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The molecular complexity of the bone marrow (BM) microenvironment and its response to stress are incompletely understood, despite its key role in the regulation of hematopoiesis. Here we map the transcriptional landscape of BM vascular, perivascular, and osteoblast niche populations at single-cell resolution at both homeostasis and under stress hematopoiesis. This analysis revealed a previously unappreciated level of cellular heterogeneity within the BM niche, identified novel cellular subsets, and resolved cellular sources of pro-hematopoietic growth factors, chemokines, and membrane-bound ligands. Under conditions of stress, our studies revealed a significant transcriptional remodeling of these niche elements, including an adipocytic skewing of the perivascular cells. Among the stress-induced changes, we observed that vascular Notch ligand delta-like ligands (Dll1,4) were downregulated. In the absence of vascular Dll4, hematopoietic stem cells (HSC) prematurely induced a myeloid transcriptional program. These findings refine our understanding of the cellular architecture of the BM niche, reveal a dynamic and heterogeneous molecular landscape that is highly sensitive to stress, and illustrate the utility of single cell transcriptomic data in systematically evaluating the regulation of hematopoiesis by discrete niche populations.

The NFIA transcription factor is a glioma tumor promoter. The mechanism by which NFIA regulates glioma cell survival and drug resistance is unknown. Here we report that the NFIA pro-survival effect is mediated via an NFIA-NFκB p65 feedforward loop. We examined effects of gain- and loss-of-function manipulations of NFIA and NFκB p65 on cell growth, apoptosis and sensitivity to chemotherapy in patient-derived GBM cells. NFIA inhibited apoptosis by activation of NFkB p65 and its downstream anti-apoptotic factors. Induction of NFκB by NFIA was required for apoptosis evasion, and inhibition of NFκB effectively reversed the NFIA anti-apoptotic effect. Conversely, NFIA knockdown decreased expression of NFκB and its downstream anti-apoptotic genes and increased baseline apoptosis. Using transcriptome- and genome-wide approach, we demonstrated that NFIA directly regulated NFκB transcription. In addition, we found that NFκB activated the NFIA transcription and that knockdown of NFIA was sufficient to attenuate the NFκB pro-survival effect, suggesting a reciprocal regulation between NFIA and NFκB in governing GBM cell growth and survival. Supporting this, NFIA and NFκB expression levels were highly correlated in human GBMs and patient-derived GBM cells. Interestingly, we found that NFIA has splice isoforms in human GBM, which significantly promoted growth of GBM cells and malignant transformation of normal astrocytes. Furthermore, NFIA and the two active isoforms potently increased activity of NFκB p65 in GBM cells. These findings suggest that GBM-active NFIA isoforms differentially affect GBM initiation and progression in part by activating NFκB p65. Collectively, these data define a novel NFIA-NFκB feedforward regulation that promotes GBM tumorigenesis and malignant behavior.

Paraoxonase-2 (PON2), a member of paraoxonase family, is an anti-oxidant enzyme that is implicated in cell survival. Recent evidence demonstrated that PON2 promotes apoptosis resistance in the selective tumor cells. Here, we report genetic regulation and role of PON2 in glioma. We showed that PON2 is highly expressed in glioblastomas (GBM) compared to non-tumor brains and that high PON2 expression is associated with poor survival in patients with GBM. Functional studies showed that knockdown of PON2 inhibits cell growth, induces apoptosis, and increases pro-apoptotic factor CHOP. Using transcriptome and transcription factor motif analysis, we showed that Nuclear Factor I-A (NFIA), a glioma-promoting transcription factor, positively regulates PON2 transcription and protein level and that PON2 and NFIA expression is highly correlated in GBM. Moreover, NFIA directly regulated PON2 transcription through binding to the PON2 promoter. Consistent with this finding, PON2 knockdown-induced growth inhibition is reversed by ectopic expression of NFIA. Furthermore, Nfia-deficient brain shows decrease in expression of PON2, providing genetic evidence that supports NFIA-PON2 regulatory relationship. Collectively, these data suggest that the pro-survival effect of PON2 is at least partly mediated by NFIA in GBM, advancing our understanding of the emerging role of PON2 in glioma.

The Nuclear Factor I-A (NFIA) transcription factor was recently identified as a glioma tumor promoter. We previously reported that NFIA inhibits chemotherapy-induced apoptosis glioblastoma (GBM) cells. However, the mechanism by which the NFIA regulates cell survival and drug resistance is not fully understood. We now report that NFIA enhances apoptosis evasion through activating the NFkB p65 and its downstream anti-apoptotic factors (TRAF1 and cIAPs). Induction of NFkB by NFIA is required to protect cells from apoptosis, and inhibition of NFkB (siRNA or inhibitors) effectively reversed NFIA anti-apoptotic effect. Conversely, NFIA knockdown decreased NFkB and anti-apoptotic gene expression and ultimately induced apoptosis. Mechanistic investigation demonstrated that NFIA positively regulates NFkB transcription and protein level. Interestingly we also found that NFkB activates the NFIA promoter and increases NFIA level and that knockdown of NFIA is sufficient to attenuate NFkB pro-survival effect, suggesting a reciprocal regulation between NFIA and NFkB. Consistent with our findings, NFIA and NFkB levels and expression are highly associated in human primary GBM and patient-derived GBM cells. Collectively, these data define previously unknown NFIA-NFkB feed-forward regulation that controls GBM cell survival and drug resistance.

The Nuclear Factor I-A (NFIA) transcription factor is a glioma tumor promoter. However, the mechanism by which NFIA regulates glioma cell survival and drug resistance is still unknown. Here we report that the NFIA pro-survival effect is mediated via an NFIA-NFκB p65 feedforward loop. We examined effects of gain- and loss-of-function manipulations of NFIA and NFκB p65 on cell growth, colony formation, apoptosis and sensitivity to chemotherapy in patient-derived glioma stem cell (GSC). Ectopic NFIA inhibited apoptosis by activation of NFkB p65 and its downstream anti-apoptotic factors. Induction of NFκB by NFIA was required for apoptosis evasion, and inhibition of NFκB effectively reversed the NFIA anti-apoptotic effect. Furthermore, overexpression of NFIA promoted colony formation of non-transformed glial cells. Conversely, NFIA knockdown decreased expression of NFκB and its downstream anti-apoptotic genes and increased baseline apoptosis. Using a genome-wide approach, we demonstrated that NFIA directly regulated NFκB transcription. Interestingly, we also found that NFκB activated the NFIA transcription and that knockdown of NFIA was sufficient to attenuate the NFκB pro-survival effect, suggesting a reciprocal regulation between NFIA and NFκB in governing GBM cell growth and survival. Supporting this, NFIA and NFκB expression levels were highly correlated in human GBMs and GSC. Collectively, these data define a novel NFIA-NFκB feedforward regulation that promotes GBM malignant behavior.

Background The nuclear factor I-A (NFIA) transcription factor promotes glioma growth and inhibits apoptosis in glioblastoma (GBM) cells. Here we report that the NFIA pro-survival effect in GBM is mediated in part via a novel NFIA-nuclear factor-kappaB (NFκB) p65 feed-forward loop. Methods We examined effects of gain- and loss-of-function manipulations of NFIA and NFκB p65 on each other's transcription, cell growth, apoptosis and sensitivity to chemotherapy in patient-derived GBM cells and established GBM cell lines. Results NFIA enhanced apoptosis evasion by activating NFκB p65 and its downstream anti-apoptotic factors tumor necrosis factor receptor-associated factor 1 (TRAF1) and cellular inhibitor of apoptosis proteins (cIAPs). Induction of NFκB by NFIA was required to protect cells from apoptosis, and inhibition of NFκB effectively reversed the NFIA anti-apoptotic effect. Conversely, NFIA knockdown decreased expression of NFκB and anti-apoptotic genes TRAF1 and cIAPs, and increased baseline apoptosis. NFIA positively regulated NFκB transcription and NFκB protein level. Interestingly, NFκB also activated the NFIA promoter and increased NFIA level, and knockdown of NFIA was sufficient to attenuate the NFκB pro-survival effect, suggesting a reciprocal regulation between NFIA and NFκB in governing GBM cell survival. Supporting this, NFIA and NFκB expression levels were highly correlated in human GBM and patient-derived GBM cells. Conclusions These data define a previously unknown NFIA-NFκB feed-forward regulation that may contribute to GBM cell survival.