Loss of function of ENT3 drives histiocytosis and inflammation through TLR-MAPK signaling.

Histiocytoses are inflammatory myeloid neoplasms often driven by somatic activating mutations in mitogen-activated protein kinase (MAPK) cascade genes. H syndrome is an inflammatory genetic disorder caused by germ line loss-of-function mutations in SLC29A3, encoding the lysosomal equilibrative nucleoside transporter 3 (ENT3). Patients with H syndrome are predisposed to develop histiocytosis, yet the mechanism is unclear. Here, through phenotypic, molecular, and functional analysis of primary cells from a cohort of patients with H syndrome, we reveal the molecular pathway leading to histiocytosis and inflammation in this genetic disorder. We show that loss of function of ENT3 activates nucleoside-sensing toll-like receptors (TLR) and downstream MAPK signaling, inducing cytokine secretion and inflammation. Importantly, MEK inhibitor therapy led to resolution of histiocytosis and inflammation in a patient with H syndrome. These results demonstrate a yet-unrecognized link between a defect in a lysosomal transporter and pathological activation of MAPK signaling, establishing a novel pathway leading to histiocytosis and inflammation.

Human Dendritic Cell Enrichment and Their Activation of T Cells.

Dendritic cells (DCs) enable the immune system to mount and modulate precisely targeted responses to various threats across the organism by bridging innate and adaptive immunity. Historically, DCs have been classified as conventional (cDC) and plasmacytoid (pDC). More recently, cDCs were acknowledged as a heterogenous population composed of several subsets. Examining the functional diversity of cDCs in healthy homeostasis and pathology requires a robust experimental pipeline, beginning with an efficient enrichment protocol in preparation for cell sorting. Unfortunately, several commercial DC enrichment kits were developed before the discovery of the more recently described DC populations. Here, we detail two approaches to enrich human blood DCs or certain DC subsets and an in vitro protocol to examine DC stimulation of naïve T cells. The methods employed here overcome many hurdles encountered while enriching human DC subsets. Basic Protocol 1 describes a method that will enrich pDC, Axl Siglec6-DC (AS-DC), cDC1, DC2, DC3, monocytes, and human HLA+ cells by crosslinking unwanted cells to erythrocytes. Basic Protocol 2 describes the enrichment of pDC, AS-DC, cDC1, and DC2 but not DC3 via a highly efficient negative magnetic selection that is valuable in circumstances where DC3 is not required. Finally, Basic Protocol 3 describes a conventional protocol to perform a Mixed leucocyte Reaction (MLR) following the isolation of these DC subsets. These methods detail the advantages and pitfalls when isolating a heterogeneous population of cells. © 2023 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Human peripheral mononuclear phagocyte enrichment Basic Protocol 2: DC enrichment of pDC, cDC1, AS-DC, and DC2 but not DC3 Basic Protocol 3: Basic mixed lymphocyte reaction protocol with sorted human DC subsets.

Activation of Oncogenic Super-Enhancers Is Coupled with DNA Repair by RAD51.

DNA double-strand breaks (DSBs) are deleterious and tumorigenic but could also be essential for DNA-based processes. Yet the landscape of physiological DSBs and their role and repair are still elusive. Here, we mapped DSBs at high resolution in cancer and non-tumorigenic cells and found a transcription-coupled repair mechanism at oncogenic super-enhancers. At these super-enhancers the transcription factor TEAD4, together with various transcription factors and co-factors, co-localizes with the repair factor RAD51 of the homologous recombination pathway. Depletion of TEAD4 or RAD51 increases DSBs at RAD51/TEAD4 common binding sites within super-enhancers and decreases expression of related genes, which are mostly oncogenes. Co-localization of RAD51 with transcription factors at super-enhancers occurs in various cell types, suggesting a broad phenomenon. Together, our findings uncover a coupling between transcription and repair mechanisms at oncogenic super-enhancers, to control the hyper-transcription of multiple cancer drivers.

Tumor Suppressor Genes within Common Fragile Sites Are Active Players in the DNA Damage Response.

The role of common fragile sites (CFSs) in cancer remains controversial. Two main views dominate the discussion: one suggests that CFS loci are hotspots of genomic instability leading to inactivation of genes encoded within them, while the other view proposes that CFSs are functional units and that loss of the encoded genes confers selective pressure, leading to cancer development. The latter view is supported by emerging evidence showing that expression of a given CFS is associated with genome integrity and that inactivation of CFS-resident tumor suppressor genes leads to dysregulation of the DNA damage response (DDR) and increased genomic instability. These two viewpoints of CFS function are not mutually exclusive but rather coexist; when breaks at CFSs are not repaired accurately, this can lead to deletions by which cells acquire growth advantage because of loss of tumor suppressor activities. Here, we review recent advances linking some CFS gene products with the DDR, genomic instability, and carcinogenesis and discuss how their inactivation might represent a selective advantage for cancer cells.

WWOX guards genome stability by activating ATM.

Common fragile sites (CFSs) tend to break upon replication stress and have been suggested to be "hot spots" for genomic instability. Recent evidence, however, implies that in the wake of DNA damage, WW domain-containing oxidoreductase (WWOX, the gene product of the FRA16D fragile site), associates with ataxia telangiectasia-mutated (ATM) and regulates its activation to maintain genomic integrity.

Transcriptional regulation of the Ikzf1 locus.

Ikaros is a critical regulator of lymphocyte development and homeostasis; thus, understanding its transcriptional regulation is important from both developmental and clinical perspectives. Using a mouse transgenic reporter approach, we functionally characterized a network of highly conserved cis-acting elements at the Ikzf1 locus. We attribute B-cell and myeloid but not T-cell specificity to the main Ikzf1 promoter. Although this promoter was unable to counter local chromatin silencing effects, each of the 6 highly conserved Ikzf1 intronic enhancers alleviated silencing. Working together, the Ikzf1 enhancers provided locus control region activity, allowing reporter expression in a position and copy-independent manner. Only 1 of the Ikzf1 enhancers was responsible for the progressive upregulation of Ikaros expression from hematopoietic stem cells to lymphoid-primed multipotent progenitors to T-cell precursors, which are stages of differentiation dependent on Ikaros for normal outcome. Thus, Ikzf1 is regulated by both epigenetic and transcriptional factors that target its enhancers in both redundant and specific fashions to provide an expression profile supportive of normal lymphoid lineage progression and homeostasis. Mutations in the Ikzf1 regulatory elements and their interacting factors are likely to have adverse effects on lymphopoiesis and contribute to leukemogenesis.

H3K9me-independent gene silencing in fission yeast heterochromatin by Clr5 and histone deacetylases.

Nucleosomes in heterochromatic regions bear histone modifications that distinguish them from euchromatic nucleosomes. Among those, histone H3 lysine 9 methylation (H3K9me) and hypoacetylation have been evolutionarily conserved and are found in both multicellular eukaryotes and single-cell model organisms such as fission yeast. In spite of numerous studies, the relative contributions of the various heterochromatic histone marks to the properties of heterochromatin remain largely undefined. Here, we report that silencing of the fission yeast mating-type cassettes, which are located in a well-characterized heterochromatic region, is hardly affected in cells lacking the H3K9 methyltransferase Clr4. We document the existence of a pathway parallel to H3K9me ensuring gene repression in the absence of Clr4 and identify a silencing factor central to this pathway, Clr5. We find that Clr5 controls gene expression at multiple chromosomal locations in addition to affecting the mating-type region. The histone deacetylase Clr6 acts in the same pathway as Clr5, at least for its effects in the mating-type region, and on a subset of other targets, notably a region recently found to be prone to neo-centromere formation. The genomic targets of Clr5 also include Ste11, a master regulator of sexual differentiation. Hence Clr5, like the multi-functional Atf1 transcription factor which also modulates chromatin structure in the mating-type region, controls sexual differentiation and genome integrity at several levels. Globally, our results point to histone deacetylases as prominent repressors of gene expression in fission yeast heterochromatin. These deacetylases can act in concert with, or independently of, the widely studied H3K9me mark to influence gene silencing at heterochromatic loci.

The role of the chromatin remodeler Mi-2beta in hematopoietic stem cell self-renewal and multilineage differentiation.

The ability of somatic stem cells to self-renew and differentiate into downstream lineages is dependent on specialized chromatin environments that keep stem cell-specific genes active and key differentiation factors repressed but poised for activation. The epigenetic factors that provide this type of regulation remain ill-defined. Here we provide the first evidence that the SNF2-like ATPase Mi-2beta of the Nucleosome Remodeling Deacetylase (NuRD) complex is required for maintenance of and multilineage differentiation in the early hematopoietic hierarchy. Shortly after conditional inactivation of Mi-2beta, there is an increase in cycling and a decrease in quiescence in an HSC (hematopoietic stem cell)-enriched bone marrow population. These cycling mutant cells readily differentiate into the erythroid lineage but not into the myeloid and lymphoid lineages. Together, these effects result in an initial expansion of mutant HSC and erythroid progenitors that are later depleted as more differentiated proerythroblasts accumulate at hematopoietic sites exhibiting features of erythroid leukemia. Examination of gene expression in the mutant HSC reveals changes in the expression of genes associated with self-renewal and lineage priming and a pivotal role of Mi-2beta in their regulation. Thus, Mi-2beta provides the hematopoietic system with immune cell capabilities as well as with an extensive regenerative capacity.

Temporal and spatial control of HGC1 expression results in Hgc1 localization to the apical cells of hyphae in Candida albicans.

The human fungal pathogen Candida albicans can undergo a morphological transition from a unicellular yeast growth form to a multicellular hyphal growth form. During hyphal growth, cell division is asymmetric. Only the apical cell divides, whereas subapical cells remain in G(1), and cell surface growth is highly restricted to the tip of the apical cell. Hgc1, a hypha-specific, G(1) cyclin-like protein, is essential for hyphal development. Here, we report, using indirect immunofluorescence, that Hgc1 is preferentially localized to the dividing apical cells of hyphae. Hgc1 protein is rapidly degraded in a cell cycle-independent manner, and the protein turnover likely occurs in both the apical and the subapical cells of hyphae. In addition to rapid protein turnover, the HGC1 transcript is also dynamically regulated during cell cycle progression in hyphal growth. It is induced upon germ tube formation in early G(1); the transcript level is reduced during the G(1)/S transition and peaks again around the G(2)/M phase in the subsequent cell cycles. Transcription from the HGC1 promoter is essential for its apical cell localization, as Hgc1 no longer exhibits preferential apical localization when expressed under the MAL2 promoter. Using fluorescence in situ hybridization, the HGC1 transcript is detected only in the apical cells of hyphae, suggesting that HGC1 is transcribed in the apical cell. Therefore, the preferential localization of Hgc1 to the apical cells of hyphae results from the dynamic temporal and spatial control of HGC1 expression.

Hyphal tip-associated localization of Cdc42 is F-actin dependent in Candida albicans.

The rho-type GTPase Cdc42 is important for the establishment and maintenance of eukaryotic cell polarity. To examine whether Cdc42 is regulated during the yeast-to-hypha transition in Candida albicans, we constructed a green fluorescence protein (GFP)-Cdc42 fusion under the ACT1 promoter and observed its localization in live C. albicans cells. As in Saccharomyces cerevisiae, GFP-Cdc42 was observed around the entire periphery of the cell. In yeast-form cells of C. albicans, it clustered to the tips and sides of small buds as well as to the mother-daughter neck region of large-budded cells. Upon hyphal induction, GFP-Cdc42 clustered to the site of hyphal evagination and remained at the tips of the hyphae. This temporal and spatial localization of Cdc42 suggests that its activity is regulated during the yeast-to-hypha transition. In addition to the accumulation at the hyphal tip, GFP-Cdc42 was also seen as a band within the hyphal tube in cells that had undergone nuclear separation. With the F-actin-assembly inhibitor latrunculin A, we found that GFP-Cdc42 accumulation at the bud site in yeast-form cells is F-actin independent, whereas GFP-Cdc42 accumulation at the hyphal tip requires F-actin. Furthermore, disruption of the F-actin cytoskeleton impaired the transcriptional induction of hypha-specific genes. Therefore, hypha formation resembles mating in Saccharomyces cerevisiae in that both require F-actin for GFP-Cdc42 localization and efficient signaling.

Hyphal elongation is regulated independently of cell cycle in Candida albicans.

The mechanism for apical growth during hyphal morphogenesis in Candida albicans is unknown. Studies from Saccharomyces cerevisiae indicate that cell morphogenesis may involve cell cycle regulation by cyclin-dependent kinase. To examine whether this is the mechanism for hyphal morphogenesis, the temporal appearance of different spindle pole body and spindle structures, the cell cycle-regulated rearrangements of the actin cytoskeleton, and the phosphorylation state of the conserved Tyr19 of Cdc28 during the cell cycle were compared and found to be similar between yeast and serum-induced hyphal apical cells. These data suggest that hyphal elongation is not mediated by altering cell cycle progression or through phosphorylation of Tyr19 of Cdc28. We have also shown that germ tubes can evaginate before spindle pole body duplication, chitin ring formation, and DNA replication. Similarly, tip-associated actin polarization in each hypha occurs before the events of the G(1)/S transition and persists throughout the cell cycle, whereas cell cycle-regulated actin assemblies come and go. We have also shown that cells in phases other than G(1) can be induced to form hyphae. Hyphae induced from G(1) cells have no constrictions, and the first chitin ring is positioned in the germ tube at various distances from the base. Hyphae induced from budded cells have a constriction and a chitin ring at the bud neck, beyond which the hyphae continue to elongate with no further constrictions. Our data suggest that hyphal elongation and cell cycle morphogenesis programs are uncoupled, and each contributes to different aspects of cell morphogenesis.