We showed previously that the histone lysine methyltransferase (HKMT) H3K27me3 (EZH2) is the catalytic subunit of Polycomb repressive complex 2 (PRC2) and is required for the maintenance of HIV-1 latency in Jurkat T cells. Here we show, by using chromatin immunoprecipitation experiments, that both PRC2 and euchromatic histone-lysine N-methyltransferase 2 (EHMT2), the G9a H3K9me2-3 methyltransferase, are highly enriched at the proviral 5′ long terminal repeat (LTR) and rapidly displaced upon proviral reactivation. Clustered regularly interspaced short palindromic repeat(s) (CRISPR)-mediated knockout of EZH2 caused depletion of both EZH2 and EHMT2, but CRISPR-mediated knockout of EHMT2 was selective for EHMT2, consistent with the failure of EHMT2 knockouts to induce latent proviruses in this system. Either (i) knockout of methyltransferase by short hairpin RNA in Jurkat T cells prior to HIV-1 infection or (ii) inhibition of the enzymes with drugs significantly reduced the levels of the resulting silenced viruses, demonstrating that both enzymes are required to establish latency. To our surprise, inhibition of EZH2 (by GSK-343 or EPZ-6438) or inhibition of EHMT2 (by UNC-0638) in the Th17 primary cell model of HIV latency or resting memory T cells isolated from HIV-1-infected patients receiving highly active antiretroviral therapy, was sufficient to induce the reactivation of latent proviruses. The methyltransferase inhibitors showed synergy with interleukin-15 and suberanilohydroxamic acid. We conclude that both PRC2 and EHMT2 are required for the establishment and maintenance of HIV-1 proviral silencing in primary cells. Furthermore, EZH2 inhibitors such as GSK-343 and EPZ-6438 and the EHMT2 inhibitor UNC-0638 are strong candidates for use as latency-reversing agents in clinical studies.

SignificanceThe molecular mechanisms leading to the creation and maintenance of the latent HIV reservoir remain incompletely understood. Unbiased shRNA screens showed that the estrogen receptor acts as a potent repressor of proviral reactivation in T cells. Antagonists of ESR-1 activate latent HIV-1 proviruses while agonists, including β-estradiol, potently block HIV reactivation. Using a well-matched set of male and female donors, we found that ESR-1 plays an important role in regulating HIV transcription in both sexes. However, women are much more responsive to estrogen and appear to harbor smaller inducible RNA reservoirs. Accounting for the impact of estrogen on HIV viral reservoirs will therefore be critical for devising curative therapies for women, a group representing 51% of global HIV infections.

Metamorphosis of anuran tadpoles is controlled by thyroid hormone (TH).Here we demonstrate that transgenic Xenopus laevis tadpoles expressing a dominant negative form of TH receptor-␣ are resistant to a wide variety of the metamorphic changes induced by TH. This result confirms that TH receptors mediate both early and late developmental programs of metamorphosis as diverse as growth in the brain, limb buds, nose and Meckel's cartilage, remodeling of the intestine, and death and resorption of the gills and tail.R ising thyroid hormone (TH) levels produced by the thyroid gland of a growing tadpole orchestrate the sequential changes of metamorphosis in the majority of tadpole organs. These morphological changes range from growth and cell differentiation (limbs) to cell death and tissue resorption (tail and gills), and include the remodeling of tadpole organs into their adult forms (intestine, skin, and brain). With the discovery that the TH receptors (TRs) are transcription factors (1, 2), these varied developmental programs have been studied as complex changes in gene expression initiated by TH (3). Despite the many experiments that prove the requirement of TH in metamorphosis, there is only one demonstration to date showing conclusively that TH acts by way of TRs in a particular metamorphic program. The asymmetrical replication of the ventral retina in Xenopus laevis is inhibited by expression of a dominant negative form of the TR (TRDN; ref. 4).All vertebrates studied to date, including X. laevis (5), have two highly conserved TR isoforms called TR␣ and TR␤. In X. laevis, tadpole TR␣ is constitutive and distributed widely in tissues even before the organism forms a thyroid gland (6). Because TR␤ is a direct response gene of TH (6, 7), the amount of TR␤ in cells increases along with the rise in endogenous TH that occurs as metamorphosis proceeds (6,8). During premetamorphosis when the early events of tadpole development (such as limb growth and DNA replication in the brain) occur, the TH concentration and the TR␤ levels are very low. TR␤ and TH rise to a peak at the climax of metamorphosis when the final changes (such as gill and tail resorption and intestinal remodeling) occur. We will show that TR␣ is required for the precocious response of young tadpoles to exogenous TH in their rearing water. We have taken advantage of the new transgenesis method (9) to express green fluorescent protein (GFP) fused to a dominant negative form of TR-␣ (GFP-TRDN␣) driven by two different widely expressed promoters. Metamorphic changes that are inhibited by the GFP-TRDN␣ include brain development, limb and Meckel's cartilage growth, intestinal remodeling, gill resorption, and death of cells in the tail including muscle. Materials and MethodsPlasmids, Transgenesis, and 3,5,3 Triiodothyronine (T3) Treatment.Constructs were prepared in pCS2 ϩ -based vectors and used either the cytomegalovirus (CMV) promoter͞enhancer (10) or a 6-kb ␣2(1) mouse collagen enhancer fused to a minimal ␣2 (1) promoter (Col; ref. 11). Constructs were made ...