DNA methylation is a conserved modification that must be precisely regulated during development to facilitate its roles in silencing transposable elements and regulating gene expression. In plants, DNA methylation changes during reproduction are widely documented and, in many cases, the underlying mechanisms are well understood. In somatic tissues, the diversity of methylation patterns are only recently emerging but they are often associated with the RNA-directed DNA methylation (RdDM) pathway. Here, we discuss advances in our understanding of how the locus-specific targeting and tissue-specific expression of RdDM proteins regulate methylation patterns, how the targeting of methylation at loci with imperfect homology expands the purview of RdDM, and how natural variation within RdDM factors impacts DNA methylation patterns.

DNA methylation shapes the epigenetic landscape of the genome, plays critical roles in regulating gene expression, and ensures transposon silencing. As is evidenced by the numerous defects associated with aberrant DNA methylation landscapes, establishing proper tissue-specific methylation patterns is critical. Yet, how such differences arise remains a largely open question in both plants and animals. Here we demonstrate that CLASSY1-4 (CLSY1-4), four locus-specific regulators of DNA methylation, also control tissue-specific methylation patterns, with the most striking pattern observed in ovules where CLSY3 and CLSY4 control DNA methylation at loci with a highly conserved DNA motif. On a more global scale, we demonstrate that specific clsy mutants are sufficient to shift the epigenetic landscape between tissues. Together, these findings reveal substantial epigenetic diversity between tissues and assign these changes to specific CLSY proteins, elucidating how locus-specific targeting combined with tissue-specific expression enables the CLSYs to generate epigenetic diversity during plant development.

The search for an HIV-1 cure has been greatly hindered by the presence of a viral reservoir that persists despite antiretroviral therapy (ART). Studies of HIV-1 latency in vivo are also complicated by the low proportion of latently infected cells in HIV-1 infected individuals. A number of models of HIV-1 latency have been developed to examine the signaling pathways and viral determinants of latency and reactivation. A primary cell model of HIV-1 latency, which incorporates the generation of primary central memory CD4 T cells (TCM), full-length virus infection (HIVNL4-3) and ART to suppress virus replication, was used to investigate the establishment of HIV latency using RNA-Seq. Initially, an investigation of host and viral gene expression in the resting and activated states of this model indicated that the resting condition was reflective of a latent state. Then, a comparison of the host transcriptome between the uninfected and latently infected conditions of this model identified 826 differentially expressed genes, many of which were related to p53 signaling. Inhibition of the transcriptional activity of p53 by pifithrin-α during HIV-1 infection reduced the ability of HIV-1 to be reactivated from its latent state by an unknown mechanism. In conclusion, this model may be used to screen latency reversing agents utilized in shock and kill approaches to cure HIV, to search for cellular markers of latency, and to understand the mechanisms by which HIV-1 establishes latency.

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.