Overexpression of arogenate dehydratase reveals an upstream point of metabolic control in phenylalanine biosynthesis.

Out of the three aromatic amino acids, the highest flux in plants is directed towards phenylalanine, which is utilized to synthesize proteins and thousands of phenolic metabolites contributing to plant fitness. Phenylalanine is produced predominantly in plastids via the shikimate pathway and subsequent arogenate pathway, both of which are subject to complex transcriptional and post-transcriptional regulation. Previously, it was shown that allosteric feedback inhibition of arogenate dehydratase (ADT), which catalyzes the final step of the arogenate pathway, restricts flux through phenylalanine biosynthesis. Here, we show that in petunia (Petunia hybrida) flowers, which typically produce high phenylalanine levels, ADT regulation is relaxed, but not eliminated. Moderate expression of a feedback-insensitive ADT increased flux towards phenylalanine, while high overexpression paradoxically reduced phenylalanine formation. This reduction could be partially, but not fully, recovered by bypassing other known metabolic flux control points in the aromatic amino acid network. Using comparative transcriptomics, reverse genetics, and metabolic flux analysis, we discovered that transcriptional regulation of the d-ribulose-5-phosphate 3-epimerase gene in the pentose phosphate pathway controls flux into the shikimate pathway. Taken together, our findings reveal that regulation within and upstream of the shikimate pathway shares control over phenylalanine biosynthesis in the plant cell.

Perturbation of H3K27me3-Associated Epigenetic Processes Increases Agrobacterium-Mediated Transformation.

Agrobacterium-mediated transformation is a core technology for basic plant science and agricultural biotechnology. Improving transformation frequency is a major goal for plant transgenesis. We previously showed that T-DNA insertions in some histone genes decreased transformation susceptibility, whereas overexpression of several Arabidopsis H2A and H4 isoforms increased transformation. Overexpression of several histone H2B and H3 isoforms had little effect on transformation frequency. However, overexpression of histone H3-11 (HTR11) enhanced transformation. HTR11 is a unique H3 variant that lacks lysine at positions 9 and 27. The modification status of these lysine residues in canonical H3 proteins plays a critical role in epigenetic determination of gene expression. We mutated histone H3-4 (HTR4), a canonical H3.3 protein that does not increase transformation when overexpressed, by replacing either or both K9 and K27 with the amino acids in HTR11 (either K9I, K27Q, or both). Overexpression of HTR4 with the K27Q but not the K9I substitution enhanced transformation. HTR4K27Q was incorporated into chromatin, and HTR4K27Q overexpression lines exhibited deregulated expression of H3K27me3-enriched genes. These results demonstrate that mutation of K27 in H3.3 is sufficient to perturb H3K27me3-dependent expression in plants as in animals and suggest a distinct epigenetic role for histone HTR11. Further, these observations implicate manipulation of H3K27me3-dependent gene expression as a novel strategy to increase transformation susceptibility.

BRD9 Is a Critical Regulator of Androgen Receptor Signaling and Prostate Cancer Progression.

Switch/sucrose-nonfermentable (SWI/SNF) chromatin-remodeling complexes are critical regulators of chromatin dynamics during transcription, DNA replication, and DNA repair. A recently identified SWI/SNF subcomplex termed GLTSCR1/1L-BAF (GBAF; or "noncanonical BAF", ncBAF) uniquely contains bromodomain-containing protein BRD9 and glioma tumor suppressor candidate region 1 (GLTSCR1) or its paralog GLTSCR1-like (GLTSCR1L). Recent studies have identified a unique dependency on GBAF (ncBAF) complexes in synovial sarcoma and malignant rhabdoid tumors, both of which possess aberrations in canonical BAF (cBAF) and Polybromo-BAF (PBAF) complexes. Dependencies on GBAF in malignancies without SWI/SNF aberrations, however, are less defined. Here, we show that GBAF, particularly its BRD9 subunit, is required for the viability of prostate cancer cell lines in vitro and for optimal xenograft tumor growth in vivo. BRD9 interacts with androgen receptor (AR) and CCCTC-binding factor (CTCF), and modulates AR-dependent gene expression. The GBAF complex exhibits overlapping genome localization and transcriptional targets as bromodomain and extraterminal domain-containing (BET) proteins, which are established AR coregulators. Our results demonstrate that GBAF is critical for coordinating SWI/SNF-BET cooperation and uncover a new druggable target for AR-positive prostate cancers, including those resistant to androgen deprivation or antiandrogen therapies. SIGNIFICANCE: Advanced prostate cancers resistant to androgen receptor antagonists are still susceptible to nontoxic BRD9 inhibitors, making them a promising alternative for halting AR signaling in progressed disease.

PBRM1 Regulates Stress Response in Epithelial Cells.

Polybromo1 (PBRM1) is a chromatin remodeler subunit highly mutated in cancer, particularly clear cell renal carcinoma. PBRM1 is a member of the SWI/SNF subcomplex, PBAF (PBRM1-Brg1/Brm-associated factors), and is characterized by six tandem bromodomains. Here we establish a role for PBRM1 in epithelial cell maintenance through the expression of genes involved in cell adhesion, metabolism, stress response, and apoptosis. In support of a general role for PBRM1 in stress response and apoptosis, we observe that loss of PBRM1 results in an increase in reactive oxygen species generation and a decrease in cellular viability under stress conditions. We find that loss of PBRM1 promotes cell growth under favorable conditions but is required for cell survival under conditions of cellular stress.

Small Molecule Targeting of Specific BAF (mSWI/SNF) Complexes for HIV Latency Reversal.

The persistence of a pool of latently HIV-1-infected cells despite combination anti-retroviral therapy treatment is the major roadblock for a cure. The BAF (mammalian SWI/SNF) chromatin remodeling complex is involved in establishing and maintaining viral latency, making it an attractive drug target for HIV-1 latency reversal. Here we report a high-throughput screen for inhibitors of BAF-mediated transcription in cells and the subsequent identification of a 12-membered macrolactam. This compound binds ARID1A-specific BAF complexes, prevents nucleosomal positioning, and relieves transcriptional repression of HIV-1. Through this mechanism, these compounds are able to reverse HIV-1 latency in an in vitro T cell line, an ex vivo primary cell model of HIV-1 latency, and in patient CD4+ T cells without toxicity or T cell activation. These macrolactams represent a class of latency reversal agents with unique mechanism of action, and can be combined with other latency reversal agents to improve reservoir targeting.