Genetic control of ovarian development.

During embryonic development, ovarian somatic cells embark on a course that is separate from male somatic cells and from indifferent precursor cells. While the former aspect of ovarian development is well known, the latter has not received much attention until recently. This review attempts to integrate the most recent work regarding the differentiation of ovarian somatic cells. The discussion of the parallel development of the testis is limited to the key differences only. Similarly, germ cell development will be introduced only inasmuch as it becomes necessary to draw attention to a particular aspect of the somatic component differentiation. Finally, while postnatal ovarian development and folliculogenesis undoubtedly provide the ultimate morphological and functional fitness tests for the ovarian somatic cells, postnatal phenotypes will be only referred to when they have already been connected to genes that are expressed during embryogenesis.

Conditional ablation of Gata4 and Fog2 genes in mice reveals their distinct roles in mammalian sexual differentiation.

Assembly of functioning testis and ovary requires a GATA4-FOG2 transcriptional complex. To define the separate roles for GATA4 and FOG2 proteins in sexual development of the testis we have ablated the corresponding genes in somatic gonadal cells. We have established that GATA4 is required for testis differentiation, for the expression of Dmrt1 gene, and for testis cord morphogenesis. While Sf1Cre-mediated excision of Gata4 permitted normal expression of most genes associated with embryonic testis development, gonadal loss of Fog2 resulted in an early partial block in male pathway and sex reversal. We have also determined that testis sexual differentiation is sensitive to the timing of GATA4 loss during embryogenesis. Our results now demonstrate that these two genes also have non-overlapping essential functions in testis development.

Fog2 excision in mice leads to premature mammary gland involution and reduced Esr1 gene expression.

The critical role for GATA family proteins in maintaining the normal (non-transformed) cell state is corroborated by the recent findings of mutations or methylation in GATA genes both in primary cancers and tumor lines including breast. Previously, microarray profiling studies determined that the highest expression of both GATA3 and ESR1 (estrogen receptor alpha) is seen in tumors associated with the most favorable survival outcomes, whereas the lowest expression of GATA3 is detected in tumor subtypes showing the worst outcomes. At this time, genes and pathways that are regulated by GATA3 in the mammary gland are not well defined. We have previously established a requirement for FOG (Friend Of GATA) cofactors during mouse development. Here we report that in the murine mammary gland Fog2 gene expression is upregulated upon pregnancy and lactation with prominent expression in the epithelial cells of the gland during post-lactational regression. Mammary-specific deletion of Fog2 identified a role for this gene during gland involution; excision of the Fog2 gene leads to the accelerated involution of the gland despite diminished levels of the remodeling enzymes. Importantly, the levels of several genes linked to the control of cancerous transformation in the breast (Esr1, Prg and Foxa1) are significantly reduced upon Fog2 excision. This implicates FOG2 in the maintenance of epithelial cell differentiation in the mammary gland and in performing a protective role in breast cancer.

Negative regulation of the Wnt-beta-catenin pathway by the transcriptional repressor HBP1.

In certain cancers, constitutive Wnt signaling results from mutation in one or more pathway components. The result is the accumulation and nuclear localization of beta-catenin, which interacts with the lymphoid enhancer factor-1 (LEF)/T-cell factor (TCF) family of HMG-box transcription factors, which activate important growth regulatory genes, including cyclin D1 and c-myc. As exemplified by APC and axin, the negative regulation of beta-catenin is important for tumor suppression. Another potential mode of negative regulation is transcriptional repression of cyclin D1 and other Wnt target genes. In mammals, the transcriptional repressors in the Wnt pathway are not well defined. We have previously identified HBP1 as an HMG-box repressor and a cell cycle inhibitor. Here, we show that HBP1 is a repressor of the cyclin D1 gene and inhibits the Wnt signaling pathway. The inhibition of Wnt signaling and growth requires a common domain of HBP1. The apparent mechanism is an inhibition of TCF/LEF DNA binding through a physical interaction with HBP1. These data suggest that the suppression of Wnt signaling by HBP1 may be a mechanism to prevent inappropriate proliferation.

The Friend of GATA proteins U-shaped, FOG-1, and FOG-2 function as negative regulators of blood, heart, and eye development in Drosophila.

Friend of GATA (FOG) proteins regulate GATA factor-activated gene transcription. During vertebrate hematopoiesis, FOG and GATA proteins cooperate to promote erythrocyte and megakaryocyte differentiation. The Drosophila FOG homologue U-shaped (Ush) is expressed similarly in the blood cell anlage during embryogenesis. During hematopoiesis, the acute myeloid leukemia 1 homologue Lozenge and Glial cells missing are required for the production of crystal cells and plasmatocytes, respectively. However, additional factors have been predicted to control crystal cell proliferation. In this report, we show that Ush is expressed in hemocyte precursors and plasmatocytes throughout embryogenesis and larval development, and the GATA factor Serpent is essential for Ush embryonic expression. Furthermore, loss of ush function results in an overproduction of crystal cells, whereas forced expression of Ush reduces this cell population. Murine FOG-1 and FOG-2 also can repress crystal cell production, but a mutant version of FOG-2 lacking a conserved motif that binds the corepressor C-terminal binding protein fails to affect the cell lineage. The GATA factor Pannier (Pnr) is required for eye and heart development in Drosophila. When Ush, FOG-1, FOG-2, or mutant FOG-2 is coexpressed with Pnr during these developmental processes, severe eye and heart phenotypes result, consistent with a conserved negative regulation of Pnr function. These results indicate that the fly and mouse FOG proteins function similarly in three distinct cellular contexts in Drosophila, but may use different mechanisms to regulate genetic events in blood vs. cardial or eye cell lineages.

FOG-2, a cofactor for GATA transcription factors, is essential for heart morphogenesis and development of coronary vessels from epicardium.

We disrupted the FOG-2 gene in mice to define its requirement in vivo. FOG-2(-/-) embryos die at midgestation with a cardiac defect characterized by a thin ventricular myocardium, common atrioventricular canal, and the tetralogy of Fallot malformation. Remarkably, coronary vasculature is absent in FOG-2(-/-) hearts. Despite formation of an intact epicardial layer and expression of epicardium-specific genes, markers of cardiac vessel development (ICAM-2 and FLK-1) are not detected, indicative of failure to activate their expression and/or to initiate the epithelial to mesenchymal transformation of epicardial cells. Transgenic reexpression of FOG-2 in cardiomyocytes rescues the FOG-2(-/-) vascular phenotype, demonstrating that FOG-2 function in myocardium is required and sufficient for coronary vessel development. Our findings provide the molecular inroad into the induction of coronary vasculature by myocardium in the developing heart.

FOG acts as a repressor of red blood cell development in Xenopus.

Members of the GATA family of zinc-finger transcription factors have critical roles in a variety of cell types. GATA-1, GATA-2 and GATA-3 are required for proliferation and differentiation of several hematopoietic lineages, whereas GATA-4, GATA-5 and GATA-6 activate cardiac and endoderm gene expression programs. Two GATA cofactors have recently been identified. Friend of GATA-1 (FOG-1) interacts with GATA-1 and is expressed principally in hematopoietic lineages, whereas FOG-2 is expressed predominantly in heart and brain. Although gene targeting experiments are consistent with an essential role for FOG-1 as an activator of GATA-1 function, reporter assays in transfected cells indicate that FOG-1 and FOG-2 can act as repressors. We have cloned a Xenopus laevis homologue of FOG that is structurally most similar to FOG-1, but is expressed predominantly in heart and brain, as well as the ventral blood island and adult spleen. Ectopic expression and explant assays demonstrate that FOG proteins can act as repressors in vivo, in part through interaction with the transcriptional co-repressor, C-terminal Binding Protein (CtBP). FOG may regulate the differentiation of red blood cells by modulating expression and activity of GATA-1 and GATA-2. We propose that the FOG proteins participate in the switch from progenitor proliferation to red blood cell maturation and differentiation.

FOG-2: A novel GATA-family cofactor related to multitype zinc-finger proteins Friend of GATA-1 and U-shaped.

GATA factors are transcriptional regulatory proteins that play critical roles in the differentiation of multiple cell types in both vertebrates and invertebrates. Recent evidence suggests that the biological activities of both mammalian and Drosophila GATA factors are controlled in part by physical interaction with multitype zinc-finger proteins, Friend of GATA-1 (FOG) and U-shaped (Ush), respectively. Here we describe a new FOG-related polypeptide, designated FOG-2, that is likely to participate in differentiation mediated by GATA factors in several tissues. Expression of FOG-2 mRNA differs from that of FOG and is largely restricted to heart, neurons, and gonads in the adult. Somewhat broader expression is evident during mouse embryonic development. Similar to FOG and Ush, FOG-2 protein interacts specifically with the amino finger of GATA factors in the yeast two-hybrid system and in mammalian cells. Remarkably, though FOG-2 is quite divergent from FOG in its primary sequence, forced expression of FOG-2 rescues terminal erythroid maturation of FOG-/- hematopoietic cells. Thus, members of the FOG family of cofactors share highly specific association with GATA factors and are substantially interchangeable with respect to some aspects of function in vivo. The interaction of GATA and FOG family members constitutes an evolutionarily conserved paradigm for transcriptional control in differentiation and organogenesis.

Regulation of differentiation by HBP1, a target of the retinoblastoma protein.

Differentiation is a coordinated process of irreversible cell cycle exit and tissue-specific gene expression. To probe the functions of the retinoblastoma protein (RB) family in cell differentiation, we isolated HBP1 as a specific target of RB and p130. Our previous work showed that HBP1 was a transcriptional repressor and a cell cycle inhibitor. The induction of HBP1, RB, and p130 upon differentiation in the muscle C2C12 cells suggested a coordinated role. Here we report that the expression of HBP1 unexpectedly blocked muscle cell differentiation without interfering with cell cycle exit. Moreover, the expression of MyoD and myogenin, but not Myf5, was inhibited in HBP1-expressing cells. HBP1 inhibited transcriptional activation by the MyoD family members. The inhibition of MyoD family function by HBP1 required binding to RB and/or p130. Since Myf5 might function upstream of MyoD, our data suggested that HBP1 probably blocked differentiation by disrupting Myf5 function, thus preventing expression of MyoD and myogenin. Consistent with this, the expression of each MyoD family member could reverse the inhibition of differentiation by HBP1. Further investigation implicated the relative ratio of RB to HBP1 as a determinant of whether cell cycle exit or full differentiation occurred. At a low RB/HBP1 ratio cell cycle exit occurred but there was no tissue-specific gene expression. At elevated RB/HBP1 ratios full differentiation occurred. Similar changes in the RB/HBP1 ratio have been observed in normal C2 differentiation. Thus, we postulate that the relative ratio of RB to HBP1 may be one signal for activation of the MyoD family. We propose a model in which a checkpoint of positive and negative regulation may coordinate cell cycle exit with MyoD family activation to give fidelity and progression in differentiation.

New perspectives on retinoblastoma family functions in differentiation.

Cell differentiation is a coordinated process that includes cell cycle exit and the expression of unique genes to specify tissue identity. The focus of this review is the recent progress in understanding the functions of the RB family (RB, p130,p107) in cell differentiation. Much work has focused on the functions of RB in G1 regulation. However, much evidence now suggests a diverse function in differentiation. For discussion, differentiation will be divided into three general steps: cell cycle exit, apoptosis protection, and tissue-specific gene expression. These processes are coordinated to provide the final and unique tissue characteristics. The RB family and targets such as E2F and HBP1 have functions in each step. While there is much knowledge on each separate step of differentiation, the mechanisms that coordinate cell cycle and tissue-specific events are still not known. New evidence suggests that this coordination contains both positive and negative regulation of tissue-specific gene expression. RB. p130, HBP1, and other proteins appear to have unexpected functions in regulating tissue-specific gene expression. The ubiquitous expressions of these proteins suggest membership in a new and general pathway to coordinate cell cycle events with tissue-specific gene expression during differentiation. The collective observations hypothesize the existence of a differentiation checkpoint to insure fidelity.

Activation and repression of p21(WAF1/CIP1) transcription by RB binding proteins.

The Cdk inhibitor p21(WAF1/CIP1) is a negative regulator of the cell cycle, although its expression is induced by a number of mitogens that promote cell proliferation. We have found that E2F1 and E2F3, transcription factors that activate genes required for cell cycle progression, are strong activators of the p21 promoter. In contrast, HBP1 (HMG-box protein-1), a novel retinoblastoma protein-binding protein, can repress the p21 promoter and inhibit induction of p21 expression by E2F. Both E2Fs and HBP1 regulate p21 transcription through cis-acting elements located between nucleotides -119 to +16 of the p21 promoter and the DNA binding domains of each of these proteins are required for activity. Sequences between -119 and -60 basepairs containing four Sp1 consensus elements and two noncanonical E2F binding sites are of major importance for E2F activation, although E2F1 and E2F3 differ in the extent of their ability to activate expression when this segment is deleted. The opposing effects of E2Fs and HBP1 on p21 promoter activity suggest that interplay between these factors may determine the level of p21 transcription in vivo.

HBP1: a HMG box transcriptional repressor that is targeted by the retinoblastoma family.

A prominent feature of cell differentiation is the initiation and maintenance of an irreversible cell cycle arrest with the complex involvement of the retinoblastoma (RB) family (RB, p130, p107). We have isolated the HBP1 transcriptional repressor as a potential target of the RB family in differentiated cells. By homology, HBP1 is a sequence-specific HMG transcription factor, of which LEF-1 is the best-characterized family member. Several features of HBP1 suggest an intriguing role as a transcriptional and cell cycle regulator in differentiated cells. First, inspection of the HBP1 protein sequence revealed two consensus RB interaction motifs (LXCXE and IXCXE). Second, HBP1 interaction was selective for RB and p130, but not p107. HBP1, RB, and p130 levels are all up-regulated with differentiation; in contrast, p107 levels decline. Third, HBP1 can function as a transcriptional repressor of the promoter for N-MYC, which is a critical cell cycle and developmental gene. Fourth, because the activation of the N-MYC promoter in cycling cells required the E2F transcription factor, we show that E2F-1 and HBP1 represent opposite transcriptional signals that can be integrated within the N-MYC promoter. Fifth, the expression of HBP1 lead to efficient cell cycle arrest. The arrest phenotype was manifested in the presence of optimal proliferation signals, suggesting that HBP1 exerted a dominant regulatory role. Taken together, the results suggest that HBP1 may represent a unique transcriptional repressor with a role in initiation and establishment of cell cycle arrest during differentiation.

Independent regulation of JNK/p38 mitogen-activated protein kinases by metabolic oxidative stress in the liver.

The stress-activated protein kinases JNK and p38 mediate increased gene expression and are activated by environmental stresses and proinflammatory cytokines. Using an in vivo model in which oxidative stress is generated in the liver by intracellular metabolism, rapid protein-DNA complex formation on stress-activated AP-1 target genes was observed. Analysis of the induced binding complexes indicates that c-fos, c-jun, and ATF-2 were present, but also two additional jun family members, JunB and JunD. Activation of JNK precedes increased AP-1 DNA binding. Furthermore, JunB was shown to be a substrate for JNK, and phosphorylation requires the N-terminal activation domain. Unexpectedly, p38 activity was found to be constitutively active in the liver and was down-regulated through selective dephosphorylation following oxidative stress. One potential mechanism for p38 dephosphorylation is the rapid stress-induced activation of the phosphatase MKP-1, which has high affinity for phosphorylated p38 as a substrate. These data demonstrate that there are mechanisms for independent regulation of the JNK and p38 mitogen-activated protein kinase signal transduction pathways after metabolic oxidative stress in the liver.

The N-terminal region of E2F-1 is required for transcriptional activation of a new class of target promoter.

Because of its expression in numerous cells, the herpes simplex virus thymidine kinase promoter (HSV-TK) is one of the best characterized promoters. Using the HSV-TK promoter as a model system, we have defined a new mode of E2F-1 transcriptional activation which utilizes the N-terminal region of E2F-1. We demonstrate that E2F-1 strongly activated HSV-TK, but in the absence of consensus E2F DNA elements. Nonetheless, E2F-1 could bind to GC-rich elements, which were conclusively identified in classic studies of HSV-TK as SP-1 sites. Second, the transcriptional activation of HSV-TK required the entire E2F-1 protein, including the N-terminal 89 amino acids. In contrast, the N-terminal 89 amino acids of E2F-1 were dispensable for transcriptional activation through consensus E2F sites. Third, we demonstrated that S phase entry is not sufficient for activation of HSV-TK by E2F-1, while the activation through consensus E2F sites is strictly linked to the cell cycle. Taken together, the activation of HSV-TK by E2F-1 proceeds by a different mechanism directed in part through the N-terminal region of E2F-1 and may be uncoupled from the known cell cycle regulatory role.

Expression of the E2F-1/DP-1 transcription factor in murine development.

Because of its critical role in the control of cell proliferation and differentiation, we postulated that E2F-1 could have a role in murine development. To this end, the organ and developmental expression of the E2F-1 transcription factor was analyzed from mid-gestation to late-stage embryogenesis. We demonstrate that the mRNA levels for E2F-1 and its heterodimeric partner DP-1 reach maximal levels in the late embryonic and early postnatal period but decline in the later postnatal and adult periods. Additionally, using high resolution in situ hybridization, high expression of E2F-1 was observed in specific cells of individual tissues, suggesting that the role of E2F-1 may be more complex than previously indicated from cell culture studies. Furthermore, the unusual pattern of E2F-1 and DP-1 developmental expression may have an essential role in certain cells and tissues in the late embryonic and early postnatal period.

[Regulatory protein factor CTCF interacts with a segment of the chicken c-myc oncogene promotor, capable of changing to a noncanonical conformation]. / Reguliatornyi belkovyi faktor CTCF vzaimodeistvuet s uchastkom promotora onkogena c-myc kur, sposobnym k perekhody v nekanonicheskuiu konformatsiiu.

To identify the regions in the chicken c-myc promoter that are necessary for the binding of a nuclear trans-acting factor CTCF--the potential oncogene activator--we used a synthetic analog of the natural binding site that contains three correctly spaced CCCTC-repeats that are known to be involved in CTCF-binding. Gel retardation experiments failed to detect any CTCF-binding activity with this synthetic site. We conclude that GC-transversions made in the regions presumed to be invalid, do in fact interfere with the protein binding. The secondary structure analysis with S1-nuclease shows the presence of an unusual DNA conformation of the CTCF-binding site in the supercoiled plasmids, that can not be detected with the artificial construction. The precise mapping of S1 nuclease cleavage reveals several hypersensitive sites in the CCCTC-zone. Thus, an altered secondary structure may be functionally important for the protein recognition in vivo.

[Differences in expression and functional organization of the rat tyrosine aminotransferase gene in two lines of Morris hepatoma, 8994 and 7777]. / Razlichiia v ékspressii, funktsional'no'i organizatsii gena tirozinaminotransferazy krys v dvukh liniiakh gepatom Morrisa 8994 i 7777.

Expression and structural organization of tyrosine aminotransferase (TAT) gene in Morris hepatoma cell line 7777 with active and glucocorticoid-inducible TAT gene and in hepatoma 8994, where TAT gene does not function were analysed. No differences in the number of receptor macromolecules, translocation and nuclear binding of hormone-receptor complexes in hormone sensitive (7777) and resistant (8994) cell lines were demonstrated. Dexamethasone increases TAT gene transcription in 7777 cell line but not 8994. Restriction analysis of TAT gene does not reveal any differences either in structural or in regulatory regions. Gel retardation assay with cloned TAT fragment (-400 b.p.) from normal hepatocytes showed identical shift of mobility in 7777 and 8994 cell lines. Moreover, 5'-flanking sequence (-890 b.p.) of TAT gene linked to the bacterial CAT gene is transiently expressed in both cell lines. We have shown that HpaII site (-105 b.p.) of TAT gene is methylated in those cells where TAT gene does not function (thymus, spleen, Zajdela ascites hepatoma) and is demethylated in TAT gene expressing hepatoma 7777 and normal rat hepatocytes. In hepatoma 8994 there are no DNAse I hypersensitive regions, typical to functioning TAT gene from hepatoma 7777 and normal hepatocytes.

[Chemical reactions of double-helical nucleic acids. XI. Interaction between kinetic parameters of chemical ligation in modified DNA-duplexes and structure of the reaction site]. / Khimicheskie reaktsii v dvuspiral'nykh nukleinovykh kislotakh. XI. Vzaimosviaz' mezhdu kineticheskimi parametrami khimicheskogo ligirovaniia v modifitsirovannykh DNK-dupleksakh i strukturoi reaktsionnogo uzla.

To characterize structural factors affecting kinetic parameters of chemical ligation, DNA duplexes with various arrangements of reacting groups in the ligation site have been synthesized and studied. The modifications included replacement of a deoxythymidine with a uridine or a nucleoside having reversed configuration at C2' and/or C3' atoms, the modified residue being phosphate donor or acceptor; introduction of an "extra" nucleotide residue; replacement of a G.C pair second to the junction with a noncomplementary G.G pair. Thermal stability of the anomalous nucleotide duplexes has been characterized. Based on analysis of the kinetic parameters of chemical ligation, a suggestion is made about the conformation of the reactive site.