Structural basis of specific DNA binding by the transcription factor ZBTB24.

ZBTB24, encoding a protein of the ZBTB family of transcriptional regulators, is one of four known genes-the other three being DNMT3B, CDCA7 and HELLS-that are mutated in immunodeficiency, centromeric instability and facial anomalies (ICF) syndrome, a genetic disorder characterized by DNA hypomethylation and antibody deficiency. The molecular mechanisms by which ZBTB24 regulates gene expression and the biological functions of ZBTB24 are poorly understood. Here, we identified a 12-bp consensus sequence [CT(G/T)CCAGGACCT] occupied by ZBTB24 in the mouse genome. The sequence is present at multiple loci, including the Cdca7 promoter region, and ZBTB24 binding is mostly associated with gene activation. Crystallography and DNA-binding data revealed that the last four of the eight zinc fingers (ZFs) (i.e. ZF5-8) in ZBTB24 confer specificity of DNA binding. Two ICF missense mutations have been identified in the ZBTB24 ZF domain, which alter zinc-binding cysteine residues. We demonstrated that the corresponding C382Y and C407G mutations in mouse ZBTB24 abolish specific DNA binding and fail to induce Cdca7 expression. Our analyses indicate and suggest a structural basis for the sequence specific recognition by a transcription factor centrally important for the pathogenesis of ICF syndrome.

ATF3-Induced Mammary Tumors Exhibit Molecular Features of Human Basal-Like Breast Cancer.

Basal-like breast cancer (BLBC) is an aggressive and deadly subtype of human breast cancer that is highly metastatic, displays stem-cell like features, and has limited treatment options. Therefore, developing and characterizing preclinical mouse models with tumors that resemble BLBC is important for human therapeutic development. ATF3 is a potent oncogene that is aberrantly expressed in most human breast cancers. In the BK5.ATF3 mouse model, overexpression of ATF3 in the basal epithelial cells of the mammary gland produces tumors that are characterized by activation of the Wnt/ß-catenin signaling pathway. Here, we used RNA-Seq and microRNA (miRNA) microarrays to better define the molecular features of BK5.ATF3-derived mammary tumors. These analyses showed that these tumors share many characteristics of human BLBC including reduced expression of Rb1, Esr1, and Pgr and increased expression of Erbb2, Egfr, and the genes encoding keratins 5, 6, and 17. An analysis of miRNA expression revealed reduced levels of Mir145 and Mir143, leading to the upregulation of their target genes including both the pluripotency factors Klf4 and Sox2 as well as the cancer stem-cell-related gene Kras. Finally, we show through knock-down experiments that ATF3 may directly modulate MIR145/143 expression. Taken together, our results indicate that the ATF3 mouse mammary tumor model could provide a powerful model to define the molecular mechanisms leading to BLBC, identify the factors that contribute to its aggressiveness, and, ultimately, discover specific genes and gene networks for therapeutic targeting.

Histone modification profiling in breast cancer cell lines highlights commonalities and differences among subtypes.

BACKGROUND: Epigenetic regulators are frequently mutated or aberrantly expressed in a variety of cancers, leading to altered transcription states that result in changes in cell identity, behavior, and response to therapy. RESULTS: To define alterations in epigenetic landscapes in breast cancers, we profiled the distributions of 8 key histone modifications by ChIP-Seq, as well as primary (GRO-seq) and steady state (RNA-Seq) transcriptomes, across 13 distinct cell lines that represent 5 molecular subtypes of breast cancer and immortalized human mammary epithelial cells. DISCUSSION: Using combinatorial patterns of distinct histone modification signals, we defined subtype-specific chromatin signatures to nominate potential biomarkers. This approach identified AFAP1-AS1 as a triple negative breast cancer-specific gene associated with cell proliferation and epithelial-mesenchymal-transition. In addition, our chromatin mapping data in basal TNBC cell lines are consistent with gene expression patterns in TCGA that indicate decreased activity of the androgen receptor pathway but increased activity of the vitamin D biosynthesis pathway. CONCLUSIONS: Together, these datasets provide a comprehensive resource for histone modification profiles that define epigenetic landscapes and reveal key chromatin signatures in breast cancer cell line subtypes with potential to identify novel and actionable targets for treatment.

Characterization of CHO XPF mutant UV41: influence of XPF heterozygosity on double-strand break-induced intrachromosomal recombination.

The UV hypersensitive CHO cell mutant UV41 is the archetypal XPF mammalian cell mutant, and was essential for cloning the human nucleotide excision repair (NER) gene XPF by DNA transfection and rescue. The ERCC1 and XPF genes encode proteins that form the heterodimer responsible for making incisions required in NER and the processing of certain types of recombination intermediates. In this study, we cloned and sequenced the CHO cell XPF cDNA, determining that the XPF mutation in UV41 is a +1 insertion in exon 8 generating a premature stop codon at amino acid position 499; however, the second allele of XPF is apparently unaltered in UV41, resulting in XPF heterozygosity. XPF expression was found to be several-fold lower in UV41 compared to its parental cell line, AA8. Using approaches we previously developed to study intrachromosomal recombination in CHO cells, we modified UV41 and its parental cell line AA8 to allow site-specific gene targeting at a Flp recombination target (FRT) in intron 3 of the endogenous adenine phosphoribosyltransferase (APRT) locus. Using FLP/FRT targeting, we integrated a plasmid containing an I-SceI endonuclease sequence into this site in the paired cell lines to generate a heteroallelic APRT duplication. Frequencies of intrachromosomal recombination between APRT heteroalleles and the structures of resulting recombinants were analyzed after I-SceI induction of site-specific double-strand breaks (DSBs) in a non-homologous insertion contained within APRT homology. Our results show that I-SceI induced a small proportion of aberrant recombinants reflecting DSB-induced deletions/rearrangements in parental, repair-proficient AA8 cells. However, in XPF mutant UV41, XPF heterozygosity is responsible for a similar, but much more pronounced genomic instability phenotype, manifested independently of DSB induction. In addition, gene conversions were suppressed in UV41 cells compared to wild-type cells. These observations suggest that UV41 exhibits a genomic instability phenotype of aberrant recombinational repair, confirming a critical role for XPF in mammalian cell recombination.

Effects of varying gene targeting parameters on processing of recombination intermediates by ERCC1-XPF.

The ERCC1-XPF structure-specific endonuclease is necessary for correct processing of homologous recombination intermediates requiring the removal of end-blocking nonhomologies. We previously showed that targeting the endogenous CHO APRT locus with plasmids designed to generate such intermediates revealed defective recombination phenotypes in ERCC1 deficient cells, including suppression of targeted insertion and vector correction recombinants and the generation of a novel class of aberrant recombinants through a deletogenic mechanism. In the present study, we examined some of the mechanistic features of ERCC1-XPF in processing recombination intermediates by varying gene targeting parameters. These included altering the distance between the double-strand break (DSB) in the targeting vector and the inactivating mutation in the APRT target gene, and changing the position of the target gene mutation relative to the DSB to result in target mutations that were either upstream or downstream from the DSB. Increasing the distance from the DSB in the targeting vector to the chromosomal target gene mutation resulted in an ERCC1 dependent decrease in the efficiency of gene targeting from intermediates presenting lengthy end-blocking nonhomologies. This decrease was accompanied by a shift in the distribution of recombinant classes away from target gene conversions to targeted insertions in both wild-type and ERCC1 deficient cells, and a dramatic increase in the proportion of aberrant recombinants in ERCC1 deficient cells. Changing the position of the target gene mutation relative to the DSB in the plasmid also altered the distribution of targeted insertion subclasses recovered in wild-type cells, consistent with two-ended strand invasion followed by resolution into crossover-type products and vector integration. Our results confirm expectations from studies of Rad10-Rad1 in budding yeast that ERCC1-XPF activity affects conversion tract length, and provide evidence for the mechanism of generation of the novel, aberrant recombinant class first described in our previous study.

Drug-tolerant cancer cells show reduced tumor-initiating capacity: depletion of CD44 cells and evidence for epigenetic mechanisms.

Cancer stem cells (CSCs) possess high tumor-initiating capacity and have been reported to be resistant to therapeutics. Vice versa, therapy-resistant cancer cells seem to manifest CSC phenotypes and properties. It has been generally assumed that drug-resistant cancer cells may all be CSCs although the generality of this assumption is unknown. Here, we chronically treated Du145 prostate cancer cells with etoposide, paclitaxel and some experimental drugs (i.e., staurosporine and 2 paclitaxel analogs), which led to populations of drug-tolerant cells (DTCs). Surprisingly, these DTCs, when implanted either subcutaneously or orthotopically into NOD/SCID mice, exhibited much reduced tumorigenicity or were even non-tumorigenic. Drug-tolerant DLD1 colon cancer cells selected by a similar chronic selection protocol also displayed reduced tumorigenicity whereas drug-tolerant UC14 bladder cancer cells demonstrated either increased or decreased tumor-regenerating capacity. Drug-tolerant Du145 cells demonstrated low proliferative and clonogenic potential and were virtually devoid of CD44(+) cells. Prospective knockdown of CD44 in Du145 cells inhibited cell proliferation and tumor regeneration, whereas restoration of CD44 expression in drug-tolerant Du145 cells increased cell proliferation and partially increased tumorigenicity. Interestingly, drug-tolerant Du145 cells showed both increases and decreases in many "stemness" genes. Finally, evidence was provided that chronic drug exposure generated DTCs via epigenetic mechanisms involving molecules such as CD44 and KDM5A. Our results thus reveal that 1) not all DTCs are necessarily CSCs; 2) conventional chemotherapeutic drugs such as taxol and etoposide may directly target CD44(+) tumor-initiating cells; and 3) DTCs generated via chronic drug selection involve epigenetic mechanisms.

Regulation of CDKN2A/B and Retinoblastoma genes in Xiphophorus melanoma.

Xiphophorus interspecies hybrids provide several well-characterized genetic models of melanoma susceptibility. The Xiphophorus CDKN2A/B gene, homologous to mammalian CDKN2A/B cyclin-dependent kinase inhibitors (p16 and p15), is a candidate tumor susceptibility gene in these models. Using real-time PCR and Western blot analysis, we analyzed expression of CDKN2A/B in spontaneous and UV-induced primary melanomas from individual backcross hybrid fish. We found that CDKN2A/B mRNA is highly expressed in melanomas (18-fold), relative to other fish tissues. Expression is also elevated, to a lesser extent (9.5-fold), in melanized skin from tumor-bearing fish. However, quantitative levels of CDKN2A/B mRNA in tumors varied considerably and positively correlated with expression of the Xmrk oncogene, suggesting possible functional interaction between Xmrk and CDKN2A/B expression. As a homolog corresponding to members of the mammalian CDKN2 family which regulate cell cycle progression at the G1 checkpoint, the CDKN2A/B p13 protein is a putative regulator of the G1 checkpoint apparatus in Xiphophorus. Since CDKN2A is often observed to be inversely regulated compared to RB in some human tumors, and is capable of transcriptionally regulating RB in human ovarian tumors, we cloned the Xiphophorus maculatus RB cDNA and analyzed RB expression by real-time PCR and Western blot analysis in the fish melanomas. These experiments were designed to ascertain whether CDKN2A/B and RB expression were inversely correlated. Our results indicate that RB mRNA was consistently expressed at only a 2-fold higher level in both tumors and melanized skin than in muscle. Qualitatively similar results were obtained for protein expression. These results collectively suggest that (i) Xmrk and CDKN2A/B may be co-regulated at the transcriptional level, and (ii) there is little, if any, alteration of RB expression in Xiphophorus melanomas.

Alternative splicing of major histocompatibility complex class II DXB transcripts in Xiphophorus fishes.

Classical MHC class II glycoproteins present peptides to T cells. In Xiphophorus fishes and in the guppy, Poecilia reticulata, a classical MHC class II B-like transcript has been identified, DAB, as well as a divergent MHC class II B-like transcript, DXB. In the two species of Xiphophorus fishes studied here, X. multilineatus and X. pygmaeus, alternative splicing of the DXB transcript was observed, but not of the classical type DAB transcripts. Two alternative splice patterns were found: a 16-codon deletion and a five-nucleotide deletion that leads to an extension of the transcript. A single DXB transcript that terminates before the transmembrane region was also observed. The alternative splice pattern and the divergence of DXB from DAB suggest that in fish, DXB may have an alternate function. Alternative splicing transcripts of DXB may allow for signaling and localization of DXB within the cell.