Polycomb Repressive Complexes in Hox Gene Regulation: Silencing and Beyond: The Functional Dynamics of Polycomb Repressive Complexes in Hox Gene Regulation.

The coordinated expression of the Hox gene family encoding transcription factors is critical for proper embryonic development and patterning. Major efforts have thus been dedicated to understanding mechanisms controlling Hox expression. In addition to the temporal and spatial sequential activation of Hox genes, proper embryonic development requires that Hox genes get differentially silenced in a cell-type specific manner as development proceeds. Factors contributing to Hox silencing include the polycomb repressive complexes (PRCs), which control gene expression through epigenetic modifications. This review focuses on PRC-dependent regulation of the Hox genes and is aimed at integrating the growing complexity of PRC functional properties in the context of Hox regulation. In particular, mechanisms underlying PRC binding dynamics as well as a series of studies that have revealed the impact of PRC on the 3D organization of the genome is discussed, which has a significant role on Hox regulation during development.

Quantitative determination by UHPLC-MS/MS of 18 common drugs of abuse and metabolites, including THC and OH-THC, in volumetric dried blood spots: a sustainable method with minimally invasive sampling.

The increased use of drugs of abuse urges forensic toxicologists to create quick, simple, minimally invasive sampling techniques for biological fluids combined with analytical methods assuring accurate results. To this purpose, a method was developed aimed at quantifying 18 drugs of abuse and metabolites in DBS. Validation of the method was conducted by spiking blank whole blood with the analytes on Capitainer® B cards. The extracts were analyzed by a targeted UHPLC-MS/MS method. Linear calibration was achieved in the range of 1-100 ng/mL for: amphetamine, MDA, MDMA, methamphetamine, cocaine, codeine, benzoylecgonine, cocaethylene, morphine, 6-MAM, buprenorphine, methadone, EDDP, ketamine, norbuprenorphine norketamine, THC, and OH-THC. Experimental LOD was found at 0.5 ng/mL for all analytes except for norbuprenorphine, THC and THC-OH which yielded a LOD of 1 ng/mL. Intra- and inter-day accuracy was satisfactory with bias% resulting within 5%. Evaluation of intra- and inter-day precision yielded CV% values within 20%, for all compounds except EDDP. Average extraction recovery calculated at low (2 ng/mL) and high (75 ng/mL) concentration levels was 63% while average matrix effect determined at the same levels was found to be within 85% - 115% for all analytes except from codeine (70%) and MDMA (131%). The method was applied to authentic blood samples spotted onto the DBS card and the minimum value detected was 1.3 ng/mL. HPLC-MS/MS proved capable to identify all the targeted analytes at low concentrations in the small blood volumes obtainable from DBS cards, which in turn confirmed to be effective and sustainable micro-sampling devices.

Inhibition of the lysine demethylase LSD1 modulates the balance between inflammatory and antiviral responses against coronaviruses.

Innate immune responses to coronavirus infections are highly cell specific. Tissue-resident macrophages, which are infected by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in patients but are inconsistently infected in vitro, exert critical but conflicting effects by secreting both antiviral type I interferons (IFNs) and tissue-damaging inflammatory cytokines. Steroids, the only class of host-targeting drugs approved for the treatment of coronavirus disease 2019 (COVID-19), indiscriminately suppress both responses, possibly impairing viral clearance. Here, we established in vitro cell culture systems that enabled us to separately investigate the cell-intrinsic and cell-extrinsic proinflammatory and antiviral activities of mouse macrophages infected with the prototypical murine coronavirus MHV-A59. We showed that the nuclear factor κB-dependent inflammatory response to viral infection was selectively inhibited by loss of the lysine demethylase LSD1, which was previously implicated in innate immune responses to cancer, with negligible effects on the antiviral IFN response. LSD1 ablation also enhanced an IFN-independent antiviral response, blocking viral egress through the lysosomal pathway. The macrophage-intrinsic antiviral and anti-inflammatory activity of Lsd1 inhibition was confirmed in vitro and in a humanized mouse model of SARS-CoV-2 infection. These results suggest that LSD1 controls innate immune responses against coronaviruses at multiple levels and provide a mechanistic rationale for potentially repurposing LSD1 inhibitors for COVID-19 treatment.

Mammalian SWI/SNF chromatin remodeling complexes promote tyrosine kinase inhibitor resistance in EGFR-mutant lung cancer.

Acquired resistance to tyrosine kinase inhibitors (TKI), such as osimertinib used to treat EGFR-mutant lung adenocarcinomas, limits long-term efficacy and is frequently caused by non-genetic mechanisms. Here, we define the chromatin accessibility and gene regulatory signatures of osimertinib sensitive and resistant EGFR-mutant cell and patient-derived models and uncover a role for mammalian SWI/SNF chromatin remodeling complexes in TKI resistance. By profiling mSWI/SNF genome-wide localization, we identify both shared and cancer cell line-specific gene targets underlying the resistant state. Importantly, genetic and pharmacologic disruption of the SMARCA4/SMARCA2 mSWI/SNF ATPases re-sensitizes a subset of resistant models to osimertinib via inhibition of mSWI/SNF-mediated regulation of cellular programs governing cell proliferation, epithelial-to-mesenchymal transition, epithelial cell differentiation, and NRF2 signaling. These data highlight the role of mSWI/SNF complexes in supporting TKI resistance and suggest potential utility of mSWI/SNF inhibitors in TKI-resistant lung cancers.

HOX13-dependent chromatin accessibility underlies the transition towards the digit development program.

Hox genes encode transcription factors (TFs) that establish morphological diversity in the developing embryo. The similar DNA-binding motifs of the various HOX TFs contrast with the wide-range of HOX-dependent genetic programs. The influence of the chromatin context on HOX binding specificity remains elusive. Here, we used the developing limb as a model system to compare the binding specificity of HOXA13 and HOXD13 (HOX13 hereafter), which are required for digit formation, and HOXA11, involved in forearm/leg development. We find that upon ectopic expression in distal limb buds, HOXA11 binds sites normally HOX13-specific. Importantly, these sites are loci whose chromatin accessibility relies on HOX13. Moreover, we show that chromatin accessibility specific to the distal limb requires HOX13 function. Based on these results, we propose that HOX13 TFs pioneer the distal limb-specific chromatin accessibility landscape for the proper implementation of the distal limb developmental program.

PRC2-Associated Chromatin Contacts in the Developing Limb Reveal a Possible Mechanism for the Atypical Role of PRC2 in HoxA Gene Expression.

Preventing inappropriate gene expression in time and space is as fundamental as triggering the activation of tissue- or cell-type-specific factors at the correct developmental stage and in the correct cells. Here, we study the impact of Polycomb repressive complex 2 (PRC2) function on HoxA gene regulation. We analyze chromatin conformation of the HoxA cluster and its regulatory regions and show that in addition to the well-known role of PRC2 in silencing Hox genes via direct binding, its function is required for the changes in HoxA long-range interactions distinguishing proximal limbs from distal limbs. This effect stems from the differential PRC2 occupancy over the HoxA cluster and, at least in part, from the ability of PRC2-bound loci to engage in long-range contacts. Unexpectedly, PRC2 also impacts chromatin conformation in a way that promotes enhancer-promoter contacts required for proper HoxA expression, pointing to a dual role of PRC2 in gene regulation.

The remote transcriptional control of Hox genes.

Since the discovery by Ed Lewis that the order of Hox genes on the chromosome reflects the partitioning of their patterning function along the anterior-posterior axis of the developing fruit fly embryo, extensive efforts have been dedicated to uncovering the regulatory events underlying the collinear expression of Hox genes. These studies have revealed various aspects of Hox regulation, including short-range and long-range transcriptional enhancers, insulator elements and non-coding RNAs. With the development of technologies allowing for high resolution probing of chromatin architecture, notably Chromosome Conformation Capture (3C)-based techniques, a clear relationship is emerging between long-range regulation of Hox genes and the three-dimensional organization of the genome. Here, we provide an overview of these studies and in particular we discuss the functional relevance of genome compartmentalization, CTCF- mediated insulation and the Polycomb Repressive Complexes in the remote control of Hox genes.