Mapping and functional characterisation of a CTCF-dependent insulator element at the 3' border of the murine Scl transcriptional domain.

The Scl gene encodes a transcription factor essential for haematopoietic development. Scl transcription is regulated by a panel of cis-elements spread over 55 kb with the most distal 3' element being located downstream of the neighbouring gene Map17, which is co-regulated with Scl in haematopoietic cells. The Scl/Map17 domain is flanked upstream by the ubiquitously expressed Sil gene and downstream by a cluster of Cyp genes active in liver, but the mechanisms responsible for delineating the domain boundaries remain unclear.

Pdgfrα and Flk1 are direct target genes of Mixl1 in differentiating embryonic stem cells.

The Mixl1 homeodomain protein plays a key role in mesendoderm patterning during embryogenesis, but its target genes remain to be identified. We compared gene expression in differentiating heterozygous Mixl1(GFP/w) and homozygous null Mixl1(GFP/Hygro) mouse embryonic stem cells to identify potential downstream transcriptional targets of Mixl1. Candidate Mixl1 regulated genes whose expression was reduced in GFP+ cells isolated from differentiating Mixl1(GFP/Hygro) embryoid bodies included Pdgfrα and Flk1.

A Runx1-Smad6 rheostat controls Runx1 activity during embryonic hematopoiesis.

The oncogenic transcription factor Runx1 is required for the specification of definitive hematopoietic stem cells (HSC) in the developing embryo. The activity of this master regulator is tightly controlled during development. The transcription factors that upregulate the expression of Runx1 also upregulate the expression of Smad6, the inhibitory Smad, which controls Runx1 activity by targeting it to the proteasome.

Epigenetic silencing of BIM in glucocorticoid poor-responsive pediatric acute lymphoblastic leukemia, and its reversal by histone deacetylase inhibition.

Glucocorticoids play a critical role in the therapy of lymphoid malignancies, including pediatric acute lymphoblastic leukemia (ALL), although the mechanisms underlying cellular resistance remain unclear. We report glucocorticoid resistance attributable to epigenetic silencing of the BIM gene in pediatric ALL biopsies and xenografts established in immune-deficient mice from direct patient explants as well as a therapeutic approach to reverse resistance in vivo. Glucocorticoid resistance in ALL xenografts was consistently associated with failure to up-regulate BIM expression after dexamethasone exposure despite confirmation of a functional glucocorticoid receptor.

Endoglin expression in blood and endothelium is differentially regulated by modular assembly of the Ets/Gata hemangioblast code.

Endoglin is an accessory receptor for TGF-beta signaling and is required for normal hemangioblast, early hematopoietic, and vascular development. We have previously shown that an upstream enhancer, Eng -8, together with the promoter region, mediates robust endothelial expression yet is inactive in blood. To identify hematopoietic regulatory elements, we used array-based methods to determine chromatin accessibility across the entire locus.

Endocrine cells develop within pancreatic bud-like structures derived from mouse ES cells differentiated in response to BMP4 and retinoic acid.

We have examined factors affecting the in vitro differentiation of Pdx1(GFP/w) ESCs to pancreatic endocrine cells. Inclusion of Bone Morphogenetic Protein 4 (BMP4) during the first four days of differentiation followed by a 24-hour pulse of retinoic acid (RA) induced the formation of GFP(+) embryoid bodies (EBs). GFP expression was restricted to E-cadherin(+) tubes and GFP bright (GFP(br)) buds, reminiscent of GFP(+) early foregut endoderm and GFP(br) pancreatic buds observed in Pdx1(GFP/w) embryos.

Real-time PCR mapping of DNaseI-hypersensitive sites using a novel ligation-mediated amplification technique.

Mapping sites within the genome that are hypersensitive to digestion with DNaseI is an important method for identifying DNA elements that regulate transcription. The standard approach to locating these DNaseI-hypersensitive sites (DHSs) has been to use Southern blotting techniques, although we, and others, have recently published alternative methods using a range of technologies including high-throughput sequencing and genomic array tiling paths. In this article, we describe a novel protocol to use real-time PCR to map DHS.

Identifying gene regulatory elements by genomic microarray mapping of DNaseI hypersensitive sites.

The identification of cis-regulatory elements is central to understanding gene transcription. Hypersensitivity of cis-regulatory elements to digestion with DNaseI remains the gold-standard approach to locating such elements. Traditional methods used to identify DNaseI hypersensitive sites are cumbersome and can only be applied to short stretches of DNA at defined locations.

Retinoic acid induces Pdx1-positive endoderm in differentiating mouse embryonic stem cells.

We have generated an embryonic stem (ES) cell line in which sequences encoding green fluorescent protein (GFP) were targeted to the locus of the pancreatic-duodenal homeobox gene (Pdx1). Analysis of chimeric embryos derived from blastocyst injection of Pdx1(GFP/w) ES cells demonstrated that the pattern of GFP expression was consistent with that reported for the endogenous Pdx1 gene. By monitoring GFP expression during the course of ES cell differentiation, we have shown that retinoic acid (RA) can regulate the commitment of ES cells to form Pdx1(+) pancreatic endoderm.