Mark Wijgerde

Heterochromatin Effects on the Frequency and Duration of LCR-Mediated Gene Transcription

Locus control regions (LCRs) are responsible for initiating and maintaining a stable tissue-specific open chromatin structure of a locus. In transgenic mice, LCRs confer high level expression on linked genes independent of position in the mouse genome. Here we show that an incomplete LCR loses this property when integrated into heterochromatic regions. Two disruption mechanisms were observed. One is classical position-effect variegation, resulting in continuous transcription in a clonal subpopulation of cells. The other is a novel mechanism resulting in intermittent gene transcription in all cells. We conclude that only a complete LCR fully overcomes heterochromatin silencing and that it controls the level of transcription by ensuring activity in all cells at all times rather than directly controlling the rate of transcription.

Chromatin interaction mechanism of transcriptional control in vivo

We have used a kinetic analysis to distinguish possible mechanisms of activation of transcription of the different genes in the human β globin locus. Based on in situ studies at the single‐cell level we have previously suggested a dynamic mechanism of single genes alternately interacting with the locus control region (LCR) to activate transcription. However, those steady‐state experiments did not allow a direct measurement of the dynamics of the mechanism and the presence of loci with in situ primary transcript signals from two β‐like genes in cis has left open the possibility that multiple genes in the locus could initiate transcription simultaneously. Kinetic assays involving removal of a block to transcription elongation in conjunction with RNA FISH show that multiple β gene primary transcript signals in cis represent a transition between alternating transcriptional periods of single genes, supporting a dynamic interaction mechanism.

Erythroid Krüppel-like factor (EKLF) is active in primitive and definitive erythroid cells and is required for the function of 5′HS3 of the β-globin locus control region

Disruption of the gene for transcription factor EKLF (erythroid Krüppel‐like factor) results in fatal anaemia caused by severely reduced expression of the adult β‐globin gene, while other erythroid‐specific genes, including the embryonic ϵ‐ and fetal γ‐globin genes, are expressed normally. Thus, EKLF is thought to be a stage‐specific factor acting through the CACC box in the β‐gene promoter, even though it is already present in embryonic red cells. Here, we show that a β‐globin gene linked directly to the locus control region (LCR) is expressed at embryonic stages, and that this is only modestly reduced in EKLF−/− embryos. Thus, embryonic β‐globin expression is not intrinsically dependent on EKLF. To investigate whether EKLF functions in the locus control region, we analysed the expression of LCR‐driven lacZ reporters. This shows that EKLF is not required for reporter activation by the complete LCR. However, embryonic expression of reporters driven by 5′HS3 of the LCR requires EKLF. This suggests that EKLF interacts directly with the CACC motifs in 5′HS3 and demonstrates that EKLF is also a transcriptional activator in embryonic erythropoiesis. Finally, we show that overexpression of EKLF results in an earlier switch from γ‐ to β‐globin expression. Adult mice with the EKLF transgene have reduced platelet counts, suggesting that EKLF levels affect the balance between the megakaryocytic and erythroid lineages. Interestingly, the EKLF transgene rescues the lethal phenotype of EKLF null mice, setting the stage for future studies aimed at the analysis of the EKLF protein and its role in β‐globin gene activation.

Ventral embryonic tissues and Hedgehog proteins induce early AGM hematopoietic stem cell development

Hematopoiesis is initiated in several distinct tissues in the mouse conceptus. The aorta-gonad-mesonephros (AGM) region is of particular interest, as it autonomously generates the first adult type hematopoietic stem cells (HSCs). The ventral position of hematopoietic clusters closely associated with the aorta of most vertebrate embryos suggests a polarity in the specification of AGM HSCs. Since positional information plays an important role in the embryonic development of several tissue systems, we tested whether AGM HSC induction is influenced by the surrounding dorsal and ventral tissues. Our explant culture results at early and late embryonic day 10 show that ventral tissues induce and increase AGM HSC activity, whereas dorsal tissues decrease it. Chimeric explant cultures with genetically distinguishable AGM and ventral tissues show that the increase in HSC activity is not from ventral tissue-derived HSCs, precursors or primordial germ cells (as was previously suggested). Rather, it is due to instructive signaling from ventral tissues. Furthermore, we identify Hedgehog protein(s) as an HSC inducing signal.

Time-point and dosage of gene inactivation determine the tumor spectrum in conditional Ptch knockouts

Mutations in Patched (PTCH) have been associated with tumors characteristic both for children [medulloblastoma (MB) and rhabdomyosarcoma (RMS)] and for elderly [basal cell carcinoma (BCC)]. The determinants of the variability in tumor onset and histology are unknown. We investigated the effects of the time-point and dosage of Ptch inactivation on tumor spectrum using conditional Ptch-knockout mice. Ptch heterozygosity induced prenatally resulted in the formation of RMS, which was accompanied by the silencing of the remaining wild-type Ptch allele. In contrast, RMS was observed neither after mono- nor biallelic postnatal deletion of Ptch. Postnatal biallelic deletion of Ptch led to BCC precancerous lesions of the gastrointestinal epithelium and mesenteric tumors. Hamartomatous gastrointestinal cystic tumors were induced by monoallelic, but not biallelic Ptch mutations, independently of the time-point of mutation induction. These data suggest that the expressivity of Ptch deficiency is largely determined by the time-point, the gene dose and mode of Ptch inactivation. Furthermore, they point to key differences in the tumorigenic mechanisms underlying adult and childhood tumors. The latter ones are unique among all tumors since their occurrence decreases rather than increases with age. A better understanding of mechanisms underlying this ontological restriction is of potential therapeutic value.

Tumor Stroma–Derived Wnt5a Induces Differentiation of Basal Cell Carcinoma of Ptch-Mutant Mice via CaMKII

Basal cell carcinoma (BCC) is the most common skin tumor in humans. Although BCCs rarely metastasize, they can cause significant morbidity due to local aggressiveness. Approximately 20% of BCCs show signs of spontaneous regression. The understanding of molecular events mediating spontaneous regression has the potential to reduce morbidity of BCC and, potentially, other tumors, if translated into tumor therapies. We show that BCCs induced in conditional Ptch(flox/flox)ERT2(+/-) knockout mice regress with time and show a more differentiated phenotype. Differentiation is accompanied by Wnt5a expression in the tumor stroma, which is first detectable at the fully developed tumor stage. Coculture experiments revealed that Wnt5a is upregulated in tumor-adjacent macrophages by soluble signals derived from BCC cells. In turn, Wnt5a induces the expression of the differentiation marker K10 in tumor cells, which is mediated by Wnt/Ca(2+) signaling in a CaMKII-dependent manner. These data support a role of stromal Wnt5a in BCC differentiation and regression, which may have important implications for development of new treatment strategies for this tumor. Taken together, our results establish BCC as an easily accessible model of tumor regression. The regression of BCC despite sustained Hedgehog signaling activity seems to be mediated by tumor-stromal interactions via Wnt5a signaling.

The Dynamics of Globin Gene Expression and Gene Therapy Vectors

Abstract: The most important level of regulation of the β‐globin genes is by activation of all of the genes by the Locus Control Region (LCR) and repression of the early genes by an as yet unknown factor acting on sequences flanking the genes. Superimposed on this is a mechanism in which the early genes (ɛ and γ) suppress the late genes (δ and β) by competition for the interaction with the LCR. Although this extra level of gene regulation is quantitatively of less importance than the direct repression mechanism, it has important implications and has provided an excellent assay system to probe the regulation of transcription at the single cell level.

Remarkably low fibroblast acid α-glucosidase activity in three adults with Pompe disease

INTRODUCTION Most adults with Pompe disease are compound heterozygotes in which one acid α-glucosidase (GAA) allele harbors the c.-32-13T>G mutation, causing partial loss of GAA, and the other allele harbors a fully deleterious mutation. The fibroblast GAA activity in these patients is usually between 5% and 25% of the average in healthy individuals. In some adult patients, however, the fibroblast GAA activity is much lower and is in the range that is normally observed in classic-infantile Pompe disease. We investigated the genotype-phenotype correlation in three such adult patients and measured the GAA activity as well as the glycogen content in muscle and fibroblasts in order to better understand the clinical course. METHODS DNA was sequenced and GAA activity and glycogen content were measured in leukocytes, fibroblasts and muscle. Muscle biopsies were microscopically analyzed and the biosynthesis of GAA in fibroblasts was analyzed by immunoblotting. GAA activity and glycogen content in fibroblasts and muscle tissue in healthy controls, adult patients with Pompe disease and classic-infantile patients were compared with those of the three index patients. RESULTS One patient had genotype c.525delT/c.671G>A (r.0/p.Arg224Gln). Two affected brothers had genotype c.569G>A/c.1447G>A (p.Arg190His/p.Gly483Arg). In all three cases the GAA activity and the glycogen content in fibroblasts were within the same range as in classic-infantile Pompe disease, but the activity and glycogen content in muscle were both within the adult range. In fibroblasts, the first step of GAA synthesis appeared unaffected but lysosomal forms of GAA were not detectable with immunoblotting. CONCLUSION Some adult patients with mutations other than c.-32-13T>G can have very low GAA activity in fibroblasts but express higher activity in muscle and store less glycogen in muscle than patients with classic-infantile Pompe disease. This might explain why these patients have a slowly progressive course of Pompe disease.

The Regulation of Human β Globin Gene Expression: The Dynamics of Transcriptional Competition in the Human β-Globin Locus

The genes of the human s-globin locus are arranged in the order in which they are expressed during development (fig. 1). This arrangement, and the involvement of the LCR in activating all of the genes gives the locus an intrinsic structural polarity. It has been suggested that this polarity could play a direct role in the regulation of the locus by affecting competition between the genes for the activating function of the LCR (Hanscombe et al., 1991).