Abstract
Abstract 3643
GATA-1 is a zinc finger transcription factor that regulates the differentiation of megakaryocytes and erythrocytes from the megakaryocyte-erythrocyte progenitor (MEP). Mutations in GATA1 are associated with hematologic malignancies of these two related lineages. Acquired mutations that lead to expression of the short isoform of GATA-1, termed GATA-1s, are associated with Acute Megakaryocytic Leukemia in children with Down syndrome (DS-AMKL). Moreover, inherited mutations in the N-finger of GATA1, such as V205M, cause a set of related diseases characterized by dyserythropoietic anemia and thrombocytopenia. These latter mutations disrupt recruitment of the essential cofactor FOG-1 and thus promote disease by interfering with the ability of the GATA-1:FOG complex to properly regulate gene expression. Despite the fact that V205 lies along the surface of the zinc finger opposite to DNA, previous studies suggest that FOG-1 may regulate the chromatin binding activity of GATA-1 at a subset of sites. In contrast to V205 mutation, the precise mechanisms by which GATA-1s contributes to disease is poorly understood. Previous studies have shown that GATA-1s uncouples megakaryocyte proliferation from differentiation, likely by an inability of GATA-1s to properly repress expression of a subset of GATA-1 target genes. We hypothesized that both inherited and acquired GATA1 mutations contribute to disease by interfering with not only target gene activation or repression, but also with GATA-1 chromatin binding. In order to define the chromatin binding activity of GATA-1 and its disease associated mutants, we performed chromatin immunoprecipitation coupled with next generation sequencing (ChIP-Seq) for wild-type GATA-1, GATA-1s and GATA-1V205G in the G1ME cell line. G1ME cells, which were derived from GATA-1 null ES cells, approximate an MEP in that they can differentiate into either erythroid cells (in the presence of EPO) or megakaryocytes (in the presence of TPO) upon reconstitution with GATA-1. We expressed GATA-1, GATA-1s, or GATA-1V205G in G1ME cells by retroviral transduction and subjected the cells to ChIP for GATA-1. The resulting DNA was sequenced on the Illumina GAII, yielding 11.8M, 10.4M, and 8.5M uniquely mapped reads. Analysis of the datasets using QuEST yielded 2367, 963, and 4130 peaks, respectively, with an FDR of <0.4%. A search for genes within 50kb of each peak in each data set revealed GATA occupancy of 1699 (GATA-1), 704 (GATA-1s), and 2757 (GATA-1V205G) genes. These results show that GATA-1s indeed binds significantly fewer genes in vivo. Surprisingly, these results also show that GATA-1V205G binds more genes, suggesting that loss of the GATA-1:FOG-1 interaction leads to increased promiscuity of GATA-1 binding to chromatin. Next, we used the non-biased motif finder MEME to identify specific transcription factor binding motifs in each data set. This analysis revealed the presence of canonical GATA binding sites in 82% (GATA-1), 65% (GATA-1s), and 85% (GATA-1V205G) of the peaks. Moreover, we identified Ets-family transcription factor binding motifs in 49%, 43%, and 41% of the peaks, respectively. Other motifs that were discovered at lower frequency include CACCC-box motifs and the Gfi1b binding site. DAVID pathway analysis of the three different GATA-1 datasets demonstrated that although most pathways are conserved among the three proteins, the “Acute Myeloid Leukemia” pathway was altered by the GATA-1s mutation. Among the genes in this pathway that are bound by GATA-1 but not GATA-1s are Kit, Grb2, and Sos1. Other genes that are bound by GATA-1, but not by GATA-1s, include Lmo2, Ikaros, Klf1, Ldb1, and Trp53. Taken together, our data report the novel discovery that the N-terminus of GATA-1, which has not previously been implicated in DNA binding, is essential for proper binding of GATA-1 to chromatin. In addition, we reveal that one key function of FOG-1 is to restrict binding of GATA-1 to a subset of loci in vivo. Future studies will focus on defining the role of the N-terminus in chromatin binding and on determining how FOG- modulates occupancy in vivo. Given that loss of the N-terminus is an essential step in leukemogenesis, the identification of GATA sites that fail to be bound by GATA-1s will provide important new insights into the mechanisms of this malignancy.
Disclosures:
No relevant conflicts of interest to declare.
Regulation of Nucleolar Chromatin by B23/Nucleophosmin Jointly Depends upon Its RNA Binding Activity and Transcription Factor UBF
