Susanna Fletcher Greer

The Class II Transactivator (CIITA) is essential to the regulation of Major Histocompatibility Class II (MHC II) genes transcription. As the “master regulator” of MHC II transcription, CIITA regulation is imperative and requires various posttranslational modifications (PTMs) in order to facilitate its role. Previously we identified various ubiquitination events on CIITA. Monoubiquitination is important for CIITA transactivity, while K63 linked ubiquitination is involved in crosstalk with ERK1/2 phosphorylation, where together they mediate cellular movement from the cytoplasm to nuclear region. Further, CIITA is also modified by degradative K48 polyubiquitination. However, the E3 ligase responsible for these modifications was unknown. We show CIITA ubiquitination and transactivity are enhanced with the histone acetyltransferase (HAT), p300/CBP associated factor (pCAF), and the E3 ligase region within pCAF is necessary for both. Additionally, pCAF mediated ubiquitination is independent of pCAF’s HAT domain, and acetylation deficient CIITA is K48 polyubiquitinated and degraded in the presence of pCAF. Lastly, we identify the histone acetyltransferase, pCAF, as the E3 ligase responsible for CIITA’s ubiquitination.

Radiation treatment is a pivotal therapy for several cancer types, including colorectal cancer. It has been shown that sublethal doses of radiation modulate gene expression, making tumor cells more susceptible to T-cell-mediated immune attack. We have recently shown that low dose radiation enhances expression of multiple death receptors (Fas, DR4 and DR5) and co-stimulatory molecules (4-1BBL and OX-40L) in colorectal cancer (CRC) cells; however, it is unclear how ionizing radiation (IR) enhances expression of these molecules mechanistically. In the present study, we elucidate the molecular mechanisms by which radiation controls expression of these molecules in CRC. Here we report that, enhanced expression of these genes following radiation treatment of CRC cells is due, in part, to changes in DNA methylation and histone acetylation. We observed that radiation (5 Gy) significantly increased histone acetylation at the promoter regions of 4-1BBL, Fas and DR5 but not OX-40L. However, radiation did not induce changes in the global levels of acetylated histone H3 suggesting specificity of IR-induced changes. Furthermore, evaluation of epigenetic controlling enzymes revealed that IR did not alter overall cellular levels of HDACs (HDAC1, HDAC2 or HDAC3) or DNMTs (DNMT1, DNMT3a, or DNMT3b). Instead, radiation decreased binding of HDAC2 and HDAC3 at the promoter regions of Fas and 4-1BBL, respectively. Radiation also resulted in reduced DNMT1 at both the Fas and 4-1BBL promoter regions but not a control gene. We conclude that single dose radiation can influence the expression of immune response relevant genes in colorectal tumor cells by altering the binding of epigenetic enzymes, and modulating histone acetylation, at specific gene promoters.

The class II transactivator (CIITA) is known as the master regulator for the major histocompatibility class II (MHC II) molecules. CIITA is dynamically regulated through a series of intricate post-translational modifications (PTMs). CIITA's role is to initiate transcription of MHC II genes, which are responsible for presenting extracellular antigen to CD4+ T-cells. In the present study, we identified extracellular signal-regulated kinase (ERK)1/2 as the kinase responsible for phosphorylating the regulatory site, Ser280, which leads to increased levels of mono-ubiquitination and an overall increase in MHC II activity. Further, we identify that CIITA is also modified by Lys63-linked ubiquitination. Lys63 ubiquitinated CIITA is concentrated in the cytoplasm and following activation of ERK1/2, CIITA phosphorylation occurs and Lys=ubiquitinated CIITA translocates to the nucleus. CIITA ubiquitination and phosphorylation perfectly demonstrates how CIITA location and activity is regulated through PTM cross-talk. Identifying CIITA PTMs and understanding how they mediate CIITA regulation is necessary due to the critical role CIITA has in the initiation of the adaptive immune response.

Transcriptional activation of major histocompatibility complex (MHC) I and II molecules by the cytokine, interferon γ (IFN-γ), is a key step in cell-mediated immunity against pathogens and tumors. Recent evidence suggests that suppression of MHC I and II expression on multiple tumor types plays important roles in tumor immunoevasion. One such tumor is malignant melanoma, a leading cause of skin cancer-related deaths. Despite growing awareness of MHC expression defects, the molecular mechanisms by which melanoma cells suppress MHC and escape from immune-mediated elimination remain unknown. Here, we analyze the dysregulation of the Janus kinase (JAK)/STAT pathway and its role in the suppression of MHC II in melanoma cell lines at the radial growth phase (RGP), the vertical growth phase (VGP) and the metastatic phase (MET). While RGP and VGP cells both express MHC II, MET cells lack not only MHC II, but also the critical transcription factors, interferon response factor (IRF) 1 and its upstream activator, signal transducer and activator of transcription 1 (STAT1). Suppression of STAT1 in vitro was also observed in patient tumor samples, suggesting STAT1 silencing as a global mechanism of MHC II suppression and immunoevasion.

Accumulating evidence shows the 26S proteasome is involved in the regulation of gene expression. We and others have demonstrated that proteasome components bind to sites of 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, although prior observations suggest both proteolytic and non-proteolytic activities. Here, we define novel, non-proteolytic, roles for each of the three 19S heterodimers, represented by the 19S ATPases Sug1, S7, and S6a, in mammalian gene expression using the inflammatory gene CIITApIV. These 19S ATPases are recruited to induced CIITApIV promoters and also associate with CIITA coding regions. Additionally, these ATPases interact with elongation factor PTEFb complex members CDK9 and Hexim-1 and with Ser5 phosphorylated RNA Pol II. Both the generation of transcripts from CIITApIV and efficient recruitment of RNA Pol II to CIITApIV are negatively impacted by siRNA mediated knockdown of these 19S ATPases. Together, these results define novel roles for 19S ATPases in mammalian gene expression and indicate roles for these ATPases in promoting transcription processes.

RGS10 is an important regulator of cell survival and chemoresistance in ovarian cancer. We recently showed that RGS10 transcript expression is suppressed during acquired chemoresistance in ovarian cancer. The suppression of RGS10 is due to DNA hypermethylation and histone deacetylation, two important mechanisms that contribute to silencing of tumor suppressor genes during cancer progression. Here, we fully investigate the molecular mechanisms of epigenetic silencing of RGS10 expression in chemoresistant A2780-AD ovarian cancer cells. We identify two important epigenetic regulators, HDAC1 and DNMT1, that exhibit aberrant association with RGS10 promoters in chemoresistant ovarian cancer cells. Knockdown of HDAC1 or DNMT1 expression, and pharmacological inhibition of DNMT or HDAC enzymatic activity, significantly increases RGS10 expression and cisplatin-mediated cell death. Finally, DNMT1 knock down also decreases HDAC1 binding to the RGS10 promoter in chemoresistant cells, suggesting HDAC1 recruitment to RGS10 promoters requires DNMT1 activity. Our results suggest that HDAC1 and DNMT1 contribute to the suppression of RGS10 during acquired chemoresistance and support inhibition of HDAC1 and DNMT1 as an adjuvant therapeutic approach to overcome ovarian cancer chemoresistance.

Sub-lethal doses of radiation can alter the phenotype of target tissue by modulating gene expression and making tumor cells more susceptible to T-cell-mediated immune attack. We have previously shown that sub-lethal tumor cell irradiation enhances killing of colorectal carcinoma cells by tumor-specific cytotoxic T cells by unknown mechanisms. Recent data from our lab indicates that irradiation of tumor cells results in the upregulation of OX40L and 41BBL, and that T cells incubated with irradiated tumor cells displayed improved CTL survival, activation and effector activity. The objective of this current study was to determine the mechanism of enhanced OX40L and 41BBL expression in human colorectal tumor cells.

One mechanism frequently utilized by tumor cells to escape immune system recognition and elimination is suppression of cell surface expression of Major Histocompatibility Class II (MHC II) molecules. Expression of MHC II is regulated primarily at the level of transcription by the Class II Transactivator, CIITA, and decreased CIITA expression is observed in multiple tumor types. We investigate here contributions of epigenetic modifications to transcriptional silencing of CIITA in variants of the human breast cancer cell line MDA MB 435. Significant increases in histone H3 lysine 27 trimethylation upon IFN-γ stimulation correlate with reductions in transcription factor recruitment to the interferon-γ inducible CIITA promoter, CIITApIV, and with significantly increased CIITApIV occupancy by the histone methyltransferase enhancer of zeste homolog 2 (EZH2). Most compelling is evidence that decreased expression of EZH2 in MDA MB 435 variants results in significant increases in CIITA and HLA-DRA mRNA expression, even in the absence of interferon-γ stimulation, as well as increased cell surface expression of MHC II. Together, these data add mechanistic insight to prior observations of increased EZH2 expression and decreased CIITA expression in multiple tumor types.

Chromatin immunoprecipitation (ChIP) assays were developed in order to comprehensively describe physiological interactions between DNA sequences, transcriptional regulators, and the modification status of associated chromatin. In ChIP assays, living cells are treated with chemical cross-linkers to covalently bind proteins to each other and to their DNA targets. Once cross-linked to associated proteins, chromatin is extracted and fragmented by sonication and protein–DNA complexes are isolated using specific antibodies against a target protein. The cross-links that bind proteins to DNA are then reversed, and purified DNA fragments are analyzed by qPCR to determine if a specific sequence is present. As DNA regulatory elements frequently rely on the interaction of multiple transcription factors and cofactors to regulate gene expression, Re-ChIP methods were developed to allow for the identification of multiple (concurrently binding) proteins on a single DNA sequence. Re-ChIP assays have enabled the analysis of multiple, simultaneous, posttranslational modifications to histones in order to determine the combinatorial pattern of modifications associated with transcriptional status of a gene. Together, ChIP and Re-ChIP have contributed to the elucidation of the epigenetic code-regulating gene expression and have enhanced our understanding of physiological binding of proteins to DNA targets. The protocols that follow describe general strategies used to perform ChIP and Re-ChIP assays for the study of specific protein–DNA interactions.