Abstract: The candidate tumor suppressor BAP1 is a deubiquitinating enzyme (DUB) involved in the regulation of cell proliferation, although the molecular mechanisms governing its function remain poorly defined. BAP1 was recently shown to interact with and deubiquitinate the transcriptional regulator host cell factor 1 (HCF-1). Here we show that BAP1 assembles multiprotein complexes containing numerous transcription factors and cofactors, including HCF-1 and the transcription factor Yin Yang 1 (YY1). Through its coiled-coil motif, BAP1 directly interacts with the zinc fingers of YY1. Moreover, HCF-1 interacts with the middle region of YY1 encompassing the glycine-lysine-rich domain and is essential for the formation of a ternary complex with YY1 and BAP1 in vivo. BAP1 activates transcription in an enzymatic-activity-dependent manner and regulates the expression of a variety of genes involved in numerous cellular processes. We further show that BAP1 and HCF-1 are recruited by YY1 to the promoter of the cox7c gene, which encodes a mitochondrial protein used here as a model of BAP1-activated gene expression. Our findings (i) establish a direct link between BAP1 and the transcriptional control of genes regulating cell growth and proliferation and (ii) shed light on a novel mechanism of transcription regulation involving ubiquitin signaling.
Abstract: Suppressed neutrophil apoptosis, a hallmark of sepsis, perpetuates inflammation and delays resolution. Myeloid nuclear differentiation antigen (MNDA) is expressed only in myeloid cells and has been implicated in cell differentiation; however, its function in mature neutrophils is not known.
Abstract: During development and erythropoiesis, globin gene expression is finely modulated through an important network of transcription factors and chromatin modifying activities. In this report we provide in vivo evidence that endogenous Ikaros is recruited to the human beta-globin locus and targets the histone deacetylase HDAC1 and the chromatin remodeling protein Mi-2 to the human gamma-gene promoters, thereby contributing to gamma-globin gene silencing at the time of the gamma- to beta-globin gene transcriptional switch. We show for the first time that Ikaros interacts with GATA-1 and enhances the binding of the latter to different regulatory regions across the locus. Consistent with these results, we show that the combinatorial effect of Ikaros and GATA-1 impairs close proximity between the locus control region and the human gamma-globin genes. Since the absence of Ikaros also affects GATA-1 recruitment to GATA-2 promoter, we propose that the combinatorial effect of Ikaros and GATA-1 is not restricted to globin gene regulation.
Abstract: Although distal regulatory regions are frequent throughout the genome, the molecular mechanisms by which they act in a promoter-specific manner remain to be elucidated. The human beta-globin locus constitutes an extremely well-established multigenic model to investigate this issue. In erythroid cells, the beta-globin locus control region (LCR) exerts distal regulatory function by influencing local chromatin organization and inducing high-level expression of individual beta-like globin genes. Moreover, in transgenic mice expressing the entire human beta-globin locus, deletion of LCR-hypersensitive site 2 (HS2) can alter beta-like globin gene expression. Here, we show that abnormal expression of human beta-like globin genes in the absence of HS2 is associated with decreased efficacy of pre-initiation complex formation at the human epsilon- and gamma-promoters, but not at the beta-promoter. This promoter-specific phenomenon is associated with reduced long-range interactions between the HS2-deleted LCR and human gamma-promoters. We also find that HS2 is dispensable for high-level human beta-gene transcription, whereas deletion of this hypersensitive site can alter locus chromatin organization; therefore the functions exerted by HS2 in transcriptional enhancement and locus chromatin organization are distinct. Overall, our data delineate one mechanism whereby a distal regulatory region provides promoter-specific transcriptional enhancement.
Abstract: Posttranslational modifications of transcription factors provide alternate protein interaction platforms that lead to varied downstream effects. We have investigated how the acetylation of EKLF plays a role in its ability to alter the beta-like globin locus chromatin structure and activate transcription of the adult beta-globin gene. By establishing an EKLF-null erythroid line whose closed beta-locus chromatin structure and silent beta-globin gene status can be rescued by retroviral infection of EKLF, we demonstrate the importance of EKLF acetylation at lysine 288 in the recruitment of CBP to the locus, modification of histone H3, occupancy by EKLF, opening of the chromatin structure, and transcription of adult beta-globin. We also find that EKLF helps to coordinate this process by the specific association of its zinc finger domain with the histone H3 amino terminus. Although EKLF interacts equally well with H3.1 and H3.3, we find that only H3.3 is enriched at the adult beta-globin promoter. These data emphasize the critical nature of lysine acetylation in transcription factor activity and enable us to propose a model of how modified EKLF integrates coactivators, chromatin remodelers, and nucleosomal components to alter epigenetic chromatin structure and stimulate transcription.
Abstract: Basal expression of lineage-specific transcription factors (TFs) in multipotent hematopoietic progenitor cells (HPCs) plays a pivotal role in normal hematopoiesis. Indeed, the interplay between lineage-specific TFs and chromatin modifying or remodeling complexes allows chromatin modifications at specific hematopoietic loci and promotes transcriptionally prone conformations. During hematopoiesis, the expression of various lineage-specific genes can be preceded by their potentiation i.e., by chromatin activation, in progenitor cells. Gene potentiation appears to counterbalance epigenetic silencing of lineage-specific genes in early progenitors, while maintaining an accessible chromatin conformation in the lineage pathway selected. Herein, we discuss the impact of lineage-specific TFs on gene potentiation and priming in normal hematopoiesis, and emphasize the complementary role of locus control region (LCR) or LCR-like structures and promoter regions in gene-specific potentiation events.
Abstract: During development, the regulated expression of tissue-specific genes can be preceded by their potentiation, that is, by chromatin activation in progenitor cells. For example, the human beta-like globin genes are potentiated in a gene- and developmental-specific manner in hematopoietic progenitors. Developmental regulation of human beta-gene expression in erythroid cells is mostly determined by transcriptional activators; however, it is not clear how gene-specific potentiation is set in hematopoietic progenitors. Using human and transgenic multipotent hematopoietic progenitors, we demonstrate that human beta-globin locus activation is characterized by TBP, NF-E2, CBP and BRG1 recruitment at both the Locus Control Region and human beta-gene promoter. Our results further indicate that in hematopoietic progenitors, EKLF influences chromatin organization at the human beta-globin locus and is instrumental for human beta-gene potentiation. Thus, we show that lineage-specific transcriptional activators expressed at basal levels in progenitor cells can participate in gene potentiation.
Abstract: Abnormal epigenetic regulation of gene expression contributes significantly to a variety of human pathologies including cancer. Deletion of hypersensitive site 2 (HS2) at the human beta-globin locus control region can lead to abnormal epigenetic regulation of globin genes in transgenic mice. Here, two HS2-deleted transgenic mouse lines were used as model to demonstrate that heritable alteration of chromatin organization at the human beta-globin locus in multipotent hematopoietic progenitors contributes to the abnormal expression of the beta-globin gene in mature erythroid cells. This alteration is characterized by specific patterns of histone covalent modifications that are inherited during erythropoiesis and, moreover, is plastic because it can be reverted by transient treatment with the histone deacetylase inhibitor Trichostatin A. Altogether, our results indicate that aberrant epigenetic regulation can be detected and modified before tissue-specific gene transcription, a finding which may lead to novel strategies for the prevention of chromatin-related pathologies.
Abstract: To study epigenetic regulation of the human beta-globin locus during hematopoiesis, we investigated patterns of histone modification and chromatin accessibility along this locus in hematopoietic progenitor cells (HPCs) derived from both humans and transgenic mice. We demonstrate that the developmentally related activation of human beta-like globin genes in humans and transgenic mice HPCs is preceded by a wave of gene-specific histone H3 hyperacetylation and K4 dimethylation. In erythroid cells, expression of beta-like globin genes is associated with histone hyperacetylation along these genes and, surprisingly, with local deacetylation at active promoters. We also show that endogenous mouse beta major and human beta-like genes are subject to different epigenetic control mechanisms in HPCs. This difference is likely due to intrinsic properties of the human beta-globin locus since, in transgenic mice, this locus is epigenetically regulated in the same manner as in human HPCs. Our results suggest that a defined pattern of histone H3 acetylation/dimethylation is important for specific activation of human globin promoters during development in human and transgenic HPCs. We propose that this transient acetylation/dimethylation is involved in gene-specific potentiation in HPCs (ie, before extensive chromatin remodeling and transcription take place in erythroid cells).
Abstract: The human beta globin locus replicates late in most cell types, but becomes early replicating in erythroid cells. Using FISH to map DNA replication timing around the endogenous beta globin locus and by applying a genetic approach in transgenic mice, we have demonstrated that both the late and early replication states are controlled by regulatory elements within the locus control region. These results also show that the pattern of replication timing is set up by mechanisms that work independently of gene transcription.
Abstract: The expression of transgene loci in mammals often occurs in a heterocellular fashion resulting in variegated patterns of expression. We have examined the effect of chromosomal integration site, copy number, and transcriptionally activating sequences on the variegation of a keratin 5-lacZ (K5Z) construct in the stratified epithelia of transgenic mice. lacZ expression in these mice is always mosaic, and the beta-gal activity per cell is usually higher in the lines with a higher proportion of expressing cells. Similar constructs, in which cDNAs were exchanged by lacZ sequences, showed no variegation. Also, when a strongly active, nonvariegating construct was coinjected with K5Z, most transgenic lines showed an almost homogeneous lacZ expression. The comparison of transgene arrays of different copies inserted at the same locus (obtained by using a lox/Cre system) showed that the reduction of copy number does not lead to an increase in the proportion of cells that express the transgene. Finally, in most of the variegating or nonexpressing lines the transgenes were located both at intermediate positions and at peritelomeric regions in the long chromosome arms. These findings suggest that the probability and efficiency of expression of K5Z genes depend on both long range chromosomal influences and on sequences in the transgene array.
Abstract: GATA-1 is a tissue-specific transcription factor that is essential for the production of red blood cells. Here we show that overexpression of GATA-1 in erythroid cells inhibits their differentiation, leading to a lethal anaemia. Using chromosome-X-inactivation of a GATA-1 transgene and chimaeric animals, we show that this defect is intrinsic to erythroid cells, but nevertheless cell nonautonomous. Usually, cell nonautonomy is thought to reflect aberrant gene function in cells other than those that exhibit the phenotype. On the basis of our data, we propose an alternative mechanism in which a signal originating from wild-type erythroid cells restores normal differentiation to cells overexpressing GATA-1 in vivo. The existence of such a signalling mechanism indicates that previous interpretations of cell-nonautonomous defects may be erroneous in some cases and may in fact assign gene function to incorrect cell types.
Abstract: Locus control regions (LCRs) alleviate chromatin-mediated transcriptional repression. Incomplete LCRs partially lose this property when integrated in transcriptionally restrictive genomic regions such as centromeres. This frequently results in position effect variegation (PEV), i.e. the suppression of expression in a proportion of the cells. Here we show that this PEV is influenced by the heterochromatic protein SUV39H1 and by the Polycomb group proteins M33 and BMI-1. A concentration variation of these proteins modulates the proportion of cells expressing human globins in a locus-dependent manner. Similarly, the transcription factors Sp1 or erythroid Krüppel-like factor (EKLF) also influence PEV, characterized by a change in the number of expressing cells and the chromatin structure of the locus. However, in contrast to results obtained in a euchromatic locus, EKLF influences the expression of the gamma- more than the beta-globin genes, suggesting that the relief of silencing is caused by the binding of EKLF to the LCR and that genes at an LCR proximal position are more likely to be in an open chromatin state than genes at a distal position.
Abstract: We have used a kinetic analysis to distinguish possible mechanisms of activation of transcription of the different genes in the human beta globin locus. Based on in situ studies at the single-cell level we have previously suggested a dynamic mechanism of single genes alternately interacting with the locus control region (LCR) to activate transcription. However, those steady-state experiments did not allow a direct measurement of the dynamics of the mechanism and the presence of loci with in situ primary transcript signals from two beta-like genes in cis has left open the possibility that multiple genes in the locus could initiate transcription simultaneously. Kinetic assays involving removal of a block to transcription elongation in conjunction with RNA FISH show that multiple beta gene primary transcript signals in cis represent a transition between alternating transcriptional periods of single genes, supporting a dynamic interaction mechanism.
Abstract: We have used gene competition to study the regulation of the human beta-globin locus in transgenic mice as a model system of a multigene locus. The locus is regulated by the locus control region (LCR), which is required for the expression of all the genes. Analysis of the locus at the single-cell level shows that the LCR appears to interact directly with the genes via a looping mechanism. This interaction is monogenic, and the level of transcription is determined by the frequency and stability of LCR/gene complex formation. These parameters are dependent both on the distance between the LCR and gene(s), and the concentration of transcription factors in the nucleus. Disturbance of complex formation leads to position effects, particularly when the locus is integrated in a heterochromatic environment.
Abstract: The most important level of the regulation of the beta-globin genes is by activation of all of the genes by the locus control region (LCR). Part of the developmental regulation of the locus is achieved by competition of the genes for the interaction with the LCR. Although this level of gene regulation is quantitatively of less importance than the direct repression mechanism for the early genes, it has important implications and has provided an excellent assay to probe the regulation of transcription at the single cell level. The results of these studies indicate that the LCR interacts with individual globin genes and that LCR/gene interactions are dynamic with complexes forming and dissociating continually. We conclude that transcription only appears to take place while the LCR and gene interact and that the level of transcription is determined by the frequency and duration of such interaction rather than by changes in the rate of transcription of the promoters. This mechanism has clear implications for the design of vectors for the purpose of gene therapy.
Abstract: The most important level of regulation of the beta-globin genes is by activation of all of the genes by the Locus Control Region (LCR) and repression of the early genes by an as yet unknown factor acting on sequences flanking the genes. Superimposed on this is a mechanism in which the early genes (epsilon and gamma) suppress the late genes (delta and beta) by competition for the interaction with the LCR. Although this extra level of gene regulation is quantitatively of less importance than the direct repression mechanism, it has important implications and has provided an excellent assay system to probe the regulation of transcription at the single cell level. These studies indicate that the LCR interacts with individual globin genes and that LCR/gene interactions are dynamic with complexes forming and dissociating continually. The levels of expression of each of the genes appear to depend on: 1) the frequency of interaction which is itself dependent on the distance of the gene to the LCR, 2) the affinity of the LCR for the gene and 3) the stability of the LCR/gene complex. The latter two are dependent on the balance of transcription factors. We conclude that transcription only appears to take place while the LCR and gene interact and that the level of transcription is determined by the frequency and duration of such interaction rather than by changes in the rate of transcription of promoters.
Abstract: Locus control regions (LCRs) are responsible for initiating and maintaining a stable tissue-specific open chromatin structure of a locus. In transgenic mice, LCRs confer high level expression on linked genes independent of position in the mouse genome. Here we show that an incomplete LCR loses this property when integrated into heterochromatic regions. Two disruption mechanisms were observed. One is classical position-effect variegation, resulting in continuous transcription in a clonal subpopulation of cells. The other is a novel mechanism resulting in intermittent gene transcription in all cells. We conclude that only a complete LCR fully overcomes heterochromatin silencing and that it controls the level of transcription by ensuring activity in all cells at all times rather than directly controlling the rate of transcription.
Abstract: Integration of retroviral genomes is a site-specific process with respect to the virus but not the host genome. Numerous chromosomal sites and various sequences can be used as targets. Nevertheless, preferential regions and integration patterns have been observed. Using a functional assay, we investigated if host structural DNA elements could be associated with retroviral integration sites. The results were that 9 of 10 distinct retroviral integration events occurred in close proximity of structural elements behaving like intrinsically bent DNA.
Abstract: Illegitimate recombination is the dominant mechanism of recombination in mammalian somatic cells. It is responsible for most genome rearrangements such as translocations, deletions and integrations. Little is known as yet about the mechanism of illegitimate recombination and the enzymes involved. Recently, it has been shown that intrinsically bent DNA, also known as curved DNA, is present at chromosomal sites of illegitimate recombination events. Here we report that KIN17, a new mouse nuclear protein, binds to the curved DNA fragments found at illegitimate recombination sites.
Abstract: Studies done in prokaryotes and eukaryotes have indicated that DNA sequence divergence decreases the frequency of homologous recombination. To determine which step(s) of homologous recombination is sensitive to DNA sequence divergence in mammalian cells we have used an assay that does not rely on the recovery of functional products. The assay is based on the acquisition by homologous recombination of endogenous LINE-1 sequences by exogenous LINE-1 sequences. In parallel experiments, we introduced into mouse cells two gapped exogenous LINE-1 sequences, one from the mouse, L1Md-A2, and the other from the rat, L1Rn-3. Although L1Rn-3 is on average less than 85% homologous to the LINE-1 elements of the mouse, the frequency of homologous recombination with endogenous LINE-1 elements obtained with L1Rn-3 was the same as the one obtained with L1Md-A2 which is on average 95% homologous to the LINE-1 elements of the mouse. The endogenous LINE-1 sequences rescued by L1Rn-3 were 8-18% divergent from L1Rn-3 sequences, whereas those rescued by L1Md-A2 were 2-5% divergent from L1Md-A2 sequences. The gap which had been introduced into the exogenous LINE-1 sequences had been precisely repaired in 50% of the recombinants obtained with L1Md-A2. None of the L1Rn-3 recombinants showed precise gap repair.(ABSTRACT TRUNCATED AT 250 WORDS)
Abstract: Illegitimate recombination is the most frequent mechanism for chromosomal rearrangements in mammalian cells, yet little is known about this process. Most of the studies to date have looked at the sequences present at illegitimate junctions. These revealed the presence of recurrent DNA motifs, none of which was consistently found. We have undertaken to determine if intrinsic DNA structures such as bent DNA elements could be a major determinant in chromosomal illegitimate recombination. Using a two dimensional electrophoretic assay we found that eight out of eight junctions, resulting from various types of chromosomal rearrangements, had migration behaviour characteristic of DNA containing intrinsically bent DNA elements. In all cases, these occurred within one kilobase of the junctions, and in most cases could be found in both participating DNA segments. We also found that these bent DNA elements were present before the recombination event. When we analysed the frequency of intrinsically bent DNA elements in random chromosomal fragments, we found it to be about one per 11 kilobases. Thus these results suggest that bent DNA is associated with chromosomal illegitimate recombination.
