Chromatin "prepattern" and histone modifiers in a fate choice for liver and pancreas

Abstract

Transcriptionally silent genes can be marked by histone modifications and regulatory proteins that indicate the genes' potential to be activated. Such marks have been identified in pluripotent cells, but it is unknown how such marks occur in descendant, multipotent embryonic cells that have restricted cell fate choices. We isolated mouse embryonic endoderm cells and assessed histone modifications at regulatory elements of silent genes that are activated upon liver or pancreas fate choices. We found that the liver and pancreas elements have distinct chromatin patterns. Furthermore, the histone acetyltransferase P300, recruited via bone morphogenetic protein signaling, and the histone methyltransferase Ezh2 have modulatory roles in the fate choice. These studies reveal a functional "prepattern" of chromatin states within multipotent progenitors and potential targets to modulate cell fate induction.

Figure 1. Distinct chromatin marks at the earliest liver and pancreas genes

ChIP for histone modifications at regulatory elements of a constitutive gene (black boxes), liver genes (green boxes), and a pancreas gene (blue boxes) in undifferentiated endoderm (open boxes) and nascent hepatoblasts (filled boxes). Liver and pancreas genes are silent in endoderm and only the liver genes become active in hepatoblasts. Signals (n≥5) over IgG were normalized by values at the Gapdh exon1 segment and shown as mean ± SD; ***p ≤ 0.001.

Figure 2. Histone acetyltranferase P300 helps promote the liver fate choice over that for ventral pancreas

(A) FACS analysis of Liv2+ hepatoblasts relative to total embryonic cells, as mean ± SEM; n = 28 WT; 21 P300+/− embryos. (B) ChIP assays as in Fig. 1 (n≥5). (C) In situ hybridization for Afp RNA in typical E9.5 sections. Largest sections of liver bud are shown. (D) (left) Immunohistochemistry for PDX1 in sections of E9.5 embryos; ventral (v.) pancreatic buds; (right) quantitation of the total number of PDX1+ cells in all sections throughout the ventral pancreatic buds; mean ± SEM.

Figure 3. The H3K27 methyltransferase gene Ezh2 restricts the extent of ventral pancreas specification

(A) ChIP assays (n=4), mean ± SD. (B) Immunohistochemistry for PDX1 in cross sections of representative E10 embryos. (C) Immunohistochemistry for NKX6.1 in cross sections of representative E11.5 embryos, mean ± SEM. (D) In situ hybridization for Afp RNA in cross sections of E10 embryos.

Figure 4. BMP4 signaling regulates hepatic specification via a pathway to SMAD4 and P300

(A) Smad4 and P300 expression by qRT-PCR, relative to levels in Liv2+ WT cells; as mean ± SEM. (B) FACS analysis of the percentage (%) of Liv2+ hepatoblasts to total embryonic cells; mean ± SEM. (C) ChIP assays (n≥4) in Liv2+ cells from WT or FoxA3^cre/+; Smad4^CA embryos; mean ± SD.