Selective Requirement for c-Myc at an Early Stage of V alpha 14i NKT Cell Development

V alpha 14 invariant (V alpha 14i) NKT cells are a subset of regulatory T cells that utilize a semi-invariant TCR to recognize glycolipids associated with monomorphic CD1d molecules. During development in the thymus, CD4(+)CD8(+) V alpha 14i NKT precursors recognizing endogenous CD1d-associated glycolipids on other CD4(+)CD8(+) thymocytes are selected to undergo a maturation program involving sequential expression of CD44 and NK-related markers such as NK1.1. The molecular requirements for V alpha 14i NKT cell maturation, particularly at early developmental stages, remain poorly understood. In this study, we show that CD4-Cre-mediated T cell-specific inactivation of c-Myc, a broadly expressed transcription factor with a wide range of biological activities, selectively impairs V alpha 14i NKT cell development without perturbing the development of conventional T cells. In the absence of c-Myc, V alpha 14i NKT cell precursors are blocked at an immature CD44(low)NK1.1(-) stage in a cell autonomous fashion. Residual c-Myc-deficient immature V alpha 14i NKT cells appear to proliferate normally, cannot be rescued by transgenic expression of BCL-2, and exhibit characteristic features of immature V alpha 14i NKT cells such as high levels of preformed IL-4 mRNA and the transcription factor promyelocytic leukemia zinc finger. Collectively our data identify c-Myc as a critical transcription factor that selectively acts early in V alpha 14i NKT cell development to promote progression beyond the CD44(low)NK1.1(-) precursor stage. The Journal of Immunology, 2009, 182: 4641-4648.

Transient recombinant protein expression in mammalian cells

Sarah Wulhfard

Transient gene expression (TGE) is a rapid method for generating recombinant proteins in mammalian cells, but the volumetric productivities for secreted proteins in transiently transfected CHO DG44 cells are typically more than an order of magnitude lower than the yields achieved with recombinant CHO-derived cell lines. The goals of the thesis are to identify the limitations to higher TGE yields in CHO DG44 cells and to find possible solutions to overcome the problems. Initially an attempt was made to enhance TGE production by increasing the amount of transfected plasmid DNA. However, this approach did not result in increased recombinant protein levels; on the contrary, transfection with an excess of plasmid DNA (> 1.25 μg/ml) had a negative impact on transgene mRNA levels and protein production. Moreover, it was also observed that recombinant protein yield was strongly dependent on the mRNA level. Therefore, three strategies aimed at increasing the amount of transgene mRNA were investigated. For the first approach, transfected cells were exposed to hypothermic conditions during the production phase. It was already known that lower temperatures increase protein production several fold in recombinant CHO DG44-derived cell lines. The second strategy involved the treatment of transfected cells with valproic acid, a histone deacetylase inhibitor that reduces the effects of gene silencing. The third approach aimed to increase transgene mRNA levels by overexpressing transcription factors and growth factors. With the first two strategies recombinant antibody yields of 60-80 mg/L were achieved whereas the untreated control transfections produced only 5-10 mg/L. Combination of the two strategies led to the production of 90 mg/L of antibody. Moreover, in the treated cultures, the steady-state level of transgene mRNA was 3-5 times higher than in the untreated cultures and remained stable up to 6 days post-transfection. Using the third approach, the increase in recombinant protein production was moderate and transgene mRNA amounts were only 2-fold higher in treated samples compared to the control. When specific proteins such as c-fos, c-jun, NF-kB, and acidic fibroblast growth factor (aFGF) were overexpressed the recombinant antibody production was 20 mg/L compared to 5 mg/L for the control transfection. Overexpression of either a transcription factor or a growth factor in combination with treatment with valproic acid allowed the recombinant protein yield to reach 90 mg/L. However, the benefit of the overexpressed factors was minimal compared to the effect of valproic acid alone. In conclusion, it was demonstrated that the level and stability of transgene mRNA are important factors for increasing volumetric yields in transiently transfected CHO DG44 cells. Furthermore, three approaches aimed to increase mRNA amounts were tested. Exposure to hypothermic conditions and treatment with valproic acid were the two best strategies tested. Both are simple, cost-effective, and scalable making transient gene expression in CHO DG44 cells a feasible alternative for rapid production of gram amounts of recombinant protein.

EPFL2009

Direct Reprogramming of Terminally Differentiated Mature B Lymphocytes to Pluripotency

Patrick Schorderet

The therapeutic potential of embryonic stem cells (ESc) is a key scientific interest, but the ethical implications of stem cell research have been controversial and are often a major detriment to the advancement of our knowledge. Recently, several landmark studies both in mouse and human have defined a set of four transcription factors (Klf4, c-Myc, Oct4 and Sox2) whose ectopic expression enables a somatic nucleus to shift back to a pluripotent state. These cells, referred to as induced pluripotent stem (iPS) cells, not only express markers characteristic of pluripotent cells such as alkaline phosphatase, SSEA1, Nanog and Oct4, but both their epigenetic state and their biological potency have been shown to be indistinguishable from those of ES cells. Although these results have been a great breakthrough in stem cell research and have brought hope for an alternative to embryo-derived ES cells, skeptics argue that these pluripotent cells derive from a small subset of remaining multipotent cells or cells that are more amenable to direct reprogramming. Following this approach, a fundamental question remains unsolved: namely, whether this process holds true in terminally differentiated cells. Also, clarifying whether cells of a distinct lineage are equally amenable to direct reprogramming throughout their developmental stages would determine if distinct prerequisites are needed and would be an important step towards understanding the biological processes involved in this transformation. Here, we used transgenic and inducible expression vectors of the previously described factors to reprogram mouse B lymphocytes. Alone, the four factors were able to convert non-terminally differentiated pro-B cells into a pluripotent state but failed to do so in cells that had undergone full B cell receptor rearrangements. Instead, expression of a myeloid transcription factor CCAAT/enhancer-binding-protein-α (C/EBPα), known to disrupt the epigenetic faith of B cells, was shown to be required. Multiple iPS lines were derived and characterized using various methods. Ultimately, they were shown to be fully pluripotent by their contribution to germline transmission. Our study provides definitive proof for the direct nuclear reprogramming of terminally differentiated adult cells to pluripotency

2008

Transposable elements and their KRAB/KAP1 controllers broadly regulate transcription in adult human cells

Flavia Marzetta

KAP1 is a universal corepressor for the large family of KRAB-ZFP proteins that coordinates epigenetic silencing of endogenous retroelements (EREs) during early embryonic development. This process is essential not just to prevent replication of EREs, but also to control their regulatory influence on host genes. Although in differentiated cells the control of EREs is believed to be mainly DNA methylation-dependent, emerging evidence suggests that KAP1-dependent, histone-based repression might remain a key regulator of ERE expression and its influence on cellular gene networks. Using a combination of transcriptional and histone modification profiling, we have investigated the impact of KAP1 epigenetic regulation on the transcriptional dynamics of human CD4+ T cells. We found that KAP1 binding on subsets of EREs is maintained in differentiated CD4+ T cells, where its locus-specific recruitment is dynamically modulated along with the T cell activation status. KAP1 depletion by RNA interference leads to a global decrease in repressive histone modifications that is accompanied by broad transcriptional alterations. Indeed, not only a large fraction of EREs become derepressed, but their deregulation correlates with illegittimate activation of nearby genes. Correspondingly, loss of KAP1 unleashes the intrinsic regulatory activities that some EREs are endowed with, suggesting a functional link between control of mobile genetic elements and protection of lineage-specific transcriptional networks. These findings establish the KRAB/KAP1 system as an important safeguard of CD4+ T cells transcriptional identity and suggest a critical role in maintaining epigenetic silencing of ERE-based regulatory sequences in adult tissues. Furthermore, our study shed light onto a previously unappreciated contribution of retrotransposon-derived sequences to the complexity of the human T cell transcriptome. A better knowledge of the physiological transcriptional activity of these mobile genetic elements, as well as their control mechanisms throughout development, would greatly help the debate on their pathologic implications.

EPFL2015