Characterizing the Interaction between the HTLV-1 Transactivator Tax-1 with Transcription Elongation Factor ELL2 and Its Impact on Viral Transactivation

The human T-cell leukemia virus type 1 (HTLV-1)-encoded transactivator and oncoprotein Tax-1 is essential for HTLV-1 replication. We recently found that Tax-1 interacts with transcription elongation factor for RNA polymerase II 2, ELL2, which enhances Tax-1-mediated transactivation of the HTLV-1 promotor. Here, we characterize the Tax-1:ELL2 interaction and its impact on viral transactivation by confocal imaging, co-immunoprecipitation, and luciferase assays. We found that Tax-1 and ELL2 not only co-precipitate, but also co-localize in dot-like structures in the nucleus. Tax-1:ELL2 complex formation occurred independently of Tax-1 point mutations, which are crucial for post translational modifications (PTMs) of Tax-1, suggesting that these PTMs are irrelevant for Tax-1:ELL2 interaction. In contrast, Tax-1 deletion mutants lacking either N-terminal (aa 1–37) or C-terminal regions (aa 150–353) of Tax-1 were impaired in interacting with ELL2. Contrary to Tax-1, the related, non-oncogenic Tax-2B from HTLV-2B did not interact with ELL2. Finally, we found that ELL2-R1 (aa 1–353), which carries an RNA polymerase II binding domain, and ELL2-R3 (aa 515–640) are sufficient to interact with Tax-1; however, only ELL2-truncations expressing R1 could enhance Tax-1-mediated transactivation of the HTLV-1 promoter. Together, this study identifies domains in Tax-1 and ELL2 being required for Tax-1:ELL2 complex formation and for viral transactivation.

Assembly and levels of P-TEFb depend on reversible phosphorylation of cyclinT1

The positive transcription elongation factor b (P-TEFb) is a critical co-activator for transcription of most cellular and viral genes, including those of HIV. While P-TEFb is regulated by 7SK snRNA in proliferating cells, it is absent in quiescent and terminally differentiated cells, which has remained unexplored. In these cells, we found that CycT1 not bound to CDK9 is rapidly degraded. Moreover, productive CycT1:CDK9 interactions require phosphorylation of two threonine residues (Thr143 and Thr149) in CycT1 by PKC. Conversely, PP1 dephosphorylates these sites. Thus, PKC inhibitors or removal of PKC by chronic activation results in P-TEFb disassembly and CycT1 degradation. This finding not only recapitulates P-TEFb depletion in resting CD4+ T cells but also in anergic T cells. Importantly, our studies reveal mechanisms of P-TEFb inactivation underlying T cell quiescence, anergy, and exhaustion as well as proviral latency and terminal differentiation of cells.

SPT6 loss Permits the Transdifferentiation of Keratinocytes into an Intestinal Fate that Recapitulates Barretts Metaplasia

Transient depletion of the transcription elongation factor SPT6 in the keratinocyte has been recently shown to inhibit epidermal differentiation and stratification; instead, they transdifferentiate into a gut-like lineage. We show here that this phenomenon of transdifferentiation recapitulates Barretts metaplasia, the only human pathophysiologic condition in which a stratified squamous epithelium that is injured due to chronic acid reflux is trans-committed into an intestinal fate. The evidence we present here not only pinpoint the keratinocyte as the cell of origin of Barretts metaplasia, but also provide mechanistic insights linking chronic acid injury, downregulation of SPT6, loss of epidermal fate and metaplastic progression. Because Barretts metaplasia in the esophagus (BE) is a pre-neoplastic condition with no preclinical human models, these findings have a profound impact on the modeling Barretts metaplasia-in-a-dish.

Elevated TEFM expression promotes growth and metastasis through activation of ROS/ERK signaling in hepatocellular carcinoma

TEFM (transcription elongation factor of mitochondria) has been identified as a novel nuclear-encoded transcription elongation factor in the transcription of mitochondrial genome. Our bioinformatics analysis of TCGA data revealed an aberrant over-expression of TEFM in hepatocellular carcinoma (HCC). We analyzed its biological effects and clinical significance in this malignancy. TEFM expression was analyzed by quantitative real-time PCR, western blot, and immunohistochemistry analysis in HCC tissues and cell lines. The effects of TEFM on HCC cell growth and metastasis were determined by cell proliferation, colony formation, flow cytometric cell cycle and apoptosis, migration, and invasion assays. TEFM expression was significantly increased in HCC tissues mainly caused by down-regulation of miR-194-5p. Its increased expression is correlated with poor prognosis of HCC patients. TEFM promoted HCC growth and metastasis both in vitro and in vivo by promoting G1–S cell transition, epithelial-to-mesenchymal transition (EMT), and suppressing cell apoptosis. Mechanistically, TEFM exerts its tumor growth and metastasis promoting effects at least partly through increasing ROS production and subsequently by activation of ERK signaling. Our study suggests that TEFM functions as a vital oncogene in promoting growth and metastasis in HCC and may contribute to the targeted therapy of HCC.

Spt4 Facilitates the Movement of RNA Polymerase II through the +2 Nucleosomal Barrier

Spt4 is a transcription elongation factor, with homologues in organisms with nucleosomes. Structural and in vitro studies implicate Spt4 in transcription through nucleosomes, yet the in vivo function of Spt4 is unclear. Here we assessed the precise position of Spt4 during transcription and the consequences of loss of Spt4 on RNA polymerase II (RNAPII) dynamics and nucleosome positioning in Saccharomyces cerevisiae. In the absence of Spt4, the spacing between gene-body nucleosomes increases and RNAPII accumulates upstream of the nucleosomal dyad, most dramatically at nucleosome +2. Spt4 associates with elongating RNAPII early in transcription and its association dynamically changes depending on nucleosome positions. Together, our data show that Spt4 regulates early elongation dynamics, participates in co-transcriptional nucleosome positioning, and promotes RNAPII movement through the gene-body nucleosomes, especially the +2 nucleosome.

Transposable element landscape changes are buffered by RNA silencing in aging Drosophila

Genetic mechanisms that repress transposable elements (TEs) in young animals decline during aging, as reflected by increased TE expression in aged animals. Does increased TE expression during aging lead to more genomic TE copies in older animals? To answer this question, we quantified TE Landscapes (TLs) via whole genome sequencing of young and aged Drosophila strains of wild-type and mutant backgrounds. We quantified TLs in whole flies and dissected brains and validated the feasibility of our approach in detecting new TE insertions in aging Drosophila genomes when natural defenses like RNA interference (RNAi) pathways are compromised. By also incorporating droplet digital PCR to validate genomic TE loads, we confirm TL changes can occur in a single lifespan of Drosophila when TEs are not suppressed. We also describe improved sequencing methods to quantify extra-chromosomal DNA circles (eccDNAs) in Drosophila as an additional source of TE copies that accumulate during aging. Lastly, to combat the natural progression of aging-associated TE expression, we show that knocking down PAF1, a conserved transcription elongation factor that antagonizes RNAi pathways , may bolster suppression of TEs during aging and extend lifespan. Our study suggests that RNAi mechanisms generally mitigate genomic TL expansion despite the increase in TE transcripts during aging.

New Cantharellus species from South Korea

In this third contribution involving new Cantharellus species from South Korea, two new species are introduced. In addition, we document a first report of the recently described Japanese Cantharellus anzutake outside of Japan based on identical ITS sequence data. Cantharellus citrinussp. nov. is introduced as a new member of subgenus Cinnabarini, to which the closely related Korean C. albovenosus and Chinese C. phloginus also belong. Cantharellus curvatussp. nov. is introduced as a new member of subgenus Parvocantharellus, in which the Korean C. koreanus was recently placed. The respective placements of the new taxa are significantly supported by a phylogenetic analysis of sequences from the transcription elongation factor (tef-1).

Essential histone chaperones collaborate to regulate transcription and chromatin integrity

SUMMARYHistone chaperones are critical for controlling chromatin integrity during transcription, DNA replication, and DNA repair. We have discovered that the physical interaction between two essential histone chaperones, Spt6 and Spn1/Iws1, is required for transcriptional accuracy and nucleosome organization. To understand this requirement, we have isolated suppressors of an spt6 mutation that disrupts the Spt6-Spn1 interaction. Several suppressors are in a third essential histone chaperone, FACT, while another suppressor is in the transcription elongation factor Spt5/DSIF. The FACT suppressors weaken FACT-nucleosome interactions and bypass the requirement for Spn1, possibly by restoring a necessary balance between Spt6 and FACT on chromatin. In contrast, the Spt5 suppressor modulates Spt6 function in a Spn1-dependent manner. Despite these distinct mechanisms, both suppressors alleviate the nucleosome organization defects caused by disruption of the Spt6-Spn1 interaction. Taken together, we have uncovered a network in which histone chaperones and other elongation factors coordinate transcriptional integrity and chromatin structure.