Differential contribution for ERK1 and ERK2 kinases in BRAFV600E-triggered phenotypes in adult mouse models.

The BRAF gene is mutated in a plethora of human cancers. The majority of such molecular lesions result in the expression of a constitutively active BRAF variant (BRAFV600E) which continuously bolsters cell proliferation. Although we recently addressed the early effects triggered by BRAFV600E-activation, the specific contribution of ERK1 and ERK2 in BRAFV600E-driven responses in vivo has never been explored. Here we describe the first murine model suitable for genetically dissecting the ERK1/ERK2 impact in multiple phenotypes induced by ubiquitous BRAFV600E-expression. We unveil that ERK1 is dispensable for BRAFV600E-dependent lifespan shortening and for BRAFV600E-driven tumor growth. We show that BRAFV600E-expression provokes an ERK1-independent lymphocyte depletion which does not rely on p21CIP1-induced cell cycle arrest and is unresponsive to ERK-chemical inhibition. Moreover, we also reveal that ERK1 is dispensable for BRAFV600E-triggered cytotoxicity in lungs and that ERK-chemical inhibition abrogates some of these detrimental effects, such as DNA damage, in Club cells but not in pulmonary lymphocytes. Our data suggest that ERK1/ERK2 contribution to BRAFV600E-driven phenotypes is dynamic and varies dependently on cell type, the biological function, and the level of ERK-pathway activation. Our findings also provide useful insights into the comprehension of BRAFV600E-driven malignancies pathophysiology as well as the consequences in vivo of novel ERK pathway-targeted anti-cancer therapies.

Protocol for the generation and purification of high-molecular-weight covalent RNA-DNA hybrids with T4 RNA ligase.

RNA-DNA covalent hybrids (RDHs) are widely employed in biology. Although RDHs can be manufactured, the synthesis of molecules longer than 120 nucleotides is challenging. Here, we present a protocol for the generation and purification of high-grade purified high-molecular-weight 5'-RNA-DNA-3' hybrids. We describe steps for preparing oligos and buffers, ligation reaction, and high-performance liquid chromatography-based RDH purification. This protocol is executable in standard molecular biology laboratories.

Telomere dynamics in human pluripotent stem cells.

Pluripotent stem cells (PSCs) are a promising source of stem cells for regenerative therapies. Stem cell function depends on telomere maintenance mechanisms that provide them with the proliferative capacity and genome stability necessary to multiply and regenerate tissues. We show here that established human embryonic stem cells (hESCs) have stable telomere length that is dependent on telomerase but not on alternative mechanisms based on homologous recombination pathways. Here, we show that human-induced pluripotent stem cells (hiPSCs) reprogrammed from somatic cells show progressive telomere lengthening until reaching a length similar to ESCs. hiPSCs also acquire telomeric chromatin marks of ESCs including decreased abundance of tri-methylated histone H3K9 and H4K20 and HP1 heterochromatic marks, as well as of the shelterin component TRF2. These chromatin features are accompanied with increased abundance of telomere transcripts or TERRAs. We also found that telomeres of both hESCs and hiPSCs are well protected from DNA damage during telomere elongation and once full telomere length is achieved, and exhibit stable genomes. Collectively, this study highlights that hiPSCs acquire ESC features during reprogramming and reveals the telomere biology in human pluripotent stem cells (hPSCs).

We show that established human embryonic stem cells (hESCs) have a maximum and stable telomere length that is dependent on telomerase but not on the alternative homologous recombination pathway or ALT. Human-induced pluripotent stem cells (hiPSCs) reprogrammed from somatic cells show progressive telomere lengthening until reaching a length similar maximum telomere length than ESCs, suggesting that telomere length is regulated by epigenetic mechanisms in human cells. In this regard, hiPSCs acquire telomeric chromatin marks characteristic of an "open chromatin" including increased abundance of telomere transcripts or TERRAs. Telomeres of both hESCs and hiPSCs are well protected during telomere elongation and exhibit stable genomes. Collectively, this study highlights that hiPSCs acquire ESC features during reprogramming and reveals the telomere biology in human pluripotent stem cells (hiPSCs).