Multiple-brooding rockfishes (Sebastes spp.) can utilize stored sperm from individual sires to fertilize consecutive broods.

Viviparous rockfishes (Sebastes spp., family Scorpaenidae) mate and store sperm in the ovaries for several months prior to fertilization, as oocytes develop for the parturition season. Although multiple paternity has been documented in single-brooding rockfishes, paternity in consecutive broods of multiple-brooding species has not been studied. Analyses of multilocus microsatellite genotypes in both residual larvae left in the ovary from a previous parturition and upcoming fertilized broods in the same ovary demonstrated evidence of the same sires in consecutive broods in chilipepper (Sebastes goodei) and speckled (Sebastes ovalis) rockfishes. One S. goodei mother showed evidence of multiple paternity from the same two sires in both consecutive broods. The ability to retain sperm, even after a parturition event, for use in subsequent broods, confers an advantage to ensure fertilization and allows for extension of the parturition season. This life-history strategy provides a bet-hedging advantage in the California Current system, an environmentally dynamic ecosystem where larval survivorship and subsequent recruitment to adult populations can vary temporally by orders of magnitude.

A review of gorgonian coral species (Cnidaria, Octocorallia, Alcyonacea) held in the Santa Barbara Museum of Natural History research collection: focus on species from Scleraxonia, Holaxonia, Calcaxonia - Part III: Suborder Holaxonia continued, and suborder Calcaxonia.

Alcyonacean (Gorgonian) coral species from Holaxonia (not previously reviewed in this three-part work), family Plexauridae, as well as species in Calcaxonia were reviewed. Specimens examined were collected from the California Bight and adjacent areas, many now held in the research collection of the Santa Barbara Museum of Natural History (SBMNH). The collection has incorporated numerous specimens collected by the Allan Hancock Foundation (AHF) 'Velero' Expeditions of 1931-1941 and 1948-1985. This historic collection displays an emphasis on species belonging to the Holaxonia, particularly gorgoniids and plexaurids. This third part of the larger work presented a thorough, in-depth discussion of at least one genus (Swiftia Duchassaing & Michelotti, 1864) in the Plexauridae found within the California Bight that has generated some taxonomic confusion; in that discussion are comments on other genera (such as Psammogorgia Verrill, 1868a, to which several species had been previously ascribed). The discussion of Swiftia includes description of a morphological trend (encompassing colony form, color and sclerite form), likely influenced by geography and ecology, not noted or discussed previously. Additionally, a preliminary discussion of the genus (Thesea Duchassaing & Michelotti, 1860) was presented; this genus, both historically and currently, has not been fully examined in California waters. Finally, a short review was given for the few species of Calcaxonia represented in the SBMNH research collection. This paper, Part III of the full review, continued and concludes the systematic examination of species represented in the SBMNH research collection begun in Part I, continued in Part II, focusing on all species of gorgonian coral held in the SBMNH research collection, known to currently inhabit the California Bight and adjacent areas.

A review of gorgonian coral species (Cnidaria, Octocorallia, Alcyonacea) held in the Santa Barbara Museum of Natural History research collection: focus on species from Scleraxonia, Holaxonia, Calcaxonia - Part II: Species of Holaxonia, families Gorgoniidae and Plexauridae.

Gorgonian coral specimens from the Holaxonia, families Gorgoniidae and Plexauridae held in the collection of the Santa Barbara Museum of Natural History (SBMNH) were reviewed and evaluated for species identification. The specimens were collected from within, and adjacent areas of, the California Bight. The SBMNH collection has encompassed within it a large percentage of specimens collected by the Allan Hancock Foundation (AHF) 'Velero' Expeditions of 1931-1941 and 1948-1985. This historic collection displays an emphasis on species belonging to the Holaxonia, particularly the gorgoniids and plexaurids; thus, this second part presents a thorough discussion of well-known genera from within the California Bight, with more extensive discussions of several genera that have historically, and currently, led to confusion (and thus, misidentification). A brief discussion of a California Bight grouping, referred to within as the "red whips," is presented; this grouping encompasses several species with very similar colony appearance across a number of genera. Two species, the gorgoniid Leptogorgiachilensis (Verrill, 1868) and the plexaurid Chromoplexauramarki (Kükenthal, 1913) each required the designation of a neotype from within the collection. A new species in the genus Eugorgia Verrill, 1868, a whip or thread-like form belonging to the family Gorgoniidae, is described. One additional plexaurid genus (Placogorgia) is discussed, a genus not commonly reported for the California Bight region. This is the first comprehensive work, in three parts, focusing on all species of gorgonian coral known to inhabit the California Bight. This paper, Part II of the full work, continues the systematic review of all species represented in the Santa Barbara Museum of Natural History research collection begun in Part I.

A review of gorgonian coral species (Cnidaria, Octocorallia, Alcyonacea) held in the Santa Barbara Museum of Natural History research collection: focus on species from Scleraxonia, Holaxonia, and Calcaxonia - Part I: Introduction, species of Scleraxonia and Holaxonia (Family Acanthogorgiidae).

Gorgonian specimens collected from the California Bight (northeastern Pacific Ocean) and adjacent areas held in the collection of the Santa Barbara Museum of Natural History (SBMNH) were reviewed and evaluated for species identification; much of this material is of historic significance as a large percentage of the specimens were collected by the Allan Hancock Foundation (AHF) 'Velero' Expeditions of 1931-1941 and 1948-1985. Examination and reorganization of this collection began early in 2002; initially, it was estimated that at most, twelve to fifteen species of gorgonian could be found within the Bight. Following collection evaluation, it was determined that at a minimum, approximately twenty three genera, encompassing some forty-plus species, of gorgonian coral have been found living within the California Bight region, often extending some distance into adjacent geographical areas both north and south. All species from the California Bight in the collection are discussed to some degree (in three separate parts, this being Part I), with digital images of both colony form and sclerite composition provided for most. Collection specimens from the suborders and families covered in Part I are not extensive, but several genera are featured that have not been previously reported for the California Bight region. Additionally, a potential new species (genus Sibogagorgia Stiasny, 1937) from the Paragorgiidae is described in Part I. Overall, the collection displays an emphasis on species belonging to the Holaxonia, particularly the plexaurids. A brief discussion of a California Bight grouping, referred to as the "red whips," is presented in Part II; this grouping encompasses several species with very similar colony appearance across a number of genera. A new species (a whip or thread-like form) in the genus Eugorgia Verrill, 1868, belonging to the Gorgoniidae, is described in Part II. The genus Swiftia Duchassaing & Michelotti, 1864 is one of the most challenging taxon groups represented; those species in the genus Swiftia collected within the California Bight are discussed fully, based on SBMNH (and other) specimens in Part III. Scanning electron microscopy images for species of Swiftia from the California coast have rarely, if ever, been published and are included, with a discussion of the geographic range of the genus in the eastern Pacific, from the southern boundary of the California Bight to the Bering Sea, Alaska. Finally, specimens of the genus Thesea Duchassaing & Michelotti, 1860, displaying a whip or thread-like body form, are discussed at a preliminary level in Part III; they also presented challenges to a clear understanding of their taxonomy. While Part I focuses on species of Scleraxonia and those of the Holaxonia in the Acanthogorgiidae family held in the SBMNH collection, all three parts taken together represent the first comprehensive work that reviews the research collection of SBMNH, which focuses on species of gorgonian coral known to inhabit the California Bight.

Dispersal of a nearshore marine fish connects marine reserves and adjacent fished areas along an open coast.

Marine species with pelagic larvae typically exhibit little population structure, suggesting long-distance dispersal and high gene flow. Directly quantifying dispersal of marine fishes is challenging but important, particularly for the design of marine protected areas (MPAs). Here, we studied kelp rockfish (Sebastes atrovirens) sampled along ~25 km of coastline in a boundary current-dominated ecosystem and used genetic parentage analysis to identify dispersal events and characterize them, because the distance between sedentary parents and their settled offspring is the lifetime dispersal distance. Large sample sizes and intensive sampling are critical for increasing the likelihood of detecting parent-offspring matches in such systems and we sampled more than 6,000 kelp rockfish and analysed them with a powerful set of 96 microhaplotype markers. We identified eight parent-offspring pairs with high confidence, including two juvenile fish that were born inside MPAs and dispersed to areas outside MPAs, and four fish born in MPAs that dispersed to nearby MPAs. Additionally, we identified 25 full-sibling pairs, which occurred throughout the sampling area and included all possible combinations of inferred dispersal trajectories. Intriguingly, these included two pairs of young-of-the-year siblings with one member each sampled in consecutive years. These sibling pairs suggest monogamy, either intentional or accidental, which has not been previously demonstrated in rockfishes. This study provides the first direct observation of larval dispersal events in a current-dominated ecosystem and direct evidence that larvae produced within MPAs are exported both to neighbouring MPAs and to proximate areas where harvest is allowed.

Another unusual new gorgonian (Anthozoa: Octocorallia: Plexauridae) from the Aleutian Islands of Alaska.

An unusual new species of plexaurid octocoral, Alaskagorgia splendicitrina, is described from a specimen collected in the far west Aleutian Island Archipelago, Alaska, USA. Unusual features that separate it from its only congener include: the vibrant yellow color of the live colony and an arborescent growth form with numerous coiling and twisting branches, the pale yellow color of the sclerites and the lack of small and densely warted double-headed sclerites. The new species is represented by only a single specimen despite extensive sampling in the region during the past several decades; the speculation is that it radiated from the much less explored region to the west.

Global survey of Klebsiella pneumoniae major porins from ertapenem non-susceptible isolates lacking carbapenemases.

PURPOSE: To understand the diversity of porin disruption in Klebsiella pneumoniae, the major outer membrane protein (OMP) porins, OmpK35 and OmpK36, were examined in a set of isolates that did not harbour traditional carbapenem-hydrolysing enzymes, but nevertheless tested non-susceptible to ertapenem. METHODS: A world-wide collection of Klebsiella pneumoniae isolates that were part of the Study for Monitoring Antimicrobial Resistance Trends (SMART) surveillance project over the years 2008-2014 were characterised with regard to their ß-lactamase gene carriage and potential permeability defects. Four hundred and eighty-seven isolates that did not carry carbapenemase genes, but were non-susceptible to ertapenem, were investigated by sequence analysis of the genes encoding OmpK35 and OmpK36. Isolates without obvious genetic lesions in either major porin gene were further examined by outer membrane protein SDS-PAGE. RESULTS: The majority of isolates, 83.0 % (404/487), exhibited clear genetic disruption in either or both of the ompK35 and ompK36 genes. Among the proportion of the collection with the highest ertapenem MIC value (>4 mg l-1), 60.5 % (115/190) showed mutation in both porin genes. Isolates without obvious genetic mutations were examined by SDS-PAGE, and 90.4 % (75/83) were found to lack or show altered expression of at least one of the major OMPs when compared to an ertapenem sensitive control strain. CONCLUSION: This study illustrates that porin deficiency in Klebsiella pneumoniae is a widespread phenomenon, and in combination with ESBLs and/or AmpC enzymes, likely accounts for the elevated ertapenem MICs observed in this study.

Panmixia in a Critically Endangered Fish: The Totoaba (Totoaba macdonaldi) in the Gulf of California.

Conservation of the evolutionary legacy of endangered species is a key component for long-term persistence. Totoaba is a long-lived fish endemic to the Gulf of California and is considered critically endangered. There is currently a debate concerning its conservation status and whether it can be used as a fishery resource. Unfortunately, basic information on biological and genetic population structure of the species is lacking. We sampled 313 individuals and employed 16 microsatellite loci and 3 mitochondrial DNA markers (16S, 547 pb; COI, 619 pb; control region, 650 pb) to assess population structure and demography of totoaba in the Gulf of California, with samples from locations that encompass nearly all of its recognized geographic distribution. We could not reject a hypothesis of panmixia for totoaba, using nuclear or mitochondrial markers. Demographic analysis of mtDNA suggests a sudden population expansion model. The results have important implications for totoaba conservation because poaching is a significant conservation challenge and could have additive negative effects over the single population of totoaba in the Gulf of California.

Genetic structure and different color morphotypes suggest the occurrence and bathymetric segregation of two incipient species of Sebastes off Argentina.

Rockfishes of the genus Sebastes are extensively distributed in the Pacific and Atlantic oceans. Although the occurrence of two morphologically similar species in the Southern Hemisphere, Sebastes oculatus and Sebastes capensis, is now clearly established, the taxonomic status and phylogeographic patterns for the genus in the region have not yet been completely resolved. In this study, we provide new insights into the taxonomy and evolutionary relationships of rockfishes inhabiting the Southwestern Atlantic Ocean, off the coast of mainland Argentina, by combining mitochondrial DNA (mtDNA) control region sequences, microsatellite data, and color pattern analyses. Differences in coloration ("dark" and "light" fish) together with bathymetric segregation between color morphotypes were evident from fish collection and literature review. In addition, the mtDNA phylogenetic analysis and Bayesian clustering analysis using microsatellite data separated the fish into two distinct groups (F ST = 0.041), most likely representing incipient species. Our results suggest that speciation-by-depth in the absence of physical barriers could be a widespread mechanism of speciation in Sebastes from both the Northern and Southern Hemispheres. Nevertheless, the degree of genetic differentiation found, added to the large number of individuals displaying high levels of admixture, points to the occurrence of incomplete reproductive barriers between color morphotypes. Beyond the taxonomic and phylogeographic implications of our findings, the occurrence of distinct groups of Sebastes off the coast of Argentina being targeted by different fisheries (angling and trawling) has consequences for the design and implementation of appropriate fishery regulations to avoid overharvest of either group.

HoxA10 influences protein ubiquitination by activating transcription of ARIH2, the gene encoding Triad1.

HoxA10 is a homeodomain transcription factor that is maximally expressed in myeloid progenitor cells. An increase in HoxA10 expression correlates with poor prognosis in human acute myeloid leukemia (AML). Consistent with this scenario, HoxA10 overexpression in murine bone marrow induces a myeloproliferative neoplasm that advances AML over time. Despite the importance of HoxA10 for leukemogenesis, few genuine HoxA10 target genes have been identified. The current study identified ARIH2, the gene encoding Triad1, as a HoxA10 target gene. We identified two distinct HoxA10-binding cis elements in the ARIH2 promoter and determined that HoxA10 activates these cis elements in myeloid cells. Triad1 has E3 ubiquitin ligase activity, and we found that HoxA10-overexpressing myeloid cells exhibited a Triad1-dependent increase in protein ubiquitination. Therefore, these studies have identified the regulation of protein ubiquitination as a novel function of Hox transcription factors. Forced overexpression of Triad1 has been show previously to inhibit colony formation by myeloid progenitor cells. In contrast, HoxA10-overexpressing myeloid progenitor cells exhibited increased proliferation in response to low doses of various cytokines. We found that Triad1 knockdown further increased cytokine-induced proliferation in HoxA10-overexpressing cells. Therefore, these studies have identified a HoxA10 target gene that antagonizes the overall influence of overexpressed HoxA10 on myeloproliferation. This result suggests that the consequences of HoxA10 overexpression reflect a balance between the target genes that facilitate and antagonize proliferation. These results have implications for understanding the mechanisms of leukemogenesis in AML with Hox overexpression.

HoxA10 activates CDX4 transcription and Cdx4 activates HOXA10 transcription in myeloid cells.

HoxA10 is a homeodomain transcription factor that influences a number of developmental processes, including hematopoiesis. During definitive hematopoiesis, expression of HoxA10 is maximal in committed myeloid progenitor cells and decreases as differentiation proceeds. Aberrantly increased expression of HoxA10 was found in bone marrow cells in a poor prognosis subset of human acute myeloid leukemia (AML). Consistent with this, AML developed in mice transplanted with HoxA10-overexpressing bone marrow. However, relatively few target genes have been identified that explain the role of HoxA10 in leukemogenesis. In the current study, we identified CDX4 as a HoxA10 target gene. Cdx4 is a homeodomain transcription factor that was also implicated in myeloid leukemogenesis. Although relatively few Cdx4 target genes have been identified, Cdx4 was known to influence HOX gene transcription. We identified a HoxA10-binding cis element in the CDX4 promoter that activated transcription. We also identified a Cdx4-binding cis element that activated the HOXA10 promoter. Therefore, increased Cdx4 expression in HoxA10-overexpressing cells augmented transcription of the endogenous HOXA10 gene. Increased endogenous HoxA10 in these cells induced additional CDX4 transcription. We found that Cdx4 influenced transcription of HoxA10 target genes in a HoxA10-dependent manner. Similarly, HoxA10 influenced transcription of HOX genes in a Cdx4-dependent manner. We previously found that HoxA10-overexpressing myeloid progenitors were hypersensitive to a variety of cytokines. In the current studies, we found that Cdx4 knockdown decreased cytokine hypersensitivity of HoxA10-overexpressing cells. Therefore, these studies identified a positive feedback relationship between HoxA10 and Cdx4, which potentially amplified the contribution of either transcription factor to the pathogenesis of AML.

Interferon consensus sequence binding protein (ICSBP) decreases beta-catenin activity in myeloid cells by repressing GAS2 transcription.

The interferon consensus sequence binding protein (ICSBP) is an interferon regulatory transcription factor, also referred to as IRF8. ICSBP acts as a suppressor of myeloid leukemia, although few target genes explaining this effect have been identified. In the current studies, we identified the gene encoding growth arrest specific 2 (GAS2) as an ICSBP target gene relevant to leukemia suppression. We find that ICSBP, Tel, and histone deacetylase 3 (HDAC3) bind to a cis element in the GAS2 promoter and repress transcription in myeloid progenitor cells. Gas2 inhibits calpain protease activity, and beta-catenin is a calpain substrate in these cells. Consistent with this, ICSBP decreases beta-catenin protein and activity in a Gas2- and calpain-dependent manner. Conversely, decreased ICSBP expression increases beta-catenin protein and activity by the same mechanism. This is of interest, because decreased ICSBP expression and increased beta-catenin activity are associated with poor prognosis and blast crisis in chronic myeloid leukemia (CML). We find that the expression of Bcr/abl (the CML oncoprotein) increases Gas2 expression in an ICSBP-dependent manner. This results in decreased calpain activity and a consequent increase in beta-catenin activity in Bcr/abl-positive (Bcr/abl(+)) cells. Therefore, these studies have identified a Gas2/calpain-dependent mechanism by which ICSBP influences beta-catenin activity in myeloid leukemia.

Rabex-5 ubiquitin ligase activity restricts Ras signaling to establish pathway homeostasis in Drosophila.

The Ras signaling pathway allows cells to translate external cues into diverse biological responses. Depending on context and the threshold reached, Ras signaling can promote growth, proliferation, differentiation, or cell survival. Failure to maintain precise control of Ras can have adverse physiological consequences. Indeed, excess Ras signaling disrupts developmental patterning and causes developmental disorders [1, 2], and in mature tissues, it can lead to cancer [3-5]. We identify Rabex-5 as a new component of Ras signaling crucial for achieving proper pathway outputs in multiple contexts in vivo. We show that Drosophila Rabex-5 restricts Ras signaling to establish organism size, wing vein pattern, and eye versus antennal fate. Rabex-5 has both Rab5 guanine nucleotide exchange factor (GEF) activity that regulates endocytic trafficking [6] and ubiquitin ligase activity [7, 8]. Surprisingly, overexpression studies demonstrate that Rabex-5 ubiquitin ligase activity, not its Rab5 GEF activity, is required to restrict wing vein specification and to suppress the eye phenotypes of oncogenic Ras expression. Furthermore, genetic interaction experiments indicate that Rabex-5 acts at the step of Ras, and tissue culture studies show that Rabex-5 promotes Ras ubiquitination. Together, these findings reveal a new mechanism for attenuating Ras signaling in vivo and suggest an important role for Rabex-5-mediated Ras ubiquitination in pathway homeostasis.

The interferon consensus sequence binding protein (ICSBP/IRF8) activates transcription of the FANCF gene during myeloid differentiation.

The interferon consensus sequence binding protein (ICSBP) is an interferon regulatory transcription factor with leukemia-suppressor activity. ICSBP regulates genes that are involved in phagocyte function, proliferation, and apoptosis. In murine models ICSBP deficiency results in a myeloproliferative disorder (MPD) with increased mature neutrophils. Over time this MPD progresses to acute myeloid leukemia (AML), suggesting that ICSBP deficiency is adequate for MPD, but additional genetic lesions are required for AML. The hypothesis of these studies is that dysregulation of key target genes predisposes to disease progression under conditions of decreased ICSBP expression. To investigate this hypothesis, we used chromatin co-immunoprecipitation to identify genes involved the ICSBP-leukemia suppressor effect. In the current studies, we identify the gene encoding Fanconi F (FANCF) as an ICSBP target gene. FancF participates in a repair of cross-linked DNA. We identify a FANCF promoter cis element, which is activated by ICSBP in differentiating myeloid cells. We also determine that DNA cross-link repair is impaired in ICSBP-deficient myeloid cells in a FancF-dependent manner. This effect is observed in differentiating cells, suggesting that ICSBP protects against the genotoxic stress of myelopoiesis. Decreased ICSBP expression is found in human AML and chronic myeloid leukemia during blast crisis (CML-BC). Our studies suggest that ICSBP deficiency may be functionally important for accumulation of chromosomal abnormalities during disease progression in these myeloid malignancies.

Impairment of ubiquitylation by mutation in Drosophila E1 promotes both cell-autonomous and non-cell-autonomous Ras-ERK activation in vivo.

Ras signaling can promote proliferation, cell survival and differentiation. Mutations in components of the Ras pathway are found in many solid tumors and are associated with developmental disorders. We demonstrate here that Drosophila tissues containing hypomorphic mutations in E1, the most upstream enzyme in the ubiquitin pathway, display cell-autonomous upregulation of Ras-ERK activity and Ras-dependent ectopic proliferation. Ubiquitylation is widely accepted to regulate receptor tyrosine kinase (RTK) endocytosis upstream of Ras. However, although the ectopic proliferation of E1 hypomorphs is dramatically suppressed by removing one copy of Ras, removal of the more upstream components Egfr, Grb2 or sos shows no suppression. Thus, decreased ubiquitylation may lead to growth-relevant Ras-ERK activation by failing to regulate a step downstream of RTK endocytosis. We further demonstrate that Drosophila Ras is ubiquitylated. Our findings suggest that Ras ubiquitylation restricts growth and proliferation in vivo. We also report our intriguing observation that complete inactivation of E1 causes non-autonomous activation of Ras-ERK in adjacent tissue, mimicking oncogenic Ras overexpression. We demonstrate that maintaining sufficient E1 function is required both cell autonomously and non-cell autonomously to prevent inappropriate Ras-ERK-dependent growth and proliferation in vivo and may implicate loss of Ras ubiquitylation in developmental disorders and cancer.

Constitutively active SHP2 cooperates with HoxA10 overexpression to induce acute myeloid leukemia.

The homeodomain transcription factor HoxA10 is maximally expressed in myeloid progenitor cells. Sustained HoxA10 expression during differentiation has been described in poor prognosis human acute myeloid leukemia (AML). Consistent with this, engineered overexpression of HoxA10 in murine bone marrow induces a myeloproliferative disorder that progresses to AML over time. This murine model suggests that HoxA10 overexpression is sufficient for myeloproliferation but that differentiation block, and therefore AML, requires acquisition of additional mutations. In myeloid progenitor cells, HoxA10 represses transcription of genes that encode phagocyte effector proteins such as gp91PHOX and p67PHOX. Tyrosine phosphorylation of HoxA10 during myelopoiesis decreases binding to these target genes. In immature myeloid cells, HoxA10 also activates transcription of the DUSP4 gene that encodes Mkp2, an anti-apoptotic protein. HoxA10 binding to the DUSP4 promoter decreases during myelopoiesis. Therefore, both myeloid-specific gene repression and DUSP4 activation by HoxA10 decrease during myelopoiesis. This results in phenotypic differentiation and facilitates apoptosis as differentiation proceeds. HoxA10 is de-phosphorylated by SHP2 protein-tyrosine phosphatase in myeloid progenitors. This mechanism maintains HoxA10 in a nonphosphorylated state in immature, but not differentiating, myeloid cells. Constitutively active SHP2 mutants have been described in human AML, which dephosphorylate HoxA10 throughout myelopoiesis. In this study, we hypothesize that constitutive SHP2 activation synergizes with HoxA10 overexpression to accelerate progression to AML. Because both HoxA10 overexpression and constitutive SHP2 activation are found in poor prognosis human AML, these studies contribute to understanding biochemical aspects of disease progression in myeloid malignancy.

Constitutive activation of SHP2 in mice cooperates with ICSBP deficiency to accelerate progression to acute myeloid leukemia.

Myeloproliferative disorders (MPDs) are characterized by cytokine hypersensitivity and apoptosis resistance. Development of a block in myeloid differentiation is associated with progression of MPD to acute myeloid leukemia (AML) and portends poor prognosis. Identifying molecular markers of this transition may suggest targets for therapeutic intervention. Interferon consensus sequence binding protein (ICSBP, also known as IRF8) is an interferon-regulatory transcription factor that functions as a leukemia tumor suppressor. In mice, ICSBP deficiency induces an MPD that progresses to AML over time, suggesting that ICSBP deficiency is sufficient for myeloproliferation, but additional genetic lesions are necessary for AML. Since activity of ICSBP is influenced by tyrosine phosphorylation state, we hypothesized that mutations in molecular pathways that regulate this process might synergize with ICSBP deficiency for progression to AML. Consistent with this, we found that constitutive activation of SHP2 protein tyrosine phosphatase synergized with ICSBP haploinsufficiency to facilitate cytokine-induced myeloproliferation, apoptosis resistance, and rapid progression to AML in a murine bone marrow transplantation model. Constitutive SHP2 activation cooperated with ICSBP deficiency to increase the number of progenitors in the bone marrow and myeloid blasts in circulation, indicating a block in differentiation. Since SHP2 activation and ICSBP deficiency may coexist in human myeloid malignancies, our studies have identified a molecular mechanism potentially involved in disease progression in such diseases.

The interferon consensus sequence-binding protein (ICSBP/IRF8) represses PTPN13 gene transcription in differentiating myeloid cells.

The interferon consensus sequence-binding protein (ICSBP/IRF8) is an interferon regulatory factor that is expressed in myeloid and B-cells. ICSBP-deficient mice develop a myeloproliferative disorder characterized by cytokine hypersensitivity and apoptosis resistance. To identify ICSBP target genes involved in these effects, we screened a CpG island microarray with chromatin that co-immunoprecipitated with ICSBP from myeloid cells. Using this technique, we identified PTPN13 as an ICSBP target gene. PTPN13 encodes Fas-associated phosphatase 1 (Fap-1), a ubiquitously expressed protein-tyrosine phosphatase. This was of interest because interaction of Fap-1 with Fas results in Fas dephosphorylation and inhibition of Fas-induced apoptosis. In this study, we found that ICSBP influenced Fas-induced apoptosis in a Fap-1-dependent manner. We also found that ICSBP interacted with a cis element in the proximal PTPN13 promoter and repressed transcription. This interaction increased during myeloid differentiation and was regulated by phosphorylation of conserved tyrosine residues in the interferon regulatory factor domain of ICSBP. ICSBP deficiency was present in human myeloid malignancies, including chronic myeloid leukemia. Therefore, these studies identified a mechanism for increased survival of mature myeloid cells in the ICSBP-deficient murine model and in human myeloid malignancies with decreased ICSBP expression.

Identification of a HoxA10 activation domain necessary for transcription of the gene encoding beta3 integrin during myeloid differentiation.

Transcription of the ITGB3 gene, which encodes beta3 integrin, increases during myeloid differentiation. alphavbeta3 integrin mediates adhesion to fibronectin or vitronectin and regulates various aspects of the inflammatory response in mature phagocytes. In these studies, we found that the homeodomain transcription factor HoxA10 interacted with a specific ITGB3 cis element and activated transcription of this gene during myeloid differentiation. We also found that increased fibronectin adhesion in differentiating myeloid cells was dependent upon this HoxA10-induced increase in beta3 integrin expression. We determined that activation of ITGB3 transcription required a HoxA10 domain that was not identical to the "hexapeptide" that mediates interaction of Hox and Pbx proteins. This activation domain was also not identical to a previously identified HoxA10 repression domain that mediates interaction with transcriptional co-repressors. Instead, this HoxA10 activation domain had homology to "PQ" protein-protein interaction domains that have been described previously in other transcription factors. Consistent with this, we found that the HoxA10 PQ-like domain recruited the CREB-binding protein (CBP) to the ITGB3 promoter. This was associated with an increase in local histone acetylation in vivo. In immature myeloid cells, we previously determined that HoxA10 repressed transcription of the CYBB and NCF2 genes, which encode the phagocyte oxidase proteins gp91(PHOX) and p67(PHOX), respectively. Therefore, our studies indicated that HoxA10 either activates or represses gene transcription at various points during myelopoiesis. Our studies also suggested that HoxA10 is a bifunctional protein that is involved in dynamic regulation of multiple aspects of phagocyte phenotype and function.

PU.1, interferon regulatory factor (IRF) 2, and the interferon consensus sequence-binding protein (ICSBP/IRF8) cooperate to activate NF1 transcription in differentiating myeloid cells.

Nf1 (neurofibromin 1) is a Ras-GAP protein that regulates cytokine-induced proliferation of myeloid cells. In previous studies, we found that the interferon consensus sequence-binding protein (ICSBP; also referred to as interferon regulatory factor 8) activates transcription of the gene encoding Nf1 (the NF1 gene) in differentiating myeloid cells. We also found that NF1 activation requires cytokine-stimulated phosphorylation of a conserved tyrosine residue in the interferon regulatory factor (IRF) domain of ICSBP/IRF8. In this study, we found that ICSBP/IRF8 cooperates with PU.1 and interferon regulatory factor 2 to activate a composite ets/IRF-cis element in the NF1 promoter. We found that PU.1 binds directly to the NF1-cis element, and DNA-bound PU.1 interacts with IRF2, recruiting IRF2 to the cis element. This interaction requires cytokine-induced phosphorylation of specific serine residues in the PU.1 PEST domain and of a conserved tyrosine residue in the IRF domain of IRF2. We found that ICSBP/IRF8 interaction with the NF1-cis element requires pre-binding of PU.1 and IRF2. The conserved IRF domain tyrosine in ICSBP/IRF8 is required for interaction with the DNA-bound PU.1-IRF2 heterodimer. NF1 deficiency in myeloid progenitor cells results in cytokine hypersensitivity and myeloproliferation. Therefore, these studies identify a target gene for the previously observed tumor-suppressor effect of PU.1. Additionally, these studies identify a tumor-suppressor function for the "oncogenic" transcription factor, IRF2.