KMT2D regulates activation, localization, and integrin expression by T-cells.

Individuals with Kabuki syndrome present with immunodeficiency; however, how pathogenic variants in the gene encoding the histone-modifying enzyme lysine methyltransferase 2D (KMT2D) lead to immune alterations remain poorly understood. Following up on our prior report of KMT2D-altered integrin expression in B-cells, we performed targeted analyses of KMT2D's influence on integrin expression in T-cells throughout development (thymocytes through peripheral T-cells) in murine cells with constitutive- and conditional-targeted Kmt2d deletion. Using high-throughput RNA-sequencing and flow cytometry, we reveal decreased expression (both at the transcriptional and translational levels) of a cluster of leukocyte-specific integrins, which perturb aspects of T-cell activation, maturation, adhesion/localization, and effector function. H3K4me3 ChIP-PCR suggests that these evolutionary similar integrins are under direct control of KMT2D. KMT2D loss also alters multiple downstream programming/signaling pathways, including integrin-based localization, which can influence T-cell populations. We further demonstrated that KMT2D deficiency is associated with the accumulation of murine CD8+ single-positive (SP) thymocytes and shifts in both human and murine peripheral T-cell populations, including the reduction of the CD4+ recent thymic emigrant (RTE) population. Together, these data show that the targeted loss of Kmt2d in the T-cell lineage recapitulates several distinct features of Kabuki syndrome-associated immune deficiency and implicates epigenetic mechanisms in the regulation of integrin signaling.

Cis-regulatory atlas of primary human CD4+ T cells.

Cis-regulatory elements (CRE) are critical for coordinating gene expression programs that dictate cell-specific differentiation and homeostasis. Recently developed self-transcribing active regulatory region sequencing (STARR-Seq) has allowed for genome-wide annotation of functional CREs. Despite this, STARR-Seq assays are only employed in cell lines, in part, due to difficulties in delivering reporter constructs. Herein, we implemented and validated a STARR-Seq-based screen in human CD4+ T cells using a non-integrating lentiviral transduction system. Lenti-STARR-Seq is the first example of a genome-wide assay of CRE function in human primary cells, identifying thousands of functional enhancers and negative regulatory elements (NREs) in human CD4+ T cells. We find an unexpected difference in nucleosome organization between enhancers and NRE: enhancers are located between nucleosomes, whereas NRE are occupied by nucleosomes in their endogenous locations. We also describe chromatin modification, eRNA production, and transcription factor binding at both enhancers and NREs. Our findings support the idea of silencer repurposing as enhancers in alternate cell types. Collectively, these data suggest that Lenti-STARR-Seq is a successful approach for CRE screening in primary human cell types, and provides an atlas of functional CREs in human CD4+ T cells.

Therapeutic sensitivity to Rac GTPase inhibition requires consequential suppression of mTORC1, AKT, and MEK signaling in breast cancer.

Rac GTPases have oncogenic roles in cell growth, survival, and migration. We tested response to the Rac inhibitor EHT1864 in a panel of breast cancer cell lines. EHT1864-induced growth inhibition was associated with dual inhibition of the PI3K/AKT/mTORC1 and MEK/ERK pathways. Breast cancer cells harboring PIK3CA mutations or HER2 overexpression were most sensitive to Rac inhibition, suggesting that such oncogenic alterations link Rac activation with PI3K/AKT/mTORC1 and MEK/ERK signaling. Interestingly, EHT1864 decreased activation of the mTORC1 substrate p70S6K earlier than AKT inhibition, suggesting that Rac may activate mTORC1/p70S6K independently of AKT. Comparison of the growth-inhibitory profile of EHT1864 to 137 other anti-cancer drugs across 656 cancer cell lines revealed significant correlation with the p70S6K inhibitor PF-4708671. We confirmed that Rac complexes contain MEK1/2 and ERK1/2, but also contain p70S6K; these interactions were disrupted by EHT1864. Pharmacokinetic profiles revealed that EHT1864 was present in mouse plasma at concentrations effective in vitro for approximately 1 h after intraperitoneal administration. EHT1864 suppressed growth of HER2+ tumors, and enhanced response to anti-estrogen treatment in ER+ tumors. Further therapeutic development of Rac inhibitors for HER2+ and PIK3CA-mutant cancers is warranted.

Pyruvic oxime nitrification and copper and nickel resistance by a Cupriavidus pauculus, an active heterotrophic nitrifier-denitrifier.

Heterotrophic nitrifiers synthesize nitrogenous gasses when nitrifying ammonium ion. A Cupriavidus pauculus, previously thought an Alcaligenes sp. and noted as an active heterotrophic nitrifier-denitrifier, was examined for its ability to produce nitrogen gas (N2) and nitrous oxide (N2O) while heterotrophically nitrifying the organic substrate pyruvic oxime [CH3-C(NOH)-COOH]. Neither N2 nor N2O were produced. Nucleotide and phylogenetic analyses indicated that the organism is a member of a genus (Cupriavidus) known for its resistance to metals and its metabolism of xenobiotics. The microbe (a Cupriavidus pauculus designated as C. pauculus strain UM1) was examined for its ability to perform heterotrophic nitrification in the presence of Cu(2+) and Ni(2+) and to metabolize the xenobiotic phenol. The bacterium heterotrophically nitrified well when either 1 mM Cu(2+) or 0.5 mM Ni(2+) was present in either enriched or minimal medium. The organism also used phenol as a sole carbon source in either the presence or absence of 1 mM Cu(2+) or 0.5 mM Ni(2+). The ability of this isolate to perform a number of different metabolisms, its noteworthy resistance to copper and nickel, and its potential use as a bioremediation agent are discussed.