Type 2 diabetes mellitus (T2DM) results from pancreatic ␤ cell failure in the setting of insulin resistance. Heterozygous mutations in the gene encoding the ␤ cell transcription factor pancreatic duodenal homeobox 1 (Pdx1) are associated with both T2DM and maturity onset diabetes of the young (MODY4), and low levels of Pdx1 accompany ␤ cell dysfunction in experimental models of glucotoxicity and diabetes. Here, we find that Pdx1 is required for compensatory ␤ cell mass expansion in response to diet-induced insulin resistance through its roles in promoting ␤ cell survival and compensatory hypertrophy. Pdx1-deficient ␤ cells show evidence of endoplasmic reticulum (ER) stress both in the complex metabolic milieu of high-fat feeding as well as in the setting of acutely reduced Pdx1 expression in the Min6 mouse insulinoma cell line. Further, Pdx1 deficiency enhances ␤ cell susceptibility to ER stressassociated apoptosis. The results of high throughput expression microarray and chromatin occupancy analyses reveal that Pdx1 regulates a broad array of genes involved in diverse functions of the ER, including proper disulfide bond formation, protein folding, and the unfolded protein response. These findings suggest that Pdx1 deficiency leads to a failure of ␤ cell compensation for insulin resistance at least in part by impairing critical functions of the ER.chromatin occupancy ͉ diabetes ͉ gene regulation ͉ islet compensation

Heterozygous mutations in the gene encoding the pancreatic homeodomain transcription factor pancreatic duodenal homeobox 1 (PDX1) are associated with maturity onset diabetes of the young, type 4 (MODY4) and type 2 diabetes. Pdx1 governs the early embryonic development of the pancreas and the later differentiation of the insulin-producing islet β cells of the endocrine compartment. We derived a Pdx1 hypomorphic allele that reveals a role for Pdx1 in the specification of endocrine progenitors. Mice homozygous for this allele displayed a selective reduction in endocrine lineages associated with decreased numbers of endocrine progenitors and a marked reduction in levels of mRNA encoding the proendocrine transcription factor neurogenin 3 (Ngn3). During development, Pdx1 occupies an evolutionarily conserved enhancer region of Ngn3 and interacts with the transcription factor one cut homeobox 1 (Hnf6) to activate this enhancer. Furthermore, mRNA levels of all 4 members of the transcription factor network that regulates Ngn3 expression, SRY-box containing gene 9 (Sox9), Hnf6, Hnf1b, and forkhead box A2 (Foxa2), were decreased in homozygous mice. Pdx1 also occupied regulatory sequences in Foxa2 and Hnf1b. Thus, Pdx1 contributes to specification of endocrine progenitors both by regulating expression of Ngn3 directly and by participating in a cross-regulatory transcription factor network during early pancreas development. These results provide insights that may be applicable to β cell replacement strategies involving the guided differentiation of ES cells or other progenitor cell types into the β cell lineage, and they suggest a molecular mechanism whereby human PDX1 mutations cause diabetes. IntroductionInsulin deficiency due to reduced pancreatic islet β cell number underlies the progression of both type 1 and type 2 diabetes, prompting efforts to develop β cell replacement therapies. The high hopes for human islet transplantation as such a therapy have been tempered by their limited availability and short-term function after transplantation, resulting in an intense focus on the development of alternate sources of β cells, through the guided differentiation of stem or progenitor cells or the transdifferentiation of more abundant mature cells. Transdifferentiation approaches have focused largely on the expression of a single or a combination of transcription factors that drive β cell development and differentiation and most have relied, at least in part, upon pancreatic duodenal homeobox 1 (Pdx1), a homeodomain transcription factor with critical regulatory roles in early pancreas development and in the mature β cell (1-5). Recent advances in the derivation of insulin-expressing β-like cells from ES cells (6-11) have also been guided by the principles of embryonic pancreas development (reviewed in ref. 12). Clinical translation of these approaches, however, will require improvements in efficiency, fidelity, and stability of generating the β cell phenotype, efforts which will likely be informed by the identification of novel...

MSY2, a germ-cell-specific member of the Y-box family of DNA-͞ RNA-binding proteins, is proposed to function as a coactivator of transcription in the nucleus and to stabilize and store maternal and paternal mRNAs in the cytoplasm. In mice lacking Msy2, a normal Mendelian ratio is observed after matings between heterozygotes with equal numbers of phenotypically normal but sterile male and female homozygotes (Msy2 ؊/؊ ). Spermatogenesis is disrupted in postmeiotic null germ cells with many misshapen and multinucleated spermatids, and no spermatozoa are detected in the epididymis. Apoptosis is increased in the testes of homozygotes, and real-time RT-PCR assays reveal large reductions in the mRNA levels of postmeiotic male germ cell mRNAs and smaller reductions of meiotic germ cell transcripts. In females, there is no apparent decrease in either the number of follicles or their morphology in ovaries obtained from 2-and 8-day-old Msy2 ؊/؊ mice. In contrast, follicle number and progression are reduced in 21-day-old Msy2 ؊/؊ ovaries. In adult Msy2 ؊/؊ females, oocyte loss increases, anovulation is observed, and multiple oocyte and follicle defects are seen. Thus, Msy2 represents one of a small number of germ-cell-specific genes whose deletion leads to the disruption of both spermatogenesis and oogenesis.he highly conserved family of Y-box proteins, expressed in organisms ranging from bacteria to humans, contains a cold-shock domain essential for nucleic-acid binding and variable N and C termini that confer binding specificity (1). As DNA-binding proteins, Y-box proteins serve as transcription coactivators, recognizing DNA motifs such as CTGATTGGC͞ TC͞TAA (2). As RNA-binding proteins, Y-box proteins help stabilize mRNAs and, depending on their concentration, can inhibit or stimulate mRNA translation (3, 4).Among DNA-͞RNA-binding proteins, the mouse Y-box protein MSY2 is one of the most abundant, constituting 0.7% ( Fig. 1) and 2% of total protein in male germ cells and fully grown oocytes, respectively (5, 6). MSY2 is the mouse ortholog of the Xenopus laevis FRGY2 (2) and human Contrin proteins (7), Y-box proteins proposed to be solely expressed in germ cells (8). In the testis, Msy2 is expressed in meiotic and postmeiotic germ cells, where it is believed to function in long-term mRNA storage and stabilization because cessation of transcription in postmeiotic germ cells necessitates posttranscriptional regulation for many mRNAs encoding late-stage germ cell and spermatozoan proteins (8). In addition, MSY2 marks specific mRNAs in the nucleus for storage in the cytoplasm, providing a linkage between transcription and mRNA storage for a subset of male germ cell mRNAs (9).In the female, MSY2 protein accumulates during oocyte growth, but after fertilization, it is totally degraded by the late two-cell stage (6). As in postmeiotic male germ cells, MSY2 is located in the cytoplasm in oocytes, but, in contrast to male germ cells, where MSY2 is soluble, Ϸ70% of MSY2 is retained after permeabilization procedures that release Ͼ70...

During spermatogenesis, male germ cells temporally synthesize many proteins as they differentiate through meiosis and become spermatozoa. The germ cell Y-box protein, MSY2, constituting Ϸ0.7% of total protein in male germ cells, binds to a consensus promoter element, and shows a general lack of RNA-binding specificity. Combining immunoprecipitation and suppressive subtractive hybridization, we identified populations of germ cell mRNAs that are not bound or bound by MSY2. The former population is enriched in cell growth and ubiquitously expressed mRNAs, whereas the latter population is enriched for stored or translationally delayed, male gamete-specific transcripts. Chromatin precipitation assays reveal that most of the MSY2 target mRNAs are transcribed from genes containing the Y-box DNA-binding motif in their promoters. In transgenic mice, mRNAs encoding exogenous GFP are directed or not directed into the MSY2-bound fraction by promoters containing or lacking the Y-box motif, respectively. We propose that MSY2 marks specific mRNAs in the nucleus for cytoplasmic storage, thereby linking transcription and mRNA storage͞translational delay in meiotic and postmeiotic male germ cells of the mouse.mRNA storage ͉ spermatogenesis ͉ transcription and translation linkage ͉ Y-box protein