OCA-B (OCT Coactivator from B cells) is highly expressed in B cells and is the first-identified tissue-specific transcriptional coactivator. Mechanistically, OCA-B is recruited to target genes through primary interactions with DNA-binding transcription factors OCT1/OCT2, as well as secondary interactions with MEF2B, and its effector functions in activating transcription involve interactions with the Mediator coactivator complex. Physiologically, OCA-B plays an essential role in antigen-stimulated Germinal Center (GC) formation and transcription of secondary immunoglobulin (Ig) genes. OCA-B also regulates bone marrow and peripheral B cell development. While GC B cell-specific inactivation of Oca-B is sufficient to cause GC defects, OCA-B function in follicular T helper (Tfh) cells also plays an important role in GC reactions. In GC B cells, OCA-B-dependent genes include known GC regulatory genes such as Bcl6, Mef2b and Irf4. In CD4+ memory T cells, OCA-B is required to maintain a subset of genes, including Il2, in a transcriptionally poised state, enabling their rapid and robust up-regulation upon repeated stimulation. Moreover, recent studies have indicated an essential role for OCA-B in B-cell malignancy and multiple autoimmune diseases, highlighting OCA-B as a potential therapeutic target for these diseases.

Supplementary Figure 1 from Inactivation of gadd45a Sensitizes Epithelial Cancer Cells to Ionizing Radiation In vivo Resulting in Prolonged Survival

The NuRD complex contains both chromatin remodeling and histone deacetylase activities. Mice lacking the MTA2 subunit of NuRD show developmental defects in pro-B, pre-B, immature B, and marginal zone B cells, and abnormal germinal center B cell differentiation during immune responses. Mta2 inactivation also causes a derepression of Igll1 and VpreB1 genes in pre-B cells. Furthermore, MTA2/NuRD interacts directly with AIOLOS/IKAROS and shows a striking overlap with AIOLOS/IKAROS target genes in human pre-B cells, suggesting a functional interdependence between MTA2/NuRD and AIOLOS. Mechanistically, MTA2 deficiency in mice leads to increased H3K27 acetylation at both Igll1 and VpreB1 promoters. Gene profiling analyses also identify distinct MTA2-dependent transcription programs in pro-B and pre-B cells. In addition, we find a strong synergy between MTA2 and OCA-B in repressing Igll1 and VpreB1 at the pre-B cell stage, and in regulating both the pre-B to immature B transition and splenic B cell development.

Soon after exposure to genotoxic reagents, mammalian cells inhibit transcription to prevent collisions with repair machinery and to mount a proper DNA damage response. However, mechanisms underlying early transcriptional inhibition are poorly understood. In this report, we show that site-specific acetylation of super elongation complex (SEC) subunit AFF1 by p300 reduces its interaction with other SEC components and impairs P-TEFb-mediated C-terminal domain phosphorylation of RNA polymerase II both in vitro and in vivo. Reexpression of wild-type AFF1, but not an acetylation mimic mutant, restores SEC component recruitment and target gene expression in AFF1 knockdown cells. Physiologically, we show that, upon genotoxic exposure, p300-mediated AFF1 acetylation is dynamic and strongly correlated with concomitant global down-regulation of transcription-and that this can be reversed by overexpression of an acetylation-defective AFF1 mutant. Therefore, we describe a mechanism of dynamic transcriptional regulation involving p300-mediated acetylation of a key elongation factor during genotoxic stress.

Histone posttranslational modification leads to downstream effects indirectly by allowing or preventing docking of effector molecules, or directly by changing the intrinsic biophysical properties of local chromatin. To date, little has been done to study posttranslational modifications that lie outside of the unstructured tail domains of histones. Core residues, and in particular arginines in H3 and H4, mediate key interactions between the histone octamer and DNA in forming the nucleosomal particle. Using mass spectrometry, we find that one of these core residues, arginine 42 of histone H3 (H3R42), is dimethylated in mammalian cells by the methyltransferases coactivator arginine methyltransferase 1 (CARM1) and protein arginine methyltransferase 6 (PRMT6) in vitro and in vivo, and we demonstrate that methylation of H3R42 stimulates transcription in vitro from chromatinized templates. Thus, H3R42 is a new, "nontail" histone methylation site with positive effects on transcription. We propose that methylation of basic histone residues at the DNA interface may disrupt histone:DNA interactions, with effects on downstream processes, notably transcription.

Although histone H3 Lys4 (H3K4) methylation is widely associated with gene activation, direct evidence for its causal role in transcription, through specific MLL family members, is scarce. Here we have purified a human MLL2 (Kmt2b) complex that is highly active in H3K4 methylation and chromatin transcription in a cell-free system. This effect requires S-adenosyl methionine and intact H3K4, thus establishing a direct and causal role for MLL2-mediated H3K4 methylation in transcription. We also show that human AKAP95, a chromatin-associated protein, physically and functionally associates with MLL complexes and directly enhances their methyltransferase activity. Ectopic AKAP95 stimulates expression of a chromosomal reporter gene in synergy with MLL1 or MLL2, whereas AKAP95 depletion impairs retinoic acid-mediated gene induction in embryonic stem cells. These results demonstrate an important role for AKAP95 in regulating histone methylation and gene expression, particularly during cell-fate transitions.

The high frequency of p53 mutation in human cancers indicates the important role of p53 in suppressing tumorigenesis. It is well established that the p53 regulates multiple, distinct, cellular functions such as cell cycle arrest and apoptosis. Despite intensive studies, little is known about which function is essential, or if multiple pathways are required, for p53-dependent tumor suppression in vivo . Using a mouse brain carcinoma model that shows high selective pressure for p53 inactivation, we found that even partially abolishing p53-dependent apoptosis by Bax inactivation was sufficient to significantly reduce the selective pressure for p53 loss. This finding is consistent with previous reports that apoptosis is the primary p53 function selected against during E μ -myc induced mouse lymphoma progression. However, unlike observed in the E μ -myc induced lymphoma model, attenuation of apoptosis is not sufficient to phenocopy the aggressive tumor progression associated with complete loss of p53 activity. We conclude apoptosis is the primary tumor suppressive p53 function and the ablation of additional p53 pleiotropic effects further exacerbates tumor progression.

The high frequency of p53 mutation in human cancers indicates the important role of p53 in suppressing tumorigenesis. It is well established that the p53 regulates multiple, distinct cellular functions such as cell-cycle arrest and apoptosis. Despite intensive studies, little is known about which function is essential, or if multiple pathways are required, for p53-dependent tumor suppression in vivo. Using a mouse brain carcinoma model that shows high selective pressure for p53 inactivation, we found that even partially abolishing p53-dependent apoptosis by Bax inactivation was sufficient to significantly reduce the selective pressure for p53 loss. This finding is consistent with previous reports that apoptosis is the primary p53 function selected against during Eμ-myc–induced mouse lymphoma progression. However, unlike observed in the Eμ-myc–induced lymphoma model, attenuation of apoptosis is not sufficient to phenocopy the aggressive tumor progression associated with complete loss of p53 activity. We conclude that apoptosis is the primary tumor suppressive p53 function and the ablation of additional p53 pleiotropic effects further exacerbates tumor progression. Mol Cancer Res; 9(4); 430–9. ©2011 AACR.

Abstract Ionizing radiation (IR) therapy is one of the most commonly used treatments for cancer patients. The responses of tumor cells to IR are often tissue specific and depend on pathway aberrations present in the tumor. Identifying molecules and mechanisms that sensitize tumor cells to IR provides new potential therapeutic strategies for cancer treatment. In this study, we used two genetically engineered mouse carcinoma models, brain choroid plexus carcinoma (CPC) and prostate, to test the effect of inactivating gadd45a, a DNA damage response p53 target gene, on tumor responses to IR. We show that gadd45a deficiency significantly increases tumor cell death after radiation. Effect on survival was assessed in the CPC model and was extended in IR-treated mice with gadd45a deficiency compared with those expressing wild-type gadd45a. These studies show a significant effect of gadd45a inactivation in sensitizing tumor cells to IR, implicating gadd45a as a potential drug target in radiotherapy management. [Cancer Res 2008;68(10):3579–83]

Dynamic changes in chromatin structure through ATP-dependent remodeling and covalent modifications on histones play important roles in transcription regulation. Among the many chromatin modifiers identified, the NuRD (nucleosome remodeling histone deacetylase) complex is unique because it possesses both nucleosome remodeling and histone deacetylase activities. To understand the biological function of the NuRD complex, we generated a knock-out mouse model of the Mta2 (metastasis-associated protein 2) gene, which encodes a NuRD-specific component. Mta2 null mice exhibited partial embryonic lethality. The surviving mice developed lupus-like autoimmune symptoms including skin lesions, bodyweight loss, glomerulonephritis, liver inflammation, and production of autoantibodies. Transplantation of bone marrow cells from Mta2 null mice recapitulated some of the symptoms including skin lesion and bodyweight loss in the recipient mice. Mta2 null T lymphocytes showed normal development but hyperproliferation upon stimulation, which correlates with hyperinduction of interleukin (IL)-2, IL-4, and interferon (IFN)-gamma. T cell hyperproliferation, but not other autoimmune symptoms, was observed in T cell-specific Mta2 knock-out mice. Mta2 null T cells produced more IL-4 and IFN-gamma under Th2 activation conditions, but normal levels of IL-4 and IFN-gamma under Th1 activation conditions. Furthermore, we found that IL-4 is a direct target gene of Mta2. Our study suggests that Mta2/NuRD is involved in modulating IL-4 and IFN-gamma expression in T cell immune responses, and gene expression in non-T cells plays an important role in controlling autoimmunity.

Malignant rhabdoid tumors (MRT) are rare aggressive cancers that occur in young children. Seventy-five percent of sporadic MRTs harbor inactivating SNF5 mutations, and mice heterozygous for an Snf5-null allele develop MRTs with partial penetrance. The diagnosis of choroid plexus carcinomas (CPC) in addition to MRTs in families with a single mutant SNF5 allele prompted us to assess the role of SNF5 loss in CPC in genetically engineered mice. With high frequency, TgT(121) mice develop CPCs that are initiated by inactivation of retinoblastoma protein (pRb) and related proteins p107 and p130. However, CPC penetrance and latency were not significantly affected by Snf5 heterozygosity, consistent with recent evidence that CPCs in SNF5 families were, in many cases, misdiagnosed MRTs. Surprisingly, although the CPC phenotype was unaffected, TgT(121);Snf5(+/-) mice developed MRTs with increased penetrance and decreased latency compared with TgT(121);Snf5(+/+) littermates. MRTs expressed the T(121) protein with a concomitant increase in mitotic activity. The predominant appearance of TgT(121);Snf5(+/-) MRTs in the spinal cord led to the discovery that these tumors likely arose from a subset of spinal cord neural progenitor cells expressing T(121) rather than from transdifferentiation of CPC. Significantly, the target cell type(s) for MRT is unknown. Hence, this study not only shows that pRb(f) and SNF5 inactivation cooperate to induce MRTs but also provides new insight into the MRT target population.

A new γ-ray self-absorption correction for a random-shaped sample is discussed to extract characteristic information of fissile material through its γ-spectrum. And it is proved by a great deal of numeric simulation tests that such a new type of correction is free from the geometry size and shape of the even sample under the condition of L⪢R>3/μ. Using the correction, multi-composition material can be calculated directly. Moreover, the correction can also be used in studying the layered sample analysis and the layered structure property can be attained directly.

Recent studies have shown the p19(ARF) tumor suppressor to be involved in the response to oncogenic stress by regulating the activity of p53. This response is mediated by antagonizing the function of Mdm2, a negative regulator of p53, indicating a pathway for tumor suppression that involves numerous genes altered in human tumors. We previously described a transgenic mouse brain tumor model in which oncogenic stress, provided by cell-specific inactivation of the pRb pathway, triggers a p53-dependent apoptotic response. This response suppresses the growth of developing tumors and thus represents a bona fide in vivo tumor suppressor activity. We further showed that EM, a transcription factor known to induce P19(ARF) expression, was required for the response. Here, we use a genetic approach to test whether p19(ARF) functions to transduce the signal from E2F1 to p53 in this tumor suppression pathway. Contrary to the currently accepted hypothesis, we show that a deficiency in P19(ARF) has no impact on p53-mediated apoptosis or tumor suppression in this system. All measures of p53 function, including the level of apoptosis induced by pRb inactivation, the expression of p21. (a p53-responsive gene), and the rate of tumor growth, were comparable in mice with and without a functional p19(ARF) gene. Thus, although p19(ARF) is required in some cell types to transmit an oncogenic response signal to p53, it is dispensable for this function in an in vivo epithelial system. These results underscore the complexity of p53 tumor suppression and further indicate the existence of distinct cell-specific pathways that respond to similar stimuli.

We examined the selective pressure for, and the impact of, p53 inactivation during epithelial tumor evolution in a transgenic brain tumor model. In TgT(121) mice, cell-specific inactivation of the pRb pathway in brain choroid plexus epithelium initiates tumorigenesis and induces p53-dependent apoptosis. We previously showed that p53 deficiency accelerates tumor growth due to diminished apoptosis. Here we show that in a p53(+/-) background, slow-growing dysplastic tissue undergoes clonal progression to solid angiogenic tumors in all animals. p53 is inactivated in all progressed tumors, with loss of the wild-type allele occurring in 90% of tumors. Moreover, similar progression occurs in 38% of TgT(121)p53(+/+) mice, also with loss of at least one p53 allele and inactivation of p53. Thus, the selective pressure for p53 inactivation, likely based on its apoptotic function, is high. Yet, in all cases, p53 inactivation correlates with progression beyond apoptosis reduction, from dysplasia to solid vascularized tumors. Hence, p53 suppresses tumor progression in this tissue by multiple mechanisms. Previous studies of fibroblasts and hematopoietic cells show that p53 deficiency can be associated with chromosomal instability, a mechanism that may drive tumor progression. To determine whether genomic gains or losses are present in tumors that progress in the absence of p53, we performed comparative genomic hybridization analysis. Surprisingly, the only detectable chromosomal imbalance was partial or complete loss of chromosome 11, which harbors the p53 gene and is thus the selected event. Flow cytometry confirmed that the majority of tumor cells were diploid. These studies indicate that loss of p53 function is frequent under natural selective pressures and furthermore that p53 loss can facilitate epithelial tumor progression by a mechanism in addition to apoptosis reduction and distinct from chromosomal instability.

The conductivity of alternating current in the xAg2O: (1 ‐ x)Rb2O:4Geo2 glass series has been measured between 5.0 K and room temperature where x is varied from 0.0 to 1.0. The typical low‐temperature conductivity region is observed below 200 K. The temperature and frequency dependence of conductivity in this region can be explained by the thermal excitation of asymmetric double well potential configurations. Further, the classical mixed mobile ion effect is essentially absent, which shows that the effect is associated with the independent movement of the mobile ions.