09-864 Sigma-AldrichAnti-JHDM1B Antibody

Both DNA and histone methylation are dysregulated in the myelodysplastic syndromes (MDS). Based on preliminary data we hypothesized that dysregulated interactions of KDM2B, let-7b and EZH2 signals lead to an aberrant epigenetic landscape. Gene expression in CD34+ cells from MDS marrows was analyzed by NanoString miR array and validated by real-time polymerase chain reaction (PCR). The functions of KDM2B, let-7b and EZH2 were characterized in myeloid cell lines and in primary MDS cells. Let-7b levels were significantly higher, and KDM2B and EZH2 expression was lower in primary CD34+ MDS marrow cells (n = 44) than in healthy controls (n = 21; pless than 0.013, and pless than 0.0001, respectively). Overexpression of let-7b reduced EZH2 and KDM2B protein levels, and decreased cells in S-phase while increasing G0/G1 cells (p = 0.0005), accompanied by decreased H3K27me3 and cyclin D1. Silencing of KDM2B increased let-7b expression. Treatment with the cyclopentanyl analog of 3-deazaadenosine, DZNep, combined with the DNA hypomethylating agent 5-azacitidine, decreased levels of EZH2, suppressed methylation of di- and tri-methylated H3K27, and increased p16 expression, associated with cell proliferation. Thus, KDM2B, via let-7b/EZH2, promotes transcriptional repression. DZNep bypassed the inhibitory KDM2B/let-7b/EZH2 axis by preventing H3K27 methylation and reducing cell proliferation. DZNep might be able to enhance the therapeutic effects of DNA hypomethylating agents such as 5-azacitidine, currently considered standard therapy for patients with MDS.

Histone methylation is the important transcription regulatory system that affects mammalian development and cell differentiation. Alterations in epigenetic gene regulation are associated with disease. Fbxl10 (F-box and leucine-rich repeat protein 10) is a JmjC domain-containing histone demethylase. Although Fbxl10 has been implicated in cell cycle regulation, cell death, senescence, and tumorigenesis, these functions are controversial and its physiological function is unclear. To determine the in vivo function of Fbxl10, in this study, we generated a homozygous mutation in the mouse Fbxl10 gene. About half of Fbxl10-deficient mice exhibit failure of neural tube closure, resulting in exencephaly and die shortly after birth. Fbxl10 deficiency also causes retinal coloboma and a curled tail with low penetrances. Fbxl10 mRNA is specifically expressed in the cranial neural folds at E8.5 embryos, and apoptosis increased in the neuroepithelium and mesenchyme of Fbxl10-deficient E9.5 embryos, consistent with neural tube defects found in Fbxl10-deficient mice. Depletion of Fbxl10 induced the increased expression of p19ARF, an inducer of apoptosis, in E8.5 embryos and mouse embryonic fibroblast cells. In addition, the number of mitotic neural progenitor cells is significantly increased in the mutant E14.5 brain. Our findings suggest that the Fbxl10 gene makes important contributions to embryonic neural development by regulating cell proliferation and cell death in mice.

Housekeeping genes frequently used in gene expression studies are highly regulated in human adipose tissue. To ensure a correct interpretation of results, it is critical to select appropriate reference genes. Subcutaneous (SC) and omental (OM) adipose tissue expression was analyzed from lean and obese subjects using whole genome complementary DNA (cDNA) microarrays to identify stably expressed genes and commercial TaqMan low density arrays (LDAs), with 16 common control genes. The best candidate gene from microarrays analysis was F-box and leucine-rich repeat protein-10 (FBXL10) (fold-change 10(-3) P < 0.01), an ubiquitous nucleolar protein evolutionarily conserved. Hypoxanthine phosphoribosyltransferase 1 (HPRT1) and importin 8 (IPO8), were the best reference genes among the 16 genes in the LDAs with coefficient of variation (CV) of 4.51 and 4.55%, respectively. However, when the LDAs data were further analyzed by the geNorm and NormFinder softwares, IPO8, a nuclear protein mediating import of proteins, was the first and the third better reference gene, respectively. IPO8 and FBXL10 were further validated by real-time PCR in additional OM and SC fat samples and primary cultured preadipocytes. According to their CV, IPO8 resulted more suitable than FBXL10 in both adipose tissue depots and SC preadipocytes, whereas FBXL10 performed better than IPO8 in OM cultured preadipocytes. Both genes expression levels did not change throughout adipogenesis. Thus, we provide clear evidence that IPO8 and FBXL10 are good candidates to use as reference genes in gene expression studies in human OM and SC adipose tissues as well as differentiated primary preadipocytes.

JHDM1B is an evolutionarily conserved and ubiquitously expressed member of the JHDM (JmjC-domain-containing histone demethylase) family. Because it contains an F-box motif, this protein is also known as FBXL10 (ref. 4). With the use of a genome-wide RNAi screen, the JHDM1B worm orthologue (T26A5.5) was identified as a gene that regulates growth. In the mouse, four independent screens have identified JHDM1B as a putative tumour suppressor by retroviral insertion analysis. Here we identify human JHDM1B as a nucleolar protein and show that JHDM1B preferentially binds the transcribed region of ribosomal DNA to repress the transcription of ribosomal RNA genes. We also show that repression of ribosomal RNA genes by JHDM1B is dependent on its JmjC domain, which is necessary for the specific demethylation of trimethylated lysine 4 on histone H3 in the nucleolus. In agreement with the notion that ribosomal RNA synthesis and cell growth are coupled processes, we show a JmjC-domain-dependent negative effect of JHDM1B on cell size and cell proliferation. Because aberrant ribosome biogenesis and the disruption of epigenetic control mechanisms contribute to cellular transformation, these results, together with the low levels of JHDM1B expression found in aggressive brain tumours, suggest a role for JHDM1B in cancer development.

The corepressor BCOR potentiates transcriptional repression by the proto-oncoprotein BCL6 and suppresses the transcriptional activity of a common mixed-lineage leukemia fusion partner, AF9. Mutations in human BCOR cause male lethal, X-linked oculofaciocardiodental syndrome. We identified a BCOR complex containing Polycomb group (PcG) and Skp-Cullin-F-box subcomplexes. The PcG proteins include RING1, RYBP, NSPC1, a Posterior Sex Combs homolog, and RNF2, an E3 ligase for the mono-ubiquitylation of H2A. BCOR complex components and mono-ubiquitylated H2A localize to BCL6 targets, indicating that the BCOR complex employs PcG proteins to expand the repertoire of enzymatic activities that can be recruited by BCL6. This also suggests that BCL6 can target PcG proteins to DNA. In addition, the BCOR complex contains components of a second ubiquitin E3 ligase, namely, SKP1 and FBXL10 (JHDM1B). We show that BCOR coimmunoprecipitates isoforms of FBXL10 which contain a JmjC domain that recently has been determined to have histone H3K36 demethylase activity. The recruitment of two distinct classes of E3 ubiquitin ligases and a histone demethylase by BCOR suggests that BCOR uses a unique combination of epigenetic modifications to direct gene silencing.