BackgroundCurrent clinical therapy of non-small cell lung cancer depends on histo-pathological classification. This approach poorly predicts clinical outcome for individual patients. Gene expression profiling holds promise to improve clinical stratification, thus paving the way for individualized therapy.Methodology and Principal FindingsA genome-wide gene expression analysis was performed on a cohort of 91 patients. We used 91 tumor- and 65 adjacent normal lung tissue samples. We defined sets of predictor genes (probe sets) with the expression profiles. The power of predictor genes was evaluated using an independent cohort of 96 non-small cell lung cancer- and 6 normal lung samples. We identified a tumor signature of 5 genes that aggregates the 156 tumor and normal samples into the expected groups. We also identified a histology signature of 75 genes, which classifies the samples in the major histological subtypes of non-small cell lung cancer. Correlation analysis identified 17 genes which showed the best association with post-surgery survival time. This signature was used for stratification of all patients in two risk groups. Kaplan-Meier survival curves show that the two groups display a significant difference in post-surgery survival time (p = 5.6E-6). The performance of the signatures was validated using a patient cohort of similar size (Duke University, n = 96). Compared to previously published prognostic signatures for NSCLC, the 17 gene signature performed well on these two cohorts.ConclusionsThe gene signatures identified are promising tools for histo-pathological classification of non-small cell lung cancer, and may improve the prediction of clinical outcome.

One of the most common regulatory elements is the GC box and the related GT/CACC box, which are widely distributed in promoters, enhancers and locus control regions of housekeeping as well as tissue-specific genes. For long it was generally thought that Sp1 is the major factor acting through these motifs. Recent discoveries have shown that Sp1 is only one of many transcription factors binding and acting through these elements. Sp1 simply represents the first identified and cloned protein of a family of transcription factors characterised by a highly conserved DNA-binding domain consisting of three zinc fingers. Currently this new family of transcription factors has at least 16 different mammalian members. Here, we will summarise and discuss recent advances that have been directed towards understanding the biological role of these proteins.

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Transcription factor Sp1 has been implicated in the expression of many genes. Moreover, it has been suggested that Sp1 is linked to the maintenance of methylation-free CpG islands, the cell cycle, and the formation of active chromatin structures. We have inactivated the mouse Sp1 gene. Sp1-/- embryos are retarded in development, show a broad range of abnormalities, and die around day 11 of gestation. In Sp1-/- embryos, the expression of many putative target genes, including cell cycle-regulated genes, is not affected, CpG islands remain methylation free, and active chromatin is formed at the globin loci. However, the expression of the methyl-CpG-binding protein MeCP2 is greatly reduced in Sp1-/- embryos. MeCP2 is thought to be required for the maintenance of differentiated cells. We suggest that Sp1 is an important regulator of this process.

Hereditary Persistence of Fetal Hemoglobin (HPFH) is characterized by persistent high levels of fetal hemoglobin (HbF) in adults. Several contributory factors, both genetic and environmental, have been identified 1, but others remain elusive. Ten of twenty-seven members from a Maltese family presented with HPFH. A genome-wide SNP scan followed by linkage analysis revealed a candidate region on chromosome 19p13.12–13. Sequencing identified a nonsense mutation in the KLF1 gene, p.K288X, ablating the DNA binding domain of this key erythroid transcriptional regulator 2. Only HPFH family members were heterozygote carriers of this mutation. Expression profiling on primary erythroid progenitors revealed down-regulation of KLF1 target genes in HPFH samples. Functional assays demonstrated that, in addition to its established role in adult globin expression, KLF1 is a critical activator of the BCL11A gene, encoding a suppressor of HbF expression 3. These observations provide a rationale for the effects of KLF1 haploinsufficiency on HbF levels.

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Three-dimensional organization of a gene locus is important for its regulation, as recently demonstrated for the ␤-globin locus. When actively expressed, the cis-regulatory elements of the ␤-globin locus are in proximity in the nuclear space, forming a compartment termed the Active Chromatin Hub (ACH). However, it is unknown which proteins are involved in ACH formation. Here, we show that EKLF, an erythroid transcription factor required for adult ␤-globin gene transcription, is also required for ACH formation. We conclude that transcription factors can play an essential role in the three-dimensional organization of gene loci. The mouse ␤-globin locus contains multiple ␤-like globin genes, arranged from 5Ј to 3Ј in order of their developmental expression (Fig. 1A). The adult-type ␤ maj -gene is transcribed at a very low level during primitive erythropoiesis in the embryonic yolk sac, but becomes expressed at high levels around day 11 of gestation (E11) when definitive erythropoiesis commences in the fetal liver (Trimborn et al. 1999). The ␤-globin locus control region (LCR) is essential for efficient globin transcription (Grosveld et al. 1987;Bender et al. 2000). It consists of a series of DNaseI hypersensitive sites (HS) located ∼50 kb upstream of the ␤ maj promoter (Fig. 1A). We have shown that the ␤-globin locus forms an Active Chromatin Hub (ACH) in erythroid cells (Tolhuis et al. 2002). The ACH is a nuclear compartment dedicated to RNA polymerase II transcription, formed by the cis-regulatory elements of the ␤-globin locus with the intervening DNA looping out. The ACH consists of the HS of the LCR, two HS located ∼60 kb upstream of the embryonic y-globin gene (5ЈHS-62/-60) and 3ЈHS1 downstream of the genes. In addition, the actively expressed globin genes are part of the ACH (Carter et al. 2002;Tolhuis et al. 2002). In erythroid precursors that do not express the globin genes yet, a substructure of the ACH, called a chromatin hub (CH) (Patrinos et al. 2004) is found, which excludes the genes and the HS at the 3Ј site of the LCR (Palstra et al. 2003).Expression of the ␤ maj -gene requires the presence of the erythroid Krüppel-like transcription factor EKLF, the erythroid-specific member of the Sp/XKLF-family (Miller and Bieker 1993). EKLF −/− mice die of anemia around E14, because of a deficit in ␤-globin expression (Nuez et al. 1995;Perkins et al. 1995). The ␤-globin locus contains a number of EKLF-binding sites, in particular in the LCR and the ␤ maj -globin promoter (Perkins 1999;Bieker 2001). Because ␤ maj -globin expression depends on the presence of EKLF, we were interested in determining whether EKLF is involved in the formation of the ACH. Results and DiscussionWe used chromatin conformation capture (3C) technology (Dekker et al. 2002) to investigate the three-dimensional conformation of the mouse ␤-globin locus in the absence of EKLF. Cells from E12.5 EKLF −/− and wild-type fetal livers were cross-linked with formaldehyde, followed by restriction enzyme digestion of the DNA. The samples were ligated ...

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