KAT6A and ENL Form an Epigenetic Transcriptional Control Module to Drive Critical Leukemogenic Gene-Expression Programs.

Epigenetic programs are dysregulated in acute myeloid leukemia (AML) and help enforce an oncogenic state of differentiation arrest. To identify key epigenetic regulators of AML cell fate, we performed a differentiation-focused CRISPR screen in AML cells. This screen identified the histone acetyltransferase KAT6A as a novel regulator of myeloid differentiation that drives critical leukemogenic gene-expression programs. We show that KAT6A is the initiator of a newly described transcriptional control module in which KAT6A-catalyzed promoter H3K9ac is bound by the acetyl-lysine reader ENL, which in turn cooperates with a network of chromatin factors to induce transcriptional elongation. Inhibition of KAT6A has strong anti-AML phenotypes in vitro and in vivo, suggesting that KAT6A small-molecule inhibitors could be of high therapeutic interest for mono-therapy or combinatorial differentiation-based treatment of AML. SIGNIFICANCE: AML is a poor-prognosis disease characterized by differentiation blockade. Through a cell-fate CRISPR screen, we identified KAT6A as a novel regulator of AML cell differentiation. Mechanistically, KAT6A cooperates with ENL in a "writer-reader" epigenetic transcriptional control module. These results uncover a new epigenetic dependency and therapeutic opportunity in AML. This article is highlighted in the In This Issue feature, p. 587.

PALP: A rapid imaging technique for stratifying ferroptosis sensitivity in normal and tumor tissues in situ.

Ferroptosis is an emerging cancer suppression strategy. However, how to select cancer patients for treating with ferroptosis inducers remains challenging. Here, we develop photochemical activation of membrane lipid peroxidation (PALP), which uses targeted lasers to induce localized polyunsaturated fatty acyl (PUFA)-lipid peroxidation for reporting ferroptosis sensitivity in cells and tissues. PALP captured by BODIPY-C11 can be suppressed by lipophilic antioxidants and iron chelation, and is dependent on PUFA-lipid levels. Moreover, we develop PALPv2, for studying lipid peroxidation on selected membranes along the z axis in live cells using two-photon microscopes. Using PALPv1, we detect PUFA-lipids in multiple tissues, and validate a PUFA-phospholipid reduction during muscle aging as previously reported. Patterns of PALPv1 signals across multiple cancer cell types in vitro and in vivo are concordant with their ferroptosis susceptibility and PUFA-phospholipid levels. We envision that PALP will enable rapid stratification of ferroptosis sensitivity in cancer patients and facilitate PUFA-lipid research.

LSD1 inhibition sustains T cell invigoration with a durable response to PD-1 blockade.

Exhausted CD8+ T cells are key targets of immune checkpoint blockade therapy and their ineffective reinvigoration limits the durable benefit in some cancer patients. Here, we demonstrate that histone demethylase LSD1 acts to enforce an epigenetic program in progenitor exhausted CD8+ T cells to antagonize the TCF1-mediated progenitor maintenance and to promote terminal differentiation. Consequently, genetic perturbation or small molecules targeting LSD1 increases the persistence of the progenitor exhausted CD8+ T cells, which provide a sustained source for the proliferative conversion to numerically larger terminally exhausted T cells with tumor-killing cytotoxicity, thereby leading to effective and durable responses to anti-PD1 therapy. Collectively, our findings provide important insights into epigenetic mechanisms that regulate T cell exhaustion and have important implications for durable immunotherapy.

KDM5A suppresses PML-RARα target gene expression and APL differentiation through repressing H3K4me2.

Epigenetic abnormalities are frequently involved in the initiation and progression of cancers, including acute myeloid leukemia (AML). A subtype of AML, acute promyelocytic leukemia (APL), is mainly driven by a specific oncogenic fusion event of promyelocytic leukemia-RA receptor fusion oncoprotein (PML-RARα). PML-RARα was reported as a transcription repressor through the interaction with nuclear receptor corepressor and histone deacetylase complexes leading to the mis-suppression of its target genes and differentiation blockage. Although previous studies were mainly focused on the connection of histone acetylation, it is still largely unknown whether alternative epigenetics mechanisms are involved in APL progression. KDM5A is a demethylase of histone H3 lysine 4 di- and tri-methylations (H3K4me2/3) and a transcription corepressor. Here, we found that the loss of KDM5A led to APL NB4 cell differentiation and retarded growth. Mechanistically, through epigenomics and transcriptomics analyses, KDM5A binding was detected in 1889 genes, with the majority of the binding events at promoter regions. KDM5A suppressed the expression of 621 genes, including 42 PML-RARα target genes, primarily by controlling the H3K4me2 in the promoters and 5' end intragenic regions. In addition, a recently reported pan-KDM5 inhibitor, CPI-455, on its own could phenocopy the differentiation effects as KDM5A loss in NB4 cells. CPI-455 treatment or KDM5A knockout could greatly sensitize NB4 cells to all-trans retinoic acid-induced differentiation. Our findings indicate that KDM5A contributed to the differentiation blockage in the APL cell line NB4, and inhibition of KDM5A could greatly potentiate NB4 differentiation.

The human mitochondrial 12S rRNA m4C methyltransferase METTL15 is required for mitochondrial function.

Mitochondrial DNA gene expression is coordinately regulated both pre- and post-transcriptionally, and its perturbation can lead to human pathologies. Mitochondrial rRNAs (mt-rRNAs) undergo a series of nucleotide modifications after release from polycistronic mitochondrial RNA precursors, which is essential for mitochondrial ribosomal biogenesis. Cytosine N4-methylation (m4C) at position 839 (m4C839) of the 12S small subunit mt-rRNA was identified decades ago; however, its biogenesis and function have not been elucidated in detail. Here, using several approaches, including immunofluorescence, RNA immunoprecipitation and methylation assays, and bisulfite mapping, we demonstrate that human methyltransferase-like 15 (METTL15), encoded by a nuclear gene, is responsible for 12S mt-rRNA methylation at m4C839 both in vivo and in vitro We tracked the evolutionary history of RNA m4C methyltransferases and identified a difference in substrate preference between METTL15 and its bacterial ortholog rsmH. Additionally, unlike the very modest impact of a loss of m4C methylation in bacterial small subunit rRNA on the ribosome, we found that METTL15 depletion results in impaired translation of mitochondrial protein-coding mRNAs and decreases mitochondrial respiration capacity. Our findings reveal that human METTL15 is required for mitochondrial function, delineate the evolution of methyltransferase substrate specificities and modification patterns in rRNA, and highlight a differential impact of m4C methylation on prokaryotic ribosomes and eukaryotic mitochondrial ribosomes.

Mettl17, a regulator of mitochondrial ribosomal RNA modifications, is required for the translation of mitochondrial coding genes.

Embryonic stem cells (ESCs) are pluripotent stem cells with the ability to self-renew and to differentiate into any cell types of the 3 germ layers. Recent studies have demonstrated that there is a strong connection between mitochondrial function and pluripotency. Here, we report that methyltransferase like (Mettl) 17, identified from the clustered regularly interspaced short palindromic repeats knockout screen, is required for proper differentiation of mouse embryonic stem cells (mESCs). Mettl17 is located in mitochondria through its N-terminal targeting sequence and specifically interacts with 12S mitochondrial ribosomal RNA (mt-rRNA) as well as small subunits of mitochondrial ribosome (MSSUs). Loss of Mettl17 affects the stability of both 12S mt-rRNA and its associated proteins of MSSUs. We further showed that Mettl17 is an S-adenosyl methionine (SAM)-binding protein and regulates mitochondrial ribosome function in a SAM-binding-dependent manner. Loss of Mettl17 leads to around 70% reduction of m4C840 and 50% reduction of m5C842 of 12S mt-rRNA, revealing the first regulator of the m4C840 and indicating a crosstalk between the 2 nearby modifications. The defects of mitochondrial ribosome caused by deletion of Mettl17 lead to the impaired translation of mitochondrial protein-coding genes, resulting in significant changes in mitochondrial oxidative phosphorylation and cellular metabolome, which are important for mESC pluripotency.-Shi, Z., Xu, S., Xing, S., Yao, K., Zhang, L., Xue, L., Zhou, P., Wang, M., Yan, G., Yang, P., Liu, J., Hu, Z., Lan, F. Mettl17, a regulator of mitochondrial ribosomal RNA modifications, is required for the translation of mitochondrial coding genes.

A multifunctional composite Fe3O4/MOF/l-cysteine for removal, magnetic solid phase extraction and fluorescence sensing of Cd(ii).

Fe3O4/MOF (metal organic framework)/l-cysteine was synthesized and applied for the removal of Cd(ii) from wastewater. The adsorption kinetics and isotherms were investigated, and the results indicated that the adsorption obeyed the pseudo-second-order kinetic model and Langmuir isotherm. The maximum adsorption capacity was calculated to be 248.24 mg g-1. Fe3O4/MOF/l-cysteine was further applied to determine trace amounts of Cd(ii) in real water samples using ICP-AES (inductively coupled plasma-atomic emission spectroscopy) based on magnetic solid-phase extraction (MSPE). The determination limit was 10.6 ng mL-1. Additionally, Fe3O4/MOF/l-cysteine can also be used as a fluorescent sensor for "turn-off" detection of Cd(ii), and the detection limit was 0.94 ng mL-1.

Nono, a Bivalent Domain Factor, Regulates Erk Signaling and Mouse Embryonic Stem Cell Pluripotency.

Nono is a component of the para-speckle, which stores and processes RNA. Mouse embryonic stem cells (mESCs) lack para-speckles, leaving the function of Nono in mESCs unclear. Here, we find that Nono functions as a chromatin regulator cooperating with Erk to regulate mESC pluripotency. We report that Nono loss results in robust self-renewing mESCs with epigenomic and transcriptomic features resembling the 2i (GSK and Erk inhibitors)-induced "ground state." Erk interacts with and is required for Nono localization to a subset of bivalent genes that have high levels of poised RNA polymerase. Nono loss compromises Erk activation and RNA polymerase poising at its target bivalent genes in undifferentiated mESCs, thus disrupting target gene activation and differentiation. These findings argue that Nono collaborates with Erk signaling to regulate the integrity of bivalent domains and mESC pluripotency.

A primary role of TET proteins in establishment and maintenance of De Novo bivalency at CpG islands.

Ten Eleven Translocation (TET) protein-catalyzed 5mC oxidation not only creates novel DNA modifications, such as 5hmC, but also initiates active or passive DNA demethylation. TETs' role in the crosstalk with specific histone modifications, however, is largely elusive. Here, we show that TET2-mediated DNA demethylation plays a primary role in the de novo establishment and maintenance of H3K4me3/H3K27me3 bivalent domains underlying methylated DNA CpG islands (CGIs). Overexpression of wild type (WT), but not catalytic inactive mutant (Mut), TET2 in low-TET-expressing cells results in an increase in the level of 5hmC with accompanying DNA demethylation at a subset of CGIs. Most importantly, this alteration is sufficient in making de novo bivalent domains at these loci. Genome-wide analysis reveals that these de novo synthesized bivalent domains are largely associated with a subset of essential developmental gene promoters, which are located within CGIs and are previously silenced due to DNA methylation. On the other hand, deletion of Tet1 and Tet2 in mouse embryonic stem (ES) cells results in an apparent loss of H3K27me3 at bivalent domains, which are associated with a particular set of key developmental gene promoters. Collectively, this study demonstrates the critical role of TET proteins in regulating the crosstalk between two key epigenetic mechanisms, DNA methylation and histone methylation (H3K4me3 and H3K27me3), particularly at CGIs associated with developmental genes.

Adsorption of azo dyes from aqueous solution by the hybrid MOFs/GO.

In this work, a hybrid of chromium(III) terephthalate metal organic framework (MIL-101) and graphene oxide (GO) was synthesized and its performance in the removal of azo dyes (Amaranth, Sunset Yellow, and Carmine) from water was evaluated. The adsorption for azo dyes on MIL-101/GO was compared with that of MIL-101, and it was found that the addition of GO enhanced the stability of MIL-101 in water and increased the adsorption capacity. The maximum adsorption capacities of MIL-101/GO were 111.01 mg g(-1) for Amaranth, 81.28 mg g(-1) for Sunset Yellow, and 77.61 mg g(-1) for Carmine. The adsorption isotherms and kinetics were investigated, showing that the adsorption fits the Freundlich isotherm and the pseudo-second-order kinetic model. The recyclability of MIL-101/GO was shown by the regeneration by acetone. The high adsorption capability and excellent reusability make MIL-101/GO a competent adsorbent for the removal dyes from aqueous solution.

Renal denervation using focused infrared fiber lasers: a potential treatment for hypertension.

BACKGROUND AND OBJECTIVE: Renal denervation has recently become of great interest as a potential treatment for resistant hypertension. Denervation techniques using radio frequency (RF) or ultrasound energy sources have already been explored in literature. In this study, we investigate the use of lasers as a potential energy source for renal denervation. In vitro studies are performed in porcine/ovine renal arteries with focused laser beams at 980 nm, 1210 nm, and 1700 nm to study the ability to damage renal nerves without causing injury to non-target tissue structures like the endothelium. Then, a 980 nm laser catheter prototype is built and used to demonstrate in vivo renal denervation in ovine renal arteries. SUBJECTS AND METHODS: This study utilizes fiber coupled infrared lasers at 980 nm, 1210 nm, and 1700 nm. In vitro laser denervation studies at 980 nm are performed in both porcine and ovine renal arteries to study the ability of focused laser beams to damage renal nerves without injuring the endothelium. In vitro studies using lasers close to the lipid absorption lines at 1210 nm and 1700 nm are also performed in porcine renal arteries to study the possibility of selectively damaging the renal nerves by targeting the lipid myelin sheaths surrounding the nerves. Then, a laser catheter prototype is designed and built for in vivo renal denervation in ovine renal arteries using the 980 nm laser (powers ranging from 2 to 4 W, 5 seconds per exposure). Histochemical evaluations of the frozen sections are performed using methylthiazolyldiphenyl-tetrazolium bromide (MTT) assay. RESULTS: Histochemical analysis of in vitro laser treatments at 980 nm in porcine and ovine renal arteries show clear evidence of laser-induced renal nerve damage without injury to the endothelium and part of the media. No evidence of selective nerve damage is observed using the 1210 nm and 1700 nm lasers with the current treatment parameters. Histochemical analysis of in vivo laser treatments in ovine renal arteries using a focused 980 nm laser show clear evidence of renal nerve damage with depths of damage extending > 1.5 mm from the artery wall. Sections with laser-induced damage to the media/adventitia at depths of > 1 mm without injury to the endothelium are also observed. CONCLUSIONS: We demonstrate the use of focused lasers as an attractive energy source for causing renal nerve damage without injury to the artery wall and thus, may have potential therapeutic applications for conditions such as resistant hypertension, where renal denervation has been shown to be a promising form of treatment.

Field trial of active remote sensing using a high-power short-wave infrared supercontinuum laser.

Field trial results of a 5 W all-fiber broadband supercontinuum (SC) laser covering the short-wave infrared (SWIR) wavelength bands from ~1.55 to 2.35 µm are presented. The SC laser is kept on a 12 story tower at the Wright Patterson Air Force Base and propagated through the atmosphere to a target 1.6 km away. Beam quality of the SC laser after propagating through 1.6 km is studied using a SWIR camera and show a near diffraction limited beam with an M(2) value of <1.3. The SC laser is used as the illumination source to perform spectral reflectance measurements of various samples at 1.6 km, and the results are seen to be in good agreement with in-lab measurements using a conventional lamp source. Spectral stability measurements are performed after atmospheric propagation through 1.6 km and show a relative variability of ~4%-8% across the spectrum depending on the atmospheric turbulence effects. Spectral stability measurements are also performed in-lab and show a relative variability of <0.6% across the spectrum.

Power scalable >25 W supercontinuum laser from 2 to 2.5 µm with near-diffraction-limited beam and low output variability.

A power scalable thulium-doped fiber-amplifier-based supercontinuum (SC) laser covering the shortwave infrared region from 2 to 2.5 µm is demonstrated. The SC laser has an average power up to 25.7 W and a spectral density of >12 dBm/nm. Power scalability of the laser is proven by showing that the SC laser maintains a nearly constant spectral output, beam quality (M(2) measurements), and output spectral stability as the SC average power is scaled from 5 to 25.7 W average output power. We verify that the SC laser beam is nearly diffraction limited with an M(2)<1.2 for all power levels. Output spectral stability measurements with power scaling show a radiometric variability of <0.8% across the entire SC spectrum.