The Pioneer Transcription Factor FoxA Maintains an Accessible Nucleosome Configuration at Enhancers for Tissue-Specific Gene Activation.

Nuclear DNA wraps around core histones to form nucleosomes, which restricts the binding of transcription factors to gene regulatory sequences. Pioneer transcription factors can bind DNA sites on nucleosomes and initiate gene regulatory events, often leading to the local opening of chromatin. However, the nucleosomal configuration of open chromatin and the basis for its regulation is unclear. We combined low and high levels of micrococcal nuclease (MNase) digestion along with core histone mapping to assess the nucleosomal configuration at enhancers and promoters in mouse liver. We find that MNase-accessible nucleosomes, bound by transcription factors, are retained more at liver-specific enhancers than at promoters and ubiquitous enhancers. The pioneer factor FoxA displaces linker histone H1, thereby keeping enhancer nucleosomes accessible in chromatin and allowing other liver-specific transcription factors to bind and stimulate transcription. Thus, nucleosomes are not exclusively repressive to gene regulation when they are retained with, and exposed by, pioneer factors.

The RNA polymerase III subunit Polr3b is required for the maintenance of small intestinal crypts in mice.

BACKGROUND & AIMS: The continuously self-renewing mammalian intestinal epithelium, with high cellular turnover, depends on adequate protein synthesis for its proliferative capacity. RNA polymerase III activity is closely related to cellular growth and proliferation. Here, we studied the role of Polr3b, a large RNA polymerase III subunit, in the mammalian intestinal epithelium. METHODS: We derived mice with an intestinal epithelium-specific hypomorphic mutation of the Polr3b gene, using VillinCre-mediated gene ablation. Phenotypic consequences of the Polr3b mutation on the intestinal epithelium in mice were assessed using histological and molecular methodologies, including genetic lineage tracing. RESULTS: The Polr3b mutation severely reduced survival and growth in mice during the first postnatal week, the period when the expansion of the intestinal epithelium, and thus the requirement for protein synthesis, are highest. The neonatal intestinal epithelium of Polr3bloxP/loxP;VillinCre mice was characterized by areas with reduced proliferation, abnormal epithelial architecture, loss of Wnt signaling and a dramatic increase in apoptotic cells in crypts. Genetic lineage tracing using Polr3bLoxP/LoxP;Rosa26-lox-stop-lox-YFP;VillinCre mice demonstrated that in surviving mutant mice, Polr3b-deficient dying crypts were progressively replaced by 'Cre-escaper' cells that had retained wild type Polr3b function. In addition, enteroids cultured from Polr3bloxP/loxP;VillinCre mice show reduced proliferative activity and increased apoptosis. CONCLUSIONS: We provide evidence for an essential role of the Pol III subunit Polr3b in orchestrating the maintenance of the intestinal crypt during early postnatal development in mice.

The transcription factor CREB has no non-redundant functions in hepatic glucose metabolism in mice.

AIMS/HYPOTHESIS: Excessive hepatic glucose production is a hallmark of insulin resistance in type 2 diabetes. The cAMP responsive transcription factor cAMP responsive element binding protein (CREB), thought to be a key activator of the hepatic gluconeogenic gene regulation programme, has been suggested as a therapeutic target to reduce glucose output by the liver. Here, we test directly the requirement for hepatocytic CREB for the maintenance of glucose homeostasis. METHODS: We derived mice with a Creb (also known as Creb1) loxP allele for conditional, cell-type specific gene ablation. Hepatocyte-specific deletion of Creb was induced by injecting Creb (loxP/loxP) mice with Cre recombinase expression adeno-associated virus. RESULTS: Strikingly, we found no difference in fed and fasted glucose levels, or in glucose, insulin and glucagon tolerance in mice fed a normal chow or a high-fat diet. In addition, mRNA levels of liver-specific genes, including several CREB target genes involved in gluconeogenesis, were not affected by CREB deficiency in the liver. CONCLUSION/INTERPRETATION: Our data show that CREB has no non-redundant functions in hepatic glucose metabolism, and is therefore not likely to be a useful target for the development of glucose-lowering drugs.