SUMMARY Recruitment of the P-TEFb kinase by HIV-1 Tat to the viral promoter triggers the phosphorylation and escape of RNA polymerase II from promoter-proximal pausing. It is unclear, however, if Tat recruits additional host factors that further stimulate HIV-1 transcription. Using a sequential affinity-purification scheme, we have identified human transcription factors/coactivators AFF4, ENL, AF9, and elongation factor ELL2 as components of the Tat-P-TEFb complex. Through the bridging functions of Tat and AFF4, P-TEFb and ELL2 combine to form a bifunctional elongation complex that greatly activates HIV-1 transcription. Without Tat, AFF4 can mediate the ELL2-P-TEFb interaction, albeit inefficiently. Tat overcomes this limitation by bringing more ELL2 to P-TEFb and stabilizing ELL2 in a process that requires active P-TEFb. The ability of Tat to enable two different classes of elongation factors to cooperate and coordinate their actions on the same polymerase enzyme explains why Tat is such a powerful activator of HIV-1 transcription.

Summary Bacteroidetes are a phylum of Gram-negative bacteria abundant in mammalian-associated polymicrobial communities, where they impact digestion, immunity and resistance to infection. Despite extensive competition at high cell density that occurs in these settings, cell contact-dependent mechanisms of interbacterial antagonism, such as the type VI secretion system (T6SS), have not been defined in this group of organisms. Herein we report the bioinformatic and functional characterization of a T6SS-like pathway in diverse Bacteroidetes. Using prominent human gut commensal and soil-associated species, we demonstrate that these systems localize dynamically within the cell, export antibacterial proteins, and target competitor bacteria. The Bacteroidetes system is a distinct pathway with marked differences in gene content and high evolutionary divergence from the canonical T6S pathway. Our findings offer a potential molecular explanation for the abundance of Bacteroidetes in polymicrobial environments, the observed stability of Bacteroidetes in healthy humans, and the barrier presented by the microbiota against pathogens.

The Super Elongation Complex (SEC), containing transcription elongation activators/coactivators P-TEFb, ELL2, AFF4/1, ENL, and AF9, is recruited by HIV-1 Tat and mixed lineage leukemia (MLL) proteins to activate the expression of HIV-1 and MLL-target genes, respectively. In the absence of Tat and MLL, however, it is unclear how SEC is targeted to RNA polymerase (Pol) II to stimulate elongation in general. Furthermore, although ENL and AF9 can bind the H3K79 methyltransferase Dot1L, it is unclear whether these bindings are required for SEC-mediated transcription. Here, we show that the homologous ENL and AF9 exist in separate SECs with similar but nonidentical functions. ENL/AF9 contacts the scaffolding protein AFF4 that uses separate domains to recruit different subunits into SEC. ENL/AF9 also exists outside SEC when bound to Dot1L, which is found to inhibit SEC function. The YEATS domain of ENL/AF9 targets SEC to Pol II on chromatin through contacting the human Polymerase-Associated Factor complex (PAFc) complex. This finding explains the YEATS domain's dispensability for leukemogenesis when ENL/AF9 is translocated to MLL, whose interactions with PAFc and DNA likely substitute for the PAFc/chromatin-targeting function of the YEATS domain.A ccumulating evidence has implicated the elongation stage of RNA polymerase II (Pol II) transcription as a major ratelimiting step for the expression of a large number of metazoan genes, especially those that control cell growth, renewal, and differentiation (1-3). During elongation, the processivity of Pol II is regulated by a set of transcription factors, which had been thought to exist as separate entities and impact on the Pol II elongation complex independently of one another. However, recent data from us and others indicate that at least two well defined transcription elongation factors of different classes reside in a single multisubunit complex termed SEC [super elongation complex, (4)] to cooperatively activate transcription (4-6).The first elongation factor found in SEC is human positive transcription elongation factor b (P-TEFb). Consisting of CDK9 (Cyclin-dependent Kinase 9) and cyclin T1 (CycT1), P-TEFb functions by phosphorylating the C-terminal domain (CTD) of the largest subunit of Pol II and negative elongation factors DSIF and NELF. These events antagonize the actions of the negative factors, release Pol II from promoter-proximal pausing, and trigger the production of full-length mRNA transcripts (7,8). The second elongation factor in SEC is ELL2, which promotes elongation by keeping the 3′ OH of nascent mRNA in alignment with the catalytic site to prevent Pol II backtracking (9). Besides P-TEFb and ELL2, SEC also contains transcription factors/coactivators ENL, AF9, AFF4 (AF5q31), AFF1 (AF4), and probably others (4-6). Among these, AFF4 is known to mediate the interaction between ELL2 and P-TEFb and maintain the integrity of SEC (5). While AFF1 has been shown to interact with AFF4 (10), it remains to be seen whether the interaction between these two homologo...

Hard ticks of the order Ixodidae serve as vectors for numerous human pathogens, including the causative agent of Lyme Disease Borrelia burgdorferi. Tick-associated microbes can influence pathogen colonization, offering the potential to inhibit disease transmission through engineering of the tick microbiota. Here, we investigate whether B. burgdorferi encounters abundant bacteria within the midgut of wild adult Ixodes scapularis, its primary vector. Through the use of controlled sequencing methods and confocal microscopy, we find that the majority of field-collected adult I. scapularis harbor limited internal microbial communities that are dominated by endosymbionts. A minority of I. scapularis ticks harbor abundant midgut bacteria and lack B. burgdorferi. We find that the lack of a stable resident midgut microbiota is not restricted to I. scapularis since extension of our studies to I. pacificus, Amblyomma maculatum, and Dermacentor spp showed similar patterns. Finally, bioinformatic examination of the B. burgdorferi genome revealed the absence of genes encoding known interbacterial interaction pathways, a feature unique to the Borrelia genus within the phylum Spirochaetes. Our results suggest that reduced selective pressure from limited microbial populations within ticks may have facilitated the evolutionary loss of genes encoding interbacterial competition pathways from Borrelia.

No abstract