New Insights into the Control of HIV-1 Transcription: When Tat Meets the 7SK snRNP and Super Elongation Complex (SEC)

Recent studies aimed at elucidating the mechanism controlling HIV-1 transcription have led to the identification and characterization of two multi-subunit complexes that both contain P-TEFb, a human transcription elongation factor and co-factor for activation of HIV-1 gene expression by the viral Tat protein. The first complex, termed the 7SK snRNP, acts as a reservoir where active P-TEFb can be withdrawn by Tat to stimulate HIV-1 transcription. The second complex, termed the super elongation complex (SEC), represents the form of P-TEFb delivered by Tat to the paused RNA polymerase II at the viral long terminal repeat during Tat transactivation. Besides P-TEFb, SEC also contains other elongation factors/co-activators, and they cooperatively stimulate HIV-1 transcription. Recent data also indicate SEC as a target for the mixed lineage leukemia (MLL) protein to promote the expression of MLL target genes and leukemogenesis. Given their roles in HIV-1/AIDS and cancer, further characterization of 7SK snRNP and SEC will help develop strategies to suppress aberrant transcriptional elongation caused by uncontrolled P-TEFb activation. As both complexes are also important for normal cellular gene expression, studying their structures and functions will elucidate the mechanisms that control metazoan transcriptional elongation in general

Mechanistic insights into the roles of P-TEFb and its novel cofactors in tumorigenesis and HIV transcription

Ongoing research in the field of transcription has given rise to the unappreciated role of elongation control as a rate limiting step for transcription, and the general transcription elongation factor P-TEFb therefore has taken the central stages. P-TEFb is composed of cyclin T1 and the cyclin-dependent kinase Cdk9. It stimulates transcription elongation by releasing the paused RNA Polymerase II (Pol II) through phosphorylating Pol II at Ser2 and antagonizing the effects of negative elongation factors. P-TEFb is not only essential for the transcription of the vast majority of cellular genes, but also critical for the expression of HIV genome. Elucidating how P-TEFb activity is controlled therefore plays a key role in advancing our understanding of cellular and disease-related transcription elongation. The first part of this thesis presents my early Ph.D work, which focusing on the identification and characterization of a La-like protein PIP7S (also named as LARP7). I have shown that PIP7S binds and stabilizes nearly all the nuclear 7SK, which leading to sequestration and inactivation of P-TEFb. Consistent with the fact that PIP7S is frequently mutated in human tumors and the Drosophila homolog of PIP7S is a tumor suppressor, loss of PIP7S function disrupts epithelial differentiation and causes P-TEFb-dependent transformation.The second part of this thesis introduces the identification of the Super Elongation Complex (SEC) and subsequent characterization of two of its subunits, AFF4 and ELL2. I have shown that through the bridging functions of AFF4 and Tat, P-TEFb and ELL2 combine to form a bifunctional elongation complex that greatly activates basal and HIV-1 transcription, respectively.The third part of this thesis continues to dissect the functions of the other two SEC subunits ENL and AF9. I have shown that the homologous ENL and AF9 exist in separate SECs with similar but non-identical functions. ENL/AF9 also exists outside SEC when bound to Dot1L, which is found to inhibit SEC function. The YEATS domain of ENL/AF9 targets SEC to Pol II on chromatin through contacting the PAFc complex

Brd4 Recruits P-TEFb to Chromosomes at Late Mitosis To Promote G1 Gene Expression and Cell Cycle Progression▿

Brd4, a bromodomain protein capable of interacting with acetylated histones, is implicated in transmitting epigenetic memory through mitosis. It also functions as an associated factor and positive regulator of P-TEFb, a Cdk9-cyclin T1 heterodimer that stimulates transcriptional elongation by phosphorylating RNA polymerase II. In the present study, experiments were performed to determine whether these two functions of Brd4 are interrelated and, if so, how they may impact cell cycle progression. Our data demonstrate that while the P-TEFb level remains constant, the Brd4-P-TEFb interaction increases dramatically in cells progressing from late mitosis to early G1. Concurrently, P-TEFb is recruited to chromosomes, beginning around mid- to late anaphase and before nuclear envelope/lamina formation and nuclear import of other general transcription factors. Importantly, the recruitment of P-TEFb depends on Brd4. Abrogation of this process through Brd4 knockdown reduces the binding of P-TEFb to and expression of key G1 and growth-associated genes, leading to G1 cell cycle arrest and apoptosis. Because P-TEFb is synonymous with productive elongation, its recruitment by Brd4 to chromosomes at late mitosis may indicate those genes whose active transcription status must be preserved across cell division

Mj-DWD, a double WAP domain-containing protein with antiviral relevance in Marsupenaeus japonicus

National Natural Science Foundation of China [30330470]; National Basic Science Research 973 program [2006CB101804]; The Main Science Foundation of Fujian [2006N0039]The Mj-DWD (Marsupenaeus japonicus' double-WAP domains) gene was originally found up-regulated in virus-resistant shrimp M. japonicus by suppression subtractive hybridization (SSH). The full-length cDNA of Mj-DWD encodes a novel protein containing a KGD (Lys-Gly-Asp) motif and double WAP domains. Performed by quantitative real-time PCR, the expression level of Mj-DWD gene was consistently maintained at a high level in the newly prepared virus-resistant shrimp compared to the normal one. In addition, the Mj-DWD gene was also found to be rapidly up-regulated by WSSV infection during the early phase. Furthermore, the recombinant Mj-DWD, expressed by Pichia pastoris, showed specific protease inhibitory activity on Bacillus subtilis. These findings suggest that Mj-DWD plays an important role in the host defence system against WSSV infection in M. japonicus, possibly through its protease inhibitory activity. (C) 2008 Elsevier Ltd. All rights reserved

robust large margin discriminant tangent analysis for face recognition

Fisher's Linear Discriminant Analysis (LDA) has been recognized as a powerful technique for face recognition. However, it could be stranded in the non-Gaussian case. Nonparametric discriminant analysis (NDA) is a typical algorithm that extends LDA from Gaussian case to non-Gaussian case. However, NDA suffers from outliers and unbalance problems, which cause a biased estimation of the extra-class scatter information. To address these two problems, we propose a robust large margin discriminant tangent analysis method. A tangent subspace-based algorithm is first proposed to learn a subspace from a set of intra-class and extra-class samples which are distributed in a balanced way on the local manifold patch near each sample point, so that samples from the same class are clustered as close as possible and samples from different classes will be separated far away from the tangent center. Then each subspace is aligned to a global coordinate by tangent alignment. Finally, an outlier detection technique is further proposed to learn a more accurate decision boundary. Extensive experiments on challenging face recognition data set demonstrate the effectiveness and efficiency of the proposed method for face recognition. Compared to other nonparametric methods, the proposed one is more robust to outliers. © 2011 Springer-Verlag London Limited.Fisher's Linear Discriminant Analysis (LDA) has been recognized as a powerful technique for face recognition. However, it could be stranded in the non-Gaussian case. Nonparametric discriminant analysis (NDA) is a typical algorithm that extends LDA from Gaussian case to non-Gaussian case. However, NDA suffers from outliers and unbalance problems, which cause a biased estimation of the extra-class scatter information. To address these two problems, we propose a robust large margin discriminant tangent analysis method. A tangent subspace-based algorithm is first proposed to learn a subspace from a set of intra-class and extra-class samples which are distributed in a balanced way on the local manifold patch near each sample point, so that samples from the same class are clustered as close as possible and samples from different classes will be separated far away from the tangent center. Then each subspace is aligned to a global coordinate by tangent alignment. Finally, an outlier detection technique is further proposed to learn a more accurate decision boundary. Extensive experiments on challenging face recognition data set demonstrate the effectiveness and efficiency of the proposed method for face recognition. Compared to other nonparametric methods, the proposed one is more robust to outliers. © 2011 Springer-Verlag London Limited

Genomic organization of the translationally controlled tumor protein (TCTP) gene from shrimp Marsupenaeus japonicus

The shrimp translationally controlled tumor protein (TCTP) gene was previously found up-regulated in response to viral infection. In order to further understand the transcriptional regulation of TCTP gene expression under viral challenge, the 5'-flanking promoter region of the TCTP gene in shrimp Marsupenaeus japonicus (Mj-TCTP) was cloned by using genomic walking method. The sequence shows that this region contains many important potential binding sites for transcription factors, such as activator protein1 (AP1), cAMP response element binding protein (CREB), Ets like transcription factor-1 (Elk-1), suggesting its expression is highly regulated. The promoter activity of this 5'-flanking region was confirmed by fusing to the enhanced green fluorescence protein (EGFP) gene and testing in Trichoplusia ni High Five cell line. Along with the introns and exons cloned in this study, the Mj-TCTP DNA sequence was reported here as the first TCTP gene with complete genomic organization in crustaceans

LIGHTWEIGHT DESIGN OF STEERING KNUCKLE BASED ON COMPREHENSIVE SENSITIVITY

In the process of designing lightweight of the steering knuckle,because of too many random variables and various factors,it is easy to result in poor optimization results. We consider the effect of the random parameter variable on stress and total volume and put forward the concept of comprehensive sensitivity. Through the dynamic simulation we get the steering knuckle bearing under three typical working conditions. By the finite element analysis,we obtain extreme load condition data and the initial static and reliability indicators. We use comprehensive sensitivity analysis theory to get the final design variables. Finally,we complete the lightweight design by establishing mathematical model. The results show that the lightweight design method based on comprehensive sensitivity can reduce steering knuckle weight and maintain good static and fatigue reliability effectively and can be applied in the engineering field

Modulation of a P-TEFb Functional Equilibrium for the Global Control of Cell Growth and Differentiation

P-TEFb phosphorylates RNA polymerase II and negative elongation factors to stimulate general transcriptional elongation. It is kept in a functional equilibrium through alternately interacting with its positive (the Brd4 protein) and negative (the HEXIM1 protein and 7SK snRNA) regulators. To investigate the physiological significance of this phenomenon, we analyzed the responses of HeLa cells and murine erythroleukemia cells (MELC) to hexamethylene bisacetamide (HMBA), which inhibits growth and induces differentiation of many cell types. For both cell types, an efficient, albeit temporary disruption of the 7SK-HEXIM1-P-TEFb snRNP and enhanced formation of the Brd4-P-TEFb complex occurred soon after the treatment started. When the P-TEFb-dependent HEXIM1 expression markedly increased as the treatment continued, the abundant HEXIM1 pushed the P-TEFb equilibrium back toward the 7SK/HEXIM1-bound state. For HeLa cells, as HMBA produced only a minor, temporary effect on their growth, the equilibrium gradually returned to its pretreatment level. In contrast, long-term treatment of MELC induced terminal division and differentiation. Concurrently, the P-TEFb equilibrium was shifted overwhelmingly toward the 7SK snRNP side. Together, these data link the P-TEFb equilibrium to the intracellular transcriptional demand and proliferative/differentiated states of cells

Protective Effects of Five Structurally Diverse Flavonoid Subgroups against Chronic Alcohol-Induced Hepatic Damage in a Mouse Model

Alcoholic liver disease (ALD) has become one of the major global health problems, with augmented morbidity and mortality. Evidence indicates that flavonoids can reduce the risk of ALD owing to their biological properties. However, the effect of structurally different flavonoid subclasses on alleviating alcohol-induced liver damage in a same model has never been studied. In this study, mice were supplemented with five kinds of flavonoid subgroups, apigenin (flavone), quercetin (flavonol), naringenin (flavanone), (-)-epigallocatechin gallate (flavanol), and genistein (isoflavone), in the same dose (0.3 mmol kg−1 body weight) and then given 50% alcohol by gastric perfusion for five consecutive weeks. The results demonstrated that genistein and naringenin had greater benefits in terms of mitigating fibrosis and apoptosis, respectively, in the liver. Lipid deposition, partial inflammatory-related factors (nuclear factor kappa B p65, cyclooxygenase-2, and interleukin-6 levels), and hepatic histopathological alterations were similarly attenuated by five kinds of flavonoids. All the flavonoids also showed different degrees of influence on protecting against alcoholic liver injury on other aspects, such as serum biochemistry makers, hepatic lipid accumulation, lipid peroxidation, antioxidant capacities, and inflammation