Major Histocompatibility Complex Class-II (MHC-II) molecules are critical regulators of adaptive immunity that present extracellular antigens required to activate CD4+ T cells. MHC-II are regulated at the level of transcription by master regulator, the Class II Transactivator (CIITA), whose association with the MHC-II promoter is necessary to initiate transcription. Recently, much research focused on novel mechanisms of transcriptional regulation of critical genes like MHC-II and CIITA; findings that the macromolecular complex of the 26S-proteasome is involved in transcription have been perhaps the most exciting as they impart novel functions to a well studied system. Proteasome is a multi-subunit complex composed of a 20S-core particle capped by a 19S-regulatory particle. The 19S contains six ATPases which are required for transcription initiation and elongation. We demonstrate that 19S ATPase-S6a inducibly associates with CIITA promoters. Decreased expression of S6a negatively impacts recruitment of the transcription factors STAT-1 and IRF-1 to the CIITA due to significant loss in histone H3 and H4 acetylation. S6a is robustly recruited to CIITA coding regions, where S6a binding coordinates with that of RNA polymerase II. RNAi mediated S6a knockdown significantly diminishes recruitment of Pol II and P-TEF-b components to CIITA coding regions, indicating S6a plays important roles in transcriptional elongation. Our research is focused on the ways in which accessibility to and transcription of DNA is regulated. While cancers are frequently linked to dysregulated gene expression, contribution of epigenetics to cancers remains unknown. To achieve metastatic ability, tumors alter gene expression to escape host immunosurveilance. MHC-II and CIITA expression are significantly downregulated in highly metastatic MDA-MB-435 breast cancer cells. This suppression correlates with elevated levels of the silencing modification H3K27me3 at CIITA and a significant reduction in Pol II recruitment. We observe elevated binding of the histone methyltransferase to CIITApIV and demonstrate this enzyme is a master regulator of CIITA gene expression. EZH2 knockdown results in significant increases in CIITA and MHC-II transcript levels in metastatic cells. In sum, transcriptional regulation by the 19S-proteasome and histone modifying enzymes represents novel mechanisms of control of mammalian gene expression and present novel therapeutic targets for manipulating MHC expression in disease.

Immune systems are fundamentally based on the differentiation of self and non-self. Unlike mammals, plants have an innate immune system responding to the pathogen only at the site of attack. One of these pathogens is Blumeria graminis f. sp. hordei which is an obligate biotrophic pathogen causing powdery mildew disease and resulting in up to 30% yield loss for both cultivated and wild barley. In this study, Pallas-01 (P-01) and Pallas-03 (P-03) barley lines were inoculated with powdery mildew race Bgh103 (64/01) resulting incompatible and compatible interactions, respectively. 6, 12, 24, 48 and 72 hour-post-inoculation (hpi) samples were used in order to define the differential gene expression of NAD malic enzyme, chloroplast lipocalin, phosphoglyceromutase (PGM), Mg chelatase and 26S protease regulatory subunit 6B homolog. In the proteomics study previously conducted in the laboratory, except for the NAD-dependent malic enzyme, the other four proteins were shown to be involved in the incompatible interaction of P-01 and Bgh103 at protein level, whereas NAD-dependent malic enzyme was changing in the compatible interaction. The expression level for both proteomics and transcriptomics were assumed to be similar. However, the level of transcript would not always reflect its protein level or correlate with the level of proteins, due to complex cellular regulation processes. Post-transcriptional modifications such as synthesis, processing, degradation and post-translational modifications are changing the level of proteins expressed, thus a parallel correlation between the protein and mRNA levels can be lost. Other possible reasons for this variation can be changes in mRNA and protein stability, efficiency of translation and protein&rsquo / s turnover rate. The transcription level changes of the genes investigated in this study are found to be differentially expressed, supporting the proteomics data indicating that these genes are possibly involved in resistance. For further investigations, genetic tools such as gene silencing with RNAi and knockout experiments are required in order to elucidate the mechanism of these candidate genes in the plant-pathogen interaction.

It is evident that components of the 26S proteasome function beyond protein degradation in the regulation of transcription. Studies in yeast implicate the 26S proteasome, specifically the 19S cap, in the epigenetic regulation of transcription. Saccharomyces cerevisiae 19S ATPases remodel chromatin by facilitating histone acetylation and methylation. However, it is unclear if the 19S ATPases play similar roles in mammalian cells. We previously found that the 19S ATPase Sug1 positively regulates transcription of the critical inflammatory gene MHC-II and that the MHC-II promoter fails to efficiently bind transcription factors upon Sug1 knockdown. MHC-II transcription is regulated by the critical coactivator CIITA. We now find that Sug1 is crucial for regulating histone H3 acetylation at the cytokine inducible MHC-II and CIITA promoters. Histone H3 acetylation is dramatically decreased upon Sug1 knockdown with a preferential loss occurring at lysine 18. Research in yeast indicates that the ortholog of Sug1, Rpt6, acts as a mediator between the activating modifications of histone H2B ubiquitination and H3 methylation. Therefore, we characterized the role the 19S proteasome plays in regulating additional activating modifications. As with acetylation, Sug1 is necessary for proper histone H3K4 and H3R17 methylation at cytokine inducible promoters. In the absence of Sug1, histone H3K4me3 and H3R17me2 are substantially inhibited. Our observation that the loss of Sug1 has no significant effect on H3K36me3 implies that Sug1’s regulation of histone modifications is localized to promoter regions as H3K4me3 but not H3K36me3 is clustered around gene promoters. Here we show that multiple H3K4 histone methyltransferase subunits bind constitutively to the inducible MHC-II and CIITA promoters and that over-expressing one subunit significantly enhances promoter activity. Furthermore, we identified a critical subunit of the H3K4 methyltransferase complex that binds multiple histone modifying enzymes, but fails to bind the CIITA promoter in the absence of Sug1, implicating Sug1 in recruiting multi-enzyme complexes responsible for initiating transcription. Finally, Sug1 knockdown maintains gene silencing as elevated levels of H3K27 trimethylation are observed upon Sug1 knockdown. Together these studies strongly implicate the 19S proteasome in mediating the initial reorganization events to relax the repressive chromatin structure surrounding inducible genes.

Major histocompatibility complex molecules (MHCII) are cell surface glycoproteins that present extracellular antigens to CD4+ T lymphocytes and initiate adaptive immune responses. Apart from their protective role, overexpression of MHCII contributes to autoimmune disorders where the immune system attacks our own tissues. Autoimmune diseases are characterized by self-reactive responses to autoantigens, promoting tissue damage, inflammation mediated by proinflammatory cytokines, autoreactive lymphocytes, and autoantibodies. MHCII molecules are tightly regulated at the level of transcription by Class II transactivator (CIITA). CIITA associates with an enhanceosome complex at MHCII promoters and regulates the expression of MHCII. It is thus crucial to understand the regulation of CIITA expression in order to regulate MHCII in autoimmune diseases. Our lab has shown that the 19S ATPases of the 26S proteasome associate with MHCII and CIITA promoters and play important roles in gene transcription, regulate covalent modifications to histones, and are involved in the assembly of activator complexes in mammalian cells. The mechanisms by which the proteasome influences transcription remain unclear. Here, we define novel roles of the 19S ATPases Sug1, S7, and S6a in expression of CIITApIV genes. These ATPases are recruited to CIITApIV promoters and coding regions, interact with the elongation factor PTEFb, and with Ser5 phosphorylated RNA Pol II. Both the generation of CIITApIV transcripts and efficient recruitment of RNA Pol II to CIITApIV are negatively impacted by knockdown of 19S ATPases. Alternatively, inflammation is also suppressed via the Regulator of G-protein signaling 10 (RGS10) in microglial cells which express high levels of RGS10 and promote homeostasis in the central nervous system. However, chronic activation of microglial cells leads to release of cytokines which cause neuroinflammation. Our investigation of roles played by RGS10 in chronically activated microglial cells indicates that RGS10 binds to promoters of IL-1β, and TNF-α and regulates these genes, while the molecular mechanism remains to be investigated. Together, our observations indicate roles for the UPS in modulating gene expression and for RGS10 in regulating proinflammatory cytokines in microglial cells, each of which provides novel therapeutic targets to combat inflammation in autoimmune and neurodegenerative diseases.

The ability of plants to sense and respond to environmental and endogenous signals is essential to their growth and development. As part of these diverse cellular functions, ubiquitin-mediated proteolysis has emerged to be an important process involved in how plant signalling pathways can be regulated in response to such cues. Of the three enzymes involved in linking ubiquitin to protein targets, E3 ubiquitin ligases are of interest as they confer substrate specificity during this ubiquitination process. The overall focal point of this research is on plant U-box (PUB) E3 ubiquitin ligases, a family that has undergone a large gene expansion possibly attributable to the regulation of biological processes unique to the plant life cycle. In Arabidopsis there are 64 predicted PUBs, many for which biological roles have yet to be determined. And as research continues to uncover PUB functions, the functional diversity in the gene family will likely expand. Specifically the focus of this research is on characterizing two ARM repeat-containing PUBs – AtPUB18 and AtPUB19. General analysis of pub18 and pub19 T-DNA insertion lines for growth defects did not yield distinct altered phenotypes. Closer inspection of selected lines showed independent gene assortment phenotypes that, with further inordinately convoluted pursuit, proved to have an AtPUB18/19-unrelated outcome. The availability of Arabidopsis microarray databases provided exploratory expression profiling as a starting point to elucidate PUB function. AtPUB19 and closely related AtPUB18 are notable for their increased expression during abiotic stresses. While condition-directed germination assays showed a decreased sensitivity to salt and ABA for pub18 pub19 double insertion lines, no related change in susceptibility to these or other abiotic stress treatments were seen with condition-directed root growth assays. Thus, this preliminary work has begun to reveal insight into the complex abiotic stress-related roles AtPUB18 and AtPUB19 have during mediation of environmental stress acclimation in Arabidopsis.

The ability of plants to sense and respond to environmental and endogenous signals is essential to their growth and development. As part of these diverse cellular functions, ubiquitin-mediated proteolysis has emerged to be an important process involved in how plant signalling pathways can be regulated in response to such cues. Of the three enzymes involved in linking ubiquitin to protein targets, E3 ubiquitin ligases are of interest as they confer substrate specificity during this ubiquitination process. The overall focal point of this research is on plant U-box (PUB) E3 ubiquitin ligases, a family that has undergone a large gene expansion possibly attributable to the regulation of biological processes unique to the plant life cycle. In Arabidopsis there are 64 predicted PUBs, many for which biological roles have yet to be determined. And as research continues to uncover PUB functions, the functional diversity in the gene family will likely expand. Specifically the focus of this research is on characterizing two ARM repeat-containing PUBs – AtPUB18 and AtPUB19. General analysis of pub18 and pub19 T-DNA insertion lines for growth defects did not yield distinct altered phenotypes. Closer inspection of selected lines showed independent gene assortment phenotypes that, with further inordinately convoluted pursuit, proved to have an AtPUB18/19-unrelated outcome. The availability of Arabidopsis microarray databases provided exploratory expression profiling as a starting point to elucidate PUB function. AtPUB19 and closely related AtPUB18 are notable for their increased expression during abiotic stresses. While condition-directed germination assays showed a decreased sensitivity to salt and ABA for pub18 pub19 double insertion lines, no related change in susceptibility to these or other abiotic stress treatments were seen with condition-directed root growth assays. Thus, this preliminary work has begun to reveal insight into the complex abiotic stress-related roles AtPUB18 and AtPUB19 have during mediation of environmental stress acclimation in Arabidopsis.