Use of tRNA gene barriers improves stability of transgene expression in CHO cells.

Instability of transgene expression is a major challenge for the biopharmaceutical industry, which can impact yields and regulatory approval. Some tRNA genes (tDNAs) can resist epigenetic silencing, the principal mechanism of expression instability, and protect adjacent genes against the spread of repressive heterochromatin. We have taken two naturally occurring clusters of human tDNAs and tested their ability to reduce epigenetic silencing of transgenes integrated into the genome of Chinese hamster ovary (CHO) cells. We find sustained improvements in productivity both in adherent CHO-K1 cells and in an industrially relevant CHO-DG44 expression system (Apollo X, FUJIFILM Diosynth Biotechnologies). We conclude that specific tDNA clusters offer potential to mitigate the widespread problem of production instability.

Effects on prostate cancer cells of targeting RNA polymerase III.

RNA polymerase (pol) III occurs in two forms, containing either the POLR3G subunit or the related paralogue POLR3GL. Whereas POLR3GL is ubiquitous, POLR3G is enriched in undifferentiated cells. Depletion of POLR3G selectively triggers proliferative arrest and differentiation of prostate cancer cells, responses not elicited when POLR3GL is depleted. A small molecule pol III inhibitor can cause POLR3G depletion, induce similar differentiation and suppress proliferation and viability of cancer cells. This response involves control of the fate-determining factor NANOG by small RNAs derived from Alu short interspersed nuclear elements. Tumour initiating activity in vivo can be reduced by transient exposure to the pol III inhibitor. Untransformed prostate cells appear less sensitive than cancer cells to pol III depletion or inhibition, raising the possibility of a therapeutic window.

The function of the conserved regulatory element within the second intron of the mammalian Csf1r locus.

The gene encoding the receptor for macrophage colony-stimulating factor (CSF-1R) is expressed exclusively in cells of the myeloid lineages as well as trophoblasts. A conserved element in the second intron, Fms-Intronic Regulatory Element (FIRE), is essential for macrophage-specific transcription of the gene. However, the molecular details of how FIRE activity is regulated and how it impacts the Csf1r promoter have not been characterised. Here we show that agents that down-modulate Csf1r mRNA transcription regulated promoter activity altered the occupancy of key FIRE cis-acting elements including RUNX1, AP1, and Sp1 binding sites. We demonstrate that FIRE acts as an anti-sense promoter in macrophages and reversal of FIRE orientation within its native context greatly reduced enhancer activity in macrophages. Mutation of transcription initiation sites within FIRE also reduced transcription. These results demonstrate that FIRE is an orientation-specific transcribed enhancer element.

Differential regulation of sense and antisense promoter activity at the Csf1R locus in B cells by the transcription factor PAX5.

OBJECTIVE: The transcription factor PAX5 is essential for the activation of B-cell-specific genes and for the silencing of myeloid-specific genes. We previously determined the molecular mechanism by which PAX5 silences the myeloid-specific colony-stimulating-factor-receptor (Csf1R) gene and showed that PAX5 directly binds to the Csf1r promoter as well as to an intronic enhancer that generates an antisense transcript in B cells. Here we examine the role of PAX5 in the regulation of sense and antisense transcription in B cells. MATERIALS AND METHODS: We performed PAX5-specific chromatin immunoprecipitation analyses across the Csfr1 locus. We investigated the role of PAX5 in regulating Csf1r sense and antisense promoter activity by transient transfections and by employing a Pax5(-/-) pro-B-cell line expressing an inducible PAX5 protein. PAX5 interacting factors were identified by pull-down experiments. The role of the transcription factor Sp3 in driving antisense promoter expression was examined in B cells from Sp3 knockout mice. RESULTS: PAX5 differentially regulates the Csf1r promoter and the promoter of the antisense transcript. PAX5 interferes with PU.1 transactivation at the sense promoter by binding to a PAX5 consensus sequence. At the antisense promoter, PAX5 does not specifically recognize DNA, but interacts with Sp3 to upregulate antisense promoter activity. Antisense promoter activation by PAX5 is dependent on the presence of its partial homeo-domain. CONCLUSIONS: We demonstrate that PAX5 regulates Csf1r in B cells by reducing the frequency of binding of the basal transcription machinery to the promoter and by activating antisense RNA expression.

Molecular and phylogenetic analyses of the complete MADS-box transcription factor family in Arabidopsis: new openings to the MADS world.

MADS-box transcription factors are key regulators of several plant development processes. Analysis of the complete Arabidopsis genome sequence revealed 107 genes encoding MADS-box proteins, of which 84% are of unknown function. Here, we provide a complete overview of this family, describing the gene structure, gene expression, genome localization, protein motif organization, and phylogenetic relationship of each member. We have divided this transcription factor family into five groups (named MIKC, Malpha, Mbeta, Mgamma, and Mdelta) based on the phylogenetic relationships of the conserved MADS-box domain. This study provides a solid base for functional genomics studies into this important family of plant regulatory genes, including the poorly characterized group of M-type MADS-box proteins. MADS-box genes also constitute an excellent system with which to study the evolution of complex gene families in higher plants.

Nonmyeloablative therapy and allogeneic hematopoietic stem cell transplantation.

The toxicities associated with conventional myeloablative therapy and allogeneic hematopoietic stem cell transplantation (SCT) limit the use of this potentially curative approach to relatively healthy young patients. The risk of treatment-related morbidity and mortality with conventional allogeneic SCT ranges from 10% to 50%, depending on the age of the patient, HLA histocompatibility, diagnosis and disease status, and presence or absence of comorbid conditions. The main goals of conventional high-dose preparative regimens are to eradicate the malignancy and induce adequate host immunosuppression to prevent graft rejection. However, accumulated data indicate that the currently used myeloablative regimens frequently do not eradicate the malignant clone, and that an immune-mediated effect between donor immunocompetent T lymphocytes and host tumor cells seems to induce a major therapeutic benefit, accounting for the significantly lower incidence of leukemic relapse seen with allogeneic SCT compared to autologous or syngeneic SCT. These observations have led to the development of newer treatment modalities focusing on the induction of host tolerance to donor cells followed by the administration of scheduled donor T-lymphocyte infusions. Preliminary clinical data are encouraging but need to be confirmed in well-designed prospective controlled trials with direct comparison to conventional allogeneic SCT and extended follow-up to determine the durability of responses and the consequences of late complications such as chronic graft-vs-host disease on the patient's quality of life.

Testicular lymphoma: a literature review and report of a case with a zoster-like cutaneous recurrence.

Testicular lymphoma accounts for approximately 1% of all cases of non-Hodgkin's lymphoma. This distinct clinical entity can have an aggressive course with a high rate of systemic relapse. We describe a unique case of a diffuse large B-cell lymphoma, T-cell-rich variant with cutaneous dermatomal zosteriform recurrence presenting as neuralgia. This case represents a unique pattern of treatment failure in this aggressive extranodal lymphoma.