Kenneth S. Zaret

Opening of Compacted Chromatin by Early Developmental Transcription Factors HNF3 (FoxA) and GATA-4

The transcription factors HNF3 (FoxA) and GATA-4 are the earliest known to bind the albumin gene enhancer in liver precursor cells in embryos. To understand how they access sites in silent chromatin, we assembled nucleosome arrays containing albumin enhancer sequences and compacted them with linker histone. HNF3 and GATA-4, but not NF-1, C/EBP, and GAL4-AH, bound their sites in compacted chromatin and opened the local nucleosomal domain in the absence of ATP-dependent enzymes. The ability of HNF3 to open chromatin is mediated by a high affinity DNA binding site and by the C-terminal domain of the protein, which binds histones H3 and H4. Thus, factors that potentiate transcription in development are inherently capable of initiating chromatin opening events.

miR-122, a mammalian liver-specific microRNA, is processed from hcr mRNA and may downregulate the high affinity cationic amino acid transporter CAT-1.

These studies show that miR-122, a 22-nucleotide microRNA, is derived from a liver-specificnon-coding polyadenylated RNA transcribed from the gene hcr. The exact sequence of miR-122as well as the adjacent secondary structure within the hcr mRNA are conserved from mammalianspecies back to fish. Levels of miR-122 in the mouse liver increase to half maximal valuesaround day 17 of embryogenesis, and reach near maximal levels of 50,000 copies per averagecell before birth. Lewis et al (2003) predicted the cationic amino acid transporter (CAT-1 orSLC7A1) as a miR-122 target. CAT-1 protein and its mRNA are expressed in all mammaliantissues but with lower levels in adult liver. Furthermore, during mouse liver development CAT-1mRNA decreases in an almost inverse correlation with miR-122. Eight potential miR-122 targetsites were predicted within the human CAT-1 mRNA, with six in the 3’-untranslated region.Using a reporter construct it was found that just three of the predicted sites, linked in a 400-nucleotide sequence from human CAT-1, acted with synergy and were sufficient to stronglyinhibit protein synthesis and reduce mRNA levels. In summary, these studies followed theaccumulation during development of miR-122 from its mRNA precursor, hcr, through toidentification of what may be a specific mRNA target, CAT-1. Link to supplemental material: http://www.landesbioscience.com/supplement/changRNA1-2-sup.pdf

Generation and Regeneration of Cells of the Liver and Pancreas

Liver and pancreas progenitors develop from endoderm cells in the embryonic foregut. Shortly after their specification, liver and pancreas progenitors rapidly acquire markedly different cellular functions and regenerative capacities. These changes are elicited by inductive signals and genetic regulatory factors that are highly conserved among vertebrates. Interest in the development and regeneration of the organs has been fueled by the intense need for hepatocytes and pancreatic β cells in the therapeutic treatment of liver failure and type I diabetes. Studies in diverse model organisms have revealed evolutionarily conserved inductive signals and transcription factor networks that elicit the differentiation of liver and pancreatic cells and provide guidance for how to promote hepatocyte and β cell differentiation from diverse stem and progenitor cell types.

Regulatory phases of early liver development: paradigms of organogenesis.

Genetic analysis, embryonic tissue explantation and in vivo chromatin studies have together identified the distinct regulatory steps that are necessary for the development of endoderm into a bud of liver tissue and, subsequently, into an organ. In this review, I discuss the acquisition of competence to express liver-specific genes by the endoderm, the control of early hepatic growth, the coordination of hepatic and vascular development and the cell differentiation that is necessary to generate a functioning liver. The regulatory mechanisms that underlie these phases are common to the development of many organ systems and might be recapitulated or disrupted during stem-cell differentiation and adult tissue pathogenesis.

Facilitators and Impediments of the Pluripotency Reprogramming Factors' Initial Engagement with the Genome

The ectopic expression of transcription factors can reprogram cell fate, yet it is unknown how the initial binding of factors to the genome relates functionally to the binding seen in the minority of cells that become reprogrammed. We report a map of Oct4, Sox2, Klf4, and c-Myc (O, S, K, and M) on the human genome during the first 48 hr of reprogramming fibroblasts to pluripotency. Three striking aspects of the initial chromatin binding events include an unexpected role for c-Myc in facilitating OSK chromatin engagement, the primacy of O, S, and K as pioneer factors at enhancers of genes that promote reprogramming, and megabase-scale chromatin domains spanned by H3K9me3, including many genes required for pluripotency, that prevent initial OSKM binding and impede the efficiency of reprogramming. We find diverse aspects of initial factor binding that must be overcome in the minority of cells that become reprogrammed.

Hepatocyte nuclear factor 4alpha controls the development of a hepatic epithelium and liver morphogenesis.

Although advances have been made in understanding cell differentiation, only rudimentary knowledge exists concerning how differentiated cells form tissues and organs. We studied liver organogenesis because the cell and tissue architecture of this organ is well defined. Approximately 60% of the adult liver consists of hepatocytes that are arranged as single-cell anastomosing plates extending from the portal region of the liver lobule toward the central vein. The basal surface of the hepatocytes is separated from adjacent sinusoidal endothelial cells by the space of Disse, where the exchange of substances between serum and hepatocytes takes place. The hepatocytes apical surface forms bile canaliculi that transport bile to the hepatic ducts. Proper liver architecture is crucial for hepatic function and is commonly disrupted in disease states, including cirrhosis and hepatitis. Here we report that hepatocyte nuclear factor 4α (Hnf4α) is essential for morphological and functional differentiation of hepatocytes, accumulation of hepatic glycogen stores and generation of a hepatic epithelium. We show that Hnf4α is a dominant regulator of the epithelial phenotype because its ectopic expression in fibroblasts induces a mesenchymal-to-epithelial transition. Most importantly, the morphogenetic parameters controlled by Hnf4α in hepatocytes are essential for normal liver architecture, including the organization of the sinusoidal endothelium.

An active tissue-specific enhancer and bound transcription factors existing in a precisely positioned nucleosomal array

Nucleosomes positioned over promoters are usually inhibitory to protein binding and activity. We analyzed at the nucleotide level of resolution the nucleosomal organization of a distal, liver-specific enhancer in various mouse tissues and found that the enhancer exists in an array of three precisely positioned nucleosomes only in liver chromatin, where the enhancer is active. In vivo footprinting reveals that essential transcription factor-binding sites are occupied on apparent nucleosome surfaces, in one case leading to a perturbed nucleosomal structure. A similar nucleosomal array is generated with an in vitro chromatin assembly system in which nucleosome positioning is dependent upon binding to the enhancer of proteins related to hepatocyte nuclear factor 3. We suggest that certain transcription factors can organize nucleosomal structures that define an active enhancer element.

Binding of the winged-helix transcription factor HNF3 to a linker histone site on the nucleosome.

The transcription factor HNF3 and linker histones H1 and H5 possess winged‐helix DNA‐binding domains, yet HNF3 and other fork head‐related proteins activate genes during development whereas linker histones compact DNA in chromatin and repress gene expression. We compared how the two classes of factors interact with chromatin templates and found that HNF3 binds DNA at the side of nucleosome cores, similarly to what has been reported for linker histone. A nucleosome structural binding site for HNF3 is occupied at the albumin transcriptional enhancer in active and potentially active chromatin, but not in inactive chromatin in vivo. While wild‐type HNF3 protein does not compact DNA extending from the nucleosome, as does linker histone, site‐directed mutants of HNF3 can compact nucleosomal DNA if they contain basic amino acids at positions previously shown to be essential for nucleosomal DNA compaction by linker histones. The results illustrate how transcription factors can possess special nucleosome‐binding activities that are not predicted from studies of factor interactions with free DNA.

Genetic programming of liver and pancreas progenitors: lessons for stem-cell differentiation.

The liver and pancreas arise from a common multipotent population of endoderm cells and share many aspects of their early development. Yet each tissue originates from multiple spatial domains of the endoderm, under the influence of different genes and inductive cues, and obtains different regenerative capacities. Emerging genetic evidence is illuminating the ability of newly specified hepatic and pancreatic progenitors to reverse their course and develop into gut progenitors. Understanding how tissue programming can be reversed and how intrinsic regenerative capacities are determined should facilitate the discovery of the basis of cellular plasticity and aid in the targeted programming and growth of stem cells.

Endothelial cell interactions initiate dorsal pancreas development by selectively inducing the transcription factor Ptf1a

Dorsal and ventral pancreatic bud development from the endoderm requires inductive interactions with diverse mesodermal cell types and the action of transcription factors expressed within the endoderm. Presently it is unclear which mesodermal interactions activate which pancreatic transcription factors, and whether such inductions are common for initiating dorsal and ventral pancreas development. Previous studies of Lammert et al. (Lammert, E., Cleaver, O. and Melton, D. (2001) Science 294, 564-567) showed that signaling from embryonic blood vessel cells, derived from the mesoderm, promotes pancreatic bud development. Using a combination of mouse Flk1-/- embryos lacking endothelial cells and tissue recombination experiments, we discovered that the initial induction of dorsal endoderm cells positive for the pancreatic and duodenal transcription factor Pdx1 does not require aorta or endothelial cell interactions, but dorsal pancreatic bud emergence and the maintenance of Pdx1 expression does. Aortal endothelial cells induce the crucial pancreatic transcription factor Ptf1a in the dorsal pancreatic endoderm; whereas the vitelline veins, which are normally adjacent to the emerging ventral pancreatic bud, are unnecessary for ventral Ptf1a induction or for ventral pancreatic bud initiation. We find that the aorta cells themselves, apart from the blood supply, cause the induction of Ptf1a in dorsal endoderm explants. Thus, endothelial cell interactions specifically promote early dorsal pancreatic development, at least in part, by inducing Ptf1a+ pancreatic progenitors. Additionally, we find that endothelial cells are necessary for the induction of both the insulin and glucagon genes.