Brain tumors are the most common solid tumors in children. Pediatric high-grade glioma (HGG) accounts for ∼8–12 % of these brain tumors and is a devastating disease as 70–90 % of patients die within 2 years of diagnosis. The failure to advance therapy for these children over the last 30 years is largely due to limited knowledge of the molecular basis for these tumors and a lack of disease models. Recently, sequencing of tumor cells revealed that histone H3 is frequently mutated in pediatric HGG, with up to 78 % of diffuse intrinsic pontine gliomas (DIPGs) carrying K27M and 36 % of non-brainstem gliomas carrying either K27M or G34R/V mutations. Although mutations in many chromatin modifiers have been identified in cancer, this was the first demonstration that histone mutations may be drivers of disease. Subsequent studies have identified high-frequency mutation of histone H3 to K36M in chondroblastomas and to G34W/L in giant cell tumors of bone, which are diseases of adolescents and young adults. Interestingly, the G34 mutations, the K36M mutations, and the majority of K27M mutations occur in genes encoding the replacement histone H3.3. Here, we review the peculiar characteristics of histone H3.3 and use this information as a backdrop to highlight current thinking about how the identified mutations may contribute to disease development.
Chromatin domain boundary elements demarcate independently regulated domains of eukaryotic genomes. While a few such boundary sequences have been studied in detail, only a small number of proteins that interact with them have been identified. One such protein is the boundary element-associated factor (BEAF), which binds to the scs boundary element of Drosophila melanogaster. It is not clear, however, how boundary elements function. In this report we show that BEAF is associated with the nuclear matrix and map the domain required for matrix association to the middle region of the protein. This region contains a predicted coiled-coil domain with several potential sites for posttranslational modification. We demonstrate that the DNA sequences that bind to BEAF in vivo are also associated with the nuclear matrix and colocalize with BEAF. These results suggest that boundary elements may function by tethering chromatin to nuclear architectural components and thereby provide a structural basis for compartmentalization of the genome into functionally independent domains.
The nucleus is a highly structured organelle and contains many functional compartments. Although the structural basis for this complex spatial organization of compartments is unknown, a major component of this organization is likely to be the non-chromatin scaffolding called nuclear matrix (NuMat). Experimental evidence over the past decades indicates that most of the nuclear functions are at least transiently associated with the NuMat, although the components of NuMat itself are poorly known. Here, we report NuMat proteome analysis from Drosophila melanogaster embryos and discuss its links with nuclear architecture and functions. In the NuMat proteome, we found structural proteins, chaperones, DNA/RNAbinding proteins, chromatin remodeling and transcription factors. This complexity of NuMat proteome is an indicator of its structural and functional significance. The eukaryotic cell nucleus contains a number of structural features like heterochromatin, nucleolus, nuclear lamina, and nuclear territories and functional features like transcription foci, replication foci, Cajal bodies, stress-induced foci, etc.(1-7). Packaging of the genome on the other hand also results in non-random distribution of a variety of structural features like chromatin domains that help to regulate expression of genes (8). How these complex structural and functional domains are established and maintained is not known. It is likely that the non-chromatin scaffolding called nuclear matrix (9) plays an important role in this process. Nuclear matrix (NuMat) 1 mainly consists of the nuclear lamina, the nucleolar remnants, and an internal nuclear meshwork of fibers of unknown constitution that can be seen by electron microscopy (10). These fibers have ultrastructural features reminiscent of intermediate filaments of the cytosol (11). The protein component of nucleoplasmic filaments, however, is largely unknown. Earlier studies on a few proteins isolated from nuclear matrix from different organisms have shown them to be DNA-and RNA-binding and structural components of the nuclear pore complex (12), enzymes (13), and nuclear membrane proteins (14). This diversity among the small number of identified NuMat constituents reflects the link of nuclear architecture with the variety of nuclear functions. The importance of NuMat constituents in nuclear processes has been apparent for a long time, and several studies have been carried out to identify the proteins of this complex structure in different organisms (15-18). Major advancements in the field of proteomics now provide further scope for extensive analysis of NuMat in this context.Here, we present a comprehensive analysis of the NuMat proteome of Drosophila melanogaster. NuMat preparations from D. melanogaster embryos were separated by one-dimensional gel electrophoresis, and the proteins were identified by LC-MS/MS. We also report a remarkable variation in the NuMat proteome depending on developmental stages, indicating a link between embryonic development and nuclear architecture. Finally, immunos...
The mitotic deacetylase complex (MiDAC) is a recently identified histone deacetylase (HDAC) complex. While other HDAC complexes have been implicated in neurogenesis, the physiological role of MiDAC remains unknown. Here, we show that MiDAC constitutes an important regulator of neural differentiation. We demonstrate that MiDAC functions as a modulator of a neurodevelopmental gene expression program and binds to important regulators of neurite outgrowth. MiDAC upregulates gene expression of pro-neural genes such as those encoding the secreted ligands SLIT3 and NETRIN1 (NTN1) by a mechanism suggestive of H4K20ac removal on promoters and enhancers. Conversely, MiDAC inhibits gene expression by reducing H3K27ac on promoter-proximal and -distal elements of negative regulators of neurogenesis. Furthermore, loss of MiDAC results in neurite outgrowth defects that can be rescued by supplementation with SLIT3 and/or NTN1. These findings indicate a crucial role for MiDAC in regulating the ligands of the SLIT3 and NTN1 signaling axes to ensure the proper integrity of neurite development.
Sequencing of cancer genomes has identified recurrent somatic mutations in histones, termed oncohistones, which are frequently poorly understood. Previously we showed that fission yeast expressing only the H3.3G34R mutant identified in aggressive pediatric glioma had reduced H3K36 trimethylation and acetylation, increased genomic instability and replicative stress, and defective homology-dependent DNA damage repair (Yadav et al., 2017). Here we show that surprisingly distinct phenotypes result from G34V (also in glioma) and G34W (giant cell tumors of bone) mutations, differentially affecting H3K36 modifications, subtelomeric silencing, genomic stability, sensitivity to irradiation, alkylating agents, hydroxyurea and influencing DNA repair. In cancer, only one of thirty alleles encoding H3 is mutated. Whilst co-expression of wild-type H3 rescues most G34 mutant phenotypes, G34R causes dominant hydroxyurea sensitivity and homologous recombination defects, and dominant subtelomeric silencing. Together, these studies demonstrate the complexity associated with different substitutions at even a single residue in H3 and highlight the utility of genetically tractable systems for their analysis.
Helicases are ubiquitous enzymes that unwind or remodel single or double-stranded nucleic acids, and that participate in a vast array of metabolic pathways. The ATP-dependent DEXH-box RNA/DNA helicase MLE was first identified as a core member of the chromatin remodeling MSL complex, responsible for dosage compensation in Drosophila males. Although this complex does not assemble in females, MLE is present. Given the multiplicity of functions attributed to its mammalian ortholog RNA helicase A, we have carried out an analysis for the purpose of determining whether MLE displays the same diversity. We have identified a number of different proteins that associate with MLE, implicating its role in specific pathways. We have documented this association in selected examples that include the spliceosome complex, Helicases are involved in all cellular transactions related to nucleic acid function and metabolism in prokaryotes and eukaryotes. These enzymes participate in transcription, RNA splicing, the stability of transcripts, and translation initiation, as well as in DNA-replication, repair, and recombination. Helicases use the energy produced by the hydrolysis of nucleotide triphosphates to catalyze the unwinding of DNA, RNA, and RNA:DNA hybrid molecules. They are also involved in protein displacement from RNA, RNA clamping, strand annealing, and RNA structure conversion (1). Eukaryotic helicases can be divided into two superfamilies, SF1 and SF2, which include three and nine families, respectively. Members of the different families can be distinguished on the basis of whether they use the hydrolysis of ATP or some other nucleotide triphosphates for energy release, whether they unwind DNA or RNA, and whether they require a 3Ј overhang or a 5Ј overhang to carry out their unwinding functions (2, 3).The Drosophila helicase maleless (MLE) 1 is a subunit of the MSL (male-specific lethal) complex that is responsible for dosage compensation -the regulatory mechanism involved in equalizing the levels of X chromosome-linked gene products between the sexes. In addition to MLE, the MSL complex contains an ubiquitin ligase (MSL2), a histone acetyl transferase (MOF), two structural proteins (MSL1 and MSL3), and one of two long non-coding RNAs (roX1 and roX2) that are necessary for its assembly and targeting. MLE is an ATP-dependent DEXH-box RNA/DNA helicase that in vitro prefers hybrid RNA:DNA or double-stranded RNA substrates with a short 3Ј overhang (4). MLE exhibits some similarity to the ATPases present in complexes that remodel chromatin by altering the positioning or the architectural relationship between histone octamers and DNA. In contrast to MLE, none of these enzymatic subunits have been shown to possess double-stranded nucleic acid unwinding activity. The absence or loss of function of MLE leads to failures in assembly and targeting of the MSL complex to its numerous sites of action along the X chromosome in males, although MSL1 and MSL2 are present at a few "high affinity" or "entry" sites (5-10). Recent biochemical ev...
Sequencing of cancer genomes has identified recurrent somatic mutations in histones, termed oncohistones, which are frequently poorly understood. Previously we showed that fission yeast expressing only the H3.3G34R mutant identified in aggressive pediatric glioma had reduced H3K36 trimethylation and acetylation, increased genomic instability and replicative stress, and defective homology-dependent DNA damage repair (Yadav et al., 2017). Here we show that surprisingly distinct phenotypes result from G34V (also in glioma) and G34W (giant cell tumors of bone) mutations, differentially affecting H3K36 modifications, subtelomeric silencing, genomic stability, sensitivity to irradiation, alkylating agents, hydroxyurea and influencing DNA repair. In cancer, only one of thirty alleles encoding H3 is mutated. Whilst co-expression of wild-type H3 rescues most G34 mutant phenotypes, G34R causes dominant hydroxyurea sensitivity and homologous recombination defects, and dominant subtelomeric silencing. Together, these studies demonstrate the complexity associated with different substitutions at even a single residue in H3 and highlight the utility of genetically tractable systems for their analysis.
Many histone co-valent modifications have been identified and shown to play key regulatory roles in eukaryotic transcription, DNA damage repair and replication. In vitro experiments designed to understand the mechanistic role of individual modifications require the availability of substantial quantities of pure histones, homogeneously modified at specific residues. We have applied the amber stop codon/suppressor tRNA strategy to the production of histone H4 acetylated at lysine 16, a particularly important isoform of this histone. Our success relies on adapting the H4 DNA sequence to the codon preference of E. coli and on preventing the premature decay of the H4 mRNA. These modifications to the original procedure render it easily applicable to the generation of any co-valently modified histone H4 isoform.
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