Collecting new targets in MODY

Transcriptional regulation is crucial in the function of the pancreatic β cell and diabetes, as evidenced by the human MODY families. Work from Fukui and colleagues (Fukui et al., 2005) and Akpinar and colleagues (Akpinar et al., 2005) in this issue of Cell Metabolism identifies a target of the MODY3 transcription factor HNF-1α that appears to function both in insulin secretion and β cell proliferation

Gene Structure, cDNA Sequence, and mRNA Distribution

The rat HNF-3 (hepatocyte nuclear factor 3) gene family encodes three transcription factors known to be important in the regulation of gene expression in liver and lung. We have cloned and characterized the mouse genes and cDNAs for HNF-3α, β, and γ and analyzed their expression patterns in various adult tissues and mouse embryonic stages. The HNF-3 proteins are highly conserved between mouse and rat, with the exception of the amino terminus of HNF-3γ, which in mouse is more similar to those of HNF-3α and β than to the amino termini of the rat HNF-3γ protein. The mouse HNF-3 genes are small and contain only two or three (HNF-3β) exons with conserved intron-exon boundaries. The proximal promoter of the mouse HNF3β gene is remarkably similar to that of the previously cloned rat HNF-3β gene, but is different from the promoters of the HNF-3α and γ genes. The mRNA distribution of the mouse HNF-3 genes was analyzed by quantitative RNase protection with gene-specific probes. While HNF-3α and β are restricted mainly to endoderm-derived tissues (lung, liver, stomach, and small intestine), HNF-3γ is more extensively expressed, being present additionally in ovary, testis, heart, and adipose tissue, but missing from lung. Transcripts for HNF-3β and α are detected most abundantly in midgestation embryos (Day 9.5), while HNF-3γ expression peaks around Day 15.5 of gestation

Structure of the Mouse Myelin P 2 Protein Gene

Myelin P 2 protein is a small (14,800 Da) protein found in peripheral and central nervous system myelin. To investigate the regulation of expression of this myelin protein, a mouse genomic library was screened with a rabbit P 2 cDNA (pSN2.2–2), and a single positive phage clone containing a 20-kb insert was obtained. This insert contained a single internal Sail restriction site and several Eco RI sites. The Eco RI fragments from this insert were subcloned into Bluescript. The rabbit P 2 cDNA (pSN2.2–2) hybridized with a 4-kb Eco RI fragment, and this 4-kb fragment was then sequenced after the creation of nested deletions. The mouse gene contained four exons: exon 1 coded for amino acids 1–24, exon 2 for amino acids 24–81, exon 3 for amino acids 82–115, and exon 4 for amino acids 116–131. The three introns were 1.2 kb, 150 bp, and 1.3 kb in length. Primer extension analysis revealed two transcription start sites at +1 and +47, consistent with the presence of two P 2 mRNAs, with the larger transcript appearing more abundant. The amino acid sequences predicted from the mouse DNA indicate that the mouse protein differs from the rabbit protein at 16 different positions, with most of the differences located in exon 3. When the gene structure of fatty acid binding protein (FABP) genes were compared, the P 2 gene had the same overall structure as others in the FABP family, suggesting a common ancestral gene for members of this family. The 5′-flanking region contains candidate TATA and CAAT sequences, as well as two AP-l binding sites that may be important in modulation of the expression of this gene.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/65134/1/j.1471-4159.1991.tb02101.x.pd

Identification of known and novel pancreas genes expressed downstream of Nkx2.2 during development

<p>Abstract</p> <p>Background</p> <p>The homeodomain containing transcription factor Nkx2.2 is essential for the differentiation of pancreatic endocrine cells. Deletion of Nkx2.2 in mice leads to misspecification of islet cell types; insulin-expressing β cells and glucagon-expressing α cells are replaced by ghrelin-expressing cells. Additional studies have suggested that Nkx2.2 functions both as a transcriptional repressor and activator to regulate islet cell formation and function. To identify genes that are potentially regulated by Nkx2.2 during the major wave of endocrine and exocrine cell differentiation, we assessed gene expression changes that occur in the absence of Nkx2.2 at the onset of the secondary transition in the developing pancreas.</p> <p>Results</p> <p>Microarray analysis identified 80 genes that were differentially expressed in e12.5 and/or e13.5 Nkx2.2^-/-embryos. Some of these genes encode transcription factors that have been previously identified in the pancreas, clarifying the position of Nkx2.2 within the islet transcriptional regulatory pathway. We also identified signaling factors and transmembrane proteins that function downstream of Nkx2.2, including several that have not previously been described in the pancreas. Interestingly, a number of known exocrine genes are also misexpressed in the Nkx2.2^-/-pancreas.</p> <p>Conclusions</p> <p>Expression profiling of Nkx2.2^-/-mice during embryogenesis has allowed us to identify known and novel pancreatic genes that function downstream of Nkx2.2 to regulate pancreas development. Several of the newly identified signaling factors and transmembrane proteins may function to influence islet cell fate decisions. These studies have also revealed a novel function for Nkx2.2 in maintaining appropriate exocrine gene expression. Most importantly, Nkx2.2 appears to function within a complex regulatory loop with Ngn3 at a key endocrine differentiation step.</p

The pluripotency factor LIN28 marks undifferentiated spermatogonia in mouse

<p>Abstract</p> <p>Background</p> <p>Life-long production of spermatozoa depends on spermatogonial stem cells. Spermatogonial stem cells exist among the most primitive population of germ cells – undifferentiated spermatogonia. Transplantation experiments have demonstrated the functional heterogeneity of undifferentiated spermatogonia. Although the undifferentiated spermatogonia can be topographically divided into A_s(single), A_pr(paired), and A_al(aligned) spermatogonia, subdivision of this primitive cell population using cytological markers would greatly facilitate characterization of their functions.</p> <p>Results</p> <p>In the present study, we show that LIN28, a pluripotency factor, is specifically expressed in undifferentiated spermatogonia (A_s, A_pr, and A_al) in mouse. <it>Ngn3 </it>also specifically labels undifferentiated spermatogonia. We used <it>Ngn3</it>-GFP knockin mice, in which GFP expression is under the control of all <it>Ngn3 </it>transcription regulatory elements. Remarkably, <it>Ngn3</it>-GFP is only expressed in ~40% of LIN28-positive A_s(single) cells. The percentage of <it>Ngn3</it>-GFP-positive clusters increases dramatically with the chain length of interconnected spermatogonia.</p> <p>Conclusion</p> <p>Our study demonstrates that LIN28 specifically marks undifferentiated spermatogonia in mice. These data, together with previous studies, suggest that the LIN28-expressing undifferentiated spermatogonia exist as two subpopulations: <it>Ngn3</it>-GFP-negative (high stem cell potential) and <it>Ngn3</it>-GFP-positive (high differentiation commitment). Furthermore, <it>Ngn3</it>-GFP-negative cells are found in chains of <it>Ngn3</it>-GFP-positive spermatogonia, suggesting that cells in the A_alspermatogonia could revert to a more primitive state.</p

Novel computational analysis of protein binding array data identifies direct targets of Nkx2.2 in the pancreas

<p>Abstract</p> <p>Background</p> <p>The creation of a complete genome-wide map of transcription factor binding sites is essential for understanding gene regulatory networks <it>in vivo</it>. However, current prediction methods generally rely on statistical models that imperfectly model transcription factor binding. Generation of new prediction methods that are based on protein binding data, but do not rely on these models may improve prediction sensitivity and specificity.</p> <p>Results</p> <p>We propose a method for predicting transcription factor binding sites in the genome by directly mapping data generated from protein binding microarrays (PBM) to the genome and calculating a moving average of several overlapping octamers. Using this unique algorithm, we predicted binding sites for the essential pancreatic islet transcription factor <it>Nkx2.2 </it>in the mouse genome and confirmed >90% of the tested sites by EMSA and ChIP. Scores generated from this method more accurately predicted relative binding affinity than PWM based methods. We have also identified an alternative core sequence recognized by the <it>Nkx2.2 </it>homeodomain. Furthermore, we have shown that this method correctly identified binding sites in the promoters of two critical pancreatic islet β-cell genes, <it>NeuroD1 </it>and <it>insulin2</it>, that were not predicted by traditional methods. Finally, we show evidence that the algorithm can also be applied to predict binding sites for the nuclear receptor <it>Hnf4α</it>.</p> <p>Conclusions</p> <p>PBM-mapping is an accurate method for predicting Nkx2.2 binding sites and may be widely applicable for the creation of genome-wide maps of transcription factor binding sites.</p

Expression of the gut-enriched Krüppel-like factor gene during development and intestinal tumorigenesis

AbstractWe examined the expression of GKLF (gut-enriched Krüppel-like factor), a recently identified zinc finger-containing transcription factor, in mice during development using the ribonuclease protection assay. In the adult, the level of GKLF transcript is abundant throughout the gastrointestinal tract. Between embryonic days 10 and 19 (E10 and E19) of development, the initial level of whole embryo GKLF transcript is low but begins to rise on E13 and peaks on E17. In the newborn, GKLF transcript level is higher in the colon than in the small intestine although the levels in both organs rise with increasing age. Expression of GKLF was also examined in the intestinal tract of the Min mouse, a model of intestinal tumorigenesis. The level of GKLF transcript is significantly decreased in the intestine of Min mice during a period of tumor formation when compared to age-matched control littermates. Our findings indicate that GKLF expression correlates with certain periods of gut development and is down-regulated during intestinal tumorigenesis, suggesting that GKLF may play a role in gut development and/or tumor formation

Foxa2 and H2A.Z Mediate Nucleosome Depletion during Embryonic Stem Cell Differentiation

SummaryNucleosome occupancy is fundamental for establishing chromatin architecture. However, little is known about the relationship between nucleosome dynamics and initial cell lineage specification. Here, we determine the mechanisms that control global nucleosome dynamics during embryonic stem (ES) cell differentiation into endoderm. Both nucleosome depletion and de novo occupation occur during the differentiation process, with higher overall nucleosome density after differentiation. The variant histone H2A.Z and the winged helix transcription factor Foxa2 both act to regulate nucleosome depletion and gene activation, thus promoting ES cell differentiation, whereas DNA methylation promotes nucleosome occupation and suppresses gene expression. Nucleosome depletion during ES cell differentiation is dependent on Nap1l1-coupled SWI/SNF and INO80 chromatin remodeling complexes. Thus, both epigenetic and genetic regulators cooperate to control nucleosome dynamics during ES cell fate decisions

Integrative genomic analysis of CREB defines a critical role for transcription factor networks in mediating the fed/fasted switch in liver

BACKGROUND: Metabolic homeostasis in mammals critically depends on the regulation of fasting-induced genes by CREB in the liver. Previous genome-wide analysis has shown that only a small percentage of CREB target genes are induced in response to fasting-associated signaling pathways. The precise molecular mechanisms by which CREB specifically targets these genes in response to alternating hormonal cues remain to be elucidated. RESULTS: We performed chromatin immunoprecipitation coupled to high-throughput sequencing of CREB in livers from both fasted and re-fed mice. In order to quantitatively compare the extent of CREB-DNA interactions genome-wide between these two physiological conditions we developed a novel, robust analysis method, termed the ‘single sample independence’ (SSI) test that greatly reduced the number of false-positive peaks. We found that CREB remains constitutively bound to its target genes in the liver regardless of the metabolic state. Integration of the CREB cistrome with expression microarrays of fasted and re-fed mouse livers and ChIP-seq data for additional transcription factors revealed that the gene expression switches between the two metabolic states are associated with co-localization of additional transcription factors at CREB sites. CONCLUSIONS: Our results support a model in which CREB is constitutively bound to thousands of target genes, and combinatorial interactions between DNA-binding factors are necessary to achieve the specific transcriptional response of the liver to fasting. Furthermore, our genome-wide analysis identifies thousands of novel CREB target genes in liver, and suggests a previously unknown role for CREB in regulating ER stress genes in response to nutrient influx