The transcription factor Cdx2 regulates inflammasome activity through expression of the NLRP3 suppressor TRIM31 to maintain intestinal homeostasis.

The intestine-specific transcription factor Cdx2 is essential for intestinal homeostasis and has been implicated in the pathogenesis of disorders including inflammatory bowel disease. However, the mechanism by which Cdx2 influences intestinal disease is not clear. Here, we present evidence supporting a novel Cdx2-TRIM31-NLRP3 (NLR family, pyrin domain containing 3) signaling pathway, which may represent a mechanistic means by which Cdx2 impacts intestinal inflammation. We found that conditional loss of Cdx function resulted in an increase in proinflammatory cytokines, including tumor necrosis factor alpha, interleukin (IL)-1ß, and IL-6, in the mouse colon. We further show that TRIM31, which encodes a suppressor of NLRP3 (a central component of the NLRP3 inflammasome complex) is a novel Cdx2 target gene and is attenuated in the colon of Cdx conditional mutants. Consistent with this, we found that attenuation of TRIM31 in Cdx mutant intestine occurs concomitant with elevated levels of NLRP3 and an increase in inflammasome products. We demonstrate that specific inhibition of NLRP3 activity significantly reduced IL-1ß and IL-6 levels and extended the life span of Cdx conditional mutants, reflecting the therapeutic potential of targeting NLRP3. Tumor necrosis factor-alpha levels were also induced independent of NLRP3, potentially via elevated activity of the proinflammatory NF-κB signaling pathway in Cdx mutants. Finally, in silico analysis of ulcerative colitis patients revealed attenuation of CDX2 and TRIM31 expression coincident with enhanced expression of proinflammatory cytokines. We conclude that the novel Cdx2-TRIM31-NLRP3 signaling pathway promotes proinflammatory cytokine expression, and its inhibition may have therapeutic potential in human intestinal diseases.

Cdx2 Regulates Intestinal EphrinB1 through the Notch Pathway.

The majority of colorectal cancers harbor loss-of-function mutations in APC, a negative regulator of canonical Wnt signaling, leading to intestinal polyps that are predisposed to malignant progression. Comparable murine APC alleles also evoke intestinal polyps, which are typically confined to the small intestine and proximal colon, but do not progress to carcinoma in the absence of additional mutations. The Cdx transcription factors Cdx1 and Cdx2 are essential for homeostasis of the intestinal epithelium, and loss of Cdx2 has been associated with more aggressive subtypes of colorectal cancer in the human population. Consistent with this, concomitant loss of Cdx1 and Cdx2 in a murine APC mutant background leads to an increase in polyps throughout the intestinal tract. These polyps also exhibit a villous phenotype associated with the loss of EphrinB1. However, the basis for these outcomes is poorly understood. To further explore this, we modeled Cdx2 loss in SW480 colorectal cancer cells. We found that Cdx2 impacted Notch signaling in SW480 cells, and that EphrinB1 is a Notch target gene. As EphrinB1 loss also leads to a villus tumor phenotype, these findings evoke a mechanism by which Cdx2 impacts colorectal cancer via Notch-dependent EphrinB1 signaling.

Role of Cdx factors in early mesodermal fate decisions.

Murine cardiac and hematopoietic progenitors are derived from Mesp1+ mesoderm. Cdx function impacts both yolk sac hematopoiesis and cardiogenesis in zebrafish, suggesting that Cdx family members regulate early mesoderm cell fate decisions. We found that Cdx2 occupies a number of transcription factor loci during embryogenesis, including key regulators of both cardiac and blood development, and that Cdx function is required for normal expression of the cardiogenic transcription factors Nkx2-5 and Tbx5 Furthermore, Cdx and Brg1, an ATPase subunit of the SWI/SNF chromatin remodeling complex, co-occupy a number of loci, suggesting that Cdx family members regulate target gene expression through alterations in chromatin architecture. Consistent with this, we demonstrate loss of Brg1 occupancy and altered chromatin structure at several cardiogenic genes in Cdx-null mutants. Finally, we provide evidence for an onset of Cdx2 expression at E6.5 coinciding with egression of cardiac progenitors from the primitive streak. Together, these findings suggest that Cdx functions in multi-potential mesoderm to direct early cell fate decisions through transcriptional regulation of several novel target genes, and provide further insight into a potential epigenetic mechanism by which Cdx influences target gene expression.

Cdx2 Regulates Gene Expression through Recruitment of Brg1-associated Switch-Sucrose Non-fermentable (SWI-SNF) Chromatin Remodeling Activity.

The packaging of genomic DNA into nucleosomes creates a barrier to transcription that can be relieved through ATP-dependent chromatin remodeling via complexes such as the switch-sucrose non-fermentable (SWI-SNF) chromatin remodeling complex. The SWI-SNF complex remodels chromatin via conformational or positional changes of nucleosomes, thereby altering the access of transcriptional machinery to target genes. The SWI-SNF complex has limited ability to bind to sequence-specific elements, and, therefore, its recruitment to target loci is believed to require interaction with DNA-associated transcription factors. The Cdx family of homeodomain transcript ion factors (Cdx1, Cdx2, and Cdx4) are essential for a number of developmental programs in the mouse. Cdx1 and Cdx2 also regulate intestinal homeostasis throughout life. Although a number of Cdx target genes have been identified, the basis by which Cdx members impact their transcription is poorly understood. We have found that Cdx members interact with the SWI-SNF complex and make direct contact with Brg1, a catalytic member of SWI-SNF. Both Cdx2 and Brg1 co-occupy a number of Cdx target genes, and both factors are necessary for transcriptional regulation of such targets. Finally, Cdx2 and Brg1 occupancy occurs coincident with chromatin remodeling at some of these loci. Taken together, our findings suggest that Cdx transcription factors regulate target gene expression, in part, through recruitment of Brg1-associated SWI-SNF chromatin remodeling activity.

Identification of novel retinoic acid target genes.

Retinoic acid is required for diverse ontogenic processes and as such identification of the genes and pathways affected by retinoic acid is critical to understanding these pleiotropic effects. The presomitic mesoderm of the E8.5 mouse embryo is composed of undifferentiated cells that are depleted of retinoic acid, yet are competent to respond to the retinoid signal. We have exploited these properties to use this tissue to identify novel retinoic acid-responsive genes, including candidate target genes, by treating E8.5 embryos with retinoic acid and assessing changes in gene expression in the presomitic mesoderm by microarray analysis. This exercise yielded a cohort of genes that were differentially expressed in response to exogenous retinoic acid exposure. Among these were a number of previously characterized retinoic acid targets, validating this approach. In addition, we recovered a number of novel candidate target genes which were confirmed as retinoic acid-responsive by independent analysis. Chromatin immunoprecipitation assays revealed retinoic acid receptor occupancy of the promoters of certain of these genes. We further confirmed direct retinoic acid regulation of the F11r gene, a new RA target, using tissue culture models. Our results reveal a significant number of potential RA targets implicated in embryonic development and offer a novel in vivo system for better understanding of retinoid-dependent transcription.

Cdx1 interacts physically with a subset of Hox proteins.

Cdx and Hox gene families encode homeodomain-containing transcription factors involved in anterior-posterior vertebral patterning. Although Cdx proteins are direct transcriptional regulators of Hox gene expression, both Hox and Cdx proteins are known to interact with other homeodomain transcription factors, leading us to speculate that Cdx and Hox proteins may also interact physically. In testing this, we found that that Cdx1 is indeed capable of associating with a subset of Hox proteins. This interaction is localized to the homeodomain region of both classes of proteins, is reliant on specific arginine residues in helix I of the Hox homeodomain, and is further modulated by N-terminal Hox sequences. More promiscuous interactions were seen with Hox proteins expressed in vivo, suggestive of bridging factors or post-translational modifications. Finally, we demonstrate that this interaction modulates Cdx-Hox transcriptional activity on a Hox-responsive element. This study is the first example of Cdx-Hox protein interactions and suggests that such complexes may modulate Hox and/or Cdx function.

Performance at different stimulus intensities with the within- and across-channel adaptive tests of temporal resolution.

OBJECTIVE: The Adaptive Tests of Temporal Resolution (ATTR©) software provides within-channel (WC) and across-channel (AC) adaptive measures of temporal resolution that are feasible for clinical applications. The purpose of the present study was to obtain normative values for young adults on two of the ATTR tests: the narrow-band noise within-channel (NBN-WC) test and the narrow-band noise across-channel (NBN-AC) test, at different stimulus intensities. DESIGN: Gap detection thresholds were measured at five sensation levels. A Latin square design was used to control for practice effects. STUDY SAMPLE: The NBN-WC group and the NBN-AC group each consisted of 25 young adults with normal hearing. RESULTS: Gap detection thresholds for both conditions decreased with increasing stimulus intensity, and stimulus intensities above 20 dB SL were not associated with large improvements in performance. Variability was larger in the NBN-AC condition. Values obtained for the NBC-WC condition were very similar to previously reported ATTR results despite equipment and design differences. CONCLUSION: Results provide normative values for NBN-WC and NBN-AC performance on the ATTR and suggest that the ATTR is a robust test for clinical use.

Evaluation of otoacoustic emissions in clinically normal alert puppies.

OBJECTIVE: To evaluate distortion product otoacoustic emission (DPOAE) measurements in puppies with normal hearing. ANIMALS: 23 clinically normal 7.5-to 10.5-week-old puppies. PROCEDURES: A cross-sectional study was performed. The DPOAE measurements were obtained with a commercially available distortion product otoacoustic measurement system and were performed in a quiet, non-sound-attenuated room. All measurements were obtained from alert puppies and were repeated 1 or 2 times to ensure that the measurements were replicable. Results that were a minimum of 8 dB higher than the noise floor were accepted. Values from the first trial in which emissions were obtained at all test frequencies were used for analysis. RESULTS: Otoacoustic emission measurements were easily obtained, robust, reliable, and consistent with auditory brainstem response and behavioral results. CONCLUSIONS AND CLINICAL RELEVANCE: Hearing screening in alert puppies can be accomplished reliably and rapidly with otoacoustic emissions testing. Results supported the possibility of the use of DPOAE measurement in hearing screening of dogs.