins (RPs). Apoptosis mediated by highly activated p53 is a typical hallmark of Cockayne syndrome cells and transcriptional abnormalities and the following activation of the RP-HDM2-p53 pathway would be a possible explanation.

Inhibition of Pol I transcription treats murine and human AML by targeting the leukemia-initiating cell population.

Despite the development of novel drugs, the prospects for many patients with acute myeloid leukemia (AML) remain dismal. This study reveals that the selective inhibitor of RNA polymerase I (Pol I) transcription, CX-5461, effectively treats aggressive AML, including mixed-lineage leukemia-driven AML, and outperforms standard chemotherapies. In addition to the previously characterized mechanism of action of CX-5461 (ie, the induction of p53-dependent apoptotic cell death), the inhibition of Pol I transcription also demonstrates potent efficacy in p53null AML in vivo. This significant survival advantage in both p53WT and p53null leukemic mice treated with CX-5461 is associated with activation of the checkpoint kinases 1/2, an aberrant G2/M cell-cycle progression and induction of myeloid differentiation of the leukemic blasts. The ability to target the leukemic-initiating cell population is thought to be essential for lasting therapeutic benefit. Most strikingly, the acute inhibition of Pol I transcription reduces both the leukemic granulocyte-macrophage progenitor and leukemia-initiating cell (LIC) populations, and suppresses their clonogenic capacity. This suggests that dysregulated Pol I transcription is essential for the maintenance of their leukemia-initiating potential. Together, these findings demonstrate the therapeutic utility of this new class of inhibitors to treat highly aggressive AML by targeting LICs.

Conditional inactivation of Upstream Binding Factor reveals its epigenetic functions and the existence of a somatic nucleolar precursor body.

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).

AMP-activated protein kinase adapts rRNA synthesis to cellular energy supply.

AMP-activated protein kinase (AMPK) senses changes in the intracellular AMP/ATP ratio, switching off energy-consuming processes and switching on catabolic pathways in response to energy depletion. Here, we show that AMPK down-regulates rRNA synthesis under glucose restriction by phosphorylating the RNA polymerase I (Pol I)-associated transcription factor TIF-IA at a single serine residue (Ser-635). Phosphorylation by AMPK impairs the interaction of TIF-IA with the TBP-containing promoter selectivity factor SL1, thereby precluding the assembly of functional transcription initiation complexes. Mutation of Ser-635 compromises down-regulation of Pol I transcription in response to low energy supply, supporting that activation of AMPK adapts rRNA synthesis to nutrient availability and the cellular energy status.

Downregulation of the upstream binding factor1 by glycogen synthase kinase3beta in myeloid cells induced to differentiate.

The upstream binding factor 1 (UBF1), one of the proteins that regulate the activity of RNA polymerase I, is downregulated in 32D myeloid cells induced to differentiate into granulocytes, either by the type 1 insulin-like growth factor (IGF-1) or the granulocytic colony stimulating factor (G-CSF). Downregulation of UBF1 is largely due to protein degradation, while mRNA levels are not affected. Inhibition of UBF1 degradation by lithium chloride (LiCl)and lactacystin suggest a role of glycogen synthase kinase beta (GSK3beta) in a proteasome-dependent degradation of UBF. GSK3beta phosphorylates in vitro and in vivo the UBF protein, which has five putative motifs for phosphorylation by GSK3beta. Elimination and/or mutations of these motifs stabilize the UBF1 protein even in cells induced to differentiate. Conversely, a stably transfected, constitutively active GSK3beta accelerates the downregulation of UBF1. We show further that activation of the differentiating protein C/EPBalpha in 32D cells transformed by the oncogenic BCR/ABL protein causes downregulation of UBF1. Finally, inhibition of differentiation of myeloid cells by a dominant negative mutant of Stat3 stabilizes the UBF1 protein, while rapamycin-induced differentiation of myeloid cells downregulates UBF1 levels. Taken together, our results indicate that the induction of granulocytic differentiation in 32D murine myeloid cells causes the degradation of UBF1, via GSK3beta and the proteasome pathway.