Evidence from oyster suggests an ancient role for Pdx in regulating insulin gene expression in animals

Hox and ParaHox genes encode transcription factors with similar expression patterns in divergent animals. The Pdx (Xlox) homeobox gene, for example, is expressed in a sharp spatial domain in the endodermal cell layer of the gut in chordates, echinoderms, annelids and molluscs. The significance of comparable gene expression patterns is unclear because it is not known if downstream transcriptional targets are also conserved. Here, we report evidence indicating that a classic transcriptional target of Pdx1 in vertebrates, the insulin gene, is a likely direct target of Pdx in Pacific oyster adults. We show that one insulin-related gene, cgILP, is co-expressed with cgPdx in oyster digestive tissue. Transcriptomic comparison suggests that this tissue plays a similar role to the vertebrate pancreas. Using ATAC-seq and ChIP, we identify an upstream regulatory element of the cgILP gene which shows binding interaction with cgPdx protein in oyster hepatopancreas and demonstrate, using a cell culture assay, that the oyster Pdx can act as a transcriptional activator through this site, possibly in synergy with NeuroD. These data argue that a classic homeodomain-target gene interaction dates back to the origin of Bilateria.

Regulation of Virulence of Entamoeba histolytica by the URE3-BP Transcription Factor

ABSTRACTIt is not understood why only some infections withEntamoeba histolyticaresult in disease. The calcium-regulated transcription factor upstream regulatory element 3-binding protein (URE3-BP) was initially identified by virtue of its role in regulating the expression of two amebic virulence genes, the Gal/GalNac lectin and ferredoxin. Here we tested whether this transcription factor has a broader role in regulating virulence. A comparison ofin vivotoin vitroparasite gene expression demonstrated that 39% ofin vivoregulated transcripts contained the URE3 motif recognized by URE3-BP, compared to 23% of all promoters (P< 0.0001). Amebae induced to express a dominant positive mutant form of URE3-BP had an increase in an elongated morphology (30% ± 6% versus 14% ± 5%;P= 0.001), a 2-fold competitive advantage at invading the intestinal epithelium (P= 0.017), and a 3-fold increase in liver abscess size (0.1 ± 0.1 g versus 0.036 ± 0.1 g;P= 0.03). These results support a role for URE3-BP in virulence regulation.IMPORTANCEAmebic dysentery and liver abscess are caused byEntamoeba histolytica. Amebae colonize the colon and cause disease by invading the intestinal epithelium. However, only one in fiveE. histolyticainfections leads to disease. The factors that govern the transition from colonization to invasion are not understood. The transcription factor upstream regulatory element 3-binding protein (URE3-BP) is a calcium-responding regulator of theE. histolyticaGal/GalNAc lectin and ferredoxin genes, both implicated in virulence. Here we discovered that inducible expression of URE3-BP changed trophozoite morphology and promoted parasite invasion in the colon and liver. These results indicate that one determinant of virulence is transcriptional regulation by URE3-BP.

Heterozygous deletion of the PU.1 locus in human AML

The transcription factor PU.1 is essential for myeloid development. Targeted disruption of an upstream regulatory element (URE) decreases PU.1 expression by 80% and leads to acute myeloid leukemia (AML) in mice. Here, we sequenced the URE sequences of PU.1 in 120 AML patients. Four polymorphisms (single nucleotide polymorphisms [SNPs]) in the URE were observed, with homozygosity in all SNPs in 37 patients. Among them, we compared samples at diagnosis and remission, and one patient with cytogenetically normal acute myeloid leukemia M2 was identified with heterozygosity in 3 of the SNPs in the URE at remission. Loss of heterozygosity was further found in this patient at 2 polymorphic sites in the 5′ promoter region and in 2 intronic sites flanking exon 4, thus suggesting loss of heterozygosity covering at least 40 kb of the PU.1 locus. Consistently, PU.1 expression in this patient was markedly reduced. Our study suggests that heterozygous deletion of the PU.1 locus can be associated with human AML.

Autoregulation of the Transcription Factor PU.1 Via Its Evolutionarily Conserved Upstream Regulatory Element Is Critical in Adult Mouse Hematopoiesis.

Abstract 1468 Poster Board I-491 Background: Levels of the Ets transcription factor PU.1 control normal hematopoietic differentiation and even modest alterations can lead to leukemia and lymphoma. Regulation of PU.1 levels at different stages of hematopoiesis requires multiple interactions between several regulatory elements and transcription factors. Our previous studies identified a potential autoregulatory mechanism of the PU.1 gene through the combined activity of the proximal promoter and an evolutionarily conserved upstream regulatory element (URE), located at –14 kb relative to the transcription start site in mice. PU.1 binds to a conserved PU.1 site in the PU.1 URE both in vitro and in vivo. Approach: To ask at which stages of hematopoietic differentiation autoregulation of PU.1 via binding to its URE might play a role, we developed a mouse model with targeted disruption of the PU.1 binding site in the PU.1 URE. Results: Targeted mutation of the PU.1 autoregulatory site in PU.1 URE abolished PU.1 binding as verified by Chromatin Immuno-precipitation (ChIP). PU.1 URE activity was manifestly reduced resulting in a variety of lineage-specific abnormalities. As shown here in adult mice, the absence of the autoregulatory PU.1 site affected PU.1 expression in a lineage dependent manner. PU.1 expression was markedly decreased in phenotypic long term hematopoietic stem cells (LT-HSC: CD150+/CD48−/ c-kit+/sca-1+/lin−) and short term HSCs (ST-HSCs: CD150−/CD48+/ c-kit+/sca-1+/lin−) and, to a lesser extent, in Common Myeloid Progenitors (CMPs: lin−/c-kit+/Sca-1−/CD34+/FcrRlow), and Megakaryocyte/Erythrocyte Progenitors (MEPs: lin−/c-kit+/Sca-1−/CD34−/FcrRhigh). Within the lymphoid linage, PU.1 levels were unchanged in Common Lymphoid Progenitors (CLPs: lin−/c-kitlow/Sca-1low /IL-7Ra+/Thy1.1−) and pre-B-cells (B220+/ CD43−), up in pro-B-cells (B220+/CD43+), and down in mature B cells. Myeloid cells appeared to be unaffected. Interestingly, while PU.1 levels were decreased in LT- and ST-HSC populations, only phenotypic LT-HSCs were reduced in number. To further analyze HSC function of PU.1 site mutated mice we performed limiting dilution transplantation assays and measured the frequency of competitive repopulation units (CRU) using the congenic Ly5.1/Ly5.2 system. Our preliminary data indicated a decrease of LT-HSC function in PU.1 site mutated mice, although their homing and engraftment functions were not affected. This was also observed in mice with targeted disruption of all three AML-1 sites that are in close proximity of the PU.1 site at the PU.1 URE. While AML-1 itself appeared not to influence LT-HSC function (M. Ichikawa, T. Asai et al. Nature Medicine, 2004), we found that the conformational changes of the URE present in mice with disrupted AML-1 binding sites, as measured by Quantitative Chromosome Conformation Capture, impede PU.1 binding to its autoregulatory site. Conclusion: PU.1 indeed autoregulates its expression via binding to the -14kb URE in a lineage specific manner in vivo. Our data point to a critical role of PU.1 autoregulation especially for long-term hematopoietic stem cell function. Disclosures: No relevant conflicts of interest to declare.

Chromosome looping at the human α-globin locus is mediated via the major upstream regulatory element (HS −40)

Previous studies in the mouse have shown that high levels of α-globin gene expression in late erythropoiesis depend on long-range, physical interactions between remote upstream regulatory elements and the globin promoters. Using quantitative chromosome conformation capture (q3C), we have now analyzed all interactions between 4 such elements lying 10 to 50 kb upstream of the human α cluster and their interactions with the α-globin promoter. All of these elements interact with the α-globin gene in an erythroid-specific manner. These results were confirmed in a mouse model of human α globin expression in which the human cluster replaces the mouse cluster in situ (humanized mouse). We have also shown that expression and all of the long-range interactions depend largely on just one of these elements; removal of the previously characterized major regulatory element (called HS −40) results in loss of all the interactions and α-globin expression. Reinsertion of this element at an ectopic location restores both expression and the intralocus interactions. In contrast to other more complex systems involving multiple upstream elements and promoters, analysis of the human α-globin cluster during erythropoiesis provides a simple and tractable model to understand the mechanisms underlying long-range gene regulation.

Genetics and Epigenetics of the PU.1 Upstream Regulatory Element: AML1 Binding Sites Are Critical for Leuekmia Induced by the AML/ETO9a Fusion Oncogene

The level of expression of the transcription factor PU.1 is a critical determinant of lineage commitment in normal hematopoiesis, and dysregulation of PU.1 leads to development of leukemia. In mice with targeted disruption of the PU.1 upstream regulatory element (URE), expression of PU.1 is decreased to 20% of wild type levels and results in development of acute myeloid leukemia (AML). These data suggests that tightly regulated PU.1 expression is important to maintain normal hematopoiesis and prevent leukemogenesis. Previously, we reported that AML1 (RUNX1) regulated PU.1 expression. Here we demonstrate that AMLl regulates PU.1 through 3 AML1 binding sites in the URE. Mice with targeted mutations in the 3 AML1 binding sites have decreased PU.1 expression in multiple hematopoietic lineages at multiple different developmental stages. Conditional targeting of AML1 in transgenic mice in which the URE homology region 2 (H2, containing all 3 AML1 binding sites) is used to drive expression of a reporter decreased reporter gene expression, suggesting that AML1 regulates PU.1 through these 3 sites in URE homology region 2. Using a second mouse model with a targeted mutation in the PU.1 binding site in the PU.1 URE (which is flanked by the 3 AML1 sites), we demonstrated that PU.1 indeed autoregulates itself through the URE. These results demonstrated that AML1 regulates PU.1 through the 3 AML1 sites in the URE. However, while low levels of PU.1 lead to leukemia, we have not observed frank leukemia development in AML1 conditional knockouts or in mice with targeted disruption of the 3 AML1 sites in the PU.1 URE. We hypothesized that this might be the case because disruption of AML1 or the AML1 sites reduces PU.1 levels to about 40% of wild type, but not as great as that found in PU.1 URE knockouts, which do progress to AML (20% of wild type). We hypothesized that downregulation of PU.1 as a result of binding of AML1/ETO fusion proteins to the URE might result in further reductions of PU.1 expression, and contribute to leukemogenesis. Therefore, we predicted that development of leukemia might be delayed in mice with mutations in the PU.1 URE AML1 DNA binding sites, and this was indeed the case in a modle using a retrovirus expressing the AML1/ETO9a form. We further explored the effect of AML1 and PU.1 binding on chromatin strucutre using chromatin immunoprecipitation (Chip) in the AML1 and PU.1 site URE knockin models, and found that AML1 is involved in H3K4me3 and H3/H4 acetylation of histone tails in the PU.1 URE, while PU.1 is involved in H3/H4 acetylation but not H3K4me3; H3K4 methylation and H3 acetylation decreased in AML1 sites mutant knockin mice and H3 acetylation decreased in PU.1 site mutant knockin mice. Mutation of the AML1 site in mice not only altered the chromatin structure of the URE region, but also interefered with the physical interaction between the URE and PU.1 promoter, as assessed by chromosome capture configuration (3C) assays. Interestingly, the AML1/ETO9a fusion oncogene has a unique role on the epigenetic status of the PU.1 URE in addition to its dominant effect on the 3 AML1 sites. AML1-ETO9 blocks the autoregulation of PU.1 through the PU.1 site in the URE. In summary, our data suggests that AML1 regulates PU.1 expression through 3 AML1 binding sites in the PU.1 URE by modifying chromatin structure in the URE region. In addition, PU.1 can autoregulate itself by facilitating similar epigenetic changes. Dysregulation of the epigenetic status by chromosome translocation products such as AML1-ETO might play an important role in leukemogenesis. First two authors contribute equally to this work.

Previously Unknown Interactions Between AML1 and MLL Provide Epigenetic Regulation of Gene Expression in Normal Hematopoiesis and in Leukemia

In all organisms, the fundamental process of transcriptional regulation requires transcription factors, which bind to DNA in response to extra-cellular signals and regulate transcription of target genes. In eukaryotes, this process also involves epigenetic regulation, which includes DNA and histone modifications. Hematopoiesis and leukemia are excellent model systems for studying the higher eukaryotic regulations of gene expression and for identifying important molecules involved in genetic and epigenetic transcriptional regulation. The Mixed-Lineage Leukemia (MLL) protein, a Set1-like H3K4 methyltransferase, and the heterodimeric transcriptional factor AML1/CBFβ are critical for definitive and adult hematopoiesis. They are required for the generation of all hematopoietic lineages and act as tumor suppressors in human leukemia. We have previously shown that the regulation of PU.1 by AML1 is mediated by 3 AML1 binding sites in the PU.1 upstream regulatory element (URE), located −14 kb relative to the transcription start site in mice (Huang et al. Nat Genet. 2008). To understand whether AML1 plays a critical role in regulating the PU.1 locus at the chromatin level, we have utilized this PU.1 regulation system as a model to study the potential interplay between AML1/CBFβ and MLL. We found that MLL binds to the evolutionarily conserved Runt-domain of AML1, enhances the formation of the AML1/CBFβ heterodimer, and blocks the ubiquitin-proteasome mediated degradation of AML1. We also found that AML1/CBFβ is required for maintenance of the H3K4-me3 histone mark at the PU.1 upstream regulatory element (URE) and promoter region, and that MLL is required for AML1 to regulate PU.1 expression. In contrast, we found that several leukemia-associated MLL fusion proteins, including MLL-AF4, MLL-AF9, MLL-ENL, and MLL-AF10, no longer stabilize AML1 but rather induce AML1 degradation and downregulate PU.1 expression. Taken together, our data indicate that MLL and some of its leukemia-associated fusion proteins physically and functionally interact with AML1. Their effects on target genes, particularly PU.1, may be critical for the normal regulation of hematopoiesis and for the aberrant regulation that underlies acute myelogenous leukemia (AML) and acute lymphoblastic leukemia (ALL).