Transient Thresholding: A Mechanism Enabling Noncooperative Transcriptional Circuitry to Form a Switch

Threshold generation in fate-selection circuits is often achieved through deterministic bistability, which requires cooperativity (i.e., nonlinear activation) and associated hysteresis. However, the Tat positive-feedback loop that controls HIV’s fate decision between replication and proviral latency lacks self-cooperativity and deterministic bistability. Absent cooperativity, it is unclear how HIV can temporarily remain in an off-state long enough for the kinetically slower epigenetic silencing mechanisms to act—expression fluctuations should rapidly trigger active positive feedback and replication, precluding establishment of latency. Here, using flow cytometry and single-cell imaging, we find that the Tat circuit exhibits a transient activation threshold. This threshold largely disappears after ∼40 h—accounting for the lack of deterministic bistability—and promoter activation shortens the lifetime of this transient threshold. Continuous differential equation models do not recapitulate this phenomenon. However, chemical reaction (master equation) models where the transcriptional transactivator and promoter toggle between inactive and active states can recapitulate the phenomenon because they intrinsically create a single-molecule threshold transiently requiring excess molecules in the inactive state to achieve at least one molecule (rather than a continuous fractional value) in the active state. Given the widespread nature of promoter toggling and transcription factor modifications, transient thresholds may be a general feature of inducible promoters

The HIV-1 Tat Protein is Monomethylated at Lysine 71 by the Lysine Methyltransferase KMT7

The HIV-1 transactivator protein Tat is a critical regulator of HIV transcription primarily enabling efficient elongation of viral transcripts. Its interactions with RNA and various host factors are regulated by ordered, transient post-translational modifications. Here, we report a novel Tat modification, monomethylation at lysine 71 (K71). We found that Lys-71 monomethylation (K71me) is catalyzed by KMT7, a methyltransferase that also targets lysine 51 (K51) in Tat. Using mass spectrometry, in vitro enzymology, and modification-specific antibodies, we found that KMT7 monomethylates both Lys-71 and Lys-51 in Tat. K71me is important for full Tat transactivation, as KMT7 knockdown impaired the transcriptional activity of wild type (WT) Tat but not a Tat K71R mutant. These findings underscore the role of KMT7 as an important monomethyltransferase regulating HIV transcription through Tat

Transient thresholding in the HIV Tat fate-selection circuit

Human Immunodeficiency Virus type 1 (HIV) is a lentivirus that infects CD4+ T cells, causing progressive immune dysfunction (AIDS). Although effective treatments exist, HIV is able to form a ‘latent reservoir’ in long-lived memory immune cells that is transcriptionally silent and invisible to both the immune system and conventional antivirals. The HIV fate decision between latency and replication is controlled by the sole HIV promoter, LTR, and its gene product and non-cooperative monomeric activator, Tat. It remains unclear how the Tat-LTR positive-feedback circuit generates an activation threshold to prevent ‘leaky’ Tat expression from transactivating the LTR, thereby enabling maintenance of the latent state. Threshold generation in gene regulatory networks (GRNs) is typically achieved through deterministic bistability, which is absent here. Without some form of non-linear activation (e.g. self-cooperativity), expression noise should trigger runaway Tat feedback and thus HIV replication. Despite this, HIV latency is apparently stable, even under strong activating conditions. Using flow cytometry and single-cell imaging, I find that HIV LTR exhibits a transient threshold in response to Tat. The molecular threshold at early times is ~40,000 Tat proteins per cell, but largely disappears after 40 hours, explaining the lack of bistability and hysteresis. Further, I demonstrate that slow ‘toggling’ between active and inactive promoter states can generate an activation threshold without cooperativity. Cellular signaling can modulate toggling frequency and thereby adjust this threshold. These results indicate a potential role for promoter toggling as a mechanism for tunable threshold generation in GRNs. Finally, I propose a class of general stochastic models for multi-step transactivation of a toggling promoter, and argue for its relevance to LTR, which is known to exhibit intrinsic bursts of transcription at multiple time scales. This work may advance the predictive modeling of Tat-LTR and similar GRNs in higher eukaryotes

Transient thresholding in the HIV Tat fate-selection circuit

Human Immunodeficiency Virus type 1 (HIV) is a lentivirus that infects CD4+ T cells, causing progressive immune dysfunction (AIDS). Although effective treatments exist, HIV is able to form a ‘latent reservoir’ in long-lived memory immune cells that is transcriptionally silent and invisible to both the immune system and conventional antivirals. The HIV fate decision between latency and replication is controlled by the sole HIV promoter, LTR, and its gene product and non-cooperative monomeric activator, Tat. It remains unclear how the Tat-LTR positive-feedback circuit generates an activation threshold to prevent ‘leaky’ Tat expression from transactivating the LTR, thereby enabling maintenance of the latent state. Threshold generation in gene regulatory networks (GRNs) is typically achieved through deterministic bistability, which is absent here. Without some form of non-linear activation (e.g. self-cooperativity), expression noise should trigger runaway Tat feedback and thus HIV replication. Despite this, HIV latency is apparently stable, even under strong activating conditions. Using flow cytometry and single-cell imaging, I find that HIV LTR exhibits a transient threshold in response to Tat. The molecular threshold at early times is ~40,000 Tat proteins per cell, but largely disappears after 40 hours, explaining the lack of bistability and hysteresis. Further, I demonstrate that slow ‘toggling’ between active and inactive promoter states can generate an activation threshold without cooperativity. Cellular signaling can modulate toggling frequency and thereby adjust this threshold. These results indicate a potential role for promoter toggling as a mechanism for tunable threshold generation in GRNs. Finally, I propose a class of general stochastic models for multi-step transactivation of a toggling promoter, and argue for its relevance to LTR, which is known to exhibit intrinsic bursts of transcription at multiple time scales. This work may advance the predictive modeling of Tat-LTR and similar GRNs in higher eukaryotes

Transient Thresholding: A Mechanism Enabling Noncooperative Transcriptional Circuitry to Form a Switch

Threshold generation in fate-selection circuits is often achieved through deterministic bistability, which requires cooperativity (i.e., nonlinear activation) and associated hysteresis. However, the Tat positive-feedback loop that controls HIV's fate decision between replication and proviral latency lacks self-cooperativity and deterministic bistability. Absent cooperativity, it is unclear how HIV can temporarily remain in an off-state long enough for the kinetically slower epigenetic silencing mechanisms to act-expression fluctuations should rapidly trigger active positive feedback and replication, precluding establishment of latency. Here, using flow cytometry and single-cell imaging, we find that the Tat circuit exhibits a transient activation threshold. This threshold largely disappears after ∼40 h-accounting for the lack of deterministic bistability-and promoter activation shortens the lifetime of this transient threshold. Continuous differential equation models do not recapitulate this phenomenon. However, chemical reaction (master equation) models where the transcriptional transactivator and promoter toggle between inactive and active states can recapitulate the phenomenon because they intrinsically create a single-molecule threshold transiently requiring excess molecules in the inactive state to achieve at least one molecule (rather than a continuous fractional value) in the active state. Given the widespread nature of promoter toggling and transcription factor modifications, transient thresholds may be a general feature of inducible promoters

Protein-Mediated and RNA-Based Origins of Replication of Extrachromosomal Mycobacterial Prophages

Bacteriophages are the most abundant biological entities in the biosphere and are a source of uncharacterized biological mechanisms and genetic tools. Here, we identify segments of phage genomes that are used for stable extrachromosomal replication in the prophage state. Autonomous replication of some of these phages requires a RepA-like protein, although most lack repA and use RNA-based systems for replication initiation. We describe a suite of plasmids based on these prophage replication functions that vary in copy number, stability, host range, and compatibility. These plasmids expand the toolbox available for genetic manipulation of Mycobacterium and other Actinobacteria, including Gordonia terrae.Temperate bacteriophages are common and establish lysogens of their bacterial hosts in which the prophage is stably inherited. It is typical for such prophages to be integrated into the bacterial chromosome, but extrachromosomally replicating prophages have been described also, with the best characterized being the Escherichia coli phage P1 system. Among the large collection of sequenced mycobacteriophages, more than half are temperate or predicted to be temperate, most of which code for a tyrosine or serine integrase that promotes site-specific prophage integration. However, within the large group of 621 cluster A temperate phages, ∼20% lack an integration cassette, which is replaced with a parABS partitioning system. A subset of these phages carry genes coding for a RepA-like protein (RepA phages), which we show here is necessary and sufficient for autonomous extrachromosomal replication. The non-RepA phages appear to replicate using an RNA-based system, as a parABS-proximal region expressing a noncoding RNA is required for replication. Both RepA and non-RepA phage-based plasmids replicate at one or two copies per cell, transform both Mycobacterium smegmatis and Mycobacterium tuberculosis, and are compatible with pAL5000-derived oriM and integration-proficient plasmid vectors. Characterization of these phage-based plasmids offers insights into the variability of lysogenic maintenance systems and provides a large suite of plasmids for actinobacterial genetics that vary in stability, copy number, compatibility, and host range

The HIV-1 Tat Protein Is Monomethylated at Lysine 71 by the Lysine Methyltransferase KMT7*

The HIV-1 transactivator protein Tat is a critical regulator of HIV transcription primarily enabling efficient elongation of viral transcripts. Its interactions with RNA and various host factors are regulated by ordered, transient post-translational modifications. Here, we report a novel Tat modification, monomethylation at lysine 71 (K71). We found that Lys-71 monomethylation (K71me) is catalyzed by KMT7, a methyltransferase that also targets lysine 51 (K51) in Tat. Using mass spectrometry, in vitro enzymology, and modification-specific antibodies, we found that KMT7 monomethylates both Lys-71 and Lys-51 in Tat. K71me is important for full Tat transactivation, as KMT7 knockdown impaired the transcriptional activity of wild type (WT) Tat but not a Tat K71R mutant. These findings underscore the role of KMT7 as an important monomethyltransferase regulating HIV transcription through Tat

BET bromodomain-targeting compounds reactivate HIV from latency via a Tat-independent mechanism.

The therapeutic potential of pharmacologic inhibition of bromodomain and extraterminal (BET) proteins has recently emerged in hematological malignancies and chronic inflammation. We find that BET inhibitor compounds (JQ1, I-Bet, I-Bet151 and MS417) reactivate HIV from latency. This is evident in polyclonal Jurkat cell populations containing latent infectious HIV, as well as in a primary T-cell model of HIV latency. Importantly, we show that this activation is dependent on the positive transcription elongation factor p-TEFb but independent from the viral Tat protein, arguing against the possibility that removal of the BET protein BRD4, which functions as a cellular competitor for Tat, serves as a primary mechanism for BET inhibitor action. Instead, we find that the related BET protein, BRD2, enforces HIV latency in the absence of Tat, pointing to a new target for BET inhibitor treatment in HIV infection. In shRNA-mediated knockdown experiments, knockdown of BRD2 activates HIV transcription to the same extent as JQ1 treatment, while a lesser effect is observed with BRD4. In single-cell time-lapse fluorescence microscopy, quantitative analyses across ~2,000 viral integration sites confirm the Tat-independent effect of JQ1 and point to positive effects of JQ1 on transcription elongation, while delaying re-initiation of the polymerase complex at the viral promoter. Collectively, our results identify BRD2 as a new Tat-independent suppressor of HIV transcription in latently infected cells and underscore the therapeutic potential of BET inhibitors in the reversal of HIV latency

BET bromodomain-targeting compounds reactivate HIV from latency via a Tat-independent mechanism

The therapeutic potential of pharmacologic inhibition of bromodomain and extraterminal (BET) proteins has recently emerged in hematological malignancies and chronic inflammation. We find that BET inhibitor compounds (JQ1, I-Bet, I-Bet151 and MS417) reactivate HIV from latency. This is evident in polyclonal Jurkat cell populations containing latent infectious HIV, as well as in a primary T-cell model of HIV latency. Importantly, we show that this activation is dependent on the positive transcription elongation factor p-TEFb but independent from the viral Tat protein, arguing against the possibility that removal of the BET protein BRD4, which functions as a cellular competitor for Tat, serves as a primary mechanism for BET inhibitor action. Instead, we find that the related BET protein, BRD2, enforces HIV latency in the absence of Tat, pointing to a new target for BET inhibitor treatment in HIV infection. In shRNA-mediated knockdown experiments, knockdown of BRD2 activates HIV transcription to the same extent as JQ1 treatment, while a lesser effect is observed with BRD4. In single-cell time-lapse fluorescence microscopy, quantitative analyses across ~2,000 viral integration sites confirm the Tat-independent effect of JQ1 and point to positive effects of JQ1 on transcription elongation, while delaying re-initiation of the polymerase complex at the viral promoter. Collectively, our results identify BRD2 as a new Tat-independent suppressor of HIV transcription in latently infected cells and underscore the therapeutic potential of BET inhibitors in the reversal of HIV latency