ChIP-seq analysis of hematopoetic progenitors

Bivalent chromatin refers to the simultaneous occurrence of transcription activation (H3K4me3)- and repression (H3K27me3)-associated histone modifications at gene promoters. This mark was first identified in ES cells and proposed to maintain genes in a poised state for future resolution to fully-active (H3K4me3-only) or fully-repressed (H3K27me3-only) states. In this report we rigorously test the poising hypothesis using a well-established developmental paradigm of hematopoietic stem cell differentiation to T lymphoid lineage committed cells. We show that bivalent chromatin is generated and resolved at specific stages of hematopoiesis. The epigenetic states from which it is generated and the states to which it is resolved vary with developmental stage, suggesting that bivalency serves different functions at each stage. Moreover, singly-marked genes do not transition to the opposing univalent state via a bivalent intermediate. Fresh ex vivo cells were used from mice to study dynamics of bivalent chromatin during hematopoietic differentiation to the T lymphoid lineage. Hematopoietic stem cells (HSC), lymphoid-primed multipotent progenitors (LMPP) and common lymphoid progenitors (CLP) were purified from bone marrow of C57BL/6J mice; early thymic precursors (ETP), double negative 2 (DN2) and double negative 3 (DN3) cells were purified from C57BL6 thymus. We analyzed at least two independent cell preparations for each subset, obtained from approximately 25 mice per preparation. Purified cells were subjected to micro-chromatin immunoprecipitation (micro-ChIP) using antibodies directed against H3K4me3 and H3K27me3. Immunoprecipitated DNA was sequenced using standard Illumina platform