A novel Xist RNA-mediated chromosome inactivation model using a mouse artificial chromosome.

OBJECTIVE: To develop a mouse artificial chromosome (MAC) carrying the mouse Xist gene (X-inactive specific transcript; Xist-MAC) as a systematic in vitro approach for investigating Xist RNA-mediated chromosome inactivation. RESULTS: Ectopic expression of the Xist gene in CHO cells led to the accumulation of Xist RNA in cis on the MAC. In addition, the introduction of Xist-MAC to embryonic stem cells from male mice via microcell-mediated chromosome transfer resulted in the accumulation of Xist RNA in cis on the MAC. Chromosomal inactivation was observed in the differentiated state. Moreover, this phenomenon was accompanied by the epigenetic modification of H3K27 trimethylation. CONCLUSIONS: We successfully generated a novel chromosome inactivation model, Xist-MAC, which will provide a valuable tool for the screening and functional analysis of X chromosome inactivation-related genes and proteins.

Loss of the fragile X mental retardation protein causes aberrant differentiation in human neural progenitor cells.

Fragile X syndrome (FXS) is caused by transcriptional silencing of the FMR1 gene during embryonic development with the consequent loss of the encoded fragile X mental retardation protein (FMRP). The pathological mechanisms of FXS have been extensively studied using the Fmr1-knockout mouse, and the findings suggest important roles for FMRP in synaptic plasticity and proper functioning of neural networks. However, the function of FMRP during early development in the human nervous system remains to be confirmed. Here we describe human neural progenitor cells (NPCs) as a model for studying FMRP functions and FXS pathology. Transcriptome analysis of the NPCs derived from FMR1-knockout human induced pluripotent stem cells (iPSCs) showed altered expression of neural differentiation markers, particularly a marked induction of the astrocyte marker glial fibrillary acidic protein (GFAP). When induced to differentiate, FMRP-deficient neurons continued to express GFAP, and showed less spontaneous calcium bursts than the parental iPSC-derived neurons. Interestingly, the aberrant expression of GFAP and the impaired firing was corrected by treatment with the protein kinase inhibitor LX7101. These findings underscore the modulatory roles of FMRP in human neurogenesis, and further demonstrate that the defective phenotype of FXS could be reversed at least partly by small molecule kinase inhibitors.

Regulation of functional KCNQ1OT1 lncRNA by ß-catenin.

Long noncoding RNAs (lncRNAs) have been implicated in many biological processes through epigenetic mechanisms. We previously reported that KCNQ1OT1, an imprinted antisense lncRNA in the human KCNQ1 locus on chromosome 11p15.5, is involved in cis-limited silencing within an imprinted KCNQ1 cluster. Furthermore, aberration of KCNQ1OT1 transcription was observed with a high frequency in colorectal cancers. However, the molecular mechanism of the transcriptional regulation and the functional role of KCNQ1OT1 in colorectal cancer remain unclear. Here, we show that the KCNQ1OT1 transcriptional level was significantly increased in human colorectal cancer cells in which ß-catenin was excessively accumulated in the nucleus. Additionally, overexpression of ß-catenin resulted in an increase in KCNQ1OT1 lncRNA-coated territory. On the other hand, knockdown of ß-catenin resulted in significant decrease of KCNQ1OT1 lncRNA-coated territory and an increase in the mRNA expression of the SLC22A18 and PHLDA2 genes that are regulated by KCNQ1OT1. We showed that ß-catenin can promote KCNQ1OT1 transcription through direct binding to the KCNQ1OT1 promoter. Our evidence indicates that ß-catenin signaling may contribute to development of colorectal cancer by functioning as a novel lncRNA regulatory factor via direct targeting of KCNQ1OT1.

Repression of hTERT transcription by the introduction of chromosome 3 into human oral squamous cell carcinoma.

Telomerase is a ribonucleoprotein enzyme that maintains telomere length. Telomerase activity is primarily attributed to the expression of telomerase reverse transcriptase (TERT). It has been reported that introduction of an intact human chromosome 3 into the human oral squamous cell carcinoma cell line HSC3 suppresses the tumorigenicity of these cells. However, the mechanisms that regulate tumorigenicity have not been elucidated. To determine whether this reduction in tumorigenicity was accompanied by a reduction in telomerase activity, we investigated the transcriptional activation of TERT in HSC3 microcell hybrid clones with an introduced human chromosome 3 (HSC3#3). HSC#3 cells showed inhibition of hTERT transcription compared to that of the parental HSC3 cells. Furthermore, cell fusion experiments showed that hybrids of HSC3 cells and cells of the RCC23 renal carcinoma cell line, which also exhibits suppression of TERT transcription by the introduction of human chromosome 3, also displayed suppressed TERT transcription. These results suggested that human chromosome 3 may carry functionally distinct, additional TERT repressor genes.

Preferential recruitment of the maternal centromere-specific histone H3 (CENH3) in oat (Avena sativa L.) × pearl millet (Pennisetum glaucum L.) hybrid embryos.

Chromosome elimination occurs frequently in interspecific hybrids between distantly related species in Poaceae. However, chromosomes from both parents behave stably in a hybrid of female oat (Avena sativa L.) pollinated by pearl millet (Pennisetum glaucum L.). To analyze the chromosome behavior in this hybrid, we cloned the centromere-specific histone H3 (CENH3) genes of oat and pearl millet and produced a pearl millet-specific anti-CENH3 antibody. Application of this antibody together with a grass species common anti-CENH3 antibody revealed the dynamic CENH3 composition of the hybrid cells before and after fertilization. Despite co-expression of CENH3 genes encoded by oat and pearl millet, only an oat-type CENH3 was incorporated into the centromeres of both species in the hybrid embryo. Oat CENH3 enables a functional centromere in pearl millet chromosomes in an oat genetic background. Comparison of CENH3 genes among Poaceae species that show chromosome elimination in interspecific hybrids revealed that the loop 1 regions of oat and pearl millet CENH3 exhibit exceptionally high similarity.

Decreased PITX1 gene expression in human cutaneous malignant melanoma and its clinicopathological significance.

BACKGROUND: The pituitary homeobox 1 (PITX1) protein is a member of the bicoid-related homeobox transcription factors and has essential roles in human development. Recently, the PITX1 gene has been considered as a tumor suppressor gene in various human cancers. OBJECTIVE: This study examined the expression of PITX1 in the development and progression of human cutaneous malignant melanoma. MATERIALS & METHODS: Immunohistochemical and/or immunofluorescence analyses were performed to examine the histological expression of PITX1 in healthy skin and 40 cutaneous malignant melanoma cases, including 10 melanoma in situ cases. RESULTS: Expression of PITX1 was shown in nuclei of melanocytes in normal skin. PITX1 expression was positive (labeling index: ≥10%) in 21 (52.5%) cases and negative (labeling index: <10%) in 19 (47.5%) of 40 cases of primary cutaneous malignant melanoma. The mean tumor thickness in PITX1-negative cases (7.11 ± 10.3 mm) was significantly higher than that in the positive cases (1.90 ± 3.19 mm) (P<0.01). The numbers of cases showing metastasis were 1 (4.76%) of 21 cases in PITX1-positive cases and 7 (36.8%) of 19 cases in PITX1-negative cases; the frequency was significantly higher in PITX1-negative cases than the positive cases (P = 0.012). Moreover, the reduction in PITX1 expression correlated significantly with clinical stage (P<0.001). Interestingly, PITX1 expression was inversely correlated with cell proliferation of cutaneous malignant melanoma (P<0.001). CONCLUSIONS: Down-regulation of PITX1 expression might contribute to the progression of cutaneous malignant melanoma via promoting cell proliferative activity.