Notch proteins are single-pass, heterodimeric, transmembrane proteins encoded by genes which are conserved from flies to humans. Notch plays a critical role in development mediated by cell-cell interaction. Upon binding of a ligand (a single pass transmembrane protein on a neighboring cell) the Notch receptor undergoes a series of proteolytic cleavages resulting in the release of the Notch intracellular domain (NICD). The NICD translocates to the nucleus and activates the transcription of target genes by turning the CSL transcription factor from a repressor to an activator 
(reviewed in Kopan et al.).
Aberrant Notch signaling has been associated with many cancers including leukemia 
, breast cancer 
, medulloblastoma 
, melanoma 
and pancreatic cancer 
. In some reports it has been described as tumorigenic while in other reports it's been described as having tumor suppressor function. In leukemia the discovery of the (7;9) chromosomal translocation 
showed that constitutively active Notch signaling can be tumorigenic. Although the translocation was later found in less than 1% of T-ALL, somatic activating mutations in Notch1 receptor were detected in over 50% of human T-ALL cases 
and 74% of tumors in a mouse leukemia model 
, showing that overexpression of activated Notch1 is indeed tumorigenic 
One possible mechanism of oncogenesis is the disruption of CSL binding homeostasis. An abundance of NICD has been shown to stoichiometrically deplete CSL from other binding partners and their associated genomic loci leading to aberrant gene regulation at those sites (10). CSL can associate with at least one partner other than Notch, p48/PTF1a 
. This disruption may lead to altered gene regulation of target genes that are important in regulating growth. A genome wide assessment of CSL in the mammalian genome has not yet been performed to assess which genes are regulated by CSL.
Furthermore, it has been demonstrated that Notch signaling is context dependent in cancer, based on its integration with other signaling pathways. The Notch pathway has been shown to crosstalk with Wnt, Cadherin and the Sonic Hedgehog pathways which have been associated with tumor formation in a variety of cancers. When Notch was activated at different stages of mesodermal differentiation, the majority of the genes regulated by Notch1 were cell type specific and dependent on the other signals 
. We wanted to assess CSL binding sites globally to examine if they are found in the regulatory region of genes mediating important signaling pathways and if CSL binding sites are distributed throughout the genome indicating that Notch signaling is integrating with signaling pathways at the level of transcription.
The molecular mechanism underlying the function of Notch1 in disease and developmental states has been investigated by identification of either the direct targets of Notch1 or the direct targets of the effector protein of Notch1 signaling CSL. An integrated systems biology approach was used to assess the direct targets of Notch1 during leukemic cell growth 
. First, differentially regulated genes were determined using microarray analysis comparing gene expression profiles of seven T-ALL cell lines treated with either DMSO or a highly active gamma secretase inhibitor. This was followed by identifying the direct targets of Notch1 in the HPB-ALL T-ALL cell line using a ChIP on chip (ChIP-chip) analysis using a spotted promoter array platform. Although the microarray analysis identified differential expression of several known direct targets of Notch signaling their ChIP-Chip analysis could not confirm promoter occupancy of Deltex1, Hes1 nor Notch3 by NICD. This may be a reflection of the array platform which only included the proximal promoter regions (−700 to +200 bp). For example, only 18% of MYC-binding sites were found to be within 1 kb of a 5′-exon using an oligonucleotide tiling array that encompasses chromosomes 21 and 22 
. Any binding site outside of the core promoter regions would be missed by this analysis which may include many well defined targets of Notch signaling.
Notch 1 does not bind DNA and therefore assessing the occupancy of Notch1 at the promoters of target genes may be limited by the technical difficulty of the Notch 1 antibody IP. The Bray group sought to assess the direct targets of Notch signaling by assessing the promoter occupancy of the Notch 1 effector protein CSL in Drosophilia DMD8 cells 
. The DMD8 cell model was used to assess global changes in mRNA expression (microarray analysis) and genome wide occupancy of CSL (Su(H) in Drosophila
) within 30 min of activating Notch using ChIP-chip analysis in hopes of identifying direct target of CSL dependent Notch signaling. Although, their genome wide promoter occupancy analysis benefited from the use of an array that tiled the Drosophila
genome to give a more complete assessment of Notch target genes, it still suffers from the general limitation of ChIP-chip technology including probe selection bias and hybridization bias. Only 262 significant Su(H) binding peaks were identified genome wide. Computational analysis of the tiling array was based on a method to detect peaks in a dense tiling array which included tiling one 50-mer every 38 bp 
. However, the array used by the Bray group included 60 base oligonucleotide probes printed for approximately every 300 bp of the genomic DNA and thus it would likely miss true positive because of the limitation of the array. A significant peak was defined as a region that was detected in five adjacent probes which corresponds to 1.5 kb region. Even though, there was a statistical enrichment of Su(H) sites in the peaks they identified, only 27% of the binding-site clusters identified computationally and 1.08% of high scoring Su(H) binding sites in noncoding regions were identified as occupied.
Assessing transcription factor binding sites genome-wide has only become possible the last few years. High-throughput sequencing combined with Chromatin Immunoprecipitation has become the gold standard for assessing transcription factor binding sights globally in vivo. It is preferred over ChIP-chip because it is an absolute rather than a relative assessment of the genomic loci bound by the protein of interest. With ChIP-seq you actually sequence the ChIP purified DNA instead of hybridizing it to set of preselected probes.
Genome wide occupancy of CSL will also be important in assessing if Notch is regulating microRNAs which are important regulators of development. MicroRNAs (miRNA are short (19–25 nucleotides in lenght) noncoding RNAs, that regulate gene expression by either inhibiting translation or marking specific mRNA for degradation 
. MiRNAs influence gene expression as broadly as transcription factors and have been shown to play a role in regulating development 
. MiRNA target predictions have indicated that miRNA may target nearly 30% of animal genes 
. Thus, its not surprising that perturbation in their homeostatic function has been associated with many diseases including cancer 
. The miR-15a and miR-16-1 genes target B cell lymphoma 2 (Bcl2), an antiapoptotic gene and thus loss of their expression has been associated with cancer 
. Some miRNAs such at the miR17–92 locus 13q31 were shown to have oncogenic potential because they are amplified in some tumor 
and their overexpression in a mouse model actually accelerated tumorigenesis 
MiRNAs play an important role in regulating hematopoiesis 
. MiR-142 was highly expressed in all hematopoietic tissues whereas miR-223 was expressed exclusively in the bone marrow which consists of hematopoietic stem cells and myeloid, erythroid and lymphoid cells at different stages 
. MiR-223 is also associated with myeloid differentiation 
. Other groups have also implicated miR-144, miR-150, and miR-155 in hematopoiesis 
. MiR-126 has been associated with megakaryocyte differentiation 
. The miR-144/451 cluster is upregulated during erythropoiesis and is under the control of the master erythrocyte regulator GATA-1 
The crosstalk between microRNA expression and Notch signaling hasbeen reported in terms of which microRNAs target the Notch pathway or its target genes. Mir-34a has been reported to downregulate Notch-1 Notch-2, and CDK6 protein expression 
. MiR-1 negatively regulates Delta-1 protein levels in mouse embryonic stem cells 
. MiR-1995b down regulates the down stream Notch target gene Hes-1 
. These miRNA have been studied as potential therapeutic targets for cancer. However, in leukemia, its not abberrent gene regulation that leads to constititutively active Notch-1 expression, its somatic activating mutations in the receptor which allows the receptor to have increase stability. Therefore, finding miRNAs that are regulated by Notch signaling may be another potential therapeutic target.
CSL is a unique transcription factor because it is bound regardless of the presence of activated Notch. A conventional ChIP with antibodies to CSL alone would be limiting because the effect of Notch signaling would not be gauged. Furthermore, it would be difficult to discern real CSL binding sites from artifacts. A sequential ChIP is a new method that allows one to assess transcription factor binding occupancy using two IgGs. A hallmark of Notch activation is the acetylation of H4. Thus, a sequential ChIP with antibody to acetylated H4 followed by antibody against CSL will identify activated Notch responsive CSL binding sites. Furthermore, CSL is a small protein 60 kDa and bound to DNA in the presence of either repressor complexes or activation complexes which are enormous in size. A sequential ChIP, especially since the first IP is against the readily accessible acetylated H4 would help reduce the complexity of the nuclear lysate to allow for optimal IP with CSL antibody in the second IP. This will possibly overcome the technical difficult associated with performing a ChIP against CSL.
Embryonic stem (ES) cells have been valuable for understanding the biology of tissue development and may serve as potential therapy for diseases such as cancer. The study of the murine hematopoietic system has resulted in the major technological advances in deriving mature tissues from embryonic stem (ES) cells 
and its further characterization will have implications beyond the study of the blood system. The induction of hematopoietic differentiation on stromal cells 
and formation of embryoid bodies (EB) 
are the two experimental systems used to generate hematopoietic precursors from embryonic stem cells in most experiments 
. We have previously modeled murine hematopoiesis using an embryonic stem cells (ES)/OP9 coculture which was shown to be a highly reproducible way to model hematopoiesis in vitro 
. The OP9 stroma cell line provides the necessary extrinsic signals for the differentiation of pluripotent ES cells first into primitive flk1+ hemangioblasts (day 4–5) and then immature hematopoietic stem and progenitor cells (day 8). We have shown that the overexpression of ligand independent Notch1 in flk1+ hemangioblasts results in an alteration of the phenotype of the day 8 hematopoietic progenitor cells characterized by cell morphology, flow cytometry and gene expression profiling.
The goal of this study is to understand the molecular mechanism underlying the phenotypic changes caused by the expression of ligand independent Notch 1. First, we performed an in silico analysis of the promoters of 148 previously identified Notch regulated genes to determine the presence of putative regulatory regions. Then, a custom ChIP-chip array was used to assess the occupancy of CSL in the promoter regions of these 148 differentially expressed genes. Finally, a comprehensive mapping of the CSL binding sites genome-wide was determined using ChIP-seq analysis. Given that miRNA have documented roles in hematopoietic development, we wanted to assess which miRNAs are regulated during normal hematopoiesis and which miRNA are differentially regulated by overexpression of Notch. Thus, we performed expression profile analysis of microRNAs, using microRNA microarray during normal hematopoiesis and in response to overexpression of ligand independent Notch1.