Dual regulation of SPI1/PU.1 transcription factor by heat shock factor 1 (HSF1) during macrophage differentiation of monocytes.

In addition to their cytoprotective role in stressful conditions, heat shock proteins (HSPs) are involved in specific differentiation pathways, for example, we have identified a role for HSP90 in macrophage differentiation of human peripheral blood monocytes that are exposed to macrophage colony-stimulating factor (M-CSF). Here, we show that deletion of the main transcription factor involved in heat shock gene regulation, heat shock factor 1 (HSF1), affects M-CSF-driven differentiation of mouse bone marrow cells. HSF1 transiently accumulates in the nucleus of human monocytes undergoing macrophage differentiation, including M-CSF-treated peripheral blood monocytes and phorbol ester-treated THP1 cells. We demonstrate that HSF1 has a dual effect on SPI1/PU.1, a transcription factor essential for macrophage differentiation and whose deregulation can lead to the development of leukemias and lymphomas. Firstly, HSF1 regulates SPI1/PU.1 gene expression through its binding to a heat shock element within the intron 2 of this gene. Furthermore, downregulation or inhibition of HSF1 impaired both SPI1/PU.1-targeted gene transcription and macrophage differentiation. Secondly, HSF1 induces the expression of HSP70 that interacts with SPI1/PU.1 to protect the transcription factor from proteasomal degradation. Taken together, HSF1 appears as a fine-tuning regulator of SPI1/PU.1 expression at the transcriptional and post-translational levels during macrophage differentiation of monocytes.

Roles of heat shock factor 1 and 2 in response to proteasome inhibition: consequence on p53 stability.

A single heat shock factor (HSF), mediating the heat shock response, exists from yeast to Drosophila, whereas several related HSFs have been found in mammals. This raises the question of the specific or redundant functions of the different members of the HSF family and in particular of HSF1 and HSF2, which are both ubiquitously expressed. Using immortalized mouse embryonic fibroblasts (iMEFs) derived from wild-type, Hsf1(-/-), Hsf2(-/-) or double-mutant mice, we observed the distinctive behaviors of these mutants with respect to proteasome inhibition. This proteotoxic stress reduces to the same extent the viability of Hsf1(-/-)- and Hsf2(-/-)-deficient cells, but through different underlying mechanisms. Contrary to Hsf2(-/-) cells, Hsf1(-/-) cells are unable to induce pro-survival heat shock protein expression. Conversely, proteasome activity is lower in Hsf2(-/-) cells and the expression of some proteasome subunits, such as Psmb5 and gankyrin, is decreased. As gankyrin is an oncoprotein involved in p53 degradation, we analyzed the status of p53 in HSF-deficient iMEFs and observed that it was strongly stabilized in Hsf2(-/-) cells. This study points a new role for HSF2 in the regulation of protein degradation and suggests that pan-HSF inhibitors could be valuable tools to reduce chemoresistance to proteasome inhibition observed in cancer therapy.

Heat shock response: lessons from mouse knockouts.

Organisms are endowed with integrated regulatory networks that transduce and amplify incoming signals into effective responses, ultimately imparting cell death and/or survival pathways. As a conserved cytoprotective mechanism from bacteria to humans, the heat shock response has been established as a paradigm for inducible gene expression, stimulating the interests of biologists and clinicians alike to tackle fundamental questions related to the molecular switches, lineage-specific requirements, unique and/or redundant roles, and even efforts to harness the response therapeutically. Gene targeting studies in mice confirm HSF1 as a master regulator required for cell growth, embryonic development, and reproduction. For example, sterility of Hsf1-null female but not null male mice established strict requirements for maternal HSF1 expression in the oocyte. Yet Hsf2 knockouts by three independent laboratories have not fully clarified the role of mammalian HSF2 for normal development, fertility, and postnatal neuronal function. In contrast, Hsf4 knockouts have provided a consistent demonstration for HSF4's critical role during lens formation. In the future, molecular analysis of HSF knockout mice will bring new insights to HSF interactions, foster better understanding of gene regulation at the genome level, lead to a better integration of the HSF pathway in life beyond heat shock, the classical laboratory challenge.

Gene expression and chromatin organization during mouse oocyte growth.

Mouse oocytes can be classified according to their chromatin organization and the presence [surrounded nucleolus (SN) oocytes] or absence [nonsurrounded nucleolus (NSN) oocytes] of a ring of Hoechst-positive chromatin around the nucleolus. Following fertilization only SN oocytes are able to develop beyond the two-cell stage. These studies indicate a correlation between SN and NSN chromatin organization and the developmental competence of the female gamete, which may depend on gene expression. In the present study, we have used the HSP70.1Luc transgene (murine HSP70.1 promoter + reporter gene firefly luciferase) to analyze gene expression in oocytes isolated from ovaries of 2-day- to 13-week-old females. Luciferase was assayed on oocytes after classification as SN or NSN type. Our data show that SN oocytes always exhibit a higher level of luciferase activity, demonstrating a higher gene expression in this category. Only after meiotic resumption, metaphase II oocytes derived from NSN or SN oocytes acquire the same level of transgene expression. We suggest that the limited availability of transcripts and corresponding proteins, excluded from the cytoplasm until GVBD in NSN oocytes, could explain why these oocytes have a lower ability to sustain embryonic development beyond the two-cell stage at which major zygotic transcription occurs. With this study we have furthered our knowledge of epigenetic regulation of gene expression in oogenesis.

Expression of an Xist promoter-luciferase construct during spermatogenesis and in preimplantation embryos: regulation by DNA methylation.

Dosage compensation for X-linked genes in mammals is accomplished by inactivating one of the two X chromosomes in females, a process involving a regulatory gene, Xist (X-inactive specific transcript). Xist maps to the X-inactivation centre and is expressed from the inactive X chromosome in female somatic cells and at the time of X inactivation during spermatogenesis in the male. In female preimplantation embryos, Xist demonstrates imprinting in that the paternal allele inherited from the sperm is preferentially expressed. This preferential paternal Xist expression is correlated with paternal X inactivation in the extraembryonic lineages at the blastocyst stage. We have analysed a 233-bp Xist promoter fragment (nt -220 to +13) for its ability to direct appropriate expression and its regulation by DNA methylation. This minimal promoter sequence directs expression of the luciferase reporter gene following injection of the construct into one-cell embryos. In vitro methylation of the construct before injection represses transcription. In six different transgenic lines, expression of the Xist promoter-luciferase transgene occurs only in the testis of the males (as for the endogenous Xist gene). The testis-specific expression is correlated with hypomethylation of the transgene, although to different extents in different lines. Following paternal transmission, expression of the Xist promoter-luciferase construct in preimplantation embryos is correlated with degree of hypomethylation in the testis and the degree of hypomethylation of the transgene in embryos at the morula stage. It is concluded that the patterns of methylation of the transgene in sperm (and in microinjected transgenes) can regulate the activity of the Xist promoter in the preimplantation embryo and thus support the hypothesis that gametic methylation patterns govern imprinted expression of the endogenous Xist gene in development.

Construction of a high-resolution genetic map encompassing the hotfoot locus.

Hotfoot (ho) is a mutation affecting posture and movement. We report a new allele associated with the insertion of a transgene and its high-resolution mapping. Analysis of the transgene revealed that two complete and two truncated copies are inserted at the ho locus. The ho locus cosegregated with D6Mit299 in 702 meioses and is confined to a 1.1-cM region between the markers D6Mit122 and D6Mit174. If the order and distances between markers are consistent with previously published mapping data, the position of the ho locus must be revised and placed approximately 30 cM from the centromere. This high-resolution genetic map is the first step towards the positional cloning of the ho mutation.

Developmental control of heat shock and chaperone gene expression. Hsp 70 genes and heat shock factors during preimplantation phase of mouse development.

Heat shock genes are found in all organisms, and synthesis of heat shock proteins is induced by various stressors in nearly all the cells forming these organisms. However, a particular situation is noticed for hsp 70 genes in mouse embryos at the beginning of their development. First, spontaneous expression of hsp 70 is observed at the onset of zygotic genome activity. Second, inducible expression is delayed until morula or early blastocyst stages. A better understanding of both these points depends on a more careful analysis of hsp 70 expression in relation to their major regulators, the heat shock factors. In this review, we will see how the development of the preimplantation embryo highlights the complexity of heat shock gene regulation involving trans-cis interactions and the cellular and nuclear environment.

Evidence for the involvement of mouse heat shock factor 1 in the atypical expression of the HSP70.1 heat shock gene during mouse zygotic genome activation.

The mouse HSP70.1 gene, which codes for a heat shock protein (hsp70), is highly transcribed at the onset of zygotic genome activation (ZGA). This expression, which occurs in the absence of stress, is then repressed. It has been claimed that this gene does not exhibit a stress response until the blastocyst stage. The promoter of HSP70.1 contains four heat shock element (HSE) boxes which are the binding sites of heat shock transcription factors (HSF). We have been studying the presence and localization of the mouse HSFs, mHSF1 and mHSF2, at different stages of embryo development. We show that mHSF1 is already present at the one-cell stage and concentrated in the nucleus. Moreover, by mutagenizing HSE sequences and performing competition experiments (in transgenic embryos with the HSP70.1 promoter inserted before a reporter gene), we show that, in contrast with previous findings, HSE boxes are involved in this spontaneous activation. Therefore, we suggest that HSF1 and HSE are important in this transient expression at the two-cell stage and that the absence of typical inducibility at this early stage of development results mainly from the high level of spontaneous transcription of this gene during the ZGA.

Quantitative control of gene expression by nucleocytoplasmic interactions in early mouse embryos: consequence for reprogrammation by nuclear transfer.

HSP 70.1 is one of the first genes to be expressed in the mouse embryo at the time of zygotic genome activation. We studied the regulation of this gene, using a transgene associating HSP 70.1 promoter and the firefly luciferase reporter gene, which allows the precise quantification of HSP 70.1 level of expression on individual embryos. In the present work, we show first that the level of HSP 70.1 expression at the two-cell stage is significantly higher (around two-fold) in embryos whose maternal cytoplasm is from C3H strain than with BALB/c strain. We verified that this difference is not an artefact of the use of transgenic embryos, of the time of first cleavage, or of in vitro culture. This regulation of HSP 70.1 level of expression is controlled by strain-specific maternal modifiers and is independent of replication, syngamy, and mitosis. Following nuclear transfer, reactivation of HSP 70.1 is also subjected to the same epigenetic influence. Only the strain-of-origin of the recipient cytoplast modulates the level of HSP 70.1 reprogrammation; the origin of donor nucleus is not significant, demonstrating the reversibility of this strain effect. These results point out the importance of the quality of recipient cytoplast in the intensity of gene reprogrammation, which may be of importance for nuclear transfer efficiency.

Progressive maturation of chromatin structure regulates HSP70.1 gene expression in the preimplantation mouse embryo.

In the widely studied model organisms, Drosophila and Xenopus, early embryogenesis involves an extended series of nuclear divisions prior to activation of the zygotic genome. The mammalian embryo differs in that the early cleavage phase is already characterized by regulated cell cycles with specific zygotic gene expression. In the mouse, where major activation of the zygotic genome occurs at the 2-cell stage, the HSP70.1 gene is among the earliest genes to be expressed. We investigated the developmentally regulated expression of this gene during the preimplantation period, using a luciferase transgene, with or without flanking scaffold attachment regions (SARs). Cleavage stage-specific modifications in expression profiles were examined in terms of histone H4 acetylation status, topoisomerase II activity, and the localisation of HMG-I/Y, a nuclear protein with known affinity for the AT-tracts of SARs. We demonstrate that HSP70.1-associated transcription factors are not limiting, and that instead, there is a progressive maturation of chromatin structure that is directly involved in HSP70.1 regulation during early mouse development.

Expression of the HSP 70.1 gene, a landmark of early zygotic activity in the mouse embryo, is restricted to the first burst of transcription.

Activation of the mouse embryonic genome at the 2-cell stage is characterized by the synthesis of several alpha-amanitin-sensitive polypeptides, some of which belong to the multigenic hsp 70 family. In the present work we show that a member of this family, the HSP 70.1 gene, is highly transcribed at the onset of zygotic genome activation. Transcription of this gene began as early as the 1-cell stage. Expression of the gene continued through the early 2-cell stage but was repressed before the completion of the second round of DNA replication. During this period we observed that the level of transcription was modulated by in vitro culture conditions. The coincidence of repression of HSP70.1 transcription with the second round of DNA replication was not found for other transcription-dependent polypeptides synthesized at the 2-cell stage.

Detection of heat shock element-binding activities by gel shift assay during mouse preimplantation development.

Heat shock gene expression is regulated by highly conserved sequence elements (HSE for "heat shock elements"). Some of heat shock genes display an atypical expression during preimplantation mouse development. We have examined the profile of HSE-binding activities (HSE-BA) in matured ovulated oocytes and during the preimplantation development by gel shift assay and quantified the data by PhosphorImager. In each of our experiments, the F9 embryonal carcinoma cell line that contains both constitutive and heat-induced activity has been used as a control. We determine the number of oocytes or embryos required to get reproducible signals and accurate quantification by PhosphorImager. Oocytes, one-cell, and two-cell embryos respond to heat shock by inducing a strong HSE-BA. At the four-cell stage, no HSE-BA can be induced by heat shock, which suggests that noninducibility of heat shock genes at this stage (when the general mechanism of transcription is well established) might result from a defect in HSF or in the mechanism of HSF activation. A progressive reappearance of the ability to induce HSE-BA by stress is observed between the eight-cell stage and the blastocyst stage, and this parallels the appearance of heat shock gene inducibility. Matured ovulated oocytes and the first cleavage stages of embryos do not contain any HSE-BA at normal temperature but we observed a HSE-BA at normal temperature at the morula stage, which is increased at the blastocyst stage. These data, which, to our knowledge, for the first time describe the profile of a DNA-binding activity during the mouse preimplantation development, could serve as a basis for the study of other transcription factors during early embryogenesis.

Sequential acquisition of transcriptional control during early embryonic development in the rabbit.

Regulation of gene expression during early embryonic development in the rabbit was investigated by quantitative assay of firefly luciferase activity obtained by microinjection of three plasmid constructs using the regulatory region of polyomavirus promoter (PrPyV) with two different enhancer sequences (wild type or mutant "embryo-responsive," ER2) coupled to this reporter gene. Following injection at the 1-cell stage maximal level of expression of these genes was reached after three cell cycles. Two important regulatory steps that progressively limited gene expression were identified: the passage through the first mitosis and the transition from maternal to zygotic control of development (MZT) described at the 8- to 16-cell stage. The completion of the first mitosis was associated with the requirement of an enhancer sequence to stimulate expression of the weak PrPyV promoter while beyond the MZT, only particular enhancer sequences, such as ER2, allowed maintainance of the expression of PrPyV promoter. In addition, comparison of expression of constructs injected in pronuclei, 2-cell embryonic nuclei, and transplanted 32-cell blastomeres revealed that the nuclear environment could be a major effector in the regulation of embryonic gene expression. A schematic view is proposed describing the sequential establishment of the regulation exerted on early embryonic gene expression in progress from the onset of the zygotic genome activity to the MZT.

Scaffold attachment regions stimulate HSP70.1 expression in mouse preimplantation embryos but not in differentiated tissues.

Eukaryotic interphase chromatin is thought to be organized into topologically discrete, independent domains acting as units upon which differential patterns of gene expression are established. Sequences which attach chromatin to in vitro preparations of a nucleoprotein matrix (scaffold attachment regions [SARs]) may act as domain boundaries, but their role remains poorly defined compared with those of other elements such as locus control regions. We have produced mice homozygous for a transgene which is transcribed as early as the activation of the embryonic genome at the two-cell stage and which is expressed ubiquitously in a number of differentiated tissues. Transgenic lines were generated in the presence or absence of flanking SAR sequences, creating an original model which enabled us to examine the effects of these elements at different developmental stages. In the preimplantation mouse embryo, flanking SARs stimulated transgene expression in a copy-dependent manner. In contrast, in the differentiated tissues of newborn and adult mice, no significant SAR-dependent increase in transgene expression was found, correlation with copy number was lost, and position effects were observed. These results suggest a limited capacity of SARs to act as insulating elements but are consistent with a proposed model of SAR-mediated chromatin opening and closing.