Definition of pRB- and p53-dependent and -independent steps in HIRA/ASF1a-mediated formation of senescence-associated heterochromatin foci

Cellular senescence is an irreversible proliferation arrest triggered by short chromosome telomeres, activated oncogenes, and cell stress and mediated by the pRB and p53 tumor suppressor pathways. One of the earliest steps in the senescence program is translocation of a histone chaperone, HIRA, into promyelocytic leukemia (PML) nuclear bodies. This relocalization precedes other markers of senescence, including the appearance of specialized domains of facultative heterochromatin called senescence-associated heterochromatin foci (SAHF) and cell cycle exit. SAHF represses expression of proliferation-promoting genes, thereby driving exit from the cell cycle. HIRA bound to another histone chaperone, ASF1a, drives formation of SAHF. Here, we show that HIRA's translocation to PML bodies occurs in response to all senescence triggers tested. Dominant negative HIRA mutants that block HIRA's localization to PML bodies prevent formation of SAHF, as does a PML-RARalpha fusion protein which disrupts PML bodies, directly supporting the idea that localization of HIRA to PML bodies is required for formation of SAHF. Significantly, translocation of HIRA to PML bodies occurs in the absence of functional pRB and p53 tumor suppressor pathways. However, our evidence indicates that downstream of HIRA's localization to PML bodies, the HIRA/ASF1a pathway cooperates with pRB and p53 to make SAHF, with the HIRA/ASF1a and pRB pathways acting in parallel. We present evidence that convergence of the HIRA/ASF1a and pRB pathways occurs through a DNAJ-domain protein, DNAJA2.     

Differentiation of human embryonic stem cells induces condensation of chromosome territories and formation of heterochromatin protein 1 foci

Abstract Human embryonic stem cells (hES) are unique in their pluripotency and capacity for self-renewal. Therefore, we have studied the differences in the level of chromatin condensation in pluripotent and all-trans retinoic acid-differentiated hES cells. Nuclear patterns of the Oct4 (6p21.33) gene, responsible for hES cell pluripotency, the C-myc (8q24.21) gene, which controls cell cycle progression, and HP1 protein (heterochromatin protein 1) were investigated in these cells. Unlike differentiated hES cells, pluripotent hES cell populations were characterized by a high level of decondensation for the territories of both chromosomes 6 (HSA6) and 8 (HSA8). The Oct4 genes were located on greatly extended chromatin loops in pluripotent hES cell nuclei, outside their respective chromosome territories. However, this phenomenon was not observed for the Oct4 gene in differentiated hES cells, for the C-myc gene in the cell types studied. The high level of chromatin decondensation in hES cells also influenced the nuclear distribution of all the variants of HP1 protein, particularly HP1α, which did not form distinct foci, as usually observed in most other cell types. Our experiments showed that unlike C-myc , the Oct4 gene and HP1 proteins undergo a high level of decondensation in hES cells. Therefore, these structures seem to be primarily responsible for hES cell pluripotency due to their accessibility to regulatory molecules. Differentiated hES cells were characterized by a significantly different nuclear arrangement of the structures studied.     

Cytological dissection of sex chromosome heterochromatin of Drosophila hydei

Prophase chromosomes of Drosophila hydei were stained with 0.5 g/ml Hoechst 33258 and examined under a fluorescence microscope. While autosomal and X chromosome heterochromatin are homogeneously fluorescent, the entirely heterochromatic Y chromosome exhibits an extremely fine longitudinal differentiation, being subdivided into 18 different regions defined by the degree of fluorescence and the presence of constrictions. Thus high resolution Hoechst banding of prophase chromosomes provides a tool comparable to polytene chromosomes for the cytogenetic analysis of the Y chromosome of D. hydei. — D. hydei heterochromatin was further characterized by Hoechst staining of chromosomes exposed to 5-bromodeoxyuridine for one round of DNA replication. After this treatment the pericentromeric autosomal heterochromatin, the X heterochromatin and the Y chromosome exhibit numerous regions of lateral asymmetry. Moreover, while the heterochromatic short arms of the major autosomes show simple lateral asymmetry, the X and the Y heterochromatin exhibit complex patterns of contralateral asymmetry. These observations, coupled with the data on the molecular content of D. hydei heterochromatin, give some insight into the chromosomal organization of highly and moderately repetitive heterochromatic DNA.     

Genetic dissection of heterochromatin in Drosophila: The role of basal X heterochromatin in meiotic sex chromosome behaviour

We have examined the female meiotic behaviour of three X chromosomes which have large deletions of the basal heterochromatin in Drosophila melanogaster. We find that most of this heterochromatin can be removed without substantially altering pairing and segregation of the two Xs. To compare the role of heterochromatin in male meiosis we have constructed individuals which carry two extra identical heterochromatic mini X chromosomes. These minis behave as univalents even though their heterochromatin is known to contain satellite DNA. We conclude therefore that this satellite DNA is not sufficient to allow effectively normal meiotic behaviour. In all other respects our results in the male extend and confirm Cooper's postulate that there exist specific pairing sites in the X heterochromatin. Thus we find no support in either female or male meiosis for the concept that satellite DNA is involved in meiotic chromosome pairing of either a chiasmate or an achiasmate kind.     

Molecular characterization of inverted pericentromeric heterochromatin of chromosome 3.

Inversion of the pericentromeric region of human chromosome 3 [inv (3) (p11q11.2)] is a rare event. Initially, this inversion was identified with staining for Q-bands by fluorescence using quinacrine (QFQ) and later characterized with staining for C-bands by CBG technique. The molecular methods of fluorescence in situ hybridization (FISH) and AluI/Giemsa and TaqI/Giemsa techniques were utilized. The findings suggest that the variable band q11.2 on chromosome 3 contains alphoid DNA sequences, which appear to be similar to those identified by conventional methods in the centromeric region (band p11).     

Molecular dissection of the human Y-chromosome

Human Y chromosome, earlier thought to be gene deficient, has attracted a great deal of attention owing to its supremacy in male sex determination and unique haplotype status in the genome. Studies on Y chromosome have shown the presence of different types of satellite DNA and several genes implicated with a variety of physical and physiological functions. The interaction of these repetitive DNA with genes in normal individuals and in patients with Y-chromosome-related genetic anomalies is still an unresolved issue and is actively being pursued. The fast changing scenario of the human genome project is likely to effect our overall understanding of the Y chromosome and Y-linked genetic anomalies in a big way. We provide a brief overview of the organization of Y chromosome with respect to several important loci encompassing both the arms and their likely involvement/modulation in genetic anomalies. The experimental approaches discussed here are envisaged to be of clinical relevance for the molecular diagnosis of the Y-linked disorders.     

Functions of chromatin remodeling factors in heterochromatin formation and maintenance

Heterochromatin is characteristically more compact than euchromatin in the eukaryotic genome. The establishment of heterochromatin is mediated by special histone modifications, recruitment and propagation of heterochromatin specific proteins, as well as formation of special primary and high order structures of chromatin. Chromatin remodeling factors are ATPases that can alter the conformation and/or positioning of nucleosomes along DNA in an ATP-dependent manner. There is increasing evidence implicating chromatin remodeling activities in heterochromatin in various organisms ranging from yeasts to humans. Chromatin remodeling factors play roles in the establishment, maintenance and epigenetic inheritance of heterochromatin, but the underlying molecular mechanisms have just begun to be investigated.     

cis-Acting determinants of heterochromatin formation on Drosophila melanogaster chromosome four

The heterochromatic domains of Drosophila melanogaster (pericentric heterochromatin, telomeres, and the fourth chromosome) are characterized by histone hypoacetylation, high levels of histone H3 methylated on lysine 9 (H3-mK9), and association with heterochromatin protein 1 (HP1). While the specific interaction of HP1 with both H3-mK9 and histone methyltransferases suggests a mechanism for the maintenance of heterochromatin, it leaves open the question of how heterochromatin formation is targeted to specific domains. Expression characteristics of reporter transgenes inserted at different sites in the fourth chromosome define a minimum of three euchromatic and three heterochromatic domains, interspersed. Here we searched for cis-acting DNA sequence determinants that specify heterochromatic domains. Genetic screens for a switch in phenotype demonstrate that local deletions or duplications of 5 to 80 kb of DNA flanking a transposon reporter can lead to the loss or acquisition of variegation, pointing to short-range cis-acting determinants for silencing. This silencing is dependent on HP1. A switch in transgene expression correlates with a switch in chromatin structure, judged by nuclease accessibility. Mapping data implicate the 1360 transposon as a target for heterochromatin formation. We propose that heterochromatin formation is initiated at dispersed repetitive elements along the fourth chromosome and spreads for approximately 10 kb or until encountering competition from a euchromatic determinant.     

Molecular dissection of the FcRbeta signaling amplifier

Human high affinity IgE receptors are expressed as two different isoforms: the tetrameric isoform, alphabetagamma(2), or the trimeric isoform, alphagamma(2). The alpha chain is the IgE binding subunit, whereas the FcRbeta and FcRgamma chains are the signaling modules. Both FcRbeta and FcRgamma contain immunoreceptor tyrosine-based activation motifs (ITAM), but the beta ITAM differs from canonical ITAMs in two ways; the spacing between the two canonical tyrosines harbors a third tyrosine, and it is one amino acid shorter than in canonical ITAMs, making it unfit to bind the tandem SH2 of Syk. We have shown that FcRbeta functions as an amplifier of the FcRgamma signaling function. However, the molecular mechanism of this amplification remains unclear. Here we show that mutation of the three tyrosines (Tyr-219, Tyr-225, and Tyr-229) in the beta ITAM essentially converts alphabetagamma(2)into an alphagamma(2) complex in terms of Lyn recruitment, FcRgamma phosphorylation, Syk activation, and calcium mobilization. Tyr-219 is the most critical residue in this regard. In addition, a detailed analysis of the dynamics of calcium mobilization suggests a possible inhibitory role for Tyr-225, which becomes apparent when Tyr-219 is mutated. Thus, the signaling amplification function of FcRbeta is mainly encoded in Tyr-219 and in its capacity to recruit Lyn. In turn, this Tyr-219-mediated Lyn recruitment enhances gamma chain phosphorylation, Syk activation, and calcium mobilization. The two other tyrosines appear to have a modulating function that remains to be fully assessed.     

Molecular dissection of a LIM domain.

LIM domains are novel sequence elements that are found in more than 60 gene products, many of which function as key regulators of developmental pathways. The LIM domain, characterized by the cysteine-rich consensus CX2CX16-23HX2CX2CX2CX16-21 CX2-3(C/H/ D), is a specific mental-binding structure that consists of two distinct zinc-binding subdomains. We and others have recently demonstrated that the LIM domain mediates protein-protein interactions. However, the sequences that define the protein-binding specificity of the LIM domain had not yet been identified. Because structural studies have revealed that the C-terminal zinc-binding module of a LIM domain displays a tertiary fold compatible with nucleic acid binding, it was of interest to determine whether the specific protein-binding activity of a LIM domain could be ascribed to one of its two zinc-binding subdomains. To address this question, we have analyzed the protein-binding capacity of a model LIM peptide, called zLIM1, that is derived from the cytoskeletal protein zyxin. These studies demonstrate that the protein-binding function of zLIM1 can be mapped to sequences contained within its N-terminal zinc-binding module. The C-terminal zinc-binding module of zLIM1 may thus remain accessible to additional interactive partners. Our results raise the possibility that the two structural subdomains of a LIM domain are capable of performing distinct biochemical functions.