Sjaak Philipsen

Transcription Factor Sp1 Is Essential for Early Embryonic Development but Dispensable for Cell Growth and Differentiation

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.

Regulation of the activity of Sp1-related transcription factors

The initiation of transcription is accomplished via interactions of many different proteins with common and gene-specific regulatory motifs. Clearly, sequence-specific transcription factors play a crucial role in the specificity of transcription initiation. A group of sequence-specific DNA-binding proteins, related to the transcription factor Sp1, has been implicated in the regulation of many different genes, since binding sites for these transcription factors (GC/GT boxes) are a recurrent motif in regulatory sequences such as promoters, enhancers and CpG islands of these genes. The simultaneous occurrence of several homologous GC/GT box-binding factors precludes a straightforward deduction of their role in transcriptional regulation. In this review, we focus on the connection between functional specificity and biochemical properties including glycosylation, phosphorylation and acetylation of Sp1-related factors.

Gene Expression-Based Classification of Non-Small Cell Lung Carcinomas and Survival Prediction

Background Current 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 Findings A 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. Conclusions The gene signatures identified are promising tools for histo-pathological classification of non-small cell lung cancer, and may improve the prediction of clinical outcome.

GATA1 Function, a Paradigm for Transcription Factors in Hematopoiesis

TRANSCRIPTIONAL CONTROL OF HEMATOPOIESIS The development of mature blood cells of distinct lineages, from the hematopoietic stem cells (HSCs), involves a progressive restriction of differentiation potential and the establishment of lineage-specific gene expression profiles (Fig. 1). The establishment of these expression profiles relies on lineagespecific transcription factors to modulate the expression of their target genes. Therefore, hematopoiesis is an excellent model system to investigate how particular transcription factors influence the establishment of lineage-specific expression profiles and how their activity is regulated. In this review we focus on the present knowledge of the biological functions of the hematopoietic transcription factor GATA1. Many aspects of its function have been revealed since its first description in 1988. Yet many new questions have surfaced, and many old questions remain to be answered. Thus, GATA1 has been in the floodlight of modern biology as a paradigm for hematopoietic transcription factors in general and GATA factors in particular.

Haploinsufficiency for the erythroid transcription factor KLF1 causes hereditary persistence of fetal hemoglobin

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 but others remain elusive. HPFH was found in 10 of 27 members from a Maltese family. We used a genome-wide SNP scan followed by linkage analysis to identify a candidate region on chromosome 19p13.12–13. Sequencing revealed a nonsense mutation in the KLF1 gene, p.K288X, which ablated the DNA-binding domain of this key erythroid transcriptional regulator. Only family members with HPFH were heterozygous carriers of this mutation. Expression profiling on primary erythroid progenitors showed that KLF1 target genes were downregulated in samples from individuals with HPFH. Functional assays suggested that, in addition to its established role in regulating adult globin expression, KLF1 is a key activator of the BCL11A gene, which encodes a suppressor of HbF expression. These observations provide a rationale for the effects of KLF1 haploinsufficiency on HbF levels.

Detailed analysis of the site 3 region of the human beta-globin dominant control region.

Four DNase I hypersensitive sites characterize the human beta‐globin Dominant Control Region (DCR) providing position independent, high levels of erythroid specific expression to linked homologous and heterologous genes when introduced into cultured cells or in transgenic mice. We have delineated the hypersensitive site located 10.5 kbp upstream of the epsilon‐globin gene by short range DNase I sensitivity mapping to a 600 bp region. Using transgenic mice and MEL cells the functional part of this region was further mapped to a 300 bp central core, which provides position independent, high level expression. It contains a number of ubiquitous and erythroid specific protein binding sites, including the previously described factors NF‐E1 (GF1) and NF‐E2. The latter binds to a dimer of the consensus binding sequence for jun/fos. The presence of this sequence is required for the function of the element, but single or multimerized copies of this site failed to give position independent, high levels of expression in transgenic mice or MEL cells. We therefore conclude that a combination of factor binding sites is necessary to allow site 3 to function as a strong transcriptional activator, resulting in position independent expression of the beta‐globin gene.

A dominant chromatin-opening activity in 5' hypersensitive site 3 of the human beta-globin locus control region.

Single‐copy human beta‐globin transgenes are very susceptible to suppression by position effects of surrounding closed chromatin. However, these position effects are overcome by a 20 kbp DNA fragment containing the locus control region (LCR). Here we show that the 6.5 kbp microlocus LCR cassette reproducibly directs full expression from independent single‐copy beta‐globin transgenes. By testing individual DNase I‐hypersensitive sites (HS) present in the microlocus cassette, we demonstrate that the 1.5 kbp 5′HS2 enhancer fragment does not direct beta‐globin expression from single‐copy transgenes. In contrast, the 1.9 kbp 5′HS3 fragment directs beta‐globin expression in five independent single‐copy transgenic mouse lines. Moreover, the 5′HS3 core element and beta‐globin proximal promoter sequences are DNase I hypersensitive in fetal liver nuclei of these expressing transgenic lines. Taken together, these results demonstrate that LCR activity is the culmination of at least two separable functions including: (i) a novel activity located in 5′HS3 that dominantly opens and remodels chromatin structure; and (ii) a recessive enhancer activity residing in 5′HS2. We postulate that the different elements of the LCR form a ‘holocomplex’ that interacts with the individual globin genes.

The beta-globin dominant control region: hypersensitive site 2.

The Dominant Control Region (DCR) of the human beta‐globin gene locus consists of four strong hypersensitive sites (HSS) upstream of the epsilon‐globin gene. Addition of these sites confers copy number dependent expression on the human beta‐globin gene in murine erythroleukaemia cells and transgenic mice, at levels comparable with the endogenous mouse globin genes. We have shown previously that a 1.9 kb fragment comprising HSS 2 accounts for 40‐50% of the full effect of the DCR. In this paper we describe a deletional analysis of HSS 2. We show that a 225 bp fragment is sufficient to direct high levels of expression of the human beta‐globin gene which is copy number dependent and integration site independent. This 225 bp fragment overlaps the major region that is hypersensitive ‘in vivo’. DNase I footprinting shows the presence of four binding sites for the erythroid specific protein NF‐E1; the three other footprinted regions display a remarkable redundancy of the sequence GGTGG and bind a number of proteins including Sp1 and the CACC box protein. The significance of these results for the regulation of globin gene expression is discussed.

Transcription factor Sp3 is essential for post-natal survival and late tooth development

Sp3 is a ubiquitously expressed transcription factor closely related to Sp1 (specificity protein 1). We have disrupted the mouse Sp3 gene by homologous recombination. Sp3‐deficient embryos are growth retarded and invariably die at birth of respiratory failure. The cause for the observed breathing defect remains obscure since only minor morphological alterations were observed in the lung, and surfactant protein expression is indistinguishable from that in wild‐type mice. Histological examinations of individual organs in Sp3−/− mice show a pronounced defect in late tooth formation. In Sp3 null mice, the dentin/enamel layer of the developing teeth is impaired due to the lack of ameloblast‐specific gene products. Comparison of the Sp1 and Sp3 knockout phenotype shows that Sp1 and Sp3 have distinct functions in vivo, but also suggests a degree of functional redundancy.

Transcriptional Regulation of BACE1, the β-Amyloid Precursor Protein β-Secretase, by Sp1

ABSTRACT Proteolytic processing of the β-amyloid precursor protein (APP) at the β site is essential to generate Aβ. BACE1, the major β-secretase involved in cleaving APP, has been identified as a type 1 membrane-associated aspartyl protease. We have cloned a 2.1-kb fragment upstream of the human BACE1 gene and identified key regions necessary for promoter activity. BACE1 gene expression is controlled by a TATA-less promoter. The region of bp −619 to +46 is the minimal promoter to control the transcription of the BACE1 gene. Several putative cis-acting elements, such as a GC box, HSF-1, a PU box, AP1, AP2, and lymphokine response element, are found in the 5′ flanking region of the BACE1 gene. Transcriptional activation and gel shift assays demonstrated that the BACE1 promoter contains a functional Sp1 response element, and overexpression of the transcription factor Sp1 potentiates BACE gene expression and APP processing to generate Aβ. Furthermore, Sp1 knockout reduced BACE1 expression. These results suggest that BACE1 gene expression is tightly regulated at the transcriptional level and that the transcription factor Sp1 plays an important role in regulation of BACE1 to process APP generating Aβ in Alzheimers disease.