Targeted chromatin ligation, a robust epigenetic profiling technique for small cell numbers.

The complexity and inefficiency of chromatin immunoprecipitation strategies restrict their sensitivity and application when examining rare cell populations. We developed a new technique that replaces immunoprecipitation with a simplified chromatin fragmentation and proximity ligation step that eliminates bead purification and washing steps. We present a simple single tube proximity ligation technique, targeted chromatin ligation, that captures histone modification patterns with only 200 cells. Our technique eliminates loss of material and sensitivity due to multiple inefficient steps, while simplifying the workflow to enhance sensitivity and create the potential for novel applications.

Cell-type-specific activation and repression of PU.1 by a complex of discrete, functionally specialized cis-regulatory elements.

The transcription factor PU.1 is critical for multiple hematopoietic lineages, but different leukocyte types require strictly distinct patterns of PU.1 regulation. PU.1 is required early for T-cell lineage development but then must be repressed by a stage-specific mechanism correlated with commitment. Other lineages require steady, low expression or upregulation. Until now, only the promoter plus a distal upstream regulatory element (URE) could be invoked to explain nearly all Sfpi1 (PU.1) activation and repression, including bifunctional effects of Runx1. However, the URE is dispensable for most Sfpi1 downregulation in early T cells, and we show that it retains enhancer activity in immature T-lineage cells even where endogenous Sfpi1 is repressed. We now present evidence for another complex of conserved noncoding elements that mediate discrete, cell-type-specific regulatory features of Sfpi1, including a myeloid cell-specific activating element and a separate, pro-T-cell-specific silencer element. These elements yield opposite, cell-type-specific responses to Runx1. T-cell-specific repression requires Runx1 acting through multiple nonconsensus sites in the silencer core. These newly characterized sites recruit Runx1 binding in early T cells in vivo and define a functionally specific scaffold for dose-dependent, Runx-mediated repression.

A gene regulatory network armature for T lymphocyte specification.

Choice of a T lymphoid fate by hematopoietic progenitor cells depends on sustained Notch-Delta signaling combined with tightly regulated activities of multiple transcription factors. To dissect the regulatory network connections that mediate this process, we have used high-resolution analysis of regulatory gene expression trajectories from the beginning to the end of specification, tests of the short-term Notch dependence of these gene expression changes, and analyses of the effects of overexpression of two essential transcription factors, namely PU.1 and GATA-3. Quantitative expression measurements of >50 transcription factor and marker genes have been used to derive the principal components of regulatory change through which T cell precursors progress from primitive multipotency to T lineage commitment. Our analyses reveal separate contributions of Notch signaling, GATA-3 activity, and down-regulation of PU.1. Using BioTapestry (www.BioTapestry.org), the results have been assembled into a draft gene regulatory network for the specification of T cell precursors and the choice of T as opposed to myeloid/dendritic or mast-cell fates. This network also accommodates effects of E proteins and mutual repression circuits of Gfi1 against Egr-2 and of TCF-1 against PU.1 as proposed elsewhere, but requires additional functions that remain unidentified. Distinctive features of this network structure include the intense dose dependence of GATA-3 effects, the gene-specific modulation of PU.1 activity based on Notch activity, the lack of direct opposition between PU.1 and GATA-3, and the need for a distinct, late-acting repressive function or functions to extinguish stem and progenitor-derived regulatory gene expression.