We developed Tethered Conformation Capture (TCC), a method for genome-wide mapping of chromatin interactions. By implementing solid-phase ligation, TCC substantially enhanced the signal-to-noise ratio and thus, enabled a detailed analysis of inter-chromosomal interactions. We identified a group of regions in each chromosome that predominantly mediate inter-chromosomal interactions. These regions are marked by high transcriptional activity, suggesting that their interactions are mediated by transcription factories. Each of these regions interacts with numerous other such regions throughout the genome in an indiscriminate fashion, partly driven by the accessibility of the partners. Therefore, it is likely that a different combination of interactions is present in different cells. Accommodating this variability, we developed a computational method to translate the TCC data into physical chromatin contacts in a population of three-dimensional genome structures. Statistical analysis of the resulting population demonstrates that the indiscriminate properties of inter-chromosomal interactions is consistent with the well-known architectural features of the human genome.

Important biological and pathologic properties are often conserved across species. Although several mouse leukemia models have been well established, the genes deregulated in both human and murine leukemia cells have not been studied systematically. We performed a serial analysis of gene expression in both human and murine MLL-ELL or MLL-ENL leukemia cells and identified 88 genes that seemed to be significantly deregulated in both types of leukemia cells, including 57 genes not reported previously as being deregulated in MLL-associated leukemias. These changes were validated by quantitative PCR. The most up-regulated genes include several HOX genes (e.g., HOX A5, HOXA9, and HOXA10) and MEIS1, which are the typical hallmark of MLL rearrangement leukemia. The most down-regulated genes include LTF, LCN2, MMP9, S100A8, S100A9, PADI4, TGFBI, and CYBB. Notably, the up-regulated genes are enriched in gene ontology terms, such as gene expression and transcription, whereas the down-regulated genes are enriched in signal transduction and apoptosis. We showed that the CpG islands of the downregulated genes are hypermethylated. We also showed that seven individual microRNAs (miRNA) from the mir-17-92 cluster, which are overexpressed in human MLL rearrangement leukemias, are also consistently overexpressed in mouse MLL rearrangement leukemia cells. Nineteen possible targets of these miRNAs were identified, and two of them (i.e., APP and RASSF2) were confirmed further by luciferase reporter and mutagenesis assays. The identification and validation of consistent changes of gene expression in human and murine MLL rearrangement leukemias provide important insights into the genetic base for MLL-associated leukemogenesis.

Myocyte enhancer factor 2 (MEF2) regulates specific gene expression in diverse developmental programs and adaptive responses. MEF2 recognizes DNA and interacts with transcription cofactors through a highly conserved N-terminal domain referred to as the MADS-box/MEF2 domain. Here we present the crystal structure of the MADS-box/MEF2 domain of MEF2A bound to DNA. In contrast to previous structural studies showing that the MEF2 domain of MEF2A is partially unstructured, the present study reveals that the MEF2 domain participates with the MADS-box in both dimerization and DNA binding as a single domain. The sequence divergence at and immediately following the C-terminal end of the MEF2 domain may allow different MEF2 dimers to recognize different DNA sequences in the flanking regions. The current structure also suggests that the ligand-binding pocket previously observed in the Cabin1-MEF2B-DNA complex and the HDAC9 (histone deacetylase 9)-MEF2B-DNA complex is not induced by cofactor binding but rather preformed by intrinsic folding. However, the structure of the ligand-binding pocket does undergo subtle but significant conformational changes upon cofactor binding. On the basis of these observations, we generated a homology model of MEF2 bound to a myocardin family protein, MASTR, that acts as a potent coactivator of MEF2-dependent gene expression. The model shows excellent shape and chemical complementarity at the binding interface and is consistent with existing mutagenesis data. The apo structure presented here can also serve as a target for virtual screening and soaking studies of small molecules that can modulate the function of MEF2 as research tools and therapeutic leads.

The biological effect of radiofrequency (RF) fields remains controversial. We address this issue by examining whether RF fields can cause changes in gene expression. We used the pulsed RF fields at a frequency of 2.45 GHz that is commonly used in telecommunication to expose cultured human HL-60 cells. We used the serial analysis of gene expression (SAGE) method to measure the RF effect on gene expression at the genome level. We observed that 221 genes altered their expression after a 2-h exposure. The number of affected genes increased to 759 after a 6-h exposure. Functional classification of the affected genes reveals that apoptosis-related genes were among the upregulated ones and the cell cycle genes among the downregulated ones. We observed no significant increase in the expression of heat shock genes. These results indicate that the RF fields at 2.45 GHz can alter gene expression in cultured human cells through non-thermal mechanism.

Enzymes that modify the epigenetic status of cells provide attractive targets for therapy in various diseases. The therapeutic development of epigenetic modulators, however, has been largely limited to direct targeting of catalytic active site conserved across multiple members of an enzyme family, which complicates mechanistic studies and drug development. Class IIa histone deacetylases (HDACs) are a group of epigenetic enzymes that depends on interaction with Myocyte Enhancer Factor-2 (MEF2) for their recruitment to specific genomic loci. Targeting this interaction presents an alternative approach to inhibiting this class of HDACs. We have used structural and functional approaches to identify and characterize a group of small molecules that indirectly target class IIa HDACs by blocking their interaction with MEF2 on DNA.Weused X-ray crystallography and 19F NMRto show that these compounds directly bind to MEF2. We have also shown that the small molecules blocked the recruitment of class IIa HDACs to MEF2-targeted genes to enhance the expression of those targets. These compounds can be used as tools to study MEF2 and class IIa HDACs in vivo and as leads for drug development.

Identification of the specific cytogenetic abnormality is one of the critical steps for classification of acute myeloblastic leukemia (AML) which influences the selection of appropriate therapy and provides information about disease prognosis. However at present, the genetic complexity of AML is only partially understood. To obtain a comprehensive, unbiased, quantitative measure, we performed serial analysis of gene expression (SAGE) on CD15 ؉ myeloid progenitor cells from 22 AML patients who had four of the most common translocations, namely t(8;21), t(15;17), t(9;11), and inv(16). The quantitative data provide clear evidence that the major change in all these translocation-carrying leukemias is a decrease in expression of the majority of transcripts compared with normal CD15 ؉ cells. From a total of 1,247,535 SAGE tags, we identified 2,604 transcripts whose expression was significantly altered in these leukemias compared with normal myeloid progenitor cells. The gene ontology of the 1,110 transcripts that matched known genes revealed that each translocation had a uniquely altered profile in various functional categories including regulation of transcription, cell cycle, protein synthesis, and apoptosis. Our global analysis of gene expression of common translocations in AML can focus attention on the function of the genes with altered expression for future biological studies as well as highlight genes͞pathways for more specifically targeted therapy.hematopoietic cell differention ͉ diagnostic microarray T he pathogenesis of acute myeloid leukemia (AML) in many patients is linked to oncogenic fusion proteins, generated as a consequence of chromosome translocations or inversions (1). Many different translocations have been described in AML, the most frequent being the t(9;11), t(15;17), t(8;21), and inv(16), which, taken together with their variants, account for Ϸ20-30% of AML cases (2, 3), although a recent analysis by Mitelman et al. (4) suggests that the proportion may be closer to 10%. These recurring translocations are now the basis for classification of some patients with AML. Despite genetic heterogeneity, there is increasing evidence for some common molecular and biological mechanisms in the genesis of AML. In particular, one of the components of each fusion protein is almost invariably a transcription factor, frequently involved in the regulation of myeloid cell differentiation (5). As a consequence, AML-associated fusion proteins function as aberrant transcriptional regulators with the potential to interfere with the normal processes of myeloid cell differentiation.Genome-wide gene expression profiling is becoming useful for the classification of many types of cancer (6, 7), including AML and acute lymphoblastic leukemia (8-15). Although AML subtypes can be distinguished by oligonucleotide microarrays, the results of analysis of different translocations between laboratories are not always similar. This lack of consistency has probably resulted from the heterogeneous nature of clinical samples (age, se...

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The recurring chromosome translocation t(11;16)(q23;p13) is detected in leukemia patients, virtually all of whom have received previous chemotherapy with topoisomerase (topo) II inhibitors. In the t(11;16), 3' CBP, on 16p13, is fused to 5' MLL, on 11q23, resulting in an MLL-CBP fusion gene that plays an important role in leukemogenesis. In this study, we cloned genomic breakpoints of the MLL and CBP genes in the t(11;16) in the SN-1 cell line and in five patients with therapy-related leukemia, all of whom had received topo II inhibitors for previous tumors. In all patients except one, both the genomic MLL-CBP and the reciprocal fusions were cloned. Genomic breakpoints in MLL occurred in the 8.3-kb breakpoint cluster region in all patients, whereas the breakpoints in CBP clustered in an 8.2-kb region of intron 3 in four patients. Genomic breakpoints in MLL occurred in intron 11 near the topo II cleavage site in the SN-1 cell line and in one patient, and they were close to LINE repetitive sequences in two other patients. In the remaining two patients, genomic breakpoints were in intron 9 in Alu repeats. Genomic breakpoints in CBP occurred in and around Alu repeats in one and two patients, respectively. In two patients, the breaks were near LINE repetitive sequences, suggesting that repetitive DNA sequences may play a role. No specific recombination motifs were identified at or near the breakpoint junctions. No topo II cleavage sites were detected in introns 2 and 3 of CBP. However, there were deletions and duplications at the breakpoints in both MLL and CBP and microhomologies or nontemplated nucleotides at most of the genomic fusion junctions, suggesting that a nonhomologous end-joining repair mechanism was involved in the t(11;16).