TATA and paused promoters active in differentiated tissues have distinct expression characteristics.

Core promoter types differ in the extent to which RNA polymerase II (Pol II) pauses after initiation, but how this affects their tissue-specific gene expression characteristics is not well understood. While promoters with Pol II pausing elements are active throughout development, TATA promoters are highly active in differentiated tissues. We therefore used a genomics approach on late-stage Drosophila embryos to analyze the properties of promoter types. Using tissue-specific Pol II ChIP-seq, we found that paused promoters have high levels of paused Pol II throughout the embryo, even in tissues where the gene is not expressed, while TATA promoters only show Pol II occupancy when the gene is active. The promoter types are associated with different chromatin accessibility in ATAC-seq data and have different expression characteristics in single-cell RNA-seq data. The two promoter types may therefore be optimized for different properties: paused promoters show more consistent expression when active, while TATA promoters have lower background expression when inactive. We propose that tissue-specific genes have evolved to use two different strategies for their differential expression across tissues.

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.

Negative elongation factor (NELF) coordinates RNA polymerase II pausing, premature termination, and chromatin remodeling to regulate HIV transcription.

A barrier to eradicating HIV infection is targeting and eliminating latently infected cells. Events that contribute to HIV transcriptional latency include repressive chromatin structure, transcriptional interference, the inability of Tat to recruit positive transcription factor b, and poor processivity of RNA polymerase II (RNAP II). In this study, we investigated mechanisms by which negative elongation factor (NELF) establishes and maintains HIV latency. Negative elongation factor (NELF) induces RNAP II promoter proximal pausing and limits provirus expression in HIV-infected primary CD4(+) T cells. Decreasing NELF expression overcomes RNAP II pausing to enhance HIV transcription elongation in infected primary T cells, demonstrating the importance of pausing in repressing HIV transcription. We also show that RNAP II pausing is coupled to premature transcription termination and chromatin remodeling. NELF interacts with Pcf11, a transcription termination factor, and diminishing Pcf11 in primary CD4(+) T cells induces HIV transcription elongation. In addition, we identify NCoR1-GPS2-HDAC3 as a NELF-interacting corepressor complex that is associated with repressed HIV long terminal repeats. We propose a model in which NELF recruits Pcf11 and NCoR1-GPS2-HDAC3 to paused RNAP II, reinforcing repression of HIV transcription and establishing a critical checkpoint for HIV transcription and latency.

Lola-I is a promoter pioneer factor that establishes de novo Pol II pausing during development.

While the accessibility of enhancers is dynamically regulated during development, promoters tend to be constitutively accessible and poised for activation by paused Pol II. By studying Lola-I, a Drosophila zinc finger transcription factor, we show here that the promoter state can also be subject to developmental regulation independently of gene activation. Lola-I is ubiquitously expressed at the end of embryogenesis and causes its target promoters to become accessible and acquire paused Pol II throughout the embryo. This promoter transition is required but not sufficient for tissue-specific target gene activation. Lola-I mediates this function by depleting promoter nucleosomes, similar to the action of pioneer factors at enhancers. These results uncover a level of regulation for promoters that is normally found at enhancers and reveal a mechanism for the de novo establishment of paused Pol II at promoters.

Combinatorial signals from CD28 differentially regulate human immunodeficiency virus transcription in T cells.

Activation through the T-cell receptor and the costimulatory receptor CD28 supports efficient HIV transcription as well as reactivation of latent provirus. To characterize critical signals associated with CD28 that regulate HIV-1 transcription, we generated a library of chimeric CD28 receptors that harbored different combinations of key tyrosine residues in the cytoplasmic tail, Tyr-173, Tyr-188, Tyr-191, and Tyr-200. We found that Tyr-191 and Tyr-200 induce HIV-1 transcription via the activation of NF-kappaB and its recruitment to the HIV-long terminal repeat. Tyr-188 modifies positive and negative signals associated with CD28. Importantly, signaling through Tyr-188, Tyr-191, and Tyr-200 is required to overcome the inhibition posed by Tyr-173. CD28 also regulates P-TEFb activity, which is necessary for HIV-1 transcription processivity, by limiting the release of P-TEFb from the HEXIM1-7SK inhibitory complex in response to T-cell receptor signaling. Our studies reveal that CD28 regulates HIV-1 provirus transcription through a complex interplay of positive and negative signals that may be manipulated to control HIV-1 transcription and replication.

The Src kinase Lck facilitates assembly of HIV-1 at the plasma membrane.

HIV type 1 (HIV-1) assembly and egress are driven by the viral protein Gag and occur at the plasma membrane in T cells. Recent evidence indicates that secretory vesicles and machinery are essential components of virus packaging in both T cells and macrophages. However, the pathways and cellular mediators of Gag targeting to the plasma membrane are not well characterized. Lck, a lymphoid specific Src kinase critical for T cell activation, is found in the plasma membrane as well as various intracellular compartments and it has been suggested to influence HIV-1 replication. To investigate Lck as a potential regulator of Gag targeting, we assessed HIV-1 replication and Gag-induced virus-like particle release in the presence and absence of Lck. Release of HIV-1 and virus-like particles was reduced in the absence of Lck. This decrease in replication was not due to altered HIV-1 infection, transcription or protein translation. However, in T cells lacking Lck, HIV-1 accumulated intracellularly. In addition, expressing Lck in HeLa cells promoted HIV-1 Gag plasma membrane localization. Palmitoylation of the Lck unique domain, which is essential for directing Lck to the plasma membrane, was critical for its effect on HIV-1 replication. Furthermore, HIV-1 Gag directly interacted with the Lck unique domain in the context of infected cells. These results indicate that Lck plays a key role in targeting HIV-1 Gag to the plasma membrane in T cells.

An Atlas of Transcription Factors Expressed in Male Pupal Terminalia of Drosophila melanogaster.

During development, transcription factors and signaling molecules govern gene regulatory networks to direct the formation of unique morphologies. As changes in gene regulatory networks are often implicated in morphological evolution, mapping transcription factor landscapes is important, especially in tissues that undergo rapid evolutionary change. The terminalia (genital and anal structures) of Drosophila melanogaster and its close relatives exhibit dramatic changes in morphology between species. While previous studies have identified network components important for patterning the larval genital disc, the networks governing adult structures during pupal development have remained uncharted. Here, we performed RNA-seq in whole Drosophila melanogaster male terminalia followed by in situ hybridization for 100 highly expressed transcription factors during pupal development. We find that the male terminalia are highly patterned during pupal stages and that specific transcription factors mark separate structures and substructures. Our results are housed online in a searchable database (https://flyterminalia.pitt.edu/) as a resource for the community. This work lays a foundation for future investigations into the gene regulatory networks governing the development and evolution of Drosophila terminalia.

Hippo Reprograms the Transcriptional Response to Ras Signaling.

Hyperactivating mutations in Ras signaling are hallmarks of carcinomas. Ras signaling mediates cell fate decisions as well as proliferation during development. It is not known what dictates whether Ras signaling drives differentiation versus proliferation. Here we show that the Hippo pathway is critical for this decision. Loss of Hippo switches Ras activation from promoting cellular differentiation to aggressive cellular proliferation. Transcriptome analysis combined with genetic tests show that this excessive proliferation depends on the synergistic induction of Ras target genes. Using ChIP-nexus, we find that Hippo signaling keeps Ras targets in check by directly regulating the expression of two key downstream transcription factors of Ras signaling: the ETS-domain transcription factor Pointed and the repressor Capicua. Our results highlight how independent signaling pathways can impinge on each other at the level of transcription factors, thereby providing a safety mechanism to keep proliferation in check under normal developmental conditions.

Genome-wide identification of Drosophila dorso-ventral enhancers by differential histone acetylation analysis.

BACKGROUND: Drosophila dorso-ventral (DV) patterning is one of the best-understood regulatory networks to date, and illustrates the fundamental role of enhancers in controlling patterning, cell fate specification, and morphogenesis during development. Histone acetylation such as H3K27ac is an excellent marker for active enhancers, but it is challenging to obtain precise locations for enhancers as the highest levels of this modification flank the enhancer regions. How to best identify tissue-specific enhancers in a developmental system de novo with a minimal set of data is still unclear. RESULTS: Using DV patterning as a test system, we develop a simple and effective method to identify tissue-specific enhancers de novo. We sample a broad set of candidate enhancer regions using data on CREB-binding protein co-factor binding or ATAC-seq chromatin accessibility, and then identify those regions with significant differences in histone acetylation between tissues. This method identifies hundreds of novel DV enhancers and outperforms ChIP-seq data of relevant transcription factors when benchmarked with mRNA expression data and transgenic reporter assays. These DV enhancers allow the de novo discovery of the relevant transcription factor motifs involved in DV patterning and contain additional motifs that are evolutionarily conserved and for which the corresponding transcription factors are expressed in a DV-biased fashion. Finally, we identify novel target genes of the regulatory network, implicating morphogenesis genes as early targets of DV patterning. CONCLUSIONS: Taken together, our approach has expanded our knowledge of the DV patterning network even further and is a general method to identify enhancers in any developmental system, including mammalian development.

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.