Lab Mission
- Elucidate the underlying molecular mechanisms of transcriptional regulation during HIV-1 latency maintenance and reactivation.
- Define fundamental principles of transcriptional regulation with broader implications to physiologic (differentiation, development) and pathological (cancer, chronic inflammation) contexts.
Transcription Regulation During HIV-1 Latency and Reactivation
HIV-1 latency and persistence in reservoir cells hamper viral eradication. Defining the mechanisms underlying regulation of latency maintenance and reactivation are paramount for devising alternative cure approaches. Using high-resolution genomics assays, we have gained previously unprecedented knowledge about the HIV-1 transcription process. Ongoing and future studies will reveal how several regulatory steps are temporally coordinated to facilitate productive transcription, the roles of nascent RNA and RNA-protein interactions, and how enhancers in the proviral genome, host and viral factors, chromatin organization, transcription factor networks and three-dimensional genome architecture collectively shape proviral fate: the decision between latency and reactivation. See below for a complete description of ongoing projects.
Ongoing Projects for Incoming Trainees (Students and Postdocs):
HIV-1 integrates semi-randomly into the human genome. Different sites of integration dictate diverse transcription levels during cell homeostasis (basal phase) and immune cell stimulation (host and viral phases). However, it remains unclear how the integration site shapes HIV-1 transcription reactivation potential. The underlying hypothesis is that the site of integration regulates one or two critical features: 1) chromatin organization, and/or 2) transcription factor density and composition in response to stimulating ligands, overall contributing to recruitment and/or activation of the RNA polymerase II transcription machinery. The site of integration could favor or preclude a poised chromatin organization that allows for low level gene expression (in the basal phase) and high level of gene expression (in the viral phase), and could impact the establishment and maintenance of the Tat positive feedback loop. Ongoing genetic and genomic approaches will allow us to disentangle the contribution(s) of the integration site to latency maintenance and reactivation.
The classical view of HIV-1 transcription activation is through regulation of the elongation step. However, this mechanism on itself does not explain how the virus achieves high levels of transcription for efficient latency reactivation. In recent work, we found that the HIV-1 Tat protein promotes transcription initiation and coordinates the transition to elongation by promoting pause release, consequently decreasing the kinetic window for premature termination. We characterized the RNA polymerase II (Pol II) pause sites at the proviral genome during latency maintenance and reactivation with nucleotide resolution. Ongoing genetic and genomic studies will enable us to precisely reveal the roles of negative and positive elongation factors in the coordination of initiation flux, pausing dwell time and productive pause release versus premature termination during transcription reactivation from latency.
It has long been believed that the nascent viral pre-mRNA structure (TAR) has the sequence and structural determinants required for recruitment of the Tat protein, which then recruits the P-TEFb kinase to facilitate transcriptional pause release. Using genomic approaches that map the location of transcriptionally engaged RNA polymerase II (Pol II) during latency maintenance and reactivation, we mapped the pause sites in the promoter-proximal regions. These data suggest a model whereby the nascent RNA length and folding dictates progressive polymerase phase transitions and discrete nascent RNA-factor interactions governing productive HIV-1 transcription during latency reactivation. By combining CRISPR-Cas9 proviral genome editing and factor acute depletion systems with genomic approaches, we will define the roles of nascent RNA sequence, length and folding in governing these critical polymerase phase transitions.
The machinery that regulates HIV-1 transcription initiation (the pre-initiation complex: PIC) throughout the multi-phase HIV-1 transcription program remains unknown. Consequently, we do not know the PIC composition before (Host phase) and after (Viral phase) synthesis of the Tat protein, whether PIC composition is remodeled by Tat and/or if Tat uniquely exploits a canonical PIC to maintain transcription in the viral phase. Ongoing genetic and biochemical approaches will help us dissect out the transcription PICs that first ignite and then sustain viral transcription during latency reactivation. Through biochemical and biophysical approaches, we aim to reveal how Tat engages with the PIC to accelerate multiple transcription checkpoints thereby facilitating initiation flux and coordinating the progression through the transcription cycle.
We identified enhancers present in the Modulatory region (M) within the proviral genome that contribute to HIV-1 transcription reactivation from latency. In mammalian genomes, enhancers communicate with promoters to control the rate of transcriptional bursting, but it remains unknown how these viral enhancers operate to maintain high transcription levels. Given that the M region has binding sites for many sequence-specific transcription factors, and the spatial proximity of the M region to the core promoter, we speculate that those transcription factors (TFs) communicate with the core promoter machinery to facilitate the flux of RNA polymerase II (Pol II) molecules to maintain transcription synchrony. Ongoing studies will help define how the viral enhancers facilitate HIV-1 transcription reactivation, and to determine how eukaryotic TFs and Tat collectively function with the viral enhancers to promote transcription reactivation from latency.
We found that HIV-1 transcription during latency reactivation promotes the formation of R-loops (DNA:RNA hybrids within a displaced single stranded DNA) throughout the provirus body and 3’- end. We are currently investigating if R-loop perturbation by overexpression of RNAse H (the enzyme that resolves them) has any functional consequences on viral gene expression and latency reactivation. Future studies will reveal the underlying molecular mechanisms and identify the machinery that facilitates R-loops metabolism.
Cell fate decisions are critical for key biological processes including T cell activation and differentiation. These cellular decisions are orchestrated through the precise spatio-temporal activation and deactivation of cell signaling–transcription programs facilitating the expression of Immediate Early Genes (IEGs). We have recently described a counterintuitive mechanism whereby the negative regulation of RNA Polymerase (Pol II) elongation facilitates transcription activation during cell stimulation (see Reference below). Ongoing and future studies will reveal how the interplay between heterochromatin formation and transcription elongation coordinates the proper activation of effector programs during immune cell fate choice and dysregulation in diseased states.
References: KAP1 negatively regulates RNA polymerase II elongation kinetics to activate signal-induced transcription
