DNA oligonucleotides that were used in competitive and quantitative PCR (qPCR) are listed inSupplementary Table S1. only at the promoter, our data strongly indicate that monoallelic BES transcription is usually activated by a mechanism that functions at the level of transcription initiation. == INTRODUCTION == Monoallelic expression of a large gene family is usually a phenomenon found in parasites that exhibit antigenic variation of cell surface proteins to evade the mammalian hosts immune system (1). It also occurs in mammalian olfactory sensory neurons, which express a single member of the large odorant receptor (OR) gene family (2). While monoallelic gene expression of a gene family is typically regulated at the level of transcription, the mechanisms enabling transcription of a single gene/allele are not well comprehended. Trypanosoma bruceiis a vector-borne lethal parasite in sub-Saharan Africa that lives freely in the human bloodstream by virtue of a dense coat of variant surface glycoprotein (VSG) expressed from a single gene. The uniform coat shields invariant membrane proteins from immune recognition (3) and is periodically altered by switchingVSGexpression to a different gene. While the parasite harbors more than 2000VSGgenes and pseudogenes,VSGexpression is restricted to 1 1 of 15 telomeric bloodstream expression sites (BESs) (4). A BES is usually a tandem array of several expression site-associated genes (ESAGs) followed by a singleVSGgene, all of which are polycistronically transcribed by a multifunctional RNA polymerase (pol) I from a promoter that is located 4060 kb upstream of theVSGgene (5). The active BES is usually transcribed outside of the nucleolus (6) in the extranucleolar expression site body (ESB), a DNase-resistant compartment that appears to limit productive transcription to a single BES (7). Silencing of gene family members is usually mediated by epigenetic mechanisms. Silent OR genes in mice are marked with histone methylations H3K9me3 and H4K20me3 that are characteristic of constitutive heterochromatin (8).Plasmodium falciparum, the parasite causing cerebral malaria, exhibits monoallelic expression of thevargene family, which encodes variants of theP. falciparumerythrocyte membrane protein 1 (9). This protein is expressed on the surface of an infected Sildenafil erythrocyte and mediates cytoadherence to endothelial host receptors keeping the infected cell in the peripheral vascular system. Deficiency of the histone deacetylase Sir2 inP. falciparumled to activation of silentvargenes and loss of monoallelicvargene expression (10,11). Accordingly, knockdown ofT. brucei RAP1, encoding a telomeric protein known to induce epigenetic gene silencing, led Sildenafil to derepression of all known BESs, detection of additional extranucleolar RNA pol I foci and formation of mixed VSG cell surface coats (12). In addition, several other factors have been implicated in BES silencing because their gene knockdowns resulted in derepression of silent BESs; these comprise the methyltransferase DOT1B (13), the chromatin remodelers TbISWI (14) and FACT (15), the histone deacetylase DAC3 (16), the nucleoplasmin-like protein NLP (17), histone H1 (18), the origin recognition complex (19), histone H3 and the histone chaperones CAF-1b and ASF1A (20) as well as the mini-chromosome maintenance-binding protein MCM-BP (21). Moreover, nucleosomes are enriched in the chromatin of silent BESs but are depleted in that of the active BES (22,23). While these studies exhibited the importance of epigenetic factors in restrictingVSGexpression to a single gene, the mechanism of transcriptional silencing is usually yet to be determined. Importantly, Sildenafil in the cited studies, matureVSGmRNA levels from derepressed BESs remained 1010 000-fold lower than that of the activeVSG. BES derepression byRAP1andDOT1Bsilencing did not affect the high expression level of the activeVSG(12,13), indicating that derepressed BESs cannot effectively compete withVSGexpression from the active BES. On the other hand, silencing eitherSPT16, encoding a FACT subunit, orNLPdid reduce the abundance of activeVSGtranscript (15,17). However, as shown in theNLPstudy, promoter-proximal gene expression of the active site was much less affected, suggesting again that in the derepressed state, BES promoters cannot efficiently compete for transcription factors from the active promoter. Rabbit Polyclonal to CNKR2 We have recently characterized the multi-subunit class I transcription factor A (CITFA) as an essential factor for RNA pol I transcription of the active BES, the large ribosomal gene unit (RRNA) and the procyclin gene units, which encode Sildenafil the major cell surface proteins of insect-stage, procyclic form (PF) trypanosomes (24). Characterization of CITFA revealed seven apparently trypanosomatid-specific proteins, termed CITFA-1 to -7, and.
