Mutations in yeast Pcf11, a conserved protein essential for mRNA 3' end processing and transcription termination, elicit the Environmental Stress Response.

The Pcf11 protein is an essential subunit of the large complex that cleaves and polyadenylates eukaryotic mRNA precursor. It has also been functionally linked to gene-looping, termination of RNA Polymerase II (Pol II) transcripts, and mRNA export. We have examined a poorly characterized but conserved domain (amino acids 142-225) of the Saccharomyces cerevisiae  Pcf11 and found that while it is not needed for mRNA 3' end processing or termination downstream of the poly(A) sites of protein-coding genes, its presence improves the interaction with Pol II and the use of transcription terminators near gene promoters. Analysis of genome-wide Pol II occupancy in cells with Pcf11 missing this region, as well as Pcf11 mutated in the Pol II CTD Interacting Domain, indicates that systematic changes in mRNA expression are mediated primarily at the level of transcription. Global expression analysis also shows that a general stress response, involving both activation and suppression of specific gene sets known to be regulated in response to a wide variety of stresses, is induced in the two pcf11 mutants, even though cells are grown in optimal conditions. The mutants also cause an unbalanced expression of cell wall-related genes that does not activate the Cell Wall Integrity pathway but is associated with strong caffeine sensitivity. Based on these findings, we propose that Pcf11 can modulate the expression level of specific functional groups of genes in ways that do not involve its well-characterized role in mRNA 3' end processing.

A combined adjuvant approach primes robust germinal center responses and humoral immunity in non-human primates.

Adjuvants and antigen delivery kinetics can profoundly influence B cell responses and should be critically considered in rational vaccine design, particularly for difficult neutralizing antibody targets such as human immunodeficiency virus (HIV). Antigen kinetics can change depending on the delivery method. To promote extended immunogen bioavailability and to present antigen in a multivalent form, native-HIV Env trimers are modified with short phosphoserine peptide linkers that promote tight binding to aluminum hydroxide (pSer:alum). Here we explore the use of a combined adjuvant approach that incorporates pSer:alum-mediated antigen delivery with potent adjuvants (SMNP, 3M-052) in an extensive head-to-head comparison study with conventional alum to assess germinal center (GC) and humoral immune responses. Priming with pSer:alum plus SMNP induces additive effects that enhance the magnitude and persistence of GCs, which correlate with better GC-TFH cell help. Autologous HIV-neutralizing antibody titers are improved in SMNP-immunized animals after two immunizations. Over 9 months after priming immunization of pSer:alum with either SMNP or 3M-052, robust Env-specific bone marrow plasma cells (BM BPC) are observed. Furthermore, pSer-modification of Env trimer reduce targeting towards immunodominant non-neutralizing epitopes. The study shows that a combined adjuvant approach can augment humoral immunity by modulating immunodominance and shows promise for clinical translation.

Host T Cell Dedifferentiation Effects Drive HIV-1 Latency Stability.

The development of therapies to eliminate the latent HIV-1 reservoir is hampered by our incomplete understanding of the biomolecular mechanism governing HIV-1 latency. To further complicate matters, recent single-cell RNA sequencing (scRNA-seq) studies reported extensive heterogeneity between latently HIV-1-infected primary T cells, implying that latent HIV-1 infection can persist in greatly differing host cell environments. We show here that transcriptomic heterogeneity is also found between latently infected T cell lines, which allowed us to study the underlying mechanisms of intercell heterogeneity at high signal resolution. Latently infected T cells exhibited a dedifferentiated phenotype, characterized by the loss of T cell-specific markers and gene regulation profiles reminiscent of hematopoietic stem cells (HSC). These changes had functional consequences. As reported for stem cells, latently HIV-1-infected T cells efficiently forced lentiviral superinfections into a latent state and favored glycolysis. As a result, metabolic reprogramming or cell redifferentiation destabilized latent infection. Guided by these findings, data mining of single-cell RNA-seq data of latently HIV-1-infected primary T cells from patients revealed the presence of similar dedifferentiation motifs. More than 20% of the highly detectable genes that were differentially regulated in latently infected cells were associated with hematopoietic lineage development (e.g., HUWE1, IRF4, PRDM1, BATF3, TOX, ID2, IKZF3, and CDK6) or were hematopoietic markers (SRGN; hematopoietic proteoglycan core protein). The data add to evidence that the biomolecular phenotype of latently HIV-1-infected cells differs from that of normal T cells and strategies to address their differential phenotype need to be considered in the design of therapeutic cure interventions. IMPORTANCE HIV-1 persists in a latent reservoir in memory CD4 T cells for the lifetime of a patient. Understanding the biomolecular mechanisms used by the host cells to suppress viral expression will provide essential insights required to develop curative therapeutic interventions. Unfortunately, our current understanding of these control mechanisms is still limited. By studying gene expression profiles, we demonstrated that latently HIV-1-infected T cells have a dedifferentiated T cell phenotype. Software-based data integration allowed the identification of drug targets that would redifferentiate viral host cells and, by extension, destabilize latent HIV-1 infection events. The importance of the presented data lies within the clear demonstration that HIV-1 latency is a host cell phenomenon. As such, therapeutic strategies must first restore proper host cell functionality to accomplish efficient HIV-1 reactivation.

Regulation of alternative polyadenylation in the yeast Saccharomyces cerevisiae by histone H3K4 and H3K36 methyltransferases.

Adjusting DNA structure via epigenetic modifications, and altering polyadenylation (pA) sites at which precursor mRNA is cleaved and polyadenylated, allows cells to quickly respond to environmental stress. Since polyadenylation occurs co-transcriptionally, and specific patterns of nucleosome positioning and chromatin modifications correlate with pA site usage, epigenetic factors potentially affect alternative polyadenylation (APA). We report that the histone H3K4 methyltransferase Set1, and the histone H3K36 methyltransferase Set2, control choice of pA site in Saccharomyces cerevisiae, a powerful model for studying evolutionarily conserved eukaryotic processes. Deletion of SET1 or SET2 causes an increase in serine-2 phosphorylation within the C-terminal domain of RNA polymerase II (RNAP II) and in the recruitment of the cleavage/polyadenylation complex, both of which could cause the observed switch in pA site usage. Chemical inhibition of TOR signaling, which causes nutritional stress, results in Set1- and Set2-dependent APA. In addition, Set1 and Set2 decrease efficiency of using single pA sites, and control nucleosome occupancy around pA sites. Overall, our study suggests that the methyltransferases Set1 and Set2 regulate APA induced by nutritional stress, affect the RNAP II C-terminal domain phosphorylation at Ser2, and control recruitment of the 3' end processing machinery to the vicinity of pA sites.

Regulation of the Ysh1 endonuclease of the mRNA cleavage/polyadenylation complex by ubiquitin-mediated degradation.

Mutation of the essential yeast protein Ipa1 has previously been demonstrated to cause defects in pre-mRNA 3' end processing and growth, but the mechanism underlying these defects was not clear. In this study, we show that the ipa1-1 mutation causes a striking depletion of Ysh1, the evolutionarily conserved endonuclease subunit of the 19-subunit mRNA Cleavage/Polyadenylation (C/P) complex, but does not decrease other C/P subunits. YSH1 overexpression rescues both the growth and 3' end processing defects of the ipa1-1 mutant. YSH1 mRNA level is unchanged in ipa1-1 cells, and proteasome inactivation prevents Ysh1 loss and causes accumulation of ubiquitinated Ysh1. Ysh1 ubiquitination is mediated by the Ubc4 ubiquitin-conjugating enzyme and Mpe1, which in addition to its function in C/P, is also a RING ubiquitin ligase. In summary, Ipa1 affects mRNA processing by controlling the availability of the C/P endonuclease and may represent a regulatory mechanism that could be rapidly deployed to facilitate reprogramming of cellular responses.

RNAP II processivity is a limiting step for HIV-1 transcription independent of orientation to and activity of endogenous neighboring promoters.

Since HIV-1 has a propensity to integrate into actively expressed genes, transcriptional interference from neighboring host promoters has been proposed to contribute to the establishment and maintenance HIV-1 latency. To gain insights into how endogenous promoters influence HIV-1 transcription we utilized a set of inducible T cell lines and characterized whether there were correlations between expression of endogenous genes, provirus and long terminal repeat architecture. We show that neighboring promoters are active but have minimal impact on HIV-1 transcription, in particular, expression of the endogenous gene did not prevent expression of HIV-1 following induction of latent provirus. We also demonstrate that releasing paused RNAP II by diminishing negative elongation factor (NELF) is sufficient to reactivate transcriptionally repressed HIV-1 provirus regardless of the integration site and orientation of the provirus suggesting that NELF-mediated RNAP II pausing is a common mechanism of maintaining HIV-1 latency.

Blimp-1, an intrinsic factor that represses HIV-1 proviral transcription in memory CD4+ T cells.

CD4(+) T cell subsets differentially support HIV-1 replication. For example, quiescent CD4(+) memory T cells are susceptible to HIV-1 infection but do not support robust HIV-1 transcription and have been implicated as the primary reservoir of latent HIV-1. T cell transcription factors that regulate maturation potentially limit HIV-1 transcription and mediate the establishment and maintenance of HIV-1 latency. We report that B lymphocyte-induced maturation protein-1 (Blimp-1), a critical regulator of B and T cell differentiation, is highly expressed in memory CD4(+) T cells compared with naive CD4(+) T cells and represses basal and Tat-mediated HIV-1 transcription. Blimp-1 binds an IFN-stimulated response element within HIV-1 provirus, and it is displaced following T cell activation. Reduction of Blimp-1 in infected primary T cells including CD4(+) memory T cells increases RNA polymerase II processivity, histone acetylation, and baseline HIV-1 transcription. Therefore, the transcriptional repressor, Blimp-1, is an intrinsic factor that predisposes CD4(+) memory T cells to latent HIV-1 infection.