Search

Down syndrome (DS), also known as trisomy 21 (T21), is associated with interferon (IFN) hypersensitivity, as well as predilections for Alzheimer’s dementia (AD) and various autoimmune diseases. IFN-α and IFN-γ receptors are encoded on chromosome 21 (Ch21). It remains unclear how other Ch21 genes contribute to the neuropathological features of DS/T21. This study tests the hypothesis that identifying IFN-stimulated response element (ISRE) control sites on Ch21 will mark novel candidate genes for DS/T21-related IFN hypersensitivity and neuropathology not previously reported to be associated with IFN functions. We performed whole chromosome searches of online databases. The general ISRE consensus and gamma interferon activation consensus sequences (GAS) were used for identifying IFN-stimulated response elements. Candidate genes were defined as those possessing two or more ISRE and/or GAS control sites within and/or upstream of the transcription start site. A literature search of gene functions was used to select the candidate genes most likely to explain neuropathology associated with IFN hypersensitivity. DOPEY2, TMEM50B, PCBP3, RCAN1, and SIM2 were found to meet the aforementioned gene search and functional criteria. These findings suggest that DOPEY2, TMEM50B, PCBP3, RCAN1, and SIM2 are genes which may be dysregulated in DS/T21 and may therefore serve as novel targets for treatments aimed at ameliorating the neuropathological features of DS/T21. Future studies should determine whether these genes are dysregulated in patients with DS, DS-related AD, and autoimmune diseases.

Invasive Aspergillosis (IA) has increased significantly in recent years in which about 90% is caused by Aspergillus fumigatus, and the mortality rate is on the rise. Its treatment has become a common problem clinically. Calcineurin (CaN) plays an important role in the morphogenesis and toxicity of Aspergillus fumigatus, making it an attractive antifungal target. Although the Calcineurin inhibitors FK506 and cyclosporine A have successfully applied in modern transplantation medicine, the systemic toxic and side effects of the two drugs have greatly limited their clinical antifungal applications. Previous studies have shown that CaN of the Aspergillus fumigatus is localized at the top and division of hyphae, and interactions of CaN with cbpA are closely related to the PxIxIT motif. The functioning of CaN is closely related to the PxIxIT motif. Calcineurin substrates or target proteins such as Crz1, SIm1, SIm2, Hph1, and CbpA all contain similar PxIxIT motifs, and similar motifs are present in the Aspergillus fumigatus KpsF gene sequence. We can find KpsF gene in the Aspergillus fumigatus genome through bioinformatics methods, which contains a similar motif in its sequence. Therefore, we can speculate that this motif may interact with Calcineurin, further illustrate the specific mechanism of pathogenesis of CaN in Aspergillus fumigatus, which will help us to have a deeper understanding of the pathogenesis of invasive aspergillosis and provide new ideas for us. By completing the construction of the Aspergillus fumigatus KpsF gene-deficient strain and over-exp strain, we found that the KpsF gene over-exp strain of Aspergillus fumigatus is significantly different from that of the control strain in morphology, and is different from the cbpA in calcium ion regulation. In the low concentration of Ca2+, KpsF may be involved in the negative feedback regulation of Calcineurin, and this negative feedback regulation is inhibited under the condition of high concentration Ca2+. Keywords: Aspergillus fumigatus, PxIxIT gene, KpsF gene, Calcineurin, Gene mutation

The morphogenesis of left-right (LR) asymmetry is a crucial phase of organogenesis. In the digestive tract, the development of anatomical asymmetry is first evident in the leftward curvature of the stomach. To elucidate the molecular events that shape this archetypal laterality, we performed transcriptome analyses of the left versus right sides of the developing stomach in frog embryos. Besides the known LR gene pitx2, the only gene found to be expressed asymmetrically throughout all stages of curvature was single-minded 2 (sim2), a Down Syndrome-related transcription factor and homolog of a Drosophila gene (sim) required for LR asymmetric looping of the fly gut. We demonstrate that sim2 functions downstream of LR patterning cues to regulate key cellular properties and behaviors in the left stomach epithelium that drive asymmetric curvature. Our results reveal unexpected convergent cooption of single-minded genes during the evolution of LR asymmetric morphogenesis, and have implications for dose-dependent roles of laterality factors in non-laterality-related birth defects.

Background Breast cancer is a leading cause of cancer-related death for women in the USA. Thus, there is an increasing need to investigate novel prognostic markers and therapeutic methods. Inflammation raises challenges in treating and preventing the spread of breast cancer. Specifically, the nuclear factor kappa b (NFκB) pathway contributes to cancer progression by stimulating proliferation and preventing apoptosis. One target gene of this pathway is PTGS2, which encodes for cyclooxygenase 2 (COX-2) and is upregulated in 40% of human breast carcinomas. COX-2 is an enzyme involved in the production of prostaglandins, which mediate inflammation. Here, we investigate the effect of Singleminded-2s (SIM2s), a transcriptional tumor suppressor that is implicated in inhibition of tumor growth and metastasis, in regulating NFκB signaling and COX-2. Methods For in vitro experiments, reporter luciferase assays were utilized in MCF7 cells to investigate promoter activity of NFκB and SIM2. Real-time PCR, immunoblotting, immunohistochemistry, and chromatin immunoprecipitation assays were performed in SUM159 and MCF7 cells. For in vivo experiments, MCF10DCIS.COM cells stably expressing SIM2s-FLAG or shPTGS2 were injected into SCID mice and subsequent tumors harvested for immunostaining and analysis. Results Our results reveal that SIM2 attenuates the activation of NFκB as measured using NFκB-luciferase reporter assay. Furthermore, immunostaining of lysates from breast cancer cells overexpressing SIM2s showed reduction in various NFκB signaling proteins, as well as pAkt, whereas knockdown of SIM2 revealed increases in NFκB signaling proteins and pAkt. Additionally, we show that NFκB signaling can act in a reciprocal manner to decrease expression of SIM2s. Likewise, suppressing NFκB translocation in DCIS.COM cells increased SIM2s expression. We also found that NFκB/p65 represses SIM2 in a dose-dependent manner, and when NFκB is suppressed, the effect on the SIM2 is negated. Additionally, our ChIP analysis confirms that NFκB/p65 binds directly to SIM2 promoter site and that the NFκB sites in the SIM2 promoter are required for NFκB-mediated suppression of SIM2s. Finally, overexpression of SIM2s decreases PTGS2 in vitro, and COX-2 staining in vivo while decreasing PTGS2 and/or COX-2 activity results in re-expression of SIM2. Conclusion Our findings identify a novel role for SIM2s in NFκB signaling and COX-2 expression.

Background Mutations in genes associated with homologous recombination (HR) increase an individual’s risk of developing triple-negative breast cancer (TNBC). Although known for their role in repairing dsDNA breaks, HR repair elements also stabilize and restart stalled replication forks. Essential to these functions are RAD51 and its paralogs, each of which has a unique role in preventing replication fork collapse and restart. However, progress toward understanding the regulation of these factors has been slow. With such a pivotal role in the maintenance of genomic integrity, furthering our understanding of this pathway through the discovery of new factors involved in HR is important. Recently, we showed that singleminded-2s (SIM2s) is stabilized in response to dsDNA breaks and is required for effective HR. Methods Initial analysis of the effect loss of SIM2s has on replication stress resolution was conducted using DNA combing assays in established breast cancer cell lines. Further analysis was conducted via immunostaining to determine the effect loss of SIM2s has on factor recruitment. In vivo confirmation was achieved through the use of a mammary epithelial cell conditional knockout mouse model before SIM2s’ role in RAD51 recruitment was determined by immunoblotting. Results Here, we show loss of SIM2s decreases replication fork stability, leading to fork collapse in response to genotoxic stress. Furthermore, loss of SIM2s results in aberrant separation of sister chromatids during mitosis, which has been previously shown to result in chromosomal fragmentation and aneuploidy. Interestingly, loss of SIM2s was shown to result in failure of RAD51 to localize to sites of replication stress in both breast cancer cell lines and primary mammary epithelial cells. Finally, we observed SIM2 is stabilized in response to genotoxic stress and interacts with RAD51, which is necessary for RAD51-DNA binding. Conclusions Together, these results show a role for SIM2s in the resolution of replication stress and further characterize the necessity of SIM2s for effective RAD51 loading in response to DNA damage or stress, ultimately promoting genomic integrity and thus preventing the accumulation of cancer-promoting mutations.

The bHLH-PAS proteins are a family of transcription factors regulating expression of a wide range of genes involved in different functions, from differentiation and development control, by oxygen and toxins sensing to circadian clock setting. In addition to the well-preserved DNA-binding bHLH and PAS domains, bHLH-PAS proteins contain long intrinsically disordered C-terminal regions, responsible for their activity regulation. Our aim was to analyse the potential connection between disordered regions of the bHLH-PAS transcription factors with posttranscriptional modifications and liquid-liquid phase separation in the context of the disease-associated missense mutations. Highly flexible disordered regions, enriched in short more ordered motives, are responsible for wide spectrum of interactions with transcriptional co-regulators. Based on our in silico analysis and taking into account fact that transcription factors functions can be modulated by posttranslational modifications and spontaneous phase separation, we assume that the location of missense mutations inducing disease states, is clearly related to sequences directly undergoing these processes or to sequences responsible for their activity regulation.

The human PIAS proteins are small ubiquitin-like modifier (SUMO) E3 ligases that participate in important cellular functions. Several of these functions depend on a conserved SUMO-interacting motif (SIM) located in the central region of all PIAS proteins (SIM1). Recently, it was determined that Siz2, a yeast homolog of PIAS proteins, possesses a second SIM at its C terminus (SIM2). Sequence alignment indicates that a SIM2 is also present in PIAS1-3, but not PIAS4. Using biochemical and structural studies, we demonstrate PIAS-SIM2 binds to SUMO1, but that phosphorylation of the PIAS-SIM2 or acetylation of SUMO1 alter this interaction in a manner distinct from what is observed for the PIAS-SIM1. We also show that the PIAS-SIM2 plays a key role in formation of a UBC9-PIAS1-SUMO1 complex. These results provide insights into how post-translational modifications selectively regulate the specificity of multiple SIMs found in the PIAS proteins by exploiting the plasticity built into the SUMO-SIM binding interface.

BackgroundBreast cancer is a leading cause of cancer-related death for women in the United States. Thus, there a need to investigate novel prognostic markers and therapeutic strategies. Inflammation raises challenges to both treating and preventing the spread of breast cancer. Specifically, the nuclear factor kappa b (NFkB) pathway contributes to cancer progression by stimulating proliferation and preventing apoptosis. One target gene of this pathway is PTGS2, the gene that encodes for cyclooxygenase 2 (COX-2), which is upregulated in 40% of human breast carcinomas. COX-2 is an enzyme involved in inflammation. Here we investigate the effect of Singleminded 2s, a transcriptional tumor suppressor that is implicated in inhibition of tumor growth and metastasis, in regulating NFkB and COX-2.MethodsWe utilized in vitro reporter assays, immunoblot analyses, qPCR and immunohistochemical analysis to dissect the relationship between NFκB, SIM2s, and COX-2. Furthermore, we utilized COX-2 targeting strategies to determine tumor suppressive activities.ResultsOur results reveal that SIM2s attenuates the activation of a NFκB via luciferase reporter assays. Furthermore, immunostaining of lysates from breast cancer cells over expressing SIM2s showed reduction in various NFκB signaling proteins, whereas knockdown of SIM2 revealed increases in the same NFκB signaling proteins. Additionally, by increasing NFκB translocation to the nucleus in DCIS.COM cells, we show that NFκB signaling can act in a reciprocal manner to decrease expression of SIM2s. Likewise, suppressing NFκB translocation in DCIS.COM cells increases SIM2s expression. We also found that NFκB/p65 represses SIM2 in via dose-dependent manner and when NFκB is suppressed the effect on the SIM2 is negated. Additionally, our CHIP analysis confirms that NFκB/p65 binds directly to SIM2 promoter site and that the NFκB sites in the SIM2 promoter are required for NFkB-mediated suppression of SIM2s. Finally, over expression of SIM2s decreases PTGS2 in vitro and COX-2 staining in vivo while decreasing PTGS2 and/or Cox-2 activity results in re-expression of SIM2. Our findings identify a novel role for SIM2s in NFκB signaling and COX-2 expression.ConclusionsThese findings provide evidence for a mechanism where SIM2s may represses COX-2 expression to provide an overall better prognosis for breast cancer patients.

The basic helix–loop–helix/Per-ARNT-SIM (bHLH-PAS) proteins are a family of transcription factors regulating expression of a wide range of genes involved in different functions, ranging from differentiation and development control by oxygen and toxins sensing to circadian clock setting. In addition to the well-preserved DNA-binding bHLH and PAS domains, bHLH-PAS proteins contain long intrinsically disordered C-terminal regions, responsible for regulation of their activity. Our aim was to analyze the potential connection between disordered regions of the bHLH-PAS transcription factors, post-transcriptional modifications and liquid-liquid phase separation, in the context of disease-associated missense mutations. Highly flexible disordered regions, enriched in short motives which are more ordered, are responsible for a wide spectrum of interactions with transcriptional co-regulators. Based on our in silico analysis and taking into account the fact that the functions of transcription factors can be modulated by posttranslational modifications and spontaneous phase separation, we assume that the locations of missense mutations inducing disease states are clearly related to sequences directly undergoing these processes or to sequences responsible for their regulation.

Transcription factors of the basic helix-loop-helix (bHLH) PER-ARNT-SIM (PAS) family generally have critical and nonredundant biological roles, but some bHLH PAS proteins compete for common cofactors or recognise similar DNA elements. Identifying factors that regulate function of bHLH PAS proteins, particularly in cells where multiple family members are coexpressed, is important for understanding bHLH PAS factor biology. This study identifies and characterises a novel interaction between melanoma-associated antigen D1 (MAGED1) and select members of the bHLH PAS transcription factor family. MAGED1 binds and positively regulates the transcriptional activity of family members SIM1, SIM2, NPAS4 and ARNT2, but does not interact with AhR, HIF1α and ARNT. This interaction is mediated by PAS repeat regions which also form the interface for bHLH PAS dimerisation, and accordingly MAGED1 is not found in complex with bHLH PAS dimers. We show that MAGED1 does not affect bHLH PAS protein levels and cannot be acting as a coactivator of transcriptionally active heterodimers, but rather appears to interact with nascent bHLH PAS proteins in the cytoplasm to enhance their function prior to nuclear import. As a selective regulator, MAGED1 may play an important role in the biology of these specific factors and in general bHLH PAS protein dynamics.

Objective. Esophageal carcinoma (EC) is a frequently common malignancy of gastrointestinal cancer in the world. This study aims to screen key genes and pathways in EC and elucidate the mechanism of it.Methods. 5 microarray datasets of EC were downloaded from Gene Expression Omnibus. Differentially expressed genes (DEGs) were screened by bioinformatics analysis. Gene Ontology (GO) enrichment, Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment, and protein-protein interaction (PPI) network construction were performed to obtain the biological roles of DEGs in EC. Quantitative real-time polymerase chain reaction (qRT-PCR) was used to verify the expression level of DEGs in EC.Results. A total of 1955 genes were filtered as DEGs in EC. The upregulated genes were significantly enriched in cell cycle and the downregulated genes significantly enriched in Endocytosis. PPI network displayed CDK4 and CCT3 were hub proteins in the network. The expression level of 8 dysregulated DEGs including CDK4, CCT3, THSD4, SIM2, MYBL2, CENPF, CDCA3, and CDKN3 was validated in EC compared to adjacent nontumor tissues and the results were matched with the microarray analysis.Conclusion. The significantly DEGs including CDK4, CCT3, THSD4, and SIM2 may play key roles in tumorigenesis and development of EC involved in cell cycle and Endocytosis.

Degree of histological differentiation is an important characteristic of cancers and may be associated with malignant potential. However, in squamous cell carcinomas, a key transcriptional factor regulating tumor differentiation is largely unknown. Chemoradiotherapy (CRT) is a standard treatment for locally advanced esophageal squamous cell carcinoma; however, the survival rate is still below 40%. From microarray data, single-minded 2 (SIM2) was overexpressed in the epithelial subtype. Here, we investigated the correlation between SIM2 expression and its clinical implication, and in vitro and in vivo functions of SIM2 in tumor differentiation and in CRT sensitivity. Although SIM2 was suppressed in cancerous tissues, SIM2-high ESCC showed a favorable prognosis in CRT. Transient SIM2 expression followed by 3D culture induced expression of differentiation markers and suppressed epithelial-mesenchymal transition- and basal-cell markers. Levels of PDPN-high tumor basal cells and of expression of genes for DNA repair and antioxidant enzymes were reduced in stable transfectants, and they showed high CDDP and H2 O2 sensitivities, and their xenografts showed a well-differentiated histology. Reduction of tumor basal cells was restored by knockdown of aryl hydrocarbon receptor nuclear translocator (ARNT) that interacted with SIM2. Together, SIM2 increases CRT sensitivity through tumor differentiation by cooperation with ARNT.

Prenatal ethanol exposure can cause extensive apoptotic neurodegeneration throughout the developing central nervous system (CNS), which results in cognitive deficits and memory decline. However, the underlying mechanisms need further study. Single-minded 2 (Sim2), a transcriptional repressor, is reportedly involved in diseases that impair learning and memory, such as Down syndrome (DS) and Alzheimer's disease. It is still unknown whether Sim2 is involved in regulating ethanol-mediated neuronal injury that might ultimately lead to neuronal dysfunction and subsequent learning and memory deficits. To study the effects of ethanol on Sim2 expression and neuronal injury, we used animal models and cell culture experiments. Our results indicated that in SH-SY5Y cells, ethanol exposure increased Sim2 expression and levels of cleaved caspase 3, which is a marker for cells undergoing apoptosis. Silencing Sim2 expression attenuated caspase 3 activation and cellular apoptosis. We also found that protein kinase A (PKA) activation induced Sim2 expression, as did ethanol. Inhibiting the PKA signaling pathway with H-89 decreased Sim2 expression and cleavage of caspase 3 that was induced by ethanol in vivo and in vitro. We further found that PKA regulated Sim2 expression at the transcriptional level. These results demonstrate that ethanol leads to increased Sim2 expression via the PKA pathway, ultimately resulting in apoptotic cell death.

The bHLH (basic helix–loop–helix) PAS (Per/Arnt/Sim) transcription factor SIM1 (single-minded 1) is important for development and function of regions of the hypothalamus that regulate energy homoeostasis and the feeding response. Low-activity SIM1 variants have been identified in individuals with severe early-onset obesity, but the underlying molecular causes of impaired function are unknown. In the present study we assess a number of human SIM1 variants with reduced activity and determine that impaired function is frequently due to defects in dimerization with the essential partner protein ARNT2 (aryl hydrocarbon nuclear translocator 2). Equivalent variants generated in the highly related protein SIM2 (single-minded 2) produce near-identical impaired function and dimerization defects, indicating that these effects are not unique to the structure of SIM1. On the basis of these data, we predict that other select SIM1 and SIM2 variants reported in human genomic databases will also be deficient in activity, and identify two new low-activity SIM1 variants (V290E and V326F) present in the population. The cumulative data is used in homology modelling to make novel observations about the dimerization interface between the PAS domains of SIM1 and ARNT2, and to define a mutational ‘hot-spot’ in SIM1 that is critical for protein function.

Down syndrome (DS) is the most common genetic aberration leading to intellectual disability. DS results from an extra copy of the long arm of human chromosome 21 (HSA21) and the increased expression of trisomic genes due to gene dosage. While expression in DS and DS models has been studied extensively at the RNA level, much less is known about expression of trisomic genes at the protein level. We have used quantitative Western blotting with antibodies to 20 proteins encoded by HSA21 to assess trisomic protein expression in lymphoblastoid cell lines (LCLs) from patients with DS and in brains from two mouse models of DS. These antibodies have recently become available and the 20 proteins largely have not been investigated previously for their potential contributions to the phenotypic features of DS. Twelve proteins had detectable expression in LCLs and three, CCT8, MX1 and PWP2, showed elevated levels in LCLs derived from patients with DS compared with controls. Antibodies against 15 proteins detected bands of appropriate sizes in lysates from mouse brain cortex. Genes for 12 of these proteins are trisomic in the Tc1 mouse model of DS, but only SIM2 and ZNF295 showed elevated expression in Tc1 cortex when compared with controls. Genes for eight of the 15 proteins are trisomic in the Ts65Dn mouse model of DS, but only ZNF294 was over expressed in cortex. Comparison of trisomic gene expression at the protein level with previous reports at the mRNA level showed many inconsistencies. These may be caused by natural inter-individual variability, differences in the age of mice analyzed, or post-transcriptional regulation of gene dosage effects. These antibodies provide resources for further investigation of the molecular basis of intellectual disability in DS.

A meta-analysis of publicly available gene expression changes in A549 cells upon treatment with anti-cancer drugs is reported. To reduce false positives, both fold-change and significance level cutoffs were used. Simulated datasets and permutation analysis were used to guide choice of ratio cutoff. Of the genes identified, FDXR is the only gene differentially expressed in six of the seven drug treatments. Though FDXR has been reported to be differentially expressed upon treatment with 5-fluorouracil and its expression correlated to long term disease survival, to our knowledge this is a first study implicating a wide effect of anti-cancer drug treatment on FDXR expression. The other genes identified which are differentially expressed in four out of the seven drug treatments are CDKN1A and PARVB which are upregulated and MYC, HBP1, LDLR, SIM2, ALX1 and GPHN which are downregulated.

The human single-minded 2 (SIM2) gene is mapped within the Down syndrome critical region (DSCR) of chromosome 21 and its short splice variant SIM2-s is suggested to be a molecular target for cancer therapy. It has been shown that SIM2-s is expressed in colon, pancreatic and prostate tumors but not in their corresponding normal tissues. In present study, we found that SIM2-s was expressed in glioma tissues as well as in glioblastoma (GBM) cell lines but not in other brain tumors or normal cortex. The invasive potential of GBM cells was significantly decreased by RNA interference targeting SIM2-s, while the proliferation was not affected. Further investigation showed that the mRNA and protein levels of matrix metalloproteinase 2 (MMP-2) were downregulated in cells treated with SIM2-s siRNA, concomitance with the upregulation of tissue inhibitor of matrix metalloproteinase 2 (TIMP-2). Moreover, the enzymatic activity of MMP-2 was clearly decreased. Our results collectively suggested that SIM2-s expressed in gliomas selectively and played a role in GBM cells invasion, which may be partly associated with the expression of MMP-2 and TIMP-2.

To investigate the mRNA expression of the dendritic spine protein drebrin in Alzheimer's disease (AD), we performed post-mortem in situ hybridization studies in brain sections from 20 AD patients and 21 controls. AD diagnosis was confirmed by decreased drebrin protein and increased Abeta(40) (+464%; P < 0.05), Abeta(42) (+369%; P < 0.0001), Abeta(42/40) ratio (+226%; P < 0.01), total tau (+2,725%; P < 0.0001), and paired helical filament tau (PHFtau; +867%; P < 0.001) compared with controls. We found significant decreases in drebrin mRNA in the parietal cortex (-27%; P < 0.01), the temporal cortex (-22%; P < 0.05), and the hippocampus (-25%; P < 0.05) of AD patients compared with controls. Cortical levels of drebrin mRNA correlated positively with soluble total tau (r(2) = +0.244) but negatively with duration of symptoms (r(2) = -0.357) and PHFtau (r(2) = -0.248). Drebrin mRNA levels were correlated to a lesser degree with the drebrin protein content (r(2) = +0.136) and with sim2 (r(2) = +0.176), a potential modulator of drebrin transcription. Our results suggest that the down-regulation of drebrin mRNA expression plays an important role in AD and is closely related to the progression of the disease.

The mamillary body (MM) is a group of hypothalamic nuclei related to memory and spatial navigation that interconnects hippocampal, thalamic, and tegmental regions. Here we demonstrate that Lhx5, a LIM-HD domain transcription factor expressed early in the developing posterior hypothalamus, is required for the generation of the MM and its derived tracts. The MM markers Foxb1, Sim2, and Lhx1 are absent in Lhx5 knock-out mice from early embryonic stages, suggesting abnormal specification of this region. This was supported by the absence of Nkx2.1 and expansion of Shh in the prospective mamillary area. Interestingly, we also found an ectopic domain expressing Lhx2 and Lhx9 along the anterio-posterior hypothalamic axis. Our results suggest that Lhx5 controls early aspects of hypothalamic development by regulating gene expression and cellular specification in the prospective MM.

The single-minded 2 gene (Sim2) plays a crucial role in the mental retardation of Down Syndrome (DS). To explore how Sim2 influences spatial memory, a DNA plasmid-encoding mouse Sim2 (mSim2) wrapped with liposome was bilaterally injected into the hippocampus of rats. The effect of overexpressing mSim2 on spatial learning was determined by a Morris water maze task. The expression of synapsin I was detected by reverse transcriptional-polymerase chain reaction (RT-PCR) analysis and immunohistochemistry, respectively. The phosphosynapsin was also examined by immunohistochemistry. As demonstrated by RT-PCR, mSim2 was overexpressed in the hippocampus of rats, and pcDNA3/mSim2-transfected rats showed longer latency to find the hidden platform compared with pcDNA3-transfected rats (P < 0.05). Synapsin I mRNA and protein expression were decreased significantly by mSim2 transfection, as demonstrated by RT-PCR and immunohistochemistry. Moreover, the expression profile of phosphosynapsin was similar to that of synapsin I. So it is concluded that Sim2 could impair the ability of learning and memory by inhibiting synaptic plasticity, and may play a crucial role in the pathogenesis of DS.

The mammillary body (MB), and its axonal projections to the thalamus (mammillothalamic tract, MTT) and the tegmentum (mammillotegmental tract, MTEG), are components of a circuit involved in spatial learning. The bHLH-PAS transcription factors SIM1 and SIM2 are co-expressed in the developing MB. We have found that MB neurons are generated and that they survive at least until E18.5 in embryos lacking both Sim1 and Sim2 (Sim1(-/-);Sim2(-/-)). However, the MTT and MTEG are histologically absent in Sim1(-/-);Sim2(-/-) embryos, and are reduced in embryos lacking Sim1 but bearing one or two copies of Sim2, indicating a contribution of the latter to the development of MB axons. We have generated, by homologous recombination, a null allele of Sim1 (Sim1(tlz)) in which the tau-lacZ fusion gene was introduced, allowing the staining of MB axons. Consistent with the histological studies, lacZ staining showed that the MTT/MTEG is barely detectable in Sim1(tlz/tlz);Sim2(+/-) and Sim1(tlz/tlz);Sim2(-/-) brains. Instead, MB axons are splayed and grow towards the midline. Slit1 and Slit2, which code for secreted molecules that induce the repulsion of ROBO1-producing axons, are expressed in the midline at the level of the MB, whereas Robo1 is expressed in the developing MB. The expression of Rig-1/Robo3, a negative regulator of Slit signalling, is upregulated in the prospective MB of Sim1/Sim2 double mutants, raising the possibility that the growth of mutant MB axons towards the midline is caused by a decreased sensitivity to SLIT. Finally, we found that Sim1 and Sim2 act along compensatory, but not hierarchical, pathways, suggesting that they play similar roles in vivo.

Human single-minded 2 (SIM2) is a member of the basic helix-loop-helix/Per-Arnt-Sim (bHLH/PAS) family of transcription factors and is associated with the etiology of Down syndrome phenotype. Here, we examined a possibility of the post-translational modification of SIM2 protein by transfecting various expression constructs followed by the analysis with immunoprecipitation and Western blotting. In fact, transient expression of SIM2 cDNA in HEK293 cells revealed poly-ubiquitination of SIM2 protein. In the stable transfectants, a proteasome inhibitor MG132 protected the poly-ubiquitinated SIM2 protein from degradation. Furthermore, in the cells co-transfected with SIM2 and each of four different E3 ubiquitin ligases, SIM2 was immunoprecipitated with the RING-IBR-RING-type E3 ubiquitin ligases, Parkin and HHARI, but it was not immunoprecipitated with other E3 ligases, such as one RING-type Siah-1 and the PHD type AIRE. A series of deletion constructs revealed that Parkin actually binds to SIM2 with the IBR (294-377)-RING2 (378-465) domains and that the sites for poly-ubiquitination of SIM2 reside within the PAS1-PAS2 region (aa 141-289). We postulated that transcription factor SIM2 and E3 ubiquitin ligase Parkin may interact each other to play an important physiological role in the brain development which is controlled by ubiquitination.

Sim2, a basic helix-loop-helix (bHLH)-PAS transcriptional repressor, is thought to be involved in some symptoms of Down's syndrome. In the course of searching for hypothetical Sim2 relatives, we isolated another bHLH-PAS factor, NXF. NXF was a novel gene and was selectively expressed in neuronal tissues. While no striking homolog of NXF was found in vertebrates, a Caenorhabditis elegans putative transcription factor, C15C8.2, showed similarity in the bHLH-PAS domain. NXF had an activation domain as a transcription activator, and Arnt-type bHLH-PAS subfamily members were identified as the heterodimer partners of NXF. The NXF/Arnt heterodimer was capable of binding and activating a subset of Sim2/Arnt target DNA variants, and Sim2 could compete with the NXF activity on the elements. We showed that Drebrin had several such NXF/Arnt binding elements on the promoter, which could be direct or indirect cross talking points between NXF (activation) and Sim2 (repression) action. Drebrin has been reported to be engaged in dendritic-cytoskeleton modulation at synapses, and such a novel NXF signaling system on neural gene promoter may be a molecular target of the adverse effects of Sim2 in the mental retardation of Down's syndrome.

We report here a cancer drug therapy use of a gene involved in Down's syndrome. Using bioinformatics approaches, we recently predicted Single Minded 2 gene ( SIM2 ) from Down's syndrome critical region to be specific to certain solid tumors. Involvement of SIM2 in solid tumors has not previously been reported. Intrigued by a possible association between a Down's syndrome gene and solid tumors, we monitored SIM2 expression in solid tumors. Isoform-specific expression of SIM2 short-form ( SIM2-s ) was seen selectively in colon, prostate, and pancreatic carcinomas but not in breast, lung, or ovarian carcinomas nor in most normal tissues. In colon tumors, SIM2-s expression was seen in early stages. Antisense inhibition of SIM2-s expression in a colon cancer cell line caused inhibition of gene expression, growth inhibition, and apoptosis. The administration of the antisense, but not the control, oligonucleotides caused a pronounced inhibition of tumor growth in nude mice with no major toxicity. Our findings provide a strong rationale for the genes-to-drugs paradigm, establish SIM2-s as a molecular target for cancer therapeutics, and may further understanding of the cancer risk of Down's syndrome patients.

The mouse genome contains two Sim genes, Sim1 and Sim2. They are presumed to be important for central nervous system (CNS) development because they are homologous to the Drosophila single-minded (sim) gene, mutations in which cause a complete loss of CNS midline cells. In the mammalian CNS, Sim2 and Sim1 are coexpressed in the paraventricular nucleus (PVN). While Sim1 is essential for the development of the PVN (J. L. Michaud, T. Rosenquist, N. R. May, and C.-M. Fan, Genes Dev. 12:3264-3275, 1998), we report here that Sim2 mutant has a normal PVN. Analyses of the Sim1 and Sim2 compound mutants did not reveal obvious genetic interaction between them in PVN histogenesis. However, Sim2 mutant mice die within 3 days of birth due to lung atelectasis and breathing failure. We attribute the diminished efficacy of lung inflation to the compromised structural components surrounding the pleural cavity, which include rib protrusions, abnormal intercostal muscle attachments, diaphragm hypoplasia, and pleural mesothelium tearing. Although each of these structures is minimally affected, we propose that their combined effects lead to the mechanical failure of lung inflation and death. Sim2 mutants also develop congenital scoliosis, reflected by the unequal sizes of the left and right vertebrae and ribs. The temporal and spatial expression patterns of Sim2 in these skeletal elements suggest that Sim2 regulates their growth and/or integrity.

The single-minded 2 (SIM2) protein is a basic helix-loop-helix transcription factor regulating central nervous system (CNS) development in Drosophila. In humans, SIM2 is located within the Down syndrome critical region on chromosome 21 and may be involved in the development of mental retardation phenotype in Down syndrome. In this study, knockout of SIM2 expression in mice resulted in a gas distention phenotype in the gastrointestinal tract. We found that SIM2 is required for the expression of all cryptdins and numerous other antimicrobial peptides (AMPs) expressed in the small intestine. The mechanism underlying how SIM2 controls AMP expression involves both direct and indirect regulations. For the cryptdin genes, SIM2 regulates their expression by modulating transcription factor 7-like 2, a crucial regulator in the Wnt/β-catenin signaling pathway, while for other AMP genes, such as RegIIIγ, SIM2 directly activates their promoter activity. Our results establish that SIM2 is a crucial regulator in controlling expression of intestinal AMPs to maintain intestinal innate immunity against microbes.

The secreted protein sonic hedgehog (Shh) is required to establish patterns of cellular growth and differentiation within ventral regions of the developing CNS. The expression of Shh in the two tissue sources responsible for this activity, the axial mesoderm and the ventral midline of the neural tube, is regulated along the anteroposterior neuraxis. Separate cis-acting regulatory sequences have been identified which direct Shh expression to distinct regions of the neural tube, supporting the view that multiple genes are involved in activating Shh transcription along the length of the CNS. We show here that the activity of one Shh enhancer, which directs reporter expression to portions of the ventral midbrain and diencephalon, overlaps both temporally and spatially with the expression of Sim2. Sim2 encodes a basic helix-loop-helix (bHLH-PAS) PAS domain containing transcriptional regulator whose Drosophila homolog, single-minded, is a master regulator of ventral midline development. Both vertebrate and invertebrate Sim family members were found sufficient for the activation of the Shh reporter as well as endogenous Shh mRNA. Although Shh expression is maintained in Sim2−/− embryos, it was determined to be absent from the rostral midbrain and caudal diencephalon of embryos carrying a dominant-negative transgene that disrupts the function of bHLH-PAS proteins. Together, these results suggest that bHLH-PAS family members are required for the regulation of Shh transcription within aspects of the ventral midbrain and diencephalon.