Control of Developmental Regulators by Polycomb in Human Embryonic Stem Cells

Mapping Genome Occupancy in Embryonic Stem Cells

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Examination of the targets of Suz12 revealed that they were remarkably enriched for genes that control development and transcription (Figure 3 and examples in Figure 1C) and that Suz12 tended to occupy large domains at these genes (Figure 4). Although only 8% of all annotated genes were occupied by Suz12, ~50% of those encoding transcription factors associated with developmental processes were occupied by Suz12. By comparison, RNA polymerase II preferentially occupied genes involved in a broad spectrum of cell proliferation functions such as nucleic acid metabolism, protein synthesis and cell cycle (Figure 3A and examples in Figure 1B; Supplemental Data, Table S10).

It was striking that Suz12 occupied many families of genes that control development and transcription (Figure 3B; Supplemental Data, Figure S7 and Table S11). These included 39 of 40 of the homeotic genes found in the Hox clusters and the majority of homeodomain genes. Suz12-bound homeodomain genes included almost all members of the DLX, IRX, LHX and PAX gene families, which regulate early developmental steps in neurogenesis, hematopoiesis, axial patterning, tissue patterning, organogenesis and cell-fate specification. Suz12 also occupied promoters for large subsets of the FOX, SOX and TBX gene families. The forkhead family of FOX genes is involved in axial patterning and tissue development from all three germ layers (Carlsson and Mahlapuu, 2002; Lehmann et al., 2003). Mutations in members of the SOX gene family alter cell-fate specification and differentiation and are linked to several developmental diseases (Schepers et al., 2002). The TBX family of genes regulates a wide variety of developmental processes such as gastrulation, early pattern formation, organogenesis and limb formation (Logan, 2003; Showell et al., 2004). Thus, the genes preferentially bound by Suz12 have functions that, when expressed, promote differentiation. This is likely to explain, at least in part, why PRC2 is essential for early development and ES cell pluripotency.

A remarkable feature of Suz12 binding at most genes encoding developmental regulators was the extensive span over which the regulator occupied the locus (Figure 4; Supplemental Data, Figure S8). For the majority (72%) of bound sites across the genome, Suz12 occupied a small region in a promoter region similar in size to those bound by RNA polymerase II (Figure 1). For the remaining bound regions, Suz12 occupancy encompassed large domains spanning 2-35 kb and extending from the promoter into the gene. A large portion of genes encoding developmental regulators (72%) exhibited these extended regions of Suz12 binding. In some cases, binding encompassed multiple contiguous genes. For instance, Suz12 binding extended ~100 kb across the entire HoxA, HoxB, HoxC and HoxD clusters but did not bind to adjacent genomic sequences, yielding a highly defined spatial pattern (Figure 4B). In contrast, clusters of unrelated genes, such as the interleukin 1-beta cluster, were not similarly bound by Suz12. Thus, genes encoding developmental regulators showed an unusual tendency to be occupied by Suz12 over large regions surronding the transcriptional start site.