Gut microbiota composition reflects disease severity and dysfunctional immune responses in patients with COVID-19.

OBJECTIVE: Although COVID-19 is primarily a respiratory illness, there is mounting evidence suggesting that the GI tract is involved in this disease. We investigated whether the gut microbiome is linked to disease severity in patients with COVID-19, and whether perturbations in microbiome composition, if any, resolve with clearance of the SARS-CoV-2 virus. METHODS: In this two-hospital cohort study, we obtained blood, stool and patient records from 100 patients with laboratory-confirmed SARS-CoV-2 infection. Serial stool samples were collected from 27 of the 100 patients up to 30 days after clearance of SARS-CoV-2. Gut microbiome compositions were characterised by shotgun sequencing total DNA extracted from stools. Concentrations of inflammatory cytokines and blood markers were measured from plasma. RESULTS: Gut microbiome composition was significantly altered in patients with COVID-19 compared with non-COVID-19 individuals irrespective of whether patients had received medication (p<0.01). Several gut commensals with known immunomodulatory potential such as Faecalibacterium prausnitzii, Eubacterium rectale and bifidobacteria were underrepresented in patients and remained low in samples collected up to 30 days after disease resolution. Moreover, this perturbed composition exhibited stratification with disease severity concordant with elevated concentrations of inflammatory cytokines and blood markers such as C reactive protein, lactate dehydrogenase, aspartate aminotransferase and gamma-glutamyl transferase. CONCLUSION: Associations between gut microbiota composition, levels of cytokines and inflammatory markers in patients with COVID-19 suggest that the gut microbiome is involved in the magnitude of COVID-19 severity possibly via modulating host immune responses. Furthermore, the gut microbiota dysbiosis after disease resolution could contribute to persistent symptoms, highlighting a need to understand how gut microorganisms are involved in inflammation and COVID-19.

miR-29b as a therapeutic agent for angiotensin II-induced cardiac fibrosis by targeting TGF-ß/Smad3 signaling.

Loss of miR-29 is associated with cardiac fibrosis. This study examined the role and therapeutic potential of miR-29 in mouse model of hypertension induced by angiotensin II (AngII). By using microRNA microarray, in situ hybridization, and real-time polymerase chain reaction, we found that AngII-induced cardiac fibrosis in the hypertensive heart and in cultured cardiac fibroblasts were associated with downregulation of miR-29a-c via a Smad3-dependent mechanism. In vitro knockdown of miR-29b enhanced but overexpression of miR-29b inhibited AngII-induced fibrosis, revealing a protective role of miR-29b in cardiac fibrosis in response to AngII. This was further demonstrated in vivo by the ability of overexpressing miR-29b in the mouse heart to prevent AngII-mediated cardiac fibrosis and cardiac dysfunction. Importantly, we also found that restored miR-29b in the established hypertensive heart was capable of blocking progressive cardiac fibrosis and improving cardiac dysfunction, demonstrating a therapeutic potential of miR-29b for chronic heart disease. Further studies revealed that targeting the transforming growth factor (TGF)-ß1 coding sequence region, thereby inhibiting TGF-ß/Smad3 signaling, could be a new mechanism by which miR-29b inhibited AngII-induced cardiac fibrosis. In conclusion, miR-29b plays a protective role in AngII-mediated cardiac remodeling and may be a therapeutic agent for cardiac fibrosis by targeting the TGF-ß/Smad3 pathway.

MicroRNA-29b inhibits diabetic nephropathy in db/db mice.

Inflammation and its consequent fibrosis are two main features of diabetic nephropathy (DN), but target therapy on these processes for DN remains yet ineffective. We report here that miR-29b is a novel therapeutic agent capable of inhibiting progressive renal inflammation and fibrosis in type 2 diabetes in db/db mice. Under diabetic conditions, miR-29b was largely downregulated in response to advanced glycation end (AGE) product, which was associated with upregulation of collagen matrix in mesangial cells via the transforming growth factor-ß (TGF-ß)/Smad3-dependent mechanism. These pathological changes were reversed by overexpressing miR-29b, but enhanced by knocking-down miR-29b. Similarly, loss of renal miR-29b was associated with progressive diabetic kidney injury, including microalbuminuria, renal fibrosis, and inflammation. Restored renal miR-29b by the ultrasound-based gene therapy was capable of attenuating diabetic kidney disease. Further studies revealed that inhibition of Sp1 expression, TGF-ß/Smad3-dependent renal fibrosis, NF-κB-driven renal inflammation, and T-bet/Th1-mediated immune response may be mechanisms associated with miR-29b treatment in db/db mice. In conclusion, miR-29b may play a protective role in diabetic kidney disease and may have therapeutic potential for diabetic kidney complication.

miR-29 inhibits bleomycin-induced pulmonary fibrosis in mice.

Loss of microRNA-29 (miR-29) is known to be a mechanism of transforming growth factor-ß (TGF-ß)-mediated pulmonary fibrosis, but the therapeutic implication of miR-29 for pulmonary fibrosis remains unexplored. The present study investigated whether miR-29 had therapeutic potential for lung disease induced by bleomycin in mice. In addition, the signaling mechanisms that regulated miR-29 expression were investigated in vivo and in vitro. We found that miR-29 was a downstream target gene of Smad3 and negatively regulated by TGF-ß/Smad signaling in fibrosis. This was evidenced by the findings that mice or pulmonary fibroblasts null for Smad3 were protected against bleomycin or TGF-ß1-induced loss of miR-29 along with fibrosis in vivo and in vitro. Interestingly, overexpression of miR-29 could in turn negatively regulated TGF-ß and connective tissue growth factor (CTGF) expression and Smad3 signaling. Therefore, Sleeping Beauty (SB)-mediated miR-29 gene transfer into normal and diseased lung tissues was capable of preventing and treating pulmonary fibrosis including inflammatory macrophage infiltration induced by bleomycin in mice. In conclusion, miR-29 is negatively regulated by TGF-ß/Smad3 and has a therapeutic potential for pulmonary fibrosis. SB-mediated miR-29 gene therapy is a non-invasive therapeutic strategy for lung disease associated with fibrosis.

Differential recruitment of methyl CpG-binding domain factors and DNA methyltransferases by the orphan receptor germ cell nuclear factor initiates the repression and silencing of Oct4.

The pluripotency gene Oct4 encodes a key transcription factor that maintains self-renewal of embryonic stem cell (ESC) and is downregulated upon differentiation of ESCs and silenced in somatic cells. A combination of cis elements, transcription factors, and epigenetic modifications, such as DNA methylation, mediates Oct4 gene expression. Here, we show that the orphan nuclear receptor germ cell nuclear factor (GCNF) initiates Oct4 repression and DNA methylation by the differential recruitment of methyl-CpG binding domain (MBD) and DNA methyltransferases (Dnmts) to the Oct4 promoter. When compared with wild-type ESCs and gastrulating embryos, Oct4 repression is lost and its proximal promoter is significantly hypomethylated in retinoic acid (RA)-differentiated GCNF(-/-) ESCs and GCNF(-/-) embryos. Efforts to characterize mediators of GCNF's repressive function and DNA methylation of the Oct4 promoter identified MBD3, MBD2, and de novo Dnmts as GCNF interacting factors. Upon differentiation, endogenous GCNF binds to the Oct4 proximal promoter and differentially recruits MBD3 and MBD2 as well as Dnmt3A. In differentiated GCNF(-/-) ESCs, recruitment of MBD3 and MBD2 as well as Dnmt3A to Oct4 promoter is lost and subsequently Oct4 repression and DNA methylation failed to occur. Hypomethylation of the Oct4 promoter is also observed in RA-differentiated MBD3(-/-) and Dnmt3A(-/-) ESCs, but not in MBD2(-/-) and Dnmt3B(-/-) ESCs. Thus, recruitment of MBD3, MBD2, and Dnmt3A by GCNF links two events: gene-specific repression and DNA methylation, which occur differentially at the Oct4 promoter. GCNF initiates the repression and epigenetic modification of Oct4 gene during ESC differentiation.

Differential recruitment of methylated CpG binding domains by the orphan receptor GCNF initiates the repression and silencing of Oct4 expression.

The pluripotent factor Oct4 is a key transcription factor that maintains embryonic stem (ES) cell self-renewal and is down-regulated upon the differentiation of ES cells and silenced in somatic cells. A combination of cis elements, transcription factors, and epigenetic modifications, such as DNA methylation, are involved in the regulation of Oct4 gene expression. Here we show that the orphan nuclear receptor GCNF initiates Oct4 repression and DNA methylation by the differential recruitment of MBD (methylated CpG binding domain) factors to the promoter. Compared with wild-type ES cells and gastrulating embryos, Oct4 repression is lost and its proximal promoter is significantly hypomethylated in RA-differentiated GCNF(-/-) ES cells. The Oct4 gene is reexpressed in some somatic cells of GCNF(-/-) embryos, showing that it has not been properly silenced coincident with reduced DNA methylation of its promoter. Efforts to characterize mediators of GCNF's repressive function and DNA methylation of the Oct4 promoter identified methyl-DNA binding proteins, MBD3 and MBD2, as GCNF-interacting factors. In P19 and ES cells, upon differentiation, endogenous GCNF binds to the Oct4 proximal promoter and differentially recruits MBD3 and MBD2. In differentiated GCNF(-/-) ES cells, recruitment of MBD3 and MBD2 to the Oct4 promoter is lost, and repression of Oct4 expression and DNA methylation fails to occur. RNA interference-mediated knockdown of MBD3 and/or MBD2 expression results in reduced Oct4 repression in differentiated P19 and ES cells. Repression of Oct4 expression and recruitment of MBD3 are maintained in de novo DNA methylation-deficient ES cells (Dnmt3A/3B-null cells), while MBD2 recruitment is lost. Thus, recruitment of MBD3 and MBD2 by GCNF links two events, gene-specific repression and DNA methylation, which occur differentially at the Oct4 promoter. GCNF initiates the repression and epigenetic modification of Oct4 gene during ES cell differentiation.

Genetic ablation of the amplified-in-breast cancer 1 inhibits spontaneous prostate cancer progression in mice.

Although the amplified-in-breast cancer 1 (AIB1; SRC-3, ACTR, or NCoA3) was defined as a coactivator for androgen receptor (AR) by in vitro studies, its role in AR-mediated prostate development and prostate cancer remained unexplored. We report here that AIB1 is expressed in the basal and stromal cells but not in the epithelial cells of the normal mouse prostates. AIB1 deficiency only slightly delayed prostate growth and had no effect on androgen-dependent prostate regeneration, suggesting an unessential role of AIB1 in AR function in the prostate. Surprisingly, when prostate tumorigenesis was induced by the SV40 transgene in transgenic adenocarcinoma of the mouse prostate (TRAMP) mice, AIB1 expression was observed in certain epithelial cells of the prostate intraepithelial neoplasia (PIN) and well-differentiated carcinoma and in almost all cells of the poorly differentiated carcinoma. After AIB1 was genetically inactivated in AIB1-/-/TRAMP mice, the progression of prostate tumorigenesis in most AIB1-/-/TRAMP mice was arrested at the well-differentiated carcinoma stage. Wild-type (WT)/TRAMP mice developed progressive, multifocal, and metastatic prostate tumors and died between 25 and 34 weeks. In contrast, AIB1-/-/TRAMP mice only exhibited PIN and early-stage well-differentiated carcinoma by 39 weeks. AIB1-/-/TRAMP prostates showed much lower cell proliferation than WT/TRAMP prostates. Most AIB1-/-/TRAMP mice could survive more than 35 weeks and died with other types of tumors or unknown reasons. Our results indicate that induction of AIB1 expression in partially transformed epithelial cells is essential for progression of prostate tumorigenesis into poorly differentiated carcinoma. Inhibition of AIB1 expression or function in the prostate epithelium may be a potential strategy to suppress prostate cancer initiation and progression.

Orphan nuclear receptor GCNF is required for the repression of pluripotency genes during retinoic acid-induced embryonic stem cell differentiation.

Embryonic stem (ES) cell pluripotency and differentiation are controlled by a network of transcription factors and signaling molecules. Transcription factors such as Oct4 and Nanog are required for self-renewal and maintain the undifferentiated state of ES cells. Decreases in the expression of these factors indicate the initiation of differentiation of ES cells. Inactivation of the gene encoding the orphan nuclear receptor GCNF showed that it plays an important role in the repression of Oct4 expression in somatic cells during early embryonic development. GCNF-/- ES cells were isolated to study the function of GCNF in the down-regulation of pluripotency genes during differentiation. Loss of repression of ES cell marker genes Oct4, Nanog, Sox2, FGF4, and Stella was observed upon treatment of GCNF-/- ES cells with retinoic acid. The loss of repression of pluripotency genes is either a direct or indirect consequence of loss of GCNF. Both the Oct4 and Nanog genes are direct targets of GCNF repression during ES cell differentiation and early mouse embryonic development. In contrast Sox2 and FGF4 are indirectly regulated by GCNF through Oct4. These findings establish a central role for GCNF in the repression of pluripotency gene expression during retinoic acid-induced ES cell differentiation.

Orphan nuclear receptor LRH-1 is required to maintain Oct4 expression at the epiblast stage of embryonic development.

Oct4 plays an essential role in maintaining the inner cell mass and pluripotence of embryonic stem (ES) cells. The expression of Oct4 is regulated by the proximal enhancer and promoter in the epiblast and by the distal enhancer and promoter at all other stages in the pluripotent cell lineage. Here we report that the orphan nuclear receptor LRH-1, which is expressed in undifferentiated ES cells, can bind to SF-1 response elements in the proximal promoter and proximal enhancer of the Oct4 gene and activate Oct4 reporter gene expression. LRH-1 is colocalized with Oct4 in the inner cell mass and the epiblast of embryos at early developmental stages. Disruption of the LRH-1 gene results in loss of Oct4 expression at the epiblast stage and early embryonic death. Using LRH-1(-/-) ES cells, we also show that LRH-1 is required to maintain Oct4 expression at early differentiation time points. In vitro and in vivo results show that LRH-1 plays an essential role in the maintenance of Oct4 expression in ES cells at the epiblast stage of embryonic development, thereby maintaining pluripotence at this crucial developmental stage prior to segregation of the primordial germ cell lineage at gastrulation.

Serum levels of WNT1-inducible signaling pathway protein-1 (WISP-1): a noninvasive biomarker of renal fibrosis in subjects with chronic kidney disease.

WNT1-inducible signaling pathway protein-1 (WISP-1) is an extracellular matrix-related protein that plays multiple roles in cellular physiology and pathology. Accumulating evidence shows that WISP-1 is involved in the process underlying fibrotic diseases. However, the correlation between WISP-1 and renal fibrosis is unknown. In this study, we hypothesized that WISP-1 levels might be correlated with renal fibrosis and could be used as a noninvasive biomarker to screen for renal fibrosis in patients with chronic kidney disease (CKD). We first measured the WISP-1 expression levels using a transforming growth factor-ß (TGF-ß)-induced renal fibrosis tubular epithelial cell (TEC) model and a mouse model of obstructive nephropathy. We then evaluated the correlation between serum WISP-1 levels and fibrosis scores in biopsy-proven renal fibrosis of patients with CKD. Based on the findings from both in vivo and in vitro studies, the levels of WISP-1 and fibrotic parameters (collagen I, fibronectin and α-smooth muscle actin) were significantly increased in the fibrotic models. Consistently, patients with focal proliferative IgA nephropathy, focal segmental glomerular sclerosis and diabetic nephropathy displayed markedly elevated serum WISP-1 levels and fibrosis scores of renal biopsies compared with normal subjects and patients with minimal change disease (P<0.05). Importantly, the serum WISP-1 levels were positively correlated with fibrosis scores in the renal biopsies of these patients (r=0.475, P=0.0001). Thus, serum WISP-1 levels may be used as a potential noninvasive biomarker of renal fibrosis in patients with CKD.

MicroRNAs in renal fibrosis.

MicroRNAs (miRNAs) are endogenous short non-coding RNAs that regulate most of important cellular processes by inhibiting gene expression through the post-transcriptional repression of their target mRNAs. In kidneys, miRNAs have been associated in renal development, homeostasis, and physiological functions. Results from clinical and experimental animal studies demonstrate that miRNAs play essential roles in the pathogenesis of various renal diseases. Chronic kidney diseases (CKD) is characterized by renal fibrosis. Transforming growth factor beta (TGF-ß) is recognized as a major mediator of renal fibrosis because it is able to stimulate the accumulation of extracellular matrix (ECM) proteins to impair normal kidney function. Recently, emerging evidence demonstrate the relationship between TGF-ß signaling and miRNAs expression during renal diseases. TGF-ß regulates expression of several microRNAs, such as miR-21, miR-192, miR-200, miR-433, and miR-29. MiR-21, miR-192, and miR-433 which are positively induced by TGF-ß signaling play a pathological role in kidney diseases. In contrast, members in both miR-29 and miR-200 families which are inhibited by TGF-ß signaling protect kidneys from renal fibrosis by suppressing the deposition of ECM and preventing epithelial-to-mesenchymal transition, respectively. Clinically, the presence of miRNAs in blood and urine has been examined to be early biomarkers for detecting renal diseases. From experimental animal studies of CKD, targeting microRNAs also provides evidence about therapeutic potential of miRNAs during renal diseases. Now, it comes to the stage to examine the exact mechanisms of miRNAs during the initiation and progression of renal diseases. Therefore, determining the function of miRNAs in renal fibrosis may facilitate the development of both early diagnosis and treatment of renal diseases.

Characterization of crab EcR and RXR homologs and expression during limb regeneration and oocyte maturation.

We report here complete coding sequences for the Uca pugilator homologs of the ecdysteroid (UpEcR) and retinoid-X receptors (UpRXR). Library screenings recovered cDNA clones containing a unique amino terminal open-reading frame (A/B domain) for each gene, most similar to insect B1 EcR and USP1/RXR isoforms. Splicing variants in the UpRXR ligand-binding domain were also identified, in a region critical for folding of Drosophila and lepidopteran USP. UpEcR and UpRXR proteins were able to associate, and both are required for binding to an ecdysteroid HRE; these interactions were not hormone-dependent. Ribonuclease protection assays (RPA) were conducted using A/B domain and 'common' (C or E) domain probes on RNA isolated from various stages of regenerating limb buds and ovaries. For several of the limb bud and ovarian stages examined, the relative level of A/B domain sequence protected was significantly less than common domain suggesting alternative amino terminal isoforms other than those isolated through cloning. This is the first report of UpEcR and UpRXR transcription during ovarian maturation, implicating the ovary as a potential target for hormonal control in Crustacea.

MicroRNA and nephropathy: emerging concepts.

Micro ribonucleic acids (miRNAs) are short noncoding RNAs that inhibit gene expression through the post-transcriptional repression of their target mRNAs. Increasing evidence shows that miRNAs have emerged as key players in diverse biologic processes. Aberrant miRNA expression is also closely related to various human diseases, including kidney diseases. From clinical and experimental animal studies, emerging evidence demonstrates a critical role for miRNAs in renal pathophysiology. Renal fibrosis is the hallmark of various chronic kidney diseases and transforming growth factor beta (TGF-ß) is recognized as a vital mediator of renal fibrosis because it can induce production of extracellular matrix proteins resulting in dysfunction of the kidneys. The relationship between TGF-ß signaling and miRNAs expression during renal diseases has been recently established. TGF-ß positively or negatively regulates expression of several miRNAs, such as miR-21, miR-192, miR-200, and miR-29. Both miR-192 and miR-21 are positively regulated by TGF-ß1/Smad3 signaling and play a pathological role in kidney diseases. Conversely, members of both miR-29 and miR-200 families are negatively regulated by TGF-ß/Smad3 and play a protective role in renal fibrosis by inhibiting the deposition of extracellular matrix and preventing epithelial-to-mesenchymal transition, respectively. Clinically, levels of miRNAs in circulation and urine may be potential biomarkers for detecting early stages of renal diseases and targeting miRNAs also provides promising therapeutic effects in rodent models of chronic kidney disease. However, mechanisms and roles of miRNAs under disease conditions remain to be explored. Thus, understanding the function of miRNAs in the pathogenesis of kidney diseases may offer an innovative approach for both early diagnosis and treatment of renal diseases.

TGF-ß/Smad signaling in kidney disease.

Chronic progressive kidney diseases typically are characterized by active renal fibrosis and inflammation. Transforming growth factor-ß1 (TGF-ß1) is a key mediator in the development of renal fibrosis and inflammation. TGF-ß1 exerts its biological effects by activating Smad2 and Smad3, which is regulated negatively by an inhibitory Smad7. In the context of fibrosis, although Smad3 is pathogenic, Smad2 and Smad7 are protective. Under disease conditions, Smads also interact with other signaling pathways, such as the mitogen-activated protein kinase and nuclear factor-κB pathways. In contrast to the pathogenic role of active TGF-ß1, latent TGF-ß1 plays a protective role in renal fibrosis and inflammation. Furthermore, recent studies have shown that TGF-ß/Smad signaling plays a regulating role in microRNA-mediated renal injury. Thus, targeting TGF-ß signaling by gene transfer of either Smad7 or microRNAs into diseased kidneys has been shown to retard progressive renal injury in a number of experimental models. In conclusion, TGF-ß/Smad signaling plays a critical role in renal fibrosis and inflammation. Advances in understanding of the mechanisms of TGF-ß/Smad signaling in renal fibrosis and inflammation during chronic kidney diseases should provide a better therapeutic strategy to combat kidney diseases.

Extra-germ cell expression of mouse nuclear receptor subfamily 6, group A, member 1 (NR6A1).

Nuclear receptor subfamily 6, group A, member 1 (NR6A1) is an orphan member of the nuclear receptor superfamily and is required for normal mouse embryonic development. In adult mice, NR6A1 is predominantly expressed in spermatogenic cells and growing oocytes of the gonads and has a role in female reproduction by modulating the transcription of the oocyte-specific genes bone morphogenetic protein 15 (Bmp15) and growth differentiation factor 9 (Gdf9). In our goal to further understand the functional role of NR6A1 during postnatal development, we generated a Nr6a1:beta-galactosidase (LacZ) knockin reporter (Nr6a1(LacZ/+)) mouse line in which the Nr6a1:LacZ fusion gene was expressed and then characterized Nr6a1 expression in these reporter mice by performing LacZ staining. Our RT-PCR analyses showed that Nr6a1 was expressed in a variety of somatic tissues (e.g., oviduct and lung) other than gonads of normal adult mice. In adult Nr6a1(LacZ/+) mice, robust LacZ staining was observed in the gametes of gonads. Strong positive LacZ staining was also observed in the sperm of the epididymis, epithelial cells of the oviduct, and bronchioles within the lung. In adult Nr6a1(LacZ/+) mice, positive LacZ staining was observed in other somatic tissues, including hippocampus, cerebral cortex, cerebellum, and thalamus of brain; pars intermedia and pars anterior of pituitary; parathyroid; and islet of pancreas. NR6A1 expression in sperm within the epididymis, epithelial cells in the oviduct, and bronchioles of the lung was further confirmed by immunohistochemical studies. Nr6a1 is expressed not only in the germ cells of mouse gonads but also in a variety of somatic tissues, including epididymis, oviduct, brain, and pituitary. The extra-germ cell expression of NR6A1 makes it a less attractive contraceptive.

Loss of orphan nuclear receptor GCNF function disrupts forebrain development and the establishment of the isthmic organizer.

The isthmic organizer, which is located at the midbrain-hindbrain boundary, is important for midbrain development. The mechanism by which the development of the organizer is initiated and maintained is not well understood. Inactivation of the gene encoding the orphan nuclear receptor, GCNF, diminishes the expression of secreted signaling molecules, Fgf8 and Wnt1, the paired box genes Pax2/5, En1/2, and homeodomain transcription factor Gbx2; all of which are essential for isthmic organizer function. In addition, full neuronal differentiation is not observed in the midbrain region of GCNF-/- embryos. Increased cell death may contribute to the loss of midbrain structure in GCNF-/- embryos. These results indicate that GCNF is required for establishment of the isthmic organizer, thereby regulating the midbrain development.

The embryonic function of germ cell nuclear factor is dependent on the DNA binding domain.

Germ cell nuclear factor (GCNF), an orphan nuclear receptor, is essential for mouse embryogenesis. GCNF specifically binds to a DR0 response element via its DNA binding domain (DBD) in vitro and functions as a repressor of gene transcription. To further study the role of GCNF during embryogenesis, we have employed a Cre/loxP strategy and generated a line of GCNF mutant mice (GCNF(lox/lox)) in which the 243-base pair DBD-encoding exon has been deleted in the germline. However, the ligand binding domain (LBD) of GCNF is still expressed at the mRNA and protein levels in the GCNF(lox/lox) mice. GCNF(lox/lox) mice die at 9.5-10.5 days postcoitum. The tailbuds of these mutant embryos protrude outside the yolk sac. Expression of Oct-4 in the somatic cells of GCNF(lox/lox) embryos at 8.25 days postcoitum was not silenced as in the GCNF(+/+) embryos. Therefore, GCNF(lox/lox) mice phenocopy the GCNF(-/-) mice. Our results indicate that the DBD is essential for the function of GCNF during early mouse embryogenesis, and that the LBD does not mediate any function independent of the DBD at this stage of embryonic development. Our results also suggest that GCNF is indeed a transcriptional factor that represses gene transcription mediated via its DBD.