Non-syndromic cleft lip and palate: could stress be a causal factor?

The aetiology of non-syndromic cleft lip and palate has as yet not been clearly defined. Familial relationships, environmental toxins and nutritional status have all been considered without conclusive results, although in some studies a potential link between non-syndromic cleft lip and palate and any one or more of these factors has been proposed. Elevated stress, particularly an extended term of traumatic stress, can lead to oxidative damage at the cellular level via hypothalamus-pituitary-adrenal (HPA) axis dysregulation, high cortisol and cytokine production. The effect of this hormonal shift is to re-direct the blood supply to the mother's muscles, thereby reducing the supply to the placenta, causing a potential nutritional deficiency which may then result in a genetic alteration in the foetus. Mothers with a child aged two years or younger who had been born with a cleft, who were members of CleftPals, a family support group, volunteered to be participants in this qualitative study. The research first called for a survey to be completed by the mother and this was then followed by an interview conducted by the researcher. The study involved families living in the three eastern States of Australia. The results suggest that physical and/or emotional stress may well be implicated in clefting. While little work has been done in considering stress as a causal factor, the existing literature suggests, as does this study, that elevated stress levels at, or soon after, conception appear to affect foetal development.

Generation and application of chicken egg-yolk antibodies.

Despite the fact that the use of chicken as immunization host brings many advantages to the production of polyclonal antibodies, the generation of egg yolk immunoglobulins (IgY) is rarely chosen. In this review, we report on the fast and efficient method for generation and affinity purification of IgY, in this case raised against the alpha-subunit of hypoxia-inducible factor-1 (HIF-1). The IgY antibody was successfully applied in a variety of methods and a number of different species for HIF-1alpha detection. In electrophoretic mobility shift assays, the IgY antibody recognized the native HIF-1 complex. The IgY antibody also detected HIF-1alpha protein on Western blots with extracts derived from human, monkey, pig, dog and mouse cell lines grown under hypoxic conditions. Immunofluorescence and immunoprecipitation experiments using the IgY antibody allowed detection and subcellular localization of HIF-1alpha in the nuclei of hypoxic cells. Chicken antibody production brings great benefit concerning the welfare of the immunized animals, due to non-invasive antibody harvesting with the added convenience of simple egg collection. An additional advantage is the fast and simple IgY isolation from egg yolk. IgY technology is a great improvement and should be considered as a good alternative to conventional polyclonal antibody production in mammals.

A transcriptional switch mediated by cofactor methylation.

We describe a molecular switch based on the controlled methylation of nucleosome and the transcriptional cofactors, the CREB-binding proteins (CBP)/p300. The CBP/p300 methylation site is localized to an arginine residue that is essential for stabilizing the structure of the KIX domain, which mediates CREB recruitment. Methylation of KIX by coactivator-associated arginine methyltransferase 1 (CARM1) blocks CREB activation by disabling the interaction between KIX and the kinase inducible domain (KID) of CREB. Thus, CARM1 functions as a corepressor in cyclic adenosine monophosphate signaling pathway via its methyltransferase activity while acting as a coactivator for nuclear hormones. These results provide strong in vivo and in vitro evidence that histone methylation plays a key role in hormone-induced gene activation and define cofactor methylation as a new regulatory mechanism in hormone signaling.

Transcriptional regulation in acute promyelocytic leukemia.

It has been 10 years since the seminal discovery that a mutant form of a retinoid acid receptor (RARalpha) is associated with acute promyelocytic leukemia (APL). This finding, coupled with the remarkable success of retinoic acid (RA), the natural ligand of RARalpha, in the treatment of APL, has made APL a unique model system in the study of oncogenic conversion of transcription factors in hematological malignancies. Indeed, subsequent basic and clinical studies showed that chromosomal translocation involving the RARalpha gene is the cytogenetic hallmark of APL and that these mutant forms of RARs are the oncogenes in APL that interfere with the proliferation and differentiation pathways controlled by both RAR and their fusion partners. However, it was not until recently that the role of aberrant transcriptional regulation in the pathogenesis of APL was revealed. In this review, we summarize the biochemical and biological mechanisms of transcriptional regulation by mutant RARs and their corresponding wild-type fusion partner PML and PLZF. These studies have been instrumental in our understanding of the process of leukemogenesis in general and have laid the scientific foundation for the novel concept of transcription therapy in the treatment of human cancer.

HATs on and beyond chromatin.

The role of histone acetylation as a key mechanism of transcriptional regulation has been well established. Recent advances suggest that histone acetyltransferases also play important roles in histone-modulated processes such as DNA replication, recombination and repair. In addition, acetylation of transcriptional cofactors and other proteins is an efficient means of regulating a diverse range of molecular interactions. As new histone acetyltransferases and substrates are rapidly emerging, it is becoming apparent that protein acetylation may rival phosphorylation as a mechanism to transduce cellular regulatory signals.

Modulation of CREB binding protein function by the promyelocytic (PML) oncoprotein suggests a role for nuclear bodies in hormone signaling.

Disaggregation of the spherical nuclear bodies termed promyelocytic (PML) oncogenic domains (PODs) is a characteristic of acute promyelocytic leukemia. Here, we demonstrate that the cAMP enhancer binding protein (CREB)-binding protein (CBP) associates with PML in vitro and is recruited to the PODs in vivo. Through its association with CBP, wild-type PML dramatically stimulates nuclear receptor transcriptional activity. These results demonstrate that a fraction of CBP is compartmentalized to the POD through its association with PML and thus suggest that PML and other POD-associated proteins may play an unexpectedly broad role in aspects of transcriptional regulation and human disease.

General applicability of chicken egg yolk antibodies: the performance of IgY immunoglobulins raised against the hypoxia-inducible factor 1alpha.

Avian embryos and neonates acquire passive immunity by transferring maternal immunoglobulins from serum to egg yolk. Despite being a convenient source of antibodies, egg yolk immunoglobulins (IgY) from immunized hens have so far received scant attention in research. Here we report the generation and rapid isolation of IgY from the egg yolk of hens immunized against the alpha subunit of the human hypoxia-inducible factor 1 (HIF-1alpha). Anti-HIF-1alpha IgY antibodies were affinity purified and tested for their performance in various applications. Abundant HIF-1alpha protein was detected by Western blot analysis in nuclear extracts derived from hypoxic cells of human, mouse, monkey, swine, and dog origin whereas in hypoxic quail and frog cells, the HIF-1alpha signal was weak or absent, respectively. In electrophoretic mobility shift assays, affinity-purified IgY antibody was shown to recognize the native HIF-1 (but not the related HIF-2) complex that specifically binds an oligonucleotide containing the HIF-1 DNA binding site. Furthermore, IgY antibody immunoprecipitated HIF-1alpha from hypoxic cell extracts. Immunofluorescence experiments using IgY antibody allowed the detection of HIF-1alpha in the nucleus of hypoxic COS-7 cells. For comparison, the application of a mouse monoclonal antibody raised against the identical HIF-1alpha fragment was more restricted. Because chicken housing is inexpensive, egg collection is noninvasive, isolation and affinity purification of IgY antibodies are fast and simple, and the applicability of IgY is widespread, immunization of hens represents an excellent alternative for the generation of polyclonal antibodies.

The coactivator TIF2 contains three nuclear receptor-binding motifs and mediates transactivation through CBP binding-dependent and -independent pathways.

The nuclear receptor (NR) coactivator TIF2 possesses a single NR interaction domain (NID) and two autonomous activation domains, AD1 and AD2. The TIF2 NID is composed of three NR-interacting modules each containing the NR box motif LxxLL. Mutation of boxes I, II and III abrogates TIF2-NR interaction and stimulation, in transfected cells, of the ligand-induced activation function-2 (AF-2) present in the ligand-binding domains (LBDs) of several NRs. The presence of an intact NR interaction module II in the NID is sufficient for both efficient interaction with NR holo-LBDs and stimulation of AF-2 activity. Modules I and III are poorly efficient on their own, but synergistically can promote interaction with NR holo-LBDs and AF-2 stimulation. TIF2 AD1 activity appears to be mediated through CBP, as AD1 could not be separated mutationally from the CBP interaction domain. In contrast, TIF2 AD2 activity apparently does not involve interaction with CBP. TIF2 exhibited the characteristics expected for a bona fide NR coactivator, in both mammalian and yeast cells. Moreover, in mammalian cells, a peptide encompassing the TIF2 NID inhibited the ligand-induced AF-2 activity of several NRs, indicating that NR AF-2 activity is either mediated by endogenous TIF2 or by coactivators recognizing a similar surface on NR holo-LBDs.

Functional interactions between retinoic acid receptor-related orphan nuclear receptor (ROR alpha) and the retinoic acid receptors in the regulation of the gamma F-crystallin promoter.

We have previously demonstrated that an everted repeat of the hexamer PuGGTCA located within the gamma F-crystallin promoter mediates activation of the murine gamma F-crystallin gene by retinoic acid and thyroid hormone receptors. Here, we show that the recently identified retinoic acid receptor-related orphan nuclear receptor (ROR alpha) is expressed in the murine lens and activates the gamma F-crystallin promoter. In contrast to the retinoic acid and thyroid hormone receptors, activation of the gamma F-crystallin promoter by ROR alpha requires binding to the single 3' half-site and spacer sequences of gamma F-crystallin hormone response element (gamma F-HRE). We further demonstrate that ROR alpha-dependent activation is repressed by the competitive binding of retinoic acid receptor/retinoid X receptor heterodimers to the gamma F-HRE in the absence of all-trans-retinoic acid. These studies suggest that the interplay of retinoid receptors and ROR alpha on the gamma F-HRE may constitute an important mechanism regulating gamma F-crystallin gene expression in the murine lens.

Heterodimeric interaction of the retinoic acid and thyroid hormone receptors in transcriptional regulation on the gamma F-crystallin everted retinoic acid response element.

Previously, we have identified a hormone response element (gamma F-HRE) composed of an everted repeat of the half-site (A/G)GGTCA motif separated by 8 base pairs that mediates retinoic acid (RA) activation of the gamma F-crystallin promoter. Here, we report that this element is bound by the thyroid hormone (T3) receptor in the form of heterodimers with either the retinoid X receptor (RXR) or the retinoic acid receptor (RAR). The T3R/RXR heterodimer binds to this element with high affinity but the transcriptional activity of the T3 receptor on this element is effectively antagonized by RAR alpha. Thus, RAR alpha exerts a dominant effect on the gamma F-HRE-everted repeat by mediating both RA activation and preventing T3 response. Although RAR/T3R heterodimers bind to the gamma F-HRE, they do not appear to be involved in transcriptional regulation since they bind with low affinity, and their ability to bind DNA is dramatically decreased by T3. Repression requires the DNA- and ligand-binding domains of RAR alpha and is consistent with a competitive DNA binding model of repression. However, in vitro binding studies indicate that RAR/RXR heterodimers form less stable interactions with the gamma F-HRE compared with T3R/RXR heterodimers; this suggests that in vivo the binding affinity of RAR/RXR heterodimers may be enhanced by accessory factors.

Isoform-specific amino-terminal domains dictate DNA-binding properties of ROR alpha, a novel family of orphan hormone nuclear receptors.

Three isoforms of a novel member of the steroid hormone nuclear receptor superfamily related to the retinoic acid receptors have been identified. The three isoforms, referred to as ROR alpha 1, ROR alpha 2, and ROR alpha 3, share common DNA- and putative ligand-binding domains but are characterized by distinct amino-terminal domains generated by alternative RNA processing. An exon encoding a functionally important subregion of the amino-terminal domain of the ROR alpha 2 isoform resides on the opposite strand of a cytochrome c-processed pseudogene. Binding site selection using in vitro-synthesized proteins reveals that the ROR alpha 1 and ROR alpha 2 isoforms bind DNA as monomers to hormone response elements composed of a 6-bp AT-rich sequence preceding a half-site core motif PuGGTCA (RORE). However, ROR alpha 1 and ROR alpha 2 display different binding specificities: ROR alpha 1 binds to and constitutively activates transcription from a large subset of ROREs, whereas ROR alpha 2 recognizes ROREs with strict specificity and displays weaker transcriptional activity. The differential DNA-binding activity of each isoform maps to their respective amino-terminal domains. Whereas truncation of the amino-terminal domain diminishes the ability of ROR alpha 1 to bind DNA, a similar deletion relaxes ROR alpha 2-binding specificity to that displayed by ROR alpha 1. Remarkably, transfer of the entire amino-terminal region of ROR alpha 1 or amino-terminal deletion of ROR alpha 2 confers RORE-binding specificities to heterologous receptors. These results demonstrate that the amino-terminal domain and the zinc finger region work in concert to confer high affinity and specific DNA-binding properties to the ROR isoforms and suggest a novel strategy to control DNA-binding activity of nuclear receptors.

Transcriptional and post-transcriptional regulation of IL-1 beta, IL-6 and TNF-alpha genes in chronic lymphocytic leukaemia.

The present study was designed to define the mechanisms of interleukin-1 beta (IL-1 beta), interleukin-6 (IL-6) and tumour necrosis factor (TNF-alpha) gene regulation in chronic lymphocytic leukaemia of B cell origin (B-CLL). By nuclear run-on analysis, all B-CLL cases displayed high levels of nuclear transcription of the IL-6 and TNF-alpha genes, whereas IL-1 beta gene transcription was only barely detectable. Upon in vitro culture for 1 h, B-CLL cells from different patients were substantially heterogeneous in terms of expression of steady state mRNA levels of IL-1 beta, IL-6 and TNF-alpha even though the pattern of nuclear transcription of these cytokines was only marginally affected by in vitro culture. mRNA stability was then examined and cytokine gene transcripts showed a half life of more than 2 h in cultured B-CLL cells and treatment with cycloheximide (CHX) did not affect cytokine transcript levels in B-CLL cells. These results indicate that: steady state levels of each mRNA do not reflect the rate of nuclear transcription of these cytokines in fresh or cultured B-CLL cells, that purification and in vitro culture of leukaemic cells may amplify cytokine gene expression in B-CLL, and that cytokine gene transcripts are relatively stable in B-CLL.

An everted repeat mediates retinoic acid induction of the gamma F-crystallin gene: evidence of a direct role for retinoids in lens development.

The vertebrate lens is a classical system for examining mechanisms of tissue determination and differentiation, yet little is known about the signaling molecules controlling its development. Here, we report that retinoic acid (RA), a substance known for its teratogenic effects on the eye and as a natural endogenous morphogenetic agent, acts as a regulator of gene expression in the lens. We have identified a novel type of RA response element (RARE) within the lens-specific mouse gamma F-crystallin promoter, consisting of two (A/G)GGTCA motifs in an everted arrangement spaced by 8 nucleotides. This element (gamma F-RARE) mediates activation of the gamma F-crystallin promoter by ligand-activated endogenous lens cell RA receptors (RARs) and confers RA responsiveness when linked to a heterologous promoter. gamma F-RARE is bound in vitro by RAR/RXR heterodimers, and both receptors cooperate in vivo to trans-activate this element. These observations demonstrate a direct effect of RA on lens-specific gene expression and reveal a novel role for retinoids in the development and homeostasis of the mammalian eye.

Acute undifferentiated leukemia with CD7+ and CD13+ immunophenotype. Lack of molecular lineage commitment and association with poor prognostic features.

The authors studied six adult patients with acute leukemia with these unusual characteristics: unclassifiable morphology and undifferentiated cytochemistry by French-American-British (FAB) criteria; concurrent expression of CD13 (and CD33) myeloid and early T-cell CD7 immune markers; no evidence of T-cell lineage commitment as determined by T-cell receptor beta (beta), gamma (gamma), and delta (delta) chain gene rearrangement study and cytoplasmic CD3 epsilon expression; and no evidence of myeloid cell lineage commitment, as shown by absent myeloid-specific c-fms proto-oncogene expression and negative myeloperoxidase ultrastructural staining (one case). Clinically, these diagnostic features matched with a poor prognosis, being associated with refractoriness to treatment, relapse and progression of disease, antecedent hematologic abnormality, and other malignancy. These cases may represent a distinct stem cell leukemia syndrome deserving immediate recognition and a nonconventional chemotherapeutic approach.

Interaction of a lens cell transcription factor with the proximal domain of the mouse gamma F-crystallin promoter.

The elements regulating lens-specific expression of the mouse gamma F-crystallin gene were examined. Here we show that mouse gamma F-crystallin sequences -67 to +45 contain a low basal level of lens-specific promoter activity and that sequences -67 to -25, which are highly conserved among different gamma-crystallin genes, are able to function as a strong transcriptional activator when duplicated and placed upstream of the TATA box. We also show that nuclear factors from lens and nonlens cells are able to form different complexes with sequences centered at -46 to -36 and demonstrate that binding of the factor from lens cells correlates with lens-specific promoter activity of the mouse gamma F-crystallin gene.

Expression of leukocyte alkaline phosphatase gene in normal and leukemic cells: regulation of the transcript by granulocyte colony-stimulating factor.

The levels of leukocyte alkaline phosphatase (LAP) messenger RNA (mRNA) are evaluated in B and T lymphocytes, monocytes, and polymorphonuclear cells (PMNs), and this transcript is found to be present only in PMNs. Precursors of the myelomonocytic pathway, represented by leukemic cells isolated from several cases of chronic myelogenous leukemia (CML) in its stable and blastic phase and acute myelogenous leukemia (AML), are devoid of LAP transcript. These data support the notion that LAP is a marker of the granulocyte terminal differentiation. Despite the absence of LAP mRNA in both the myeloid and the lymphoid precursors, nuclear run-on experiments show constitutive transcription of the LAP gene in leukemic cells obtained from AML, CML, as well as acute lymphoblastic leukemia (ALL) and B-cell chronic lymphocytic leukemia (B-CLL). In CML and in chronic myelo-monocytic leukemia (CMML) PMNs, granulocyte colony-stimulating factor (G-CSF) specifically accumulates LAP mRNA without showing a substantial increase in the rate of transcription of the LAP gene. Once increased by G-CSF, LAP mRNA is very stable, showing a half-life of more than 4 hours in the presence of actinomycin-D. G-CSF is suggested to play a pivotal role in the modulation of LAP transcript in PMNs.