Search

Transmission electron microscopy (TEM) has been essential in defining the structural organization of the cell due to its ability to image cell structures at molecular resolution. However, the absence of colour has made it very difficult to compare the distributions and relationships of two or more types of biomolecules simultaneously if they lack clear morphological distinctions. Furthermore, single-channel information limits functional analysis, particularly in the nucleoplasm, where fibrillar material could be chromatin, ribonucleic acid or protein. Where specific stains exist to discriminate among these molecules, they cannot be combined because conventional TEM is a single-channel technology. A potential path around this barrier is through electron spectroscopic imaging (ESI). ESI can map the distributions of chemical elements within an ultrathin section. Here, we present methods to stain specific molecules with elements that ESI can visualize to enable multichannel electron microscopy.

SUMMARYCells use multiple strategies to compartmentalize functions through a combination of membrane- bound and membraneless organelles. The latter represent complex assemblies of biomolecules that coalesce into a dense phase through low affinity, multivalent interactions and undergo rapid exchange with the surrounding dilute phase. We describe a liquid-like state for the lysine methyltransferase KMT5C characterized by diffusion within heterochromatin condensates but lacking appreciable nucleoplasmic exchange. Retention was strongly correlated with reduction of condensate surface area, suggesting formation of a liquid droplet with high connectivity. This behavior mapped to a discrete domain whose activity was dependent on multiple short linear motifs. Moreover, it was strikingly resilient to marked phylogenetic differences or targeted changes in intrinsic disorder, charge, sequence, and architecture. Collectively, these findings show that a limited number of sequence features can dominantly encode multivalency, localization, and dynamic behavior within heterochromatin condensates to confer protein retention without progression to a gel or solid.

Elucidation of non-canonical protein functions can identify novel tissue homeostasis pathways. Herein, we describe a role for the Bcl-2 family member BAD in postnatal mammary gland morphogenesis. In Bad3SA knock-in mice, where BAD cannot undergo phosphorylation at 3 key serine residues, pubertal gland development is delayed due to aberrant tubulogenesis of the ductal epithelium. Proteomic and RPPA analyses identify that BAD regulates focal adhesions and the mRNA translation repressor, 4E-BP1. These results suggest that BAD modulates localized translation that drives focal adhesion maturation and cell motility. Consistent with this, cells within Bad3SA organoids contain unstable protrusions with decreased compartmentalized mRNA translation and focal adhesions, and exhibit reduced cell migration and tubulogenesis. Critically, protrusion stability is rescued by 4E-BP1 depletion. Together our results confirm an unexpected role of BAD in controlling localized translation and cell migration during mammary gland development., Preventing phosphorylation of BAD (3SA) in mouse models and human cells inhibits mammary gland development, acting by disrupting 4E-BP1- mediated translation and affecting focal adhesion/protrusion stability, cell migration and ductal tubulogenesis.

Summary The association of nuclear DNA with histones to form chromatin is essential for temporal and spatial control of eukaryotic genomes. In this study, we examined the physical state of condensed chromatin in vitro and in vivo. Our in vitro studies demonstrate that self-association of nucleosomal arrays under a wide range of solution conditions produces supramolecular condensates in which the chromatin is physically constrained and solid-like. By measuring DNA mobility in living cells, we show that condensed chromatin also exhibits solid-like behavior in vivo. Representative heterochromatin proteins, however, display liquid-like behavior and coalesce around the solid chromatin scaffold. Importantly, euchromatin and heterochromatin show solid-like behavior even under conditions that produce limited interactions between chromatin fibers. Our results reveal that condensed chromatin exists in a solid-like state whose properties resist external forces and create an elastic gel and provides a scaffold that supports liquid-liquid phase separation of chromatin binding proteins.

Background Combined MD/PhD programs provide a structured path for physician-scientist training, but assessment of their success within Canada is limited by a lack of quantitative data. We collected outcomes data for graduates of Canadian MD/PhD programs. Methods We developed and implemented a Web-based survey consisting of 41 questions designed to collect outcomes data for Canadian MD/PhD program alumni from 8 Canadian universities who had graduated before September 2015. Respondents were categorized into 2 groups according to whether they had or had not completed all training. Results Of the 186 eligible alumni of MD/PhD programs, 139 (74.7%) completed the survey. A total of 136/138 respondents (98.6%) had completed or were currently completing residency training, and 66/80 (82%) had completed at least 1 postgraduate fellowship. Most (58 [83%]) of the 70 respondents who had completed all training were appointed as faculty at academic institutions, and 37 (53%) had been principal investigators on at least 1 recent funded project. Among the 58 respondents appointed at academic institutions, 44/57 (77%) dedicated at least 20% of their time to research, and 25/57 (44%) dedicated at least 50% to research. During their combined degree, 102/136 respondents (75.0%) published 3 or more first-author papers, and 133/136 (97.8%) matched with their first choice of specialty. The median length of physician-scientist training was 13.5 years. Most respondents graduated with debt despite having been supported by Canadian Institutes of Health Research MD/PhD studentships. Interpretation Most Canadian MD/PhD program alumni pursued careers consistent with their physician-scientist training, which indicates that these programs are meeting their primary objective. Nevertheless, our findings highlight that a minority of these positions are research intensive; this finding warrants further study. Our data provide a baseline for future monitoring of the output of Canadian MD/PhD programs.

The PAX (paired box) family is a collection of metazoan transcription factors defined by the paired domain, which confers sequence-specific DNA-binding. Ancestral PAX proteins also contained a homeodomain, which can communicate with the paired domain to modulate DNA-binding. In the present study, we sought to identify determinants of this functional interaction using the paralogous PAX3 and 7 proteins. First, we evaluated a group of heterologous paired domains and homeodomains for the ability to bind DNA cooperatively through formation of a ternary complex (paired domain:homeodomain:DNA). This revealed that capacity for ternary complex formation was unique to the PAX3 and 7 homeodomains and therefore not simply a consequence of DNA-binding. We also found PAX3 and 7 were distinguished by an extended region of conservation N-terminal to the homeodomain (NTE). Phylogenetic analyses established the NTE was restricted to PAX3/7 orthologs of segmented metazoans, indicating it arose in a bilaterian precursor prior to separation of deuterostomes and protostomes. In DNA-binding assays, presence of the NTE caused a decrease in monomeric binding by the PAX3 homeodomain that reflected a lack of secondary structure in 1D-1H-NMR. Nevertheless, this inhibitory effect could be overcome by homeodomain dimerization or cooperative binding with the paired domain, establishing that protein interactions could induce homeodomain folding in the presence of the NTE. Strikingly, the PAX7 counterpart did not impair homeodomain binding, revealing inherent differences that could account for its distinct target profile in vivo. Collectively, these findings identify critical determinants of PAX3 and 7 activity, which contribute to their functional diversification.

Progress in the treatment of adult high-grade gliomas (HGG), including chemoradiation with concurrent and adjuvant temozolomide for glioblastoma, has not translated into significant therapeutic advances for pediatric HGG, where overall survival has plateaued at 15% to 20%, especially when considering specialized pediatric treatment in tertiary care centers, maximal safe neurosurgical resection, optimized delivery of involved field radiation, and improvements in supportive care. However, recent advances in our understanding of pediatric HGG, including the application of next-generation sequencing and DNA methylation profiling, have identified mutations in the histone variant H3.3 and canonical H3.1 genes, respectively. These mutations are relatively specific to neuroanatomic compartments (cortex, midline structures, thalamus, brainstem) and are often associated with other mutations, especially in specific growth factor receptor tyrosine kinases. Targeting epigenetic pathways affected by these histone mutations, alone or in combination with small molecule inhibitors of growth factor receptor signaling pathways, will inform new treatment strategies for pediatric HGG and should be incorporated into novel cooperative group clinical trial designs.

Chromatin undergoes a rapid ATP-dependent, ATM and H2AX-independent decondensation when DNA damage is introduced by laser microirradiation. Although the detailed mechanism of this decondensation remains to be determined, the kinetics of decondensation are similar to the kinetics of poly(ADP-ribosyl)ation. We used laser microirradiation to introduce DNA strand breaks into living cells expressing a photoactivatable GFP-tagged histone H2B. We find that poly(ADP-ribosyl)ation mediated primarily by poly(ADP-ribose) polymerase 1 (PARP1) is responsible for the rapid decondensation of chromatin at sites of DNA damage. This decondensation of chromatin correlates temporally with the displacement of histones, which is sensitive to PARP inhibition and is transient in nature. Contrary to the predictions of the histone shuttle hypothesis, we did not find that histone H1 accumulated on poly(ADP-ribose) (PAR) in vivo. Rather, histone H1, and to a lessor extent, histones H2A and H2B were rapidly depleted from the sites of PAR accumulation. However, histone H1 returns to chromatin and the chromatin recondenses. Thus, the PARP-dependent relaxation of chromatin closely correlates with histone displacement.

The transcription factor PAX3 is essential for myogenesis and neural crest development, and is one of several genes mutated in human Waardenburg syndrome. Analysis of disease-causing missense mutations in PAX3 has established the interdependence of its two DNA-binding domains, the paired domain (PD) and the homeodomain (HD), as well as defects in localization and mobility. Paradoxically, mutants that retained DNA binding activity exhibited the greatest defects in localization and mobility, regardless of the domain in which they reside. In the present study, structure-function analyses were used to determine the mechanistic basis of this effect. In the context of the isolated DNA-binding domains, HD mutants adopted an increase in mobility proportional to their loss in DNA binding, while PD mutants continued to display the inverse relationship observed in the full-length protein. At the structural level, this reflected an unexpected dependence on base-specific contacts in the PD, whereas HD mobility was more severely affected by loss of backbone contacts, as has been observed with other DNA-binding proteins. This requires that the HD switch to a base-specific mode in the full-length protein. Moreover, both domains underwent substantial reduction in mobility and altered localization when in a contiguous polypeptide with the endogenous linker segment. Notably, although the HD conferred localization to heterochromatin, this activity was masked when linked to the PD, despite the absence of determinants for subnuclear compartmentalization in the PD or linker. Last, the propensity for PAX3 heterochromatin localization was modulated by sequences at the amino and carboxy termini, supporting a model in which alternate conformations lead to unmasking of the HD. These data indicate that the PD and the HD functionally interact in vivo and behave as a single binding module whose mobility and localization are dependent on sequence-specific contacts.

Antimitotic agents such as taxanes (paclitaxel and docetaxel) have greatly advanced the treatment of breast cancer, although variable patient response and drug toxicity are major limitations. Lack of validated predictive markers for taxane responsiveness precludes a priori identification of patients who are most likely to respond to treatment; thus, a subset of patients endure toxic side effects with marginal benefit. Mechanistic insights into taxane therapeutic activity may lead to rational therapeutic improvements. In this paper we report that the proapoptotic BH3-only protein Bad has a major role in taxane-induced cell death in vitro, and clinically is a prognostic indicator for overall survival of breast cancer patients after adjuvant taxane chemotherapy. Unexpectedly, Bad did not induce the mitochondrial apoptotic machinery in response to taxane treatment. Instead, Bad indirectly facilitated cell death by stimulating cellular proliferation. As dividing cells are the targets of taxane therapy, Bad-stimulated proliferation may be a marker of taxane sensitivity. Our studies indicate that quantification of Bad protein levels may have value as a diagnostic tool. They also suggest that cells expressing Bad are more sensitive to taxanes because of their altered cell cycle dynamics and reveal a clinically relevant proliferative role of Bad in breast cancer.

The granzyme B gene is activated upon cytotoxic T cell stimulation and the protein is a key inducer of apoptosis in target cells. Previous studies have identified important proximal regulatory regions but these proved insufficient to drive expression in vivo. We identified a DNase1 hypersensitive site (HS2) 3.9kb upstream of the transcription start site that was present in stimulated but not resting CD8+ cells. The CTL line CTLL R8 was stably transfected with GFP reporter constructs and showed consistently higher fluorescence values when HS2 was included. In transgenic mice the presence of the relevant region of DNA resulted in inducible, CTL-specific transcription of the transgene in all transgenic founder lines analyzed. Deletion of HS2 resulted in a 10-fold reduction in expression. This is the first report of a major distal regulatory element in the control of granzyme B transcription.

Mutations in the transcription factor PAX3 cause Waardenburg syndrome (WS) in humans and the mouse Splotch mutant, which display similar neural crest-derived defects. Previous characterization of disease-causing mutations revealed pleiotropic effects on PAX3 DNA binding and transcriptional activity. In this study, we evaluated the impact of disease alleles on PAX3 localization and mobility. Immunofluorescence analyses indicated that the majority of PAX3 occupies the interchromatin space, with only sporadic colocalization with sites of transcription. Interestingly, PAX3 disease alleles fell into two distinct categories when localization and dynamics in fluorescence recovery after photobleaching (FRAP) were assessed. The first group (class I), comprising N47H, G81A and V265F exhibit a diffuse distribution and markedly increased mobility when compared with wild-type PAX3. In contrast, the G42R, F45L, S84F, Y90H and R271G mutants (class II) display evidence of subnuclear compartmentalization and mobility intermediate between wild-type PAX3 and class I proteins. However, unlike class I mutants, which retain DNA binding, class II proteins are deficient for this activity, indicating that DNA binding is not a primary determinant of PAX3 distribution and movement. Importantly, class I properties prevail when combined with a class II mutation, which taken with the proximity of the two mutant classes within the PAX3 protein, suggests class I mutants act by perturbing PAX3 conformation. Together, these results establish that altered localization and dynamics play a key role in PAX3 dysfunction and that loss of the underlying determinants represents the principal defect for a subset of Waardenburg mutations.

In the midst of an increasingly detailed understanding of the molecular basis of genome regulation, we still only vaguely understand the relationship between molecular biochemistry and the structure of the chromatin inside of cells. The centromere is a structurally and functionally unique region of each chromosome and provides an example in which the molecular understanding far exceeds the understanding of the structure and function relationships that emerge on the chromosomal scale. The centromere is located at the primary constriction of the chromosome. During entry into mitosis, the centromere specifies the assembly site of the kinetochore, the structure that binds to microtubules to enable transport of the chromosomes into daughter cells. The epigenetic contributions to the molecular organization and function of the centromere are reviewed in the context of structural mechanisms of chromatin function.

Cytotoxic lymphocytes induce target cell apoptosis via two major pathways: Fas/FasL and granule exocytosis. The latter pathway has largely been defined by the roles of the pore-forming protein perforin and by the serine proteinases granzymes A and B. Upon entry into target cells, the granzymes cleave substrates that ultimately result in cell death. To gain further insight into granzyme B function, we have identified novel substrates. SDS-PAGE analysis of S100 cell lysates identified a 51 kDa protein that was cleaved by granzyme B. Mass spectrometry analysis revealed that this fragment was the microtubule protein, alpha-tubulin, which was confirmed by western blotting. In addition, two-dimensional gel analysis showed that the truncated form of alpha-tubulin had a more basic isoelectric point than the full-length molecule, suggesting that granzyme B removed the acidic C-terminus. Site-directed mutagenesis within this region of alpha-tubulin revealed the granzyme B recognition site, which is conserved in a subset of alpha-tubulin isoforms. Significantly, we showed that alpha-tubulin was cleaved in target cells undergoing apoptosis as induced by cytotoxic T lymphocytes. Therefore, in addition to its role in the activation of mitochondria during apoptosis, these results suggest a role for granzyme B in the dismantling of the cytoskeleton.

To date, the majority of the research regarding eukaryotic transcription factors has focused on characterizing their function primarily through in vitro methods. These studies have revealed that transcription factors are essentially modular structures, containing separate regions that participate in such activities as DNA binding, protein–protein interaction, and transcriptional activation or repression. To fully comprehend the behavior of a given transcription factor, however, these domains must be analyzed in the context of the entire protein, and in certain cases the context of a multiprotein complex. Furthermore, it must be appreciated that transcription factors function in the nucleus, where they must contend with a variety of factors, including the nuclear architecture, chromatin domains, chromosome territories, and cell-cycle-associated processes. Recent examinations of transcription factors in the nucleus have clarified the behavior of these proteins in vivo and have increased our understanding of how gene expression is regulated in eukaryotes. Here, we review the current knowledge regarding sequence-specific transcription factor compartmentalization within the nucleus and discuss its impact on the regulation of such processes as activation or repression of gene expression and interaction with coregulatory factors.Key words: transcription, subnuclear localization, chromatin, gene expression, nuclear architecture.

Proper re-establishment of heterochromatin after each round of DNA replication is critical to the preservation of cell identity. Paired box 3 (PAX3), a transcription factor important in embryonic development, was found to mediate the formation of pericentromeric heterochromatin. However, how PAX3 recognizes the heterochromatic environment and re-establishes it after DNA replication remains unclear.Cell-cycle synchronization, fluorescence microscopic analyses, and co-immunoprecipitation were used to analyze the heterochromatic localization of PAX3 in HEK 293 cells and NIH 3T3 cells.We found that PAX3 binds pericentromeric heterochromatin during middle-to-late S phase. Loading of PAX3 onto pericentromeric heterochromatin requires poly(ADP-ribose) polymerase 1 (PARP1). Furthermore, loss of PAX3 or PARP1 delays cell-cycle progression through the S phase.Our results reveal how PAX3 recognizes and maintains pericentromeric heterochromatin at the S phase of the cell cycle.

Dectin-1 is an innate antifungal C-type lectin receptor necessary for protective antifungal immunity. We recently discovered that Dectin-1 is involved in controlling fungal infections of the gastrointestinal (GI) tract, but how this C-type lectin receptor mediates these activities is unknown. Here, we show that Dectin-1 is essential for driving fungal-specific CD4(+) T-cell responses in the GI tract. Loss of Dectin-1 resulted in abrogated dendritic cell responses in the mesenteric lymph nodes (mLNs) and defective T-cell co-stimulation, causing substantial increases in CD4(+) T-cell apoptosis and reductions in the cellularity of GI-associated lymphoid tissues. CD8(+) T-cell responses were unaffected by Dectin-1 deficiency. These functions of Dectin-1 have significant implications for our understanding of intestinal immunity and susceptibility to fungal infections.

Neural tube defects (NTDs) such as spina bifida and anencephaly are common congenital malformations in humans (1/1,000 births) that result from failure of the neural tube to close during embryogenesis. The etiology of NTDs is complex, with both genetic and environmental contributions; the genetic component has been extensively studied with mouse models. Loop-tail (Lp) is a semidominant mutation on mouse chromosome 1 (ref. 4). In the two known Lp alleles (Lp, Lpm1Jus), heterozygous mice exhibit a characteristic looped tail, and homozygous embryos show a completely open neural tube in the hindbrain and spinal region, a condition similar to the severe craniorachischisis defect in humans. Morphological and neural patterning studies indicate a role for the Lp gene product in controlling early morphogenesis and patterning of both axial midline structures and the developing neural plate. The 0.6-cM/0.7-megabase (Mb) Lp interval is delineated proximally by D1Mit113/Apoa2/Fcer1g and distally by Fcer1a/D1Mit149/Spna1 and contains a minimum of 17 transcription units. One of these genes, Ltap, encodes a homolog of Drosophila Strabismus/Van Gogh (Stbm/Vang), a component of the frizzled/dishevelled tissue polarity pathway. Ltap is expressed broadly in the neuroectoderm throughout early neurogenesis and is altered in two independent Lp alleles, identifying this gene as a strong candidate for Lp.

The mammalian Pax gene family comprises nine members that are characterized by a conserved DNA-binding motif, the paired domain, which was originally described in the Drosophila protein paired. Both loss- and gain-of-function studies reveal that Pax genes carry out essential roles during embryogenesis, and in some instances, may function as master regulatory genes. This review focuses on both genetic and biochemical aspects of the Pax family, and emphasizes important differences in the activity of individual Pax genes and their protein products.Key words: Pax, paired domain, homeodomain, development, gene regulation.

We have identified alternatively spliced isoforms of murine Pax-3 and Pax-7 which differ by the presence or absence of a single glutamine residue in a linker region which separates two distinct DNA-binding subdomains within the paired domain. By reverse transcription-PCR, these isoforms of Pax-3 and Pax-7 (Q+ and Q-) were detected at similar levels through multiple developmental stages in the early mouse embryo. DNA-binding studies using the Q+ and Q- isoforms of Pax-3 revealed that this alternative splicing event had no major effect on the ability of these isoforms to bind to an oligonucleotide specific for the Pax-3 homeodomain (P2) or to a paired domain recognition sequence (e5) that interacts primarily with the N-terminal subdomain of the paired domain. However, DNA-binding studies with sequences (P6CON and CD19-2/A) containing consensus elements for both the N-terminal and C-terminal subdomains revealed that the Q- isoform binds to these sequences with a two- to fivefold-higher affinity; further mutation of the GTCAC core N-terminal subdomain recognition motif of CD19-2/A generated binding sites with a high degree of specificity for the Q- isoform. These differences in DNA binding in vitro were also reflected in the enhanced ability of the Q- isoform to stimulate transcription of a reporter containing multiple copies of CD19-2/A upstream of the thymidine kinase basal promoter. In support of the observations made with these naturally occurring Pax-3 isoforms, introducing a glutamine residue at the analogous position in PAX6 caused a fivefold reduction in binding to P6CON and a complete loss of binding to CD19-2/A and to the C-terminal subdomain-specific probe 5aCON. These studies therefore provide direct evidence for a role for the paired-domain linker region in DNA target site selection, and they identify novel isoforms of Pax-3 and Pax-7 that have the potential to mediate distinct functions in the developing embryo.

In P388/VCR-10 cells, resistance to cytotoxic drugs is caused by the overexpression of the mdr3 gene, in absence of gene amplification. The gene is transcriptionally activated following integration of a full-length mouse mammary tumor virus (MMTV) within intron I, upstream of the coding region of the gene. This integration results in the production of MMTV/mdr3 fusion transcripts that originate from the antisense 5′ LTR of the provirus. The mechanism of mdr3 activation in these cells remains unclear since it cannot be accounted for either by activation from the normal MMTV promoter or by activation of the mdr3 promoter by MMTV enhancer sequences. Subcloning and sequence analysis of the genomic region encompassing the 5′ LTR of the provirus with adjacent mdr3 sequences up to exon 2 showed that the LTR had not undergone small rearrangements or deletions. Transfections of fusion plasmids containing this genomic fragment and the reporter gene luciferase showed the presence of transcriptionally active sequences in that region. Deletions of 5′ and 3′ sequences from this fragment have shown that the antisense LTR itself has little contribution to the activation of the luciferase gene, whereas the mdr3 derived sequences that include part of intron I and the beginning of exon 2 strongly activated luciferase expression when inserted in either orientations upstream of the reporter gene. These results suggest the presence of an activator element within intron I of mdr3 capable of activating transcription from a cryptic start site present in the antisense MMTV LTR. Derepression of this activator sequence within intron I by a mechanism involving integration of a transposable element may be a prerequesite to transcriptional activation of the gene which is silent in the parental P388 cells. Further support for a derepression mechanism of activation in P388 cells is provided by the identification of independent genomic rearrangements in the 5′ region of mdr3 in additional MDR P388 derivatives analyzed in this study.

Epigenetic modifications are defined as heritable changes in gene expression that are not encoded in deoxyribonucleic acid (DNA). Despite the importance of epigenetics in tumorigenesis, there is a paucity of information regarding the epigenetic profiles of oropharyngeal squamous cell carcinoma (OPSCC).The objective of this study was to identify epigenetic signatures associated with human papillomavirus (HPV)-positive and -negative OPSCC.We collected demographic, pathologic, and survival data from 44 patients with advanced-stage OPSCC treated with surgery and chemoradiation at the University of Alberta between January 2006 and December 2008. Tumour specimen from these patients were retreived and sectioned for immunohistochemical analysis. Double immunofluorescence staining was performed with p16 (HPV surrogate) and a panel of epigenetic markers, namely, histone methyl-lysines 4, 9, and 27 and H4 methyl-lysine 20. Correlation between p16 and epigenetic markers was measured using Metamorph and Image J software.Forty-one percent of patients were p16 positive. No statistically significant differences were found between p16-positive and -negative patients in terms of age at diagnosis, tumour subsite, or smoking history. We found significant differences in histone methylation between p16-positive and -negative tumours. OPSCC tumours positive for p16 had global elevations of histone H4 monomethylated lysine 20 (H4K20me1) and H3 trimethylated lysine 27 (H3K27me3) with depletions of H4 trimethylated lysine 20 (H4K20me3). In contrast, p16-negative tumours had depleted levels of H4K20me1 and H3K27me3 with high levels of H4K20me3.HPV-positive and -negative OPSCCs have distinct epigenetic profiles representing broad gene expression differences between these tumours.

Anti-mitotic agents such as paclitaxel and docetaxel are widely used for the treatment of breast, ovarian and lung cancers. Although paclitaxel induces apoptosis, this drug also modulates autophagy. How autophagy affects paclitaxel activity, is unclear. We discovered that paclitaxel inhibited autophagy through two distinct mechanisms dependent on cell cycle stage. In mitotic cells, paclitaxel blocked activation of the class III phosphatidyl inositol 3 kinase, Vps34, a critical initiator of autophagosome formation. In non-mitotic paclitaxel-treated cells, autophagosomes were generated but their movement and maturation was inhibited. Chemically or genetically blocking autophagosome formation diminished paclitaxel-induced cell death suggesting that autophagosome accumulation sensitized cells to paclitaxel toxicity. In line with these observations, we identified that primary breast tumors that expressed diminished levels of autophagy-initiating genes were resistant to taxane therapy, identifying possible mechanisms and prognostic markers of clinical chemotherapeutic resistance.

In the midst of an increasingly detailed understanding of the molecular basis of genome regulation, we still only vaguely understand the relationship between molecular biochemistry and the structure of the chromatin inside of cells. The centromere is a structurally and functionally unique region of each chromosome and provides an example in which the molecular understanding far exceeds the understanding of the structure and function relationships that emerge on the chromosomal scale. The centromere is located at the primary constriction of the chromosome. During entry into mitosis, the centromere specifies the assembly site of the kinetochore, the structure that binds to microtubules to enable transport of the chromosomes into daughter cells. The epigenetic contributions to the molecular organization and function of the centromere are reviewed in the context of structural mechanisms of chromatin function.

Axenfeld –R ieger ocular dysgenesis is associated with mutations of the human PITX2 and FOXC1 genes, which encode transcription factors of the homeodomain and forkhead types, respectively. We have identified a functional link between FOXC1 and PITX2 which we propose underpins the similar Axenfeld–Rieger phenotype caused by mutations of these genes. FOXC1 and PITX2A physically interact, and this interaction requires crucial functional domains on both proteins: the C-terminal activation domain of FOXC1 and the homeodomain of PITX2. Immunofluorescence further shows PITX2A and FOXC1 to be colocalized within a common nuclear subcompartment. Furthermore, PITX2A can function as a negative regulator of FOXC1 transactivity. This work ties both proteins into a common pathway and offers an explanation of why increased FOXC1 gene dosage produces a phenotype resembling that of PITX2 deletions and mutations. Ocular phenotypes arise despite the deregulated expression of FOXC1-target genes through mutations in FOXC1 or PITX2. Ultimately, PITX2 loss of function mutations have a compound effect: the reduced expression of PITX2-target genes coupled with the extensive activation of FOXC1-regulated targets. Our findings indicate that the functional interaction between FOXC1 and PITX2A underlies the sensitivity to FOXC1 gene dosage in Axenfeld– Rieger syndrome and related anterior segment dysgeneses.

Histone methylation is unique among post-translational histone modifications by virtue of its stability. It is thought to be a relatively stable and heritable epigenetic mark for gene-specific regulation. In this study, we use quantitative in situ approaches to investigate the cell cycle dynamics of methylated isoforms of histone H3 lysine 9. Contrary to the expected stability of trimethylated lysines, our results for trimethylated lysine 9 (tMeK9) of H3 demonstrate that the genomic content of this methylation undergoes significant changes as cells progress through mitosis. Unexpectedly, there is a loss of tMeK9 that appears to reflect a robust demethylase activity that is active during the period between anaphase and cytokinesis. Subsequent investigations of mitoses in tMeK9-deficient cells revealed defects in chromosome congression and segregation that are distinct from the increased cohesion at centromeres previously reported in association with the loss of tMeK9. Collectively, these results identify a mitosis-specific trimethylation of Lys9 in pericentromeric heterochromatin that functions in the faithful segregation of chromosomes.

Summary The paired box protein Pax3 is an essential regulator of muscle and neural crest-derived cell types, including melanocytes. Within this lineage, Pax3 has been shown to regulate the genes encoding microphthalmia-associated transcription factor (Mitf) and tyrosinase-related protein-1 (Trp-1), despite each having dissimilar Pax3 recognition sequences. We have, therefore, examined the structural requirements for Pax3 binding to the MITF and TRP-1 promoter elements, focusing on the contribution of the paired domain and homeodomain to Pax3 target site recognition. Unexpectedly, although the MITF element is characterized by suboptimal recognition motifs for the paired domain and homeodomain, it sustains a higher level of Pax3 binding than TRP-1, which contains a canonical paired domain site. The basis for this difference involves a context-dependent cooperative binding event requiring both the paired domain and homeodomain, while the paired domain alone is sufficient for TRP-1 recognition. Significantly, the analysis of Waardenburg syndrome mutations reveals marked disparity in their effects on MITF and TRP-1 binding that further underscores mechanistic differences in their interaction with Pax3. Importantly, these mutations also exert distinct effects on the ability of Pax3 to regulate reporter genes fused to either the MITF or TRP-1 promoters. Our results, therefore, establish that Pax3 can regulate target genes through alternate modes of DNA recognition that are differentially impacted by disease-causing mutations, which together have important implications for understanding Pax3-regulated gene networks.

Histone methylation acts as an epigenetic regulator of chromatin activity through the modification of arginine and lysine residues on histones H3 and H4. In the case of lysine, this includes the formation of mono-, di-, or trimethyl groups, each of which is presumed to represent a distinct functional state at the cellular level. To examine the potential developmental roles of these modifications, we determined the global patterns of lysine methylation involving K9 on histone H3 and K20 on histone H4 in midgestation mouse embryos. For each lysine target site, we observed distinct subnuclear distributions of the mono- and trimethyl versions in 10T1/2 cells that were conserved within primary cultures and within the 3D-tissue architecture of the embryo. Interestingly, three of these modifications, histone H3 trimethyl K9, histone H4 monomethyl K20, and histone H4 trimethyl K20 exhibited marked differences in their distribution within the neuroepithelium. Specifically, both histone H3 trimethyl K9 and H4 monomethyl K20 were elevated in proliferating cells of the neural tube, which in the case of the K9 modification was limited to mitotic cells on the luminal surface. In contrast, histone H4 trimethyl K20 was progressively lost from these medial regions and became enriched in differentiating neurons in the ventrolateral neural tube. The inverse relationship of histone H4 K20 methyl derivatives is even more striking during skeletal and cardiac myogenesis where the accumulation of the trimethyl modification in pericentromeric heterochromatin suggests a role in gene silencing in postmitotic muscle cells. Importantly, our results establish that histone lysine methylation occurs in a highly dynamic manner that is consistent with their function in an epigenetic program for cell division and differentiation.

BACKGROUND: The discovery of variant histone H3.3 somatic driver mutations in pediatric high grade gliomas (pHGG) has provided important insights into their pathogenesis. The K27M and G34V/R mutants are usually localized to different neuroanatomic compartments (deep midline/brainstem or superficial/cortical, respectively) and have significant differences in their gene expression profiles. For example, there is a marked increase in the expression of the homeobox gene DLX2 and a corresponding decrease in the expression of the myelin transcription factor, MYT1. When neural progenitors that are mutant for both DLX1 and DLX2 are transplanted into a wild-type background in the developing mouse forebrain, they differentiate into cells of the oligodendrocyte lineage, supporting a role for the DLX homeobox genes in neuronal-glial cell fate decisions. METHODS: Bioinformatics approaches were used to identify homeodomain consensus TAAT/ATTA tetranucleotide DNA binding motifs in regulatory regions of genes required for oligodendrocyte differentiation, Modified chromatin immunoprecipitation (ChIP) assays using a DLX2 antibody were followed by qPCR. ChIP-reChIP assays were performed using antibodies that recognize specific histone modifications at active or repressed regulatory regions of chromatin. Electrophoretic mobility shift assays (EMSA) were performed using recombinant DLX2 protein and oligonucleotide probes from ChIP-specified promoter regions. Reporter gene assays provided functional assessment of protein-DNA interactions in vitro. Target gene expression was assessed comparing wild-type (WT) and DLX1/DLX2 mutant forebrain and retina. RESULTS: Homeodomain binding sites were localized to the promoters of several genes, including Olig2, Myt1 and Nkx2.2 in silico. ChIP assays confirmed promoter occupancy by DLX2 in several regions of these gene promoters. ChIPseq experiments are in progress. EMSA studies demonstrated specific DLX2: promoter complexes. Of interest, luciferase assays showed repression of Olig2 and Nkx2.2 reporter gene expression, consistent with co-occupancy of H3K27me3 in WT and increased target gene expression in the DLX1/DLX2 double KO embryonic tissues. CONCLUSIONS: DLX homeobox genes are necessary for tangential migration and differentiation of inhibitory interneurons in the developing CNS. Collectively, our results support a role for these transcription factors in controlling neural progenitor specification by activating GABAergic and inhibiting oligodendroglial cell fates through transcriptional repression of a suite of genes required for oligodendrocyte differentiation. Understanding how pHGG co-opt these neurodevelopmental programs will lead to novel pharmacologic approaches that promote glioma differentiation. SECONDARY CATEGORY: Pediatrics.

Off-pump coronary artery bypass (OP-CAB) graft surgery is being used with increasing frequency. This study was designed to compare OP-CAB outcomes with conventional surgical revascularization using cardiopulmonary bypass (CPB) in patients with varying risk categories at a high-volume center.Between 1/1/1999 and 1/31/2001, bypass surgery was performed on 1,312 patients, including 348 OP-CAB cases and 964 CPB cases. Compared to CPB cases, OP-CAB patients were more likely to be female and had a lower incidence of three vessel coronary artery disease, prior percutaneous intervention, and prior bypass surgery. Postoperatively, OP-CAB patients had a lower incidence of renal failure and prolonged ventilatory support, as well as a lower composite endpoint of inhospital mortality, perioperative myocardial infarction, cerebrovascular accident, and/or renal failure. In addition, OP-CAB patients required fewer transfusions and had a shorter total length of hospital stay. In general, morbidity and mortality increased in both OP-CAB and CPB groups with increasing Parsonnet score.OP-CAB surgery is a safe and effective alternative to conventional coronary artery bypass graft (CABG) surgery, with a lower incidence of major in-hospital adverse clinical events and a decreased requirement for medical resources. Adverse OP-CAB outcomes correlate well with pre-operative Parsonnet Score.

Toll-like receptors (TLRs) mediate detection of a broad range of pathogens and pathogen-derived products including LPS, peptidoglycan, bacterial lipopeptides, and lipoteichoic acid. Recent evidence indicates that the broad specificity of TLRs may be a consequence of the interactions between different TLRs. In this report, we demonstrate that while a constitutively active TLR4 homodimer can induce the production of pro-inflammatory cytokines, homodimers of TLR2 and TLR6 cannot. However, when co-expressed in the same cell, constitutively active TLR2 and TLR6 strongly induce cytokine production, indicating that these TLRs require partners to productively signal. Since TLR4 signals as a homodimer, while TLR2 and TLR6 do not, it is clear that, despite the conservation of their cytoplasmic signaling domains, the mechanisms by which they initiate signaling are different. We have localized the region of TLR4 that mediates its ability to signal as a homodimer to the membrane-proximal half of the cytoplasmic tail of the receptor.

Background Macrophages sense microorganisms through activation of members of the Toll-like receptor family, which initiate signals linked to transcription of many inflammation associated genes. In this paper we examine whether the signal from Toll-like receptors [TLRs] is sustained for as long as the ligand is present, and whether responses to different TLR agonists are additive. Results RAW264 macrophage cells were doubly-transfected with reporter genes in which the IL-12p40, ELAM or IL-6 promoter controls firefly luciferase, and the human IL-1β promoter drives renilla luciferase. The resultant stable lines provide robust assays of macrophage activation by TLR stimuli including LPS [TLR4], lipopeptide [TLR2], and bacterial DNA [TLR9], with each promoter demonstrating its own intrinsic characteristics. With each of the promoters, luciferase activity was induced over an 8 hr period, and thereafter reached a new steady state. Elevated expression required the continued presence of agonist. Sustained responses to different classes of agonist were perfectly additive. This pattern was confirmed by measuring inducible cytokine production in the same cells. While homodimerization of TLR4 mediates responses to LPS, TLR2 appears to require heterodimerization with another receptor such as TLR6. Transient expression of constitutively active forms of TLR4 or TLR2 plus TLR6 stimulated IL-12 promoter activity. The effect of LPS, a TLR4 agonist, was additive with that of TLR2/6 but not TLR4, whilst that of lipopeptide, a TLR2 agonist, was additive with TLR4 but not TLR2/6. Actions of bacterial DNA were additive with either TLR4 or TLR2/6. Conclusions These findings indicate that maximal activation by any one TLR pathway does not preclude further activation by another, suggesting that common downstream regulatory components are not limiting. Upon exposure to a TLR agonist, macrophages enter a state of sustained activation in which they continuously sense the presence of a microbial challenge.

The mammalian Pax gene family comprises nine members that are characterized by a conserved DNA-binding motif, the paired domain, which was originally described in the Drosophila protein paired. Both loss- and gain-of-function studies reveal that Pax genes carry out essential roles during embryogenesis, and in some instances, may function as master regulatory genes. This review focuses on both genetic and biochemical aspects of the Pax family, and emphasizes important differences in the activity of individual Pax genes and their protein products.

Loop-tail (Lp) is a semidominant mutation that maps to the distal portion of mouse Chromosome (Chr) 1 and is an established model for neural tube defects (NTDs). Homozygous embryos exhibit an open neural tube from the caudal midbrain to the tip of the tail that results from over-differentiation of the floor plate. To facilitate the positional cloning of the Lp gene, both cDNA selection and assignment of sequence-tagged-sites from the human transcript map have been used to identify genes within the Lp interval. Together with previous physical mapping, this has allowed the placement of 13 transcription units within an approximately 1-Mb region that spans the Lp genetic interval, and eight of these genes map to the nonrecombinant interval. This map includes genes that encode proteins involved in protein sorting and targeting (Tim23 and Copa), ion transport (Atp1a2, Atp1a4, and Girk3), transcription (Nhlh1), immune regulation (Cd48 and Fcer1alpha), cell adhesion (R88252), apoptosis (Pea15), and several of unknown function (H326, Kiaa0253, and Estm34). Expression analysis by Northern blotting indicated that a subset of these genes are expressed preferentially in the developing nervous system. Finally, this region of mouse Chr 1 represents a conserved linkage group with genes on human chromosome 1q21, a region that is frequently altered in human cancers and that harbors loci for several genetic conditions. Consequently, analysis of the Lp interval may provide important tools to understand how the corresponding region of human Chr 1 contributes to disease, in addition to defining a key gene product required for neurulation.

Pax3 contains two structurally independent DNA-binding domains, a paired domain (PD) and a homeodomain (HD). Biochemical and mutagenesis studies have shown that both domains are functionally interdependent. In particular, it has been shown that the PD can regulate the DNA-binding specificity and dimerization potential of the HD. To delineate Pax3 protein segments that are involved in the regulation of HD DNA-binding, a series of chimeric proteins were created in which the HD and linker region were gradually replaced with corresponding sequences from a heterologous HD protein, Phox. Characterization of chimeric proteins by electrophoretic mobility shift analysis (EMSA) suggests that a portion of the linker region contributes to the functional interaction between the PD and HD. In addition, stepwise removal of sequences from the Pax3 PD was used to define regions within this domain that are involved in the regulation of HD DNA-binding. EMSA of these proteins in the context of the chimeric Pax3/Phox backbone provided two key findings: (i) the C-terminal subdomain of the PD does not play a major role in the regulation of HD DNA-binding and (ii) the N-terminal subdomain and, in particular, the second alpha-helix are essential for modulation of HD DNA-binding. Significantly, deletion of helix 2 was found to be sufficient to uncouple regulation of HD DNA-binding by the PD.

The Pax-3 protein contains two DNA-binding domains, a paired domain and a homeodomain. Mutations in Pax-3 cause Waardenburg syndrome (WS) in humans and the mouse Splotch (Sp) phenotype. In the Sp-delayed mouse, a mutation in the Pax-3 paired domain (G9R) abrogates the DNA-binding activity of both the paired domain and the homeodomain, suggesting that they may functionally interact. To investigate this possibility further, we have analyzed the DNA-binding properties of additional point mutants in the Pax-3 paired domain and homeodomain that occur in WS patients (F12L, N14H, G15S, P17L, R23L, G48A, S51F and G66D in the paired domain, V47F and R53G in the homeodomain), the Pax-1 un mutation (G15A) and a substitution associated with Peters' anomaly in the PAX-6 gene (R23G). Within the paired domain, seven of 10 mutations were found to abrogate DNA-binding by the paired domain. Remarkably, these seven mutations also affected DNA binding by the homeodomain, causing either a complete loss (P17L and G66D), a reduction (R23G, R23L, G15S and G15A) or an increase in DNA-binding activity (N14H). In addition, the effect of paired domain mutations occurred at the level of monomer formation by the homeodomain, while the dimerization potential of this domain seemed unaffected in mutants where it could be analyzed. Furthermore, while both homeodomain mutations were found to abolish DNA binding by this domain, the R53G mutation also abrogated DNA binding by the paired domain. The important observation that independent mutations in either domain can affect DNA binding by the other in the intact Pax-3 protein strongly suggests that the two domains are not functionally independent but bind DNA through cooperative interactions. Modeling the deleterious mutations on the three-dimensional structure of the paired domain of Drosophila Prd shows that these mutations cluster at the DNA interface, thus suggesting that a series of DNA contacts are essential for DNA binding by both the paired domain and the homeodomain of Pax-3.

Pax-3 contains two structurally independent DNA-binding domains, a paired-domain and a homeodomain. Their functional interdependence has been suggested by the analysis of the Sp-delayed (Spd) mouse mutant, in which a glycine to arginine substitution at position 9 of the paired-domain abrogates DNA binding by both domains. This glycine is located in the beta-turn portion of a beta-hairpin motif, and the requirement for this structure was investigated by mutagenesis at this and neighboring positions. At position 9, only substitution with proline increased DNA binding by the paired-domain and homeodomain above the level observed with the Spd arginine mutation, suggesting that the beta-turn is necessary for the function of both DNA-binding domains. Alanine scanning mutagenesis also identified a number of flanking residues important for DNA binding by both domains, emphasizing the requirement of the beta-hairpin for the interaction of Pax-3 with DNA. Furthermore, we show that these mutations reduce binding by the homeodomain at the monomeric level and do not impair dimerization on a TAAT(N)2ATTA consensus motif. In contrast, the wild-type paired-domain was found to prevent dimerization on consensus motifs with 3-base pair spacing of the type TAAT(N)3ATTA. Importantly, both the deleterious effect of the Spd mutation on homeodomain DNA binding and the loss of dimerization on TAAT(N)3ATTA motifs can be transferred to a heterologous homeodomain from the human phox protein. Moreover, the presence of the paired-domain affects sequence discrimination within the 3-base pair spacer in this context. These analyses establish that the beta-hairpin motif is essential for paired-domain and homeodomain DNA binding, and suggest a novel mechanism by which the paired-domain can influence sequence specificity of the homeodomain within the Pax-3 polypeptide.

The murine Pax-3 protein contains two DNA-binding domains, a paired domain and a homeodomain, and alterations in the Pax-3 gene are responsible for the neural tube defects observed in the Splotch (Sp) mouse mutant. Of five Sp alleles, Splotch-delayed (Spd) is the only one that encodes a full-length Pax-3 protein, containing a single glycine-to-arginine substitution within the paired domain. To better understand the consequence of this mutation on Pax-3 function, we have analyzed the DNA-binding properties of wild-type and Spd Pax-3, using oligonucleotides that bind primarily to the paired domain (e5) or exclusively to the homeodomain (P2). Wild-type Pax-3 was found to bind e5 in a specific manner. In contrast, the Spd mutation reduced binding of Pax-3 to e5 17-fold, revealing a defect in DNA binding by the paired domain. Surprisingly, the Spd mutation also drastically reduced the homeodomain-specific binding to P2 by 21-fold when compared with the wild-type protein. Interestingly, a deletion which removes the Spd mutation was found to restore P2-binding activity, suggesting that within the full-length Pax-3 protein, the paired domain and homeodomain may interact. We conclude, therefore, that the Spd mutation is phenotyically expressed in vitro by a defect in the DNA-binding properties of Pax-3. Furthermore, it is apparent that the paired domain and homeodomain of Pax-3 do not function as independent domains, since a mutation in the former impairs the DNA-binding activity of the latter.

Fecal samples were collected from individually caged White Leghorn hens in two experiments to determine the relationship of dietary and fecal levels of calcium and phosphorus. Data indicated that fecal levels of both calcium and phosphorus increased with increased dietary levels and could be directly altered as their dietary counterparts were varied.

Tissue specimens obtained from a normal healthy aorta and from a plaque damaged aortal portion were sliced to a thickness of ∼10 μm. They were washed thoroughly with a saline solution to remove traces of hemoglobin and had a neutral grayish-white appearance with no evident chromophores. The samples were supported between CaF2 plates while being measured. Transmittance (7) measurements were performed over the 300–2500-nm spectral region by placing the samples in the entrance port of an integrating sphere reflectometer, an arrangement that captured all the forward scattered beam. The specimens were placed next in an integrating sphere with center-mounted sample and the combined transmittance plus reflectance (T + R) of each was measured over the same wavelength range. The measured absorptance [A = 1− (T + R)] spectra of both normal tissue and tissue containing plaque revealed no discrete absorption features in the 250-700-nm region. In general, absorptance increased toward shorter wavelengths and the greatest difference between the absorptance of normal aortal tissue and plaque occurred in the 250–350-nm region. These results indicate that laser angioplasty may be improved by the use of UV lasers that operate in the 250–350-nm region and can interact more strongly with plaque than with the surrounding arterial wall.

This study was designed to utilize our guinea pig bioassay method for irritant response to address the question of whether or not ozone and sulfur dioxide appeared to react to form sulfuric acid in the respiratory tract. Animals were exposed to 0.2, 0.4, and 0.8 ppm of each gas alone and to the combination at concentrations of 0.4 and 0.8 ppm. In these experiments sulfur dioxide alone produced no statistically significant alterations in respiration. All concentrations of ozone produced an increase in respiratory frequency. At the two higher concentrations the increase in frequency was accompanied by a decrease in compliance. The response to the combinations was the same as the response to those levels of ozone alone. No sulfuric acid was detected in the chamber atmosphere. The biological data suggest that none was formed in the lung.