Retinoid regulated association of transcriptional co-regulators and the polycomb group protein SUZ12 with the retinoic acid response elements of Hoxa1, RARbeta(2), and Cyp26A1 in F9 embryonal carcinoma cells

Hox gene expression is activated by all-trans retinoic acid (RA), through binding to retinoic acid receptor-retinoid X receptor (RAR-RXR) heterodimers bound at RA response elements (RAREs) of target genes. The RARs and RXRs each have three isotypes (alpha, beta, and gamma), which are encoded by distinct genes. Hox genes are also repressed by polycomb group proteins (PcG), though how these proteins are targeted is unclear. We used chromatin immunoprecipitation assays to investigate the association of RXRalpha, RARgamma, cofactors, and the PcG protein SUZ12 with the Hoxa1, RARbeta2, and Cyp26A1 RAREs in F9 embryonal carcinoma cells (teratocarcinoma stem cells) during RA treatment. We demonstrate that RARgamma and RXRalpha are associated with RAREs prior to and during RA treatment. pCIP, p300, and RNA polymerase II levels increased at target RAREs upon exposure to RA. Conversely, SUZ12 was found associated with all RAREs studied and these associations were attenuated by treatment with RA. Upon RA removal, SUZ12 re-associated with RAREs. H3ac, H3K4me2, and H3K27me3 marks were simultaneously detected at target loci, indicative of a bivalent domain chromatin structure. During RA mediated differentiation, H3K27me3 levels decreased at target RAREs whereas H3ac and H3K4me2 levels remained constant. These studies provide insight into the dynamics of association of co-regulators with RAREs and demonstrate a novel link between RA signaling and PcG repression.     

HOXC5 and HOXC8 expression are selectively turned on in human cervical cancer cells compared to normal keratinocytes

A growing number of data have sustained the involvement of homeobox genes expression deregulation in cancer. In this study, we have performed an exhaustive survey of the expression of the 39 class I HOX genes expressed in normal and malignant human cervix keratinocytes. Using RT-PCR, we observed that the vast majority (34/39) of HOX genes are expressed in normal keratinocytes. Only HOXA2, HOXA7, HOXC5, HOXC8 and HOXD12 were found to be silent. Interestingly, this pattern is conserved in the transformed keratinocytes (SiHa cells) except for the appearance of HOXC5 and HOXC8 mRNA. The HOXC5 and HOXC8 expression was also observed in two other transformed keratinocytes cell lines of independent origins, Eil-8 and 18-11S3, and confirmed by in situ hybridization. Our data add weight to the body of evidence attributing to a specific adult tissue a particular combination of expressed HOX genes and suggest that HOXC5 and/or HOXC8 could be involved in the process leading to the transformation of cervical keratinocytes.     

Expression of homeobox genes in oral squamous cell carcinoma cell lines treated with all‐trans retinoic acid

Oral squamous cell carcinoma (OSCC) may arise from potentially malignant oral lesions. All‐trans retinoic acid (atRA), which plays a role in cell growth and differentiation, has been studied as a possible chemotherapeutic agent in the prevention of this progression. While the mechanism by which atRA suppresses cell growth has not been completely elucidated, it is known that homeobox genes are atRA targets. To determine if these genes are involved in the atRA‐mediated OSCC growth inhibition, PCR array was performed to evaluate the expression of 84 homeobox genes in atRA‐sensitive SCC‐25 cells compared to atRA‐resistant SCC‐9 cells following 7 days with atRA treatment. Results showed that the expression of 8 homeobox genes was downregulated and expression of 4 was upregulated in SCC‐25 cells but not in SCC‐9 cells. Gene expression levels were confirmed for seven of these genes by RT‐qPCR. Expression of three genes that showed threefold downregulation was evaluated in SCC‐25 cells treated with atRA for 3, 5, and 7 days. Three different patterns of atRA‐dependent gene expression were observed. ALX1 showed downregulation only on day 7. DLX3 showed reduced expression on day 3 and further reduced on day 7. TLX1 showed downregulation only on days 5 and 7. Clearly the expression of homeobox genes is modulated by atRA in OSCC cell lines. However, the time course of this modulation suggests that these genes are not direct targets of atRA mediating OSCC growth suppression. Instead they appear to act as downstream effectors of atRA signaling. J. Cell. Biochem. 111: 1437–1444, 2010. © 2010 Wiley‐Liss, Inc.     

Variable Requirements for DNA-Binding Proteins at Polycomb-Dependent Repressive Regions in Human HOX Clusters

Polycomb group (PcG)-mediated repression is an evolutionarily conserved process critical for cell fate determination and maintenance of gene expression during embryonic development. However, the mechanisms underlying PcG recruitment in mammals remain unclear since few regulatory sites have been identified. We report two novel prospective PcG-dependent regulatory elements within the human HOXB and HOXC clusters and compare their repressive activities to a previously identified element in the HOXD cluster. These regions recruited the PcG proteins BMI1 and SUZ12 to a reporter construct in mesenchymal stem cells and conferred repression that was dependent upon PcG expression. Furthermore, we examined the potential of two DNA-binding proteins, JARID2 and YY1, to regulate PcG activity at these three elements. JARID2 has differential requirements, whereas YY1 appears to be required for repressive activity at all 3 sites. We conclude that distinct elements of the mammalian HOX clusters can recruit components of the PcG complexes and confer repression, similar to what has been seen in Drosophila. These elements, however, have diverse requirements for binding factors, which, combined with previous data on other loci, speaks to the complexity of PcG targeting in mammals.     

Regulation of H3K27me3 and H3K4me3 during early porcine embryonic development

The epigenetic marks H3K27me3 and H3K4me3 are important repressive and permissive histone modifications, respectively, which are involved in gene regulation such as Hox gene expression during embryonic development. In this study, we investigated the global levels of these two histone modifications. We also investigated the expression of H3K27me3's methyltransferase (EZH2), EZH2 co‐factors (EED and SUZ12) and demethylases (JMJD3 and UTX), as well as H3K4me3's methylases (ASH1L and MLL1) and demethylase (RBP2) in porcine pre‐implantation embryos. In addition, the expression of Hox genes, HOXA2, HOXA3, HOXA7, HOXA10, HOXB4, HOXB7, HOXC8, HOXD8, and HOXD10 was investigated. We found that global levels of H3K27me3 decreased from the 1‐ to the 4‐cell stage, corresponding to the time of major embryonic genome activation. Subsequently, the levels increased in hatched blastocysts, particularly in the trophectoderm. The expression levels of EZH2, EED, SUZ12, JMJD3, and UTX correlated well with these findings. The global levels of H3K4me3 decreased from the 1‐cell to the morula stage and increased in hatched blastocysts, especially in trophectoderm. A peak in expression of ASH1L was seen at the 4‐cell stage, but overall, expression of ASH1L, MLL1, and RBP2 correlated poorly with H3K4me3. HOXA3, A7, and B4 were expressed in 4‐cell embryos, and HOXA7, A10, B4, and D8 were expressed in hatched blastocysts, and did not correlate well to global methylation of H3K27me3 or H3K4me3. Thus, H3K4me3 may play a role in early porcine embryonic genome activation, whereas, H3K27me3 may be involved in initial cell lineage segregation in the blastocyst. Mol. Reprod. Dev. 77: 540–549, 2010. © 2010 Wiley‐Liss, Inc.     

Differential regulation by retinoic acid of the homeobox genes of the four HOX loci in human embryonal carcinoma cells.

We studied the expression of 38 human homeobox genes belonging to the four HOX complex loci in embryonal carcinoma (EC) cells induced to differentiate by culturing them in a medium containing retinoic acid (RA). Genes located at the 3' end of each one of the four HOX loci are activated by RA in a sequential order colinear with their 3' to 5' arrangement in the cluster: 3' HOX genes respond early to the drug while upstream genes respond progressively later. Among the genes located at the 5' end of HOX loci RNase protection analysis reveals that one HOX3 gene and four HOX4 genes are weakly expressed in EC stem cells and downregulated upon treatment with 10(-5) M RA. While activation of early responding genes does not require continuous protein synthesis, the observed timing and polarity of gene activation is disrupted in the absence of protein synthesis.     

Mutually exclusive antiproliferative effect of cell line-specific HOX inhibition in epithelial ovarian cancer cell lines: SKOV-3 vs RMUG-S

We aimed to discover cell line-specific overexpressed HOX genes responsible for chemoresistance and to identify the mechanisms behind HOX-induced cell line-specific chemoresistance in EOC. Ten HOX genes and eight EOC cell lines were tested for any cell line-specific overexpression that presents a mutually exclusive pattern. Cell viability was evaluated after treatment with cisplatin and/or siRNA for cell line-specific overexpressed HOX genes. Immunohistochemical (IHC) staining for HOXB9 was performed in 84 human EOC tissues. HOXA10 and HOXB9 were identified as cell line-specific overexpressed HOX genes for SKOV-3 and RMUG-S, respectively. Inhibiting the expression of cell line-specific HOX genes, but not of other HOX genes, significantly decreased cell viability. In SKOV-3 cells, cell viability decreased to 46.5% after initial 10 µM cisplatin treatment; however, there was no further decrease upon additional treatment with HOXA10 siRNA. In contrast, cell viability did not significantly decrease upon cisplatin treatment in RMUG-S cells, but decreased to 65.5% after additional treatment with HOXB9 siRNA. In both cell lines, inhibiting cell line-specific HOX expression enhanced apoptosis but suppressed the expression of epithelial-mesenchymal transition (EMT) markers such as vimentin, MMP9, and Oct4. IHC analysis showed that platinum-resistant cancer tissues more frequently had high HOXB9 expression than platinum-sensitive cancer tissues. HOXB9, which is overexpressed in RMUG-S but not in SKOV-3 cells, appeared to be associated with cell line-specific platinum resistance in RMUG-S. Inhibiting HOXB9 overexpression in RMUG-S cells may effectively eliminate platinum-resistant ovarian cancer cells by facilitating apoptosis and inhibiting EMT.