Cell autonomous expression of BCL6 is required to maintain lineage identity of mouse CCR6+ ILC3s.

Innate lymphoid cells (ILC) are similar to T helper (Th) cells in expression of cytokines and transcription factors. For example, RORγt is the lineage-specific transcription factor for both ILC3 and Th17 cells. However, the ILC counterpart for BCL6-expressing T follicular helper (Tfh) cells has not been defined. Here, we report that in the ILC compartment, BCL6 is selectively co-expressed with not only CXCR5 but also RORγt and CCR6 in ILC3 from multiple tissues. BCL6-deficient ILC3 produces enhanced levels of IL-17A and IL-22. More importantly, phenotypic and single-cell ATAC-seq analysis show that absence of BCL6 in mature ILC3 increases the numbers of ILC1 and transitional cells co-expressing ILC3 and ILC1 marker genes. A lineage-tracing experiment further reveals BCL6+ ILC3 to ILC1 trans-differentiation under steady state. Finally, microbiota promote BCL6 expression in colonic CCR6+ ILC3 and thus reinforce their stability. Collectively, our data have demonstrated that CCR6+ ILC3 have both Th17 and Tfh programs and that BCL6 expression in these cells functions to maintain their lineage identity.

Matrix association region/scaffold attachment region: the crucial player in defining the positions of chromosome breaks mediated by bile acid-induced apoptosis in nasopharyngeal epithelial cells.

BACKGROUND: It has been found that chronic rhinosinusitis (CRS) increases the risk of developing nasopharyngeal carcinoma (NPC). CRS can be caused by gastro-oesophageal reflux (GOR) that may reach nasopharynx. The major component of refluxate, bile acid (BA) has been found to be carcinogenic and genotoxic. BA-induced apoptosis has been associated with various cancers. We have previously demonstrated that BA induced apoptosis and gene cleavages in nasopharyngeal epithelial cells. Chromosomal cleavage occurs at the early stage of both apoptosis and chromosome rearrangement. It was suggested that chromosome breaks tend to cluster in the region containing matrix association region/scaffold attachment region (MAR/SAR). This study hypothesised that BA may cause chromosome breaks at MAR/SAR leading to chromosome aberrations in NPC. This study targeted the AF9 gene located at 9p22 because 9p22 is a deletion hotspot in NPC. METHODS: Potential MAR/SAR sites were predicted in the AF9 gene by using MAR/SAR prediction tools. Normal nasopharyngeal epithelial cells (NP69) and NPC cells (TWO4) were treated with BA at neutral and acidic pH. Inverse-PCR (IPCR) was used to identify chromosome breaks in SAR region (contains MAR/SAR) and non-SAR region (does not contain MAR/SAR). To map the chromosomal breakpoints within the AF9 SAR and non-SAR regions, DNA sequencing was performed. RESULTS: In the AF9 SAR region, the gene cleavage frequencies of BA-treated NP69 and TWO4 cells were significantly higher than those of untreated control. As for the AF9 non-SAR region, no significant difference in cleavage frequency was detected between untreated and BA-treated cells. A few breakpoints detected in the SAR region were mapped within the AF9 region that was previously reported to translocate with the mixed lineage leukaemia (MLL) gene in an acute lymphoblastic leukaemia (ALL) patient. CONCLUSIONS: Our findings suggest that MAR/SAR may be involved in defining the positions of chromosomal breakages induced by BA. Our report here, for the first time, unravelled the relation of these BA-induced chromosomal breakages to the AF9 chromatin structure.

Matrix association region/scaffold attachment region (MAR/SAR) sequence: its vital role in mediating chromosome breakages in nasopharyngeal epithelial cells via oxidative stress-induced apoptosis.

BACKGROUND: Oxidative stress is known to be involved in most of the aetiological factors of nasopharyngeal carcinoma (NPC). Cells that are under oxidative stress may undergo apoptosis. We have previously demonstrated that oxidative stress-induced apoptosis could be a potential mechanism mediating chromosome breakages in nasopharyngeal epithelial cells. Additionally, caspase-activated DNase (CAD) may be the vital player in mediating the chromosomal breakages during oxidative stress-induced apoptosis. Chromosomal breakage occurs during apoptosis and chromosome rearrangement. Chromosomal breakages tend to cluster in certain regions, such as matrix association region/scaffold attachment region (MAR/SAR). We hypothesised that oxidative stress-induced apoptosis may result in chromosome breaks preferentially at the MAR/SAR sites. The AF9 gene at 9p22 was targeted in this study because 9p22 is a deletion site commonly found in NPC. RESULTS: By using MAR/SAR recognition signature (MRS), potential MAR/SAR sites were predicted in the AF9 gene. The predicted MAR/SAR sites precisely match to the experimentally determined MAR/SARs. Hydrogen peroxide (H2O2) was used to induce apoptosis in normal nasopharyngeal epithelial cells (NP69) and NPC cells (HK1). Nested inverse polymerase chain reaction was employed to identify the AF9 gene cleavages. In the SAR region, the gene cleavage frequency of H2O2-treated cells was significantly higher than that of the non-treated cells. A few chromosomal breakages were detected within the AF9 region which was previously found to be involved in the mixed lineage leukaemia (MLL)-AF9 translocation in an acute lymphoblastic leukaemia patient. As for the non-SAR region, no significant difference in the gene cleavage frequency was found between the untreated control and H2O2-treated cells. Furthermore, H2O2-induced cleavages within the SAR region were reduced by caspase-3 inhibitor, which indirectly inhibits CAD. CONCLUSIONS: These results reaffirm our previous findings that oxidative stress-induced apoptosis could be one of the potential mechanisms underlying chromosome breakages in nasopharyngeal epithelial cells. MAR/SAR may play a vital role in defining the location of chromosomal breakages mediated by oxidative stress-induced apoptosis, where CAD is the major nuclease.

Oxidative stress-induced chromosome breaks within the ABL gene: a model for chromosome rearrangement in nasopharyngeal carcinoma.

BACKGROUND: The mechanism underlying chromosome rearrangement in nasopharyngeal carcinoma (NPC) remains elusive. It is known that most of the aetiological factors of NPC trigger oxidative stress. Oxidative stress is a potent apoptotic inducer. During apoptosis, chromatin cleavage and DNA fragmentation occur. However, cells may undergo DNA repair and survive apoptosis. Non-homologous end joining (NHEJ) pathway has been known as the primary DNA repair system in human cells. The NHEJ process may repair DNA ends without any homology, although region of microhomology (a few nucleotides) is usually utilised by this DNA repair system. Cells that evade apoptosis via erroneous DNA repair may carry chromosomal aberration. Apoptotic nuclease was found to be associated with nuclear matrix during apoptosis. Matrix association region/scaffold attachment region (MAR/SAR) is the binding site of the chromosomal DNA loop structure to the nuclear matrix. When apoptotic nuclease is associated with nuclear matrix during apoptosis, it potentially cleaves at MAR/SAR. Cells that survive apoptosis via compromised DNA repair may carry chromosome rearrangement contributing to NPC tumourigenesis. The Abelson murine leukaemia (ABL) gene at 9q34 was targeted in this study as 9q34 is a common region of loss in NPC. This study aimed to identify the chromosome breakages and/or rearrangements in the ABL gene in cells undergoing oxidative stress-induced apoptosis. RESULTS: In the present study, in silico prediction of MAR/SAR was performed in the ABL gene. More than 80% of the predicted MAR/SAR sites are closely associated with previously reported patient breakpoint cluster regions (BCR). By using inverse polymerase chain reaction (IPCR), we demonstrated that hydrogen peroxide (H2O2)-induced apoptosis in normal nasopharyngeal epithelial and NPC cells led to chromosomal breakages within the ABL BCR that contains a MAR/SAR. Intriguingly, we detected two translocations in H2O2-treated cells. Region of microhomology was found at the translocation junctions. This observation is consistent with the operation of microhomology-mediated NHEJ. CONCLUSIONS: Our findings suggested that oxidative stress-induced apoptosis may participate in chromosome rearrangements of NPC. A revised model for oxidative stress-induced apoptosis mediating chromosome rearrangement in NPC is proposed.

Bile acids at neutral and acidic pH induce apoptosis and gene cleavages in nasopharyngeal epithelial cells: implications in chromosome rearrangement.

BACKGROUND: Chronic rhinosinusitis (CRS) increases the risk of developing nasopharyngeal carcinoma (NPC) while nasopharyngeal reflux is known to be one of the major aetiological factors of CRS. Bile acid (BA), the component of gastric duodenal contents, has been recognised as a carcinogen. BA-induced apoptosis was suggested to be involved in human malignancies. Cells have the potential and tendency to survive apoptosis. However, cells that evade apoptosis upon erroneous DNA repair may carry chromosome rearrangements. Apoptotic nuclease, caspase-activated deoxyribonuclease (CAD) has been implicated in mediating translocation in leukaemia. We hypothesised that BA-induced apoptosis may cause chromosome breaks mediated by CAD leading to chromosome rearrangement in NPC. This study targeted the AF9 gene located at 9p22 because 9p22 is one of the most common deletion sites in NPC. METHODS: We tested the ability of BA at neutral and acidic pH in inducing phosphatidylserine (PS) externalisation, reactive oxygen species (ROS) production, mitochondrial membrane potential (MMP) disruption, and caspase 3/7 activity in normal nasopharyngeal epithelial (NP69) and NPC (TWO4) cells. Inverse-PCR (IPCR) was employed to detect AF9 gene cleavages. To investigate the role of CAD in mediating these cleavages, caspase inhibition was performed. IPCR bands representing AF9 cleaved fragments were sequenced. RESULTS: BA-treated cells showed higher levels of PS externalisation, ROS production, MMP loss and caspase 3/7 activity than untreated control cells. The effect of BA in the induction of these intracellular events was enhanced by acid. BA at neutral and acidic pH also induced significant cleavage of the AF9 gene. These BA-induced gene cleavages were inhibited by Z-DEVD-FMK, a caspase-3 inhibitor. Intriguingly, a few chromosome breaks were identified within the AF9 region that was previously reported to participate in reciprocal translocation between the mixed lineage leukaemia (MLL) and AF9 genes in an acute lymphoblastic leukaemia (ALL) patient. CONCLUSIONS: These findings suggest a role for BA-induced apoptosis in mediating chromosome rearrangements in NPC. In addition, CAD may be a key player in chromosome cleavages mediated by BA-induced apoptosis. Persistent exposure of sinonasal tract to gastric duodenal refluxate may increase genomic instability in surviving cells.

Potential role of oxidative stress-induced apoptosis in mediating chromosomal rearrangements in nasopharyngeal carcinoma.

BACKGROUND: Genetic aberrations have been identified in nasopharyngeal carcinoma (NPC), however, the underlying mechanism remains elusive. There are increasing evidences that the apoptotic nuclease caspase-activated deoxyribonuclease (CAD) is one of the players leading to translocation in leukemia. Oxidative stress, which has been strongly implicated in carcinogenesis, is a potent apoptotic inducer. Most of the NPC etiological factors are known to induce oxidative stress. Although apoptosis is a cell death process, cells possess the potential to survive apoptosis upon DNA repair. Eventually, the surviving cells may carry rearranged chromosomes. We hypothesized that oxidative stress-induced apoptosis may cause chromosomal breaks mediated by CAD. Upon erroneous DNA repair, cells that survive apoptosis may harbor chromosomal rearrangements contributing to NPC pathogenesis. This study focused on the AF9 gene at 9p22, a common deletion region in NPC. We aimed to propose a possible model for molecular mechanism underlying the chromosomal rearrangements in NPC. RESULTS: In the present study, we showed that hydrogen peroxide (H2O2) induced apoptosis in NPC (HK1) and normal nasopharyngeal epithelial (NP69) cells, as evaluated by flow cytometric analyses. Activity of caspases 3/7 was detected in H2O2-treated cells. This activity was inhibited by caspase inhibitor (CI). By nested inverse polymerase chain reaction (IPCR), we demonstrated that oxidative stress-induced apoptosis in HK1 and NP69 cells resulted in cleavages within the breakpoint cluster region (BCR) of the AF9 gene. The gene cleavage frequency detected in the H2O2-treated cells was found to be significantly higher than untreated control. We further found that treatment with CI, which indirectly inhibits CAD, significantly reduced the chromosomal breaks in H2O2-cotreated cells. Intriguingly, a few breakpoints were mapped within the AF9 region that was previously reported to translocate with the mixed lineage leukemia (MLL) gene in acute lymphoblastic leukemia (ALL) patient. CONCLUSIONS: In conclusion, our findings suggested that oxidative stress-induced apoptosis could be one of the mechanisms underlying the chromosomal rearrangements in NPC. CAD may play an important role in chromosomal cleavages mediated by oxidative stress-induced apoptosis. A potential model for oxidative stress-induced apoptosis mediating chromosomal rearrangements in NPC is proposed.