DIFFERENTIAL CELLULAR REQUIREMENTS FOR ICP4-MEDIATED ACTIVATION OF HSV-1 EARLY AND LATE GENES

The herpes simplex virus type 1 immediate-early protein, ICP4, is the major transcriptional activator of viral early and late genes. ICP4 has been shown to bind DNA and interact with components of the general transcription machinery to activate or repress viral transcription, depending on promoter context. Microarray and northern blot analysis indicated that the abundance of the basal Pol II transcription machinery was significantly altered at late times of infection. Because viral early and late genes have very different promoter architectures, the cellular requirements for ICP4-mediated activation of early and late genes may differ. In testing this hypothesis using tk and gC as representative early and late promoters respectively, the general transcription factor TFIIA was found not to be required for ICP4 activation of the late gC promoter but was essential for activation of the early tk promoter. An intact INR element was required for TFIIA to be dispensable for ICP4 activation of the gC promoter. In the presence of TFIIA, ICP4 overcame the requirement for an intact INR on the gC promoter enhancing ICP4 activation of an INR-mutated gC promoter. When examining the binding properties of TFIIA, ICP4 and TBP/TFIID on early, late, and INR-mutated late promoters, ICP4 could only effectively substitute for TFIIA in stabilizing the binding of TFIID to the TATA box of the late promoter containing a functional INR. ICP4 required the additional activities of TFIIA to stabilize the binding of TFIID to the TATA box of an INR-mutated late promoter. Additionally, microarray and northern blot analysis indicated that TFIIA expression was reduced at late times of infection. The decrease in TFIIA expression during infection, the ability of ICP4 to substitute for TFIIA in stabilizing TFIID binding to an INR-containing late promoter, and its dispensability for activation of late but not early genes suggests a mechanism for the shutoff of early gene expression while allowing the continued expression of late genes

Testing in Mice the Hypothesis That Melanin Is Protective in Malaria Infections

Malaria has had the largest impact of any infectious disease on shaping the human genome, exerting enormous selective pressure on genes that improve survival in severe malaria infections. Modern humans originated in Africa and lost skin melanization as they migrated to temperate regions of the globe. Although it is well documented that loss of melanization improved cutaneous Vitamin D synthesis, melanin plays an evolutionary ancient role in insect immunity to malaria and in some instances melanin has been implicated to play an immunoregulatory role in vertebrates. Thus, we tested the hypothesis that melanization may be protective in malaria infections using mouse models. Congenic C57BL/6 mice that differed only in the gene encoding tyrosinase, a key enzyme in the synthesis of melanin, showed no difference in the clinical course of infection by Plasmodium yoelii 17XL, that causes severe anemia, Plasmodium berghei ANKA, that causes severe cerebral malaria or Plasmodium chabaudi AS that causes uncomplicated chronic disease. Moreover, neither genetic deficiencies in vitamin D synthesis nor vitamin D supplementation had an effect on survival in cerebral malaria. Taken together, these results indicate that neither melanin nor vitamin D production improve survival in severe malaria

An integrated expression atlas of miRNAs and their promoters in human and mouse

MicroRNAs (miRNAs) are short non-coding RNAs with key roles in cellular regulation. As part of the fifth edition of the Functional Annotation of Mammalian Genome (FANTOM5) project, we created an integrated expression atlas of miRNAs and their promoters by deep-sequencing 492 short RNA (sRNA) libraries, with matching Cap Analysis Gene Expression (CAGE) data, from 396 human and 47 mouse RNA samples. Promoters were identified for 1,357 human and 804 mouse miRNAs and showed strong sequence conservation between species. We also found that primary and mature miRNA expression levels were correlated, allowing us to use the primary miRNA measurements as a proxy for mature miRNA levels in a total of 1,829 human and 1,029 mouse CAGE libraries. We thus provide a broad atlas of miRNA expression and promoters in primary mammalian cells, establishing a foundation for detailed analysis of miRNA expression patterns and transcriptional control regions

Differential Cellular Requirements for Activation of Herpes Simplex Virus Type 1 Early (tk) and Late (gC) Promoters by ICP4

The herpes simplex virus type 1 immediate-early protein, ICP4, activates the transcription of viral early and late genes and is essential for viral growth. It has been shown to bind DNA and interact with components of the general transcription machinery to activate or repress viral transcription, depending upon promoter context. Since early and late gene promoters have different architectures and cellular metabolism may be very different at early and late times after infection, the cellular requirements for ICP4-mediated activation of early and late genes may differ. This hypothesis was tested using tk and gC as representative early and late promoters, respectively. Nuclear extracts and phosphocellulose column fractions derived from nuclear extracts were able to reconstitute basal and ICP4-activated transcription of both promoters in vitro. When examining the contribution of the general transcription factors on the ability of ICP4 to activate transcription, the fraction containing the general transcription factor TFIIA was not essential for ICP4 activation of the gC promoter, but it was required for efficient activation of the tk promoter. The addition of recombinant TFIIA restored the ability of ICP4 to efficiently activate the tk promoter, but it had no net effect on activation of the gC promoter. The dispensability of TFIIA for ICP4 activation of the gC promoter required an intact INR element. In addition, microarray and Northern blot analysis indicated that TFIIA abundance may be reduced at late times of infection. This decrease in TFIIA expression during infection and its dispensability for activation of late but not early genes suggest one of possibly many mechanisms for the transition from viral early to late gene expression

Stabilized Binding of TBP to the TATA Box of Herpes Simplex Virus Type 1 Early (tk) and Late (gC) Promoters by TFIIA and ICP4▿

We have recently shown that ICP4 has a differential requirement for the general transcription factor TFIIA in vitro (S. Zabierowski and N. DeLuca, J. Virol. 78:6162-6170, 2004). TFIIA was dispensable for ICP4 activation of a late promoter (gC) but was required for the efficient activation of an early promoter (tk). An intact INR element was required for proficient ICP4 activation of the late promoter in the absence of TFIIA. Because TFIIA is known to stabilize the binding of both TATA binding protein (TBP) and TFIID to the TATA box of core promoters and ICP4 has been shown to interact with TFIID, we tested the ability of ICP4 to stabilize the binding of either TBP or TFIID to the TATA box of representative early, late, and INR-mutated late promoters (tk, gC, and gC8, respectively). Utilizing DNase I footprinting analysis, we found that ICP4 was able to facilitate TFIIA stabilized binding of TBP to the TATA box of the early tk promoter. Using mutant ICP4 proteins, the ability to stabilize the binding of TBP to both the wild-type and the INR-mutated gC promoters was located in the amino-terminal region of ICP4. When TFIID was substituted for TBP, ICP4 could stabilize the binding of TFIID to the TATA box of the wild-type gC promoter. ICP4, however, could not effectively stabilize TFIID binding to the TATA box of the INR-mutated late promoter. The additional activities of TFIIA were required to stabilize the binding of TFIID to the INR-mutated late promoter. Collectively, these data suggest that TFIIA may be dispensable for ICP4 activation of the wild-type late promoter because ICP4 can substitute for TFIIA's ability to stabilize the binding of TFIID to the TATA box. In the absence of a functional INR, ICP4 can no longer stabilize TFIID binding to the TATA box of the late promoter and requires the additional activities of TFIIA. The stabilized binding of TFIID by TFIIA may in turn allow ICP4 to more efficiently activate transcription from non-INR containing promoters

Bringing Neural Cell Therapies to the Clinic: Past and Future Strategies

Cell replacement therapy in the nervous system has a rich history, with ∼40 years of research and ∼30 years of clinical experience. There is compelling evidence that appropriate cells can integrate and function in the dysfunctioning human nervous system, but the clinical results are mixed in practice. A number of factors conspire to vary patient outcome: the indication, cell source, patient selection, and team performing transplantation are all variables that can affect efficacy. Most early clinical trials have used fetal cells, a limited cell source that resists scale and standardization. Direct fetal cell transplantation creates significant challenges to commercialization that is the ultimate goal of an effective cell therapy. One approach to help scale and standardize fetal cell preparations is the expansion of neural cells in vitro. Expansion is achieved by transformation or through the application of mitogens before cryopreservation. Recently, neural cells derived from pluripotent stem cells have provided a scalable alternative. Pluripotent stem cells are desirable for manufacturing but present alternative concerns and manufacturing obstacles. All cell sources require robust and reproducible manufacturing to make nervous system cell replacement therapy an option for patients. Here, we discuss the challenges and opportunities for cell replacement in the nervous system. In this review, we give an overview of completed and ongoing neural cell transplantation clinical trials, and we discuss the challenges and opportunities for future cell replacement trials with a particular focus on pluripotent stem cell-derived therapies

Active Notch1 confers a transformed phenotype to primary human melanocytes

The importance of mitogen-activated protein kinase signaling in melanoma is underscored by the prevalence of activating mutations in N-Ras and B-Raf, yet clinical development of inhibitors of this pathway has been largely ineffective, suggesting that alternative oncogenes may also promote melanoma. Notch is an interesting candidate that has only been correlated with melanoma development and progression; a thorough assessment of tumor-initiating effects of activated Notch on human melanocytes would clarify the mounting correlative evidence and perhaps identify a novel target for an otherwise untreatable disease. Analysis of a substantial panel of cell lines and patient lesions showed that Notch activity is significantly higher in melanomas than their nontransformed counterparts. The use of a constitutively active, truncated Notch transgene construct (N(IC)) was exploited to determine if Notch activation is a "driving" event in melanocytic transformation or instead a "passenger" event associated with melanoma progression. N(IC)-infected melanocytes displayed increased proliferative capacity and biological features more reminiscent of melanoma, such as dysregulated cell adhesion and migration. Gene expression analyses supported these observations and aided in the identification of MCAM, an adhesion molecule associated with acquisition of the malignant phenotype, as a direct target of Notch transactivation. N(IC)-positive melanocytes grew at clonal density, proliferated in limiting media conditions, and also exhibited anchorage-independent growth, suggesting that Notch alone is a transforming oncogene in human melanocytes, a phenomenon not previously described for any melanoma oncogene. This new information yields valuable insight into the basic epidemiology of melanoma and launches a realm of possibilities for drug intervention in this deadly disease