Global Analysis of Gene Networks Involved in Hematopoiesis.

The hematopoietic system consists of more than ten differentiated cell types, which are derived from a single type of hematopoietic stem cell. Conceptually and experimentally, the determination of lineage choice, and the plasticity, extent, and robustness of lineage specific patterns in gene expression are current topics of intensive interest and investigation. To approach to these issues, we compared global analyses of a large number of genes in more than 10 primary cells cell types including stem cells, myeloid cells, erythroid cells and lymphocytes as well as certain cell lines.

We are carrying out this global work on multiple different platforms in parallel:

Gene expression. For these analyses we have used Affymetrix oligonucleoide arrays (U133) for cDNA quantitation, Affymetrix oligionucleotide arrays covering the ENCODE regions of the human genome, a PCR fragment Chromosome 22.2 genomic tiling array and a PCR fragment promoter array. U133 chip analyses were performed on the RNA samples extracted from all the cell types in this study. PCA analysis of gene expression patterns of these cell types shows that there is a pervasive lineage specific pattern of expression of most genes, and this lineage difference accounts for a substantial fraction of the total variation in gene expression between samples. Our data provides a guide for investigating the transcriptional control of both major lineage specific and broadly expressed genes for many hematopoietic cell types.

DNA-Protein interaction. More than 100 transcription factors (TFs) were studied by western-blotting, using NB4 promyelocytic cells, neutrophils or monocytes differentiated from NB4 cells, primary human PMNs, K562 and Hela cells. Our result showed that many TFs are myeloid specific, their protein expression level are very different from the mRNA level of mature cells. To further study the regulatory mechanisms, chromatin-IP analysis has been done on microarrays (Chip-chip). Immunoprecipitaiton studies of NF-KB, and CEBPE were carried out with chromosome 22 genomic tiling arrays and promoter arrays. Many gene targets of these two TFs were discovered. Importantly, we found that these two TFs not only bind to the promoter regions of their target gene, but also the introns and inter-genic sequences. This, and related results from other groups, shows an unexpected complexity in transcriptional control mechanisms.

Proteomics. ICAT mass spectroscopy and DIGE have been used for protein expression analysis of 6 cell types. These studies also have generated a large amount of data, which are being analyzed.

Our analyses are producing a wealth of information on the molecular anatomy of human hematopoietic development, especially myeloid differentiation. We are exploring the construction of network maps of hematopoiesis based on these data.