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Repressive KRAB domain-containing zinc-finger proteins (KRAB-ZFPs) are abundant in mammalian genomes and contribute both to the silencing of transposable elements (TEs) and to the regulation of developmental stage- and cell type-specific gene expression. Here we describe studies of zinc finger protein 92 (Zfp92), an X-linked KRAB-ZFP that is highly expressed in pancreatic islets of adult mice, by analyzing global Zfp92 knockout (KO) mice. Physiological, transcriptomic and genome-wide chromatin binding studies indicate that the principal function of ZFP92 in mice is to bind to and suppress the activity of B1/Alu type of SINE elements and modulate the activity of surrounding genomic entities. Deletion of Zfp92 leads to changes in expression of select LINE and LTR retroelements and genes located in the vicinity of ZFP92-bound chromatin. The absence of Zfp92 leads to altered expression of specific genes in islets, adipose and muscle that result in modest sex-specific alterations in blood glucose homeostasis, body mass and fat accumulation. In islets, Zfp92 influences blood glucose concentration in postnatal mice via transcriptional effects on Mafb, whereas in adipose and muscle, it regulates Acacb, a rate-limiting enzyme in fatty acid metabolism. In the absence of Zfp92, a novel TE-Capn11 fusion transcript is overexpressed in islets and several other tissues due to de-repression of an IAPez TE adjacent to ZFP92-bound SINE elements in intron 3 of the Capn11 gene. Together, these studies show that ZFP92 functions both to repress specific TEs and to regulate the transcription of specific genes in discrete tissues.

Insm1, Neurod1, and Pax6 are essential for the formation and function of pancreatic endocrine cells. Here, we report comparative immunohistochemical, transcriptomic, functional enrichment, and RNA splicing analyses of these genes using gene knock-out mice. Quantitative immunohistochemical analysis confirmed that elimination of each of these three factors variably impairs the proliferation, survival, and differentiation of endocrine cells. Transcriptomic analysis revealed that each factor contributes uniquely to the transcriptome although their effects were overlapping. Functional enrichment analysis revealed that genes downregulated by the elimination of Insm1, Neurod1, and Pax6 are commonly involved in mRNA metabolism, chromatin organization, secretion, and cell cycle regulation, and upregulated genes are associated with protein degradation, autophagy, and apoptotic process. Elimination of Insm1, Neurod1, and Pax6 impaired expression of many RNA-binding proteins thereby altering RNA splicing events, including for Syt14 and Snap25, two genes required for insulin secretion. All three factors are necessary for normal splicing of Syt14, and both Insm1 and Pax6 are necessary for the processing of Snap25. Collectively, these data provide new insights into how Insm1, Neurod1, and Pax6 contribute to the formation of functional pancreatic endocrine cells.

Gene network transitions in embryos and other fate-changing contexts involve combinations of transcription factors. A subset of fate-changing transcription factors act as pioneers; they scan and target nucleosomal DNA and initiate cooperative events that can open the local chromatin. However, a gap has remained in understanding how molecular interactions with the nucleosome contribute to the chromatin-opening phenomenon. Here we identified a short α-helical region, conserved among FOXA pioneer factors, that interacts with core histones and contributes to chromatin opening in vitro. The same domain is involved in chromatin opening in early mouse embryos for normal development. Thus, local opening of chromatin by interactions between pioneer factors and core histones promotes genetic programming., M.I. was supported by postdoctoral fellowships from Japan Society for the Promotion of Science Foundation (H26-683), Naito Foundation (RYU10000032), Astellas Foundation for Research on Metabolic Disorders (K0076) and Uehara Memorial Foundation (H24-20124). P.S. was supported by grant nos. SAF2016-75531-R (MICINN/FEDER, UE) and B2017/BMD-3724 (Comunidad de Madrid). The research was supported by NIH grant no. GM36477 to K.S.Z.

Transcription factors positively and/or negatively impact gene expression by recruiting coregulatory factors, which interact through protein-protein binding. Here we demonstrate that mouse pancreas size and islet β-cell function are controlled by the ATP-dependent Swi/Snf chromatin remodeling coregulatory complex that physically associates with Pdx1, a diabetes-linked transcription factor essential to pancreatic morphogenesis and adult islet cell function and maintenance. Early embryonic deletion of just the Swi/Snf Brg1 ATPase subunit reduced multipotent pancreatic progenitor cell proliferation and resulted in pancreas hypoplasia. In contrast, removal of both Swi/Snf ATPase subunits, Brg1 and Brm, was necessary to compromise adult islet β-cell activity, which included whole-animal glucose intolerance, hyperglycemia, and impaired insulin secretion. Notably, lineage-tracing analysis revealed Swi/Snf-deficient β-cells lost the ability to produce the mRNAs for Ins and other key metabolic genes without effecting the expression of many essential islet-enriched transcription factors. Swi/Snf was necessary for Pdx1 to bind to the Ins gene enhancer, demonstrating the importance of this association in mediating chromatin accessibility. These results illustrate how fundamental the Pdx1:Swi/Snf coregulator complex is in the pancreas, and we discuss how disrupting their association could influence type 1 and type 2 diabetes susceptibility.

Beta cell replacement strategies hold promise for permanently treating type 1 diabetes. In Cell Stem Cell, Xiao et al. (2018) restore pancreatic beta cell mass and normalize blood glucose in diabetic mice by reprogramming pancreatic alpha to beta cells using Pdx1- and Mafa-expressing adeno-associated virus infused into the pancreatic duct.

Gene trap vectors developed for genome-wide mutagenesis can be used to study factors governing the expression of exons inserted throughout the genome. For example, entrapment vectors consisting of a partial 3'-terminal exon [i.e. a neomycin resistance gene (Neo), a poly(A) site, but no 3' splice site] were typically expressed following insertion into introns, from cellular transcripts that spliced to cryptic 3' splice sites present either within the targeting vector or in the adjacent intron. A vector (U3NeoSV1) containing the wild-type Neo sequence preferentially disrupted genes that spliced in-frame to a cryptic 3' splice site in the Neo coding sequence and expressed functional neomycin phosphotransferase fusion proteins. Removal of the cryptic Neo 3' splice site did not reduce the proportion of clones with inserts in introns; rather, the fusion transcripts utilized cryptic 3' splice sites present in the adjacent intron or generated by virus integration. However, gene entrapment with U3NeoSV2 was considerably more random than with U3NeoSV1, consistent with the widespread occurrence of potential 3' splice site sequences in the introns of cellular genes. These results clarify the mechanisms of gene entrapment by U3 gene trap vectors and illustrate features of exon definition required for 3' processing and polyadenylation of cellular transcripts.

Cre/LoxP has broad utility for studying the function, development and oncogenic transformation of pancreatic cells in mice. Here we provide an overview of the many different Cre driver lines that are available for such studies. We, discuss how variegated expression, transgene silencing, and recombination in undesired cell types have conspired to limit the performance of these lines sometimes leading to serious experimental concerns. We also discuss. preferred strategies for achieving high fidelity driver lines and remind investigators of the continuing need for caution when interpreting results obtained from any Cre/LoxP-based experiment performed in mice.

The present study describes a genome-wide method for biallelic mutagenesis in mammalian cells. Novel poly(A) gene trap vectors, which contain features for direct cloning vector–cell fusion transcripts and for post-entrapment genome engineering, were used to generate a library of 979 mutant ES cells. The entrapment mutations generally disrupted gene expression and were readily transmitted through the germline, establishing the library as a resource for constructing mutant mice. Cells homozygous for most entrapment loci could be isolated by selecting for enhanced expression of an inserted neomycin-resistance gene that resulted from losses of heterozygosity (LOH). The frequencies of LOH measured at 37 sites in the genome ranged from 1.3 3 10 � 5 to 1.2 3 10 � 4 per cell and increased with increasing distance from the centromere, implicating mitotic recombination in the process. The ease and efficiency of obtaining homozygous mutations will (i) facilitate genetic studies of gene function in cultured cells, (ii) permit genome-wide studies of recombination events that result in LOH and mediate a type of chromosomal instability important in carcinogenesis, and (iii) provide new strategies for phenotype-driven mutagenesis screens in mammalian cells.

Background Retroviruses have a diploid genome and recombine at high frequency. Recombinant proviruses can be generated when two genetically different RNA genomes are packaged into the same retroviral particle. It was shown in several studies that recombinant proviruses could be generated in each round of HIV-1 replication, whereas the recombination rates of SNV and Mo-MuLV are 5 to 10-fold lower. The reason for these differences is not clear. One possibility is that these retroviruses may differ in their ability to copackage genomic RNAs produced at different chromosomal loci.