Progenitor cells and their mature progeny elicit substantially different properties from genomes that are usually identical. Analysis of gene knockouts in vivo
and lineage-specific cis
-elements in vitro
support the idea that key regulatory TFs participate materially in differentiation, using mechanisms that are diverse and incompletely understood. Our identification of the CDX2 recognition motif at enhancers active in differentiated Caco-2 cells prompted detailed investigation of a TF whose highly restricted tissue expression, activity at intestine-specific gene promoters in vitro
, and embryonic requirement encompass the properties of a “master regulator” (Beck, 2004
; Gao et al., 2009
). Findings in Cdx2-null mice support this designation in the adult intestine and reveal its requirement for cellular functions, gene expression, and active enhancer chromatin in distinct states. We also present evidence for dynamic CDX2 associations in a regenerative tissue, including labile interactions with chromatin and other TFs that correlate strongly with gene expression in dividing and mature cells (). The sum of these results provides a comprehensive view of the actions and mechanisms of a critical lineage-specific TF.
Model for dynamic CDX2 function during intestinal differentiation
Dual activity in progenitor and differentiated cells is a feature of several lineage-restricted TFs. For example, Pu.1 functions in blood progenitor commitment and proliferation as well as terminal macrophage differentiation (DeKoter et al., 1998
), much as MITF does in melanocyte proliferation, survival, and maturation (McGill et al., 2002
). In development, the worm FoxA homolog PHA4 functions throughout pharynx formation, as do Pax6 and its homologs in the metazoan eye (Ashery-Padan and Gruss, 2001
). Even in the face of such examples, it is unclear if these factors directly control tissue-specific genes throughout a cell’s ontogeny, if their genome associations are stable across cellular transitions, and if Cdx2’s distinct activities in dividing and differentiated cells reflect a general property of lineage-determining TFs. The muscle regulator MyoD, for example, functions in both myoblast progenitors and mature myotubes, and its binding to DNA was recently reported to vary little during differentiation (Cao et al., 2010
). In contrast, redistribution of CDX2 during intestinal cell maturation exposes diversity among mechanisms of key TFs. Investigation of other regulators, ideally coupled with delineation of chromatin modifications, will resolve whether their associations with DNA are largely invariant, as reported for MyoD, or dynamic, as we observe with CDX2.
In C. elegans
pharynx development, PHA4 controls early and late genes by binding unique target sites with different affinities. Early genes contain high-affinity sites and bind first; late targets carry sites with distinct sequences and lower affinity, and engage only after PHA4 levels increase late in development (Gaudet and Mango, 2002
). CDX2 levels change little during intestinal differentiation and the consensus CDX2 motifs we identified in different conditions are almost identical (Supplemental Fig. S4
), indicating a limited role for primary DNA sequence or TF concentrations in directing occupancy. Hence, post-translational modifications, alternative partners, chromatin structure, or a combination of these factors, likely underlie CDX2’s cell state-specific complexes. Known post-translational alterations of CDX2 do not affect DNA affinity in vitro
(Gross et al., 2005
; Rings et al., 2001
) but could in principle affect alternative complexes. Biochemical analysis of modifications on CDX2 and its partner proteins might advance understanding of the mechanisms underlying nucleosome lability at target enhancers. In identifying GATA6 and HNF4A as state-specific CDX2 partners at differentially modified regions of chromatin, we take an important step toward characterizing tissue-restricted TFs that produce or interact with altered chromatin states to enable intestinal cell differentiation. Future work might determine if the factors are co-dependent or restructure chromatin in an ordered hierarchy; understanding these mechanisms will help define the networks that control intestinal cell transitions (Davidson and Levine, 2008
). Although findings in mutant mice corroborate our conclusions, the bulk of the present analysis was conducted in a tumor cell line that replicates the distinction between progenitor and differentiated states only partially. Primary intestinal crypt and villus cells would provide a physiologic model to test these ideas.
Studies in cancer cell lines paradoxically implicate CDX2 as both an oncogene and a tumor suppressor (Aoki et al., 2003
; Guo et al., 2004
); context-dependent CDX2 occupancy and function may account for distinct roles in cell proliferation and differentiation and help explain the discrepancy. Contextual genome occupancy and cooperation with other TFs might also underlie CDX2 requirements in embryonic axial patterning and trophectoderm formation. Indeed, CDX2 binding in the corresponding tissues suggests remarkable diversity in target loci (our unpublished data), underscoring the idea that TF activities are strongly influenced by cellular context. Another TF with many target genes, PPARG, was recently shown to utilize different binding sites and partners in two cell types, adipocytes and macrophages (Lefterova et al., 2010
The continually renewing gut epithelium, a frequent target of malignant transformation, is an ideal model system to study transcriptional mechanisms of differentiation. Our integrated approach toward genome-wide chromatin analysis, TF binding, and mRNA expression can also be extended to uncover mechanisms of lineage-specific gene regulation in other tissues.