Characterization and engineering of a plastic-degrading aromatic polyesterase.

Poly(ethylene terephthalate) (PET) is one of the most abundantly produced synthetic polymers and is accumulating in the environment at a staggering rate as discarded packaging and textiles. The properties that make PET so useful also endow it with an alarming resistance to biodegradation, likely lasting centuries in the environment. Our collective reliance on PET and other plastics means that this buildup will continue unless solutions are found. Recently, a newly discovered bacterium, Ideonella sakaiensis 201-F6, was shown to exhibit the rare ability to grow on PET as a major carbon and energy source. Central to its PET biodegradation capability is a secreted PETase (PET-digesting enzyme). Here, we present a 0.92 Å resolution X-ray crystal structure of PETase, which reveals features common to both cutinases and lipases. PETase retains the ancestral α/ß-hydrolase fold but exhibits a more open active-site cleft than homologous cutinases. By narrowing the binding cleft via mutation of two active-site residues to conserved amino acids in cutinases, we surprisingly observe improved PET degradation, suggesting that PETase is not fully optimized for crystalline PET degradation, despite presumably evolving in a PET-rich environment. Additionally, we show that PETase degrades another semiaromatic polyester, polyethylene-2,5-furandicarboxylate (PEF), which is an emerging, bioderived PET replacement with improved barrier properties. In contrast, PETase does not degrade aliphatic polyesters, suggesting that it is generally an aromatic polyesterase. These findings suggest that additional protein engineering to increase PETase performance is realistic and highlight the need for further developments of structure/activity relationships for biodegradation of synthetic polyesters.

PARP3 is a sensor of nicked nucleosomes and monoribosylates histone H2B(Glu2).

PARP3 is a member of the ADP-ribosyl transferase superfamily that we show accelerates the repair of chromosomal DNA single-strand breaks in avian DT40 cells. Two-dimensional nuclear magnetic resonance experiments reveal that PARP3 employs a conserved DNA-binding interface to detect and stably bind DNA breaks and to accumulate at sites of chromosome damage. PARP3 preferentially binds to and is activated by mononucleosomes containing nicked DNA and which target PARP3 trans-ribosylation activity to a single-histone substrate. Although nicks in naked DNA stimulate PARP3 autoribosylation, nicks in mononucleosomes promote the trans-ribosylation of histone H2B specifically at Glu2. These data identify PARP3 as a molecular sensor of nicked nucleosomes and demonstrate, for the first time, the ribosylation of chromatin at a site-specific DNA single-strand break.

Linker histone subtypes are not generalized gene repressors.

Antibodies to the six chicken histone H1 subtypes and the variant histone H5 have been used in immunoprecipitations of crosslinked chromatin fragments (xChIPs) to map linker histones across the ß-globin locus and the widely expressed glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and carbonic anhydrase (CA) genes in three cell types: 15-day embryo chicken erythrocytes, 15-day embryo chicken brain and the early erythroid cell line HD24. In erythrocytes, where the ß-adult and ß-hatching genes are active, the H1.01, H1.11L and H1.11R subtypes are substantially depleted throughout the ß-globin locus and the neighboring heterochromatin, in contrast to the other four subtypes, in particular the more abundant H5. Active genes therefore carry high levels of some but not all linker histone subtypes. The situation is similar in HD24 cells, except that substantial depletions are found at the promoters of the adult ß(A) and embryonic ß(ρ) and ß(ε) genes, despite these genes not yet being active in HD24 cells. The distributions in the brain tissue are characterised by the absence of H1.02, H1.03 and H5 from the hypersensitive site HS3 and from the ß-adult 3' enhancer for the H1.11L and H1.11R subtypes. The data show that although linker histone subtypes play distinct cell-type specific roles in gene regulation, their widespread distribution indicates they are not intrinsically inhibitory to basic chromatin transactions.

USP22, an hSAGA subunit and potential cancer stem cell marker, reverses the polycomb-catalyzed ubiquitylation of histone H2A.

Initial studies of the mammalian hSAGA transcriptional coactivator complex identified the acetyltransferase hGCN5/PCAF as the only known enzymatic subunit. Recently we demonstrated that the ubiquitin hydrolase USP22 comprises a second enzymatic subunit of hSAGA, and that is required for activator-driven transcription. USP22 is expressed with polycomb ubiquitin ligases in an 11 gene signature that defines therapy-resistant tumors. At the biochemical level, these Polycomb proteins function as global transcriptional repressors by catalyzing the ubiquitylation of histone H2A. In yeast, the USP22 homolog functions as a transcriptional coactivator by removing ubiquitin from a distinct core histones, H2B. Given that USP22 is expressed in cancer as part of an 11 gene signature that includes transcriptional repressors which ubiquitylate H2A, it seemed possible that USP22 might activate transcription in part via the deubiquitylation of this same substrate. As reported here, biochemical analysis of the substrate specificity of USP22 reveals that it deubiquitylates histone H2A in addition to H2B. This finding supports a model in which the H2A ubiquitin hydrolase USP22 is coordinately expressed with Polycomb H2A ubiquitin ligases in order that the transcription of certain critical transforming genes be maintained in the face of the global repression mediated by Polycomb.

The putative cancer stem cell marker USP22 is a subunit of the human SAGA complex required for activated transcription and cell-cycle progression.

Polycomb genes encode critical regulators of both normal stem cells and cancer stem cells. A gene signature that includes Polycomb genes and additional genes coregulated with Polycomb genes was recently identified. The expression of this signature has been reported to identify tumors with the cancer stem cell phenotypes of aggressive growth, metastasis, and therapy resistance. Most members of this 11 gene signature encode proteins with well-defined roles in human cancer. However, the function of the signature member USP22 remains unknown. We report that USP22 is a previously uncharacterized subunit of the human SAGA transcriptional cofactor complex. Within SAGA, USP22 deubiquitylates histone H2B. Furthermore, USP22 is recruited to specific genes by activators such as the Myc oncoprotein, where it is required for transcription. In support of a functional role within the Polycomb/cancer stem cell signature, USP22 is required for appropriate progression through the cell cycle.

Developmental activation of the lysozyme gene in chicken macrophage cells is linked to core histone acetylation at its enhancer elements.

Native chromatin IP assays were used to define changes in core histone acetylation at the lysozyme locus during developmental maturation of chicken macrophages and stimulation to high-level expression by lipo-polysaccharide. In pluripotent precursors the lysozyme gene (Lys) is inactive and there is no acetylation of core histones at the gene, its promoter or at the upstream cis-control elements. In myeloblasts, where there is a very low level of Lys expression, H4 acetylation appears at the cis-control elements but not at the Lys gene or its promoter: neither H3 nor H2B become significantly acetylated in myeloblasts. In mature macrophages, Lys expression increases 5-fold: H4, H2B and H2A.Z are all acetylated at the cis-control elements but H3 remains unacetylated except at the -2.4 S silencer. Stimulation with LPS increases Lys expression a further 10-fold: this is accompanied by a rise in H3 acetylation throughout the cis-control elements; H4 and H2B acetylation remain substantial but acetylation at the Lys gene and its promoter remains low. Acetylation is thus concentrated at the cis-control elements, not at the Lys gene or its immediate promoter. H4 acetylation precedes H3 acetylation during development and H3 acetylation is most directly linked to high-level Lys expression.

The replacement histone H2A.Z in a hyperacetylated form is a feature of active genes in the chicken.

The replacement histone H2A.Z is variously reported as being linked to gene expression and preventing the spread of heterochromatin in yeast, or concentrated at heterochromatin in mammals. To resolve this apparent dichotomy, affinity-purified antibodies against the N-terminal region of H2A.Z, in both a triacetylated and non-acetylated state, are used in native chromatin immmuno-precipitation experiments with mononucleosomes from three chicken cell types. The hyperacetylated species concentrates at the 5' end of active genes, both tissue specific and housekeeping but is absent from inactive genes, while the unacetylated form is absent from both active and inactive genes. A concentration of H2A.Z is also found at insulators under circumstances implying a link to barrier activity but not to enhancer blocking. Although acetylated H2A.Z is widespread throughout the interphase genome, at mitosis its acetylation is erased, the unmodified form remaining. Thus, although H2A.Z may operate as an epigenetic marker for active genes, its N-terminal acetylation does not.

Biochemical observation of the rapid mobility of nuclear HMGB1.

Formaldehyde-crosslinked and sonicated chromatin fragments were obtained from 15-day chicken embryo erythrocytes and purified on caesium chloride gradients. Polyclonal antibodies raised against chicken HMGB1 were used to immuno-precipitate fragments carrying HMGB1 in two protocols: (1) affinity purified antibodies covalently coupled to agarose beads and (2) diluted antiserum. The DNA of the antibody-bound chromatin was quantified and its sequence content assessed by quantitative real-time PCR to give values of the absolute enrichments generated. Amplicons were monitored within the active beta-globin locus, in the adjacent heterochromatin, in the lysozyme locus (containing an active housekeeping gene and the inactive lysozyme gene) and at the promoter of the inactive ovalbumin gene. For all amplicons the Bound/Input ratio was close to unity, implying no preferential location of HMGB1 on the chromatin. This initially unexpected result can now be understood in the light of the exceptional mobility of HMGB1 revealed by FLIP experiments showing that only 1-2 s are needed for HMGB1 to cross the nucleus: crosslinking times of 1 min were used in the present experiments.

Zygotic nucleosome assembly protein-like 1 has a specific, non-cell autonomous role in hematopoiesis.

Nucleosome assembly proteins (NAPs) bind core histones, facilitate chromatin remodeling, and can act as transcriptional coactivators. We previously described the isolation of a Xenopus NAP1-like (xNAP1L) cDNA, which encodes a member of this protein family. Its zygotic expression is restricted to neural cells, the outer cells of the ventral blood island (VBIs), and the ectoderm overlying the blood precursors. Here, we report that depletion of zygotic xNAP1L in embryos produces no obvious morphologic phenotype, but ablates alpha-globin mRNA expression in the VBIs. Transcript levels of the hematopoietic precursor genes SCL and Xaml (Runx-1) are also reduced in the VBIs. SCL expression can be rescued by injection of xNAP1L mRNA into the ectoderm, showing that the effect of xNAP1L can be non-cell autonomous. Fli1 and Hex, genes expressed in hemangioblasts but subsequently endothelial markers, were unaffected, suggesting that xNAP1L is required for the hematopoietic lineage specifically. Our data are consistent with a requirement for xNAP1L upstream of SCL, and injection of SCL mRNA into xNAP1L-depleted embryos rescues alpha-globin expression. Thus, xNAP1L, which belongs to a family of proteins previously believed to have general roles, has a specific function in hematopoiesis.

Spatial distribution of di- and tri-methyl lysine 36 of histone H3 at active genes.

Methylation of lysine 4 of histone H3 (K4/H3) is linked to transcriptional activity, whereas methylation of K9/H3 is tightly associated with gene inactivity. These are well characterized sites of methylation within histones, but there are numerous other, less characterized, sites of modification. In Saccharomyces cerevisiae, methylation of K36/H3 has been linked to active genes, but little is known about this methylation in higher eukaryotes. Here we analyzed for the first time the levels and spatial distribution of di- and tri-methyl (di- and tri-Me) K36/H3 in metazoan genes. We analyzed chicken genes that are developmentally regulated, constitutively active, or inactive. We found that active genes contain high levels of these modifications compared with inactive genes. Furthermore, in actively transcribed regions the levels of di- and tri-Me K36/H3 peak toward the 3' end of the gene. This is in striking contrast to the distributions of di- and tri-Me K4/H3, which peak early in actively transcribed regions. Thus, di/tri-Me K4/H3 and di/tri-Me K36/H3 are both useful markers of active genes, but their genic distribution indicates differing roles. Our data suggest that the unique spatial distribution of di- and tri-Me K36/H3 plays a role in transcriptional termination and/or early RNA processing.

Native chromatin immunoprecipitation.

Chromatin immunoprecipitation (ChIP) is a technique widely used for determining the genomic location of modified histones and other chromatin-associated factors. Here we describe the methodology we have used in our laboratory for the immunoprecipitation of chromatin isolated from cells in the absence of crosslinking. Chromatin released from nuclei by micrococcal nuclease digestion is centrifuged through sucrose gradients to allow selection of mono- or dinucleosomes. This allows a protein or modification at a particular gene or locus to be mapped at higher resolution than in a crosslinked ChIP experiment. Two methods for the immunoprecipitation of chromatin are described: a large-scale fractionation by which it is possible to visualize the proteins of the immunoprecipitate by polyacrylamide gel electrophoresis, PAGE and a small-scale method that is more appropriate when the quantity of chromatin is limited. The sequence content of DNA extracted from the immunoprecipitated chromatin is analyzed by hybridization of Southern or slot blots, or by quantitative polymerase chain reaction. Enrichment of particular sequences in the immunoprecipitated fraction reveals the presence and extent of the modification at this location.

Histone H3 lysine 4 methylation patterns in higher eukaryotic genes.

Lysine residues within histones can be mono-, di - or tri-methylated. In Saccharomyces cerevisiae tri-methylation of Lys 4 of histone H3 (K4/H3) correlates with transcriptional activity, but little is known about this methylation state in higher eukaryotes. Here, we examine the K4/H3 methylation pattern at the promoter and transcribed region of metazoan genes. We analysed chicken genes that are developmentally regulated, constitutively active or inactive. We found that the pattern of K4/H3 methylation shows similarities to S. cerevisiae. Tri-methyl K4/H3 peaks in the 5' transcribed region and active genes can be discriminated by high levels of tri-methyl K4/H3 compared with inactive genes. However, our results also identify clear differences compared to yeast, as significant levels of K4/H3 methylation are present on inactive genes within the beta-globin locus, implicating this modification in maintaining a 'poised' chromatin state. In addition, K4/H3 di-methylation is not genome-wide and di-methylation is not uniformly distributed throughout the transcribed region. These results indicate that in metazoa, di- and tri-methylation of K4/H3 is linked to active transcription and that significant differences exist in the genome-wide methylation pattern as compared with S. cerevisiae.

A short-range gradient of histone H3 acetylation and Tup1p redistribution at the promoter of the Saccharomyces cerevisiae SUC2 gene.

Chromatin immunoprecipitation assays are used to map H3 and H4 acetylation over the promoter nucleosomes and the coding region of the Saccharomyces cerevisiae SUC2 gene, under repressed and derepressed conditions, using wild type and mutant strains. In wild type cells, a high level of H3 acetylation at the distal end of the promoter drops sharply toward the proximal nucleosome that covers the TATA box, a gradient that become even steeper on derepression. In contrast, substantial H4 acetylation shows no such gradient and extends into the coding region. Overall levels of both H3 and H4 acetylation rise on derepression. Mutation of GCN5 or SNF2 lead to substantially reduced SUC2 expression; in gnc5 there is no reduction in basal H3 acetylation, but large reductions occur on derepression. SNF2 mutation has little effect on H3 acetylation, so SAGA and SWI/SNF recruitment seem to be independent events. H4 acetylation is little affected by either GCN5 or SNF2 mutation. In a double snf2/gcn5 mutant (very low SUC2 expression), H3 acetylation is at the minimal level, but H4 acetylation remains largely unaffected. Transcription is thus linked to H3 but not H4 acetylation. Chromatin immunoprecipitation assays show that Tup1p is evenly distributed over the four promoter nucleosomes in repressed wild type cells but redistributes upstream on derepression, a movement probably linked to its conversion from a repressor to an activator.

Xenopus nucleosome assembly protein becomes tissue-restricted during development and can alter the expression of specific genes.

Nucleosome assembly proteins have been identified in all eukaryotic species investigated to date and their suggested roles include histone shuttle, histone acceptor during transcriptional chromatin remodelling and cell cycle regulator. To examine the role of these proteins during early development we have isolated the cDNA encoding Xenopus NAP1L, raised an antibody against recombinant xNAP1L and examined the expression pattern of this mRNA and protein. Expression in adults is predominantly in ovaries. This maternal protein remains a major component of xNAP1L within the embryo until swimming tadpole stages. xNAP1L mRNA is initially throughout the embryo but by gastrula stages it is predominantly in the presumptive ectoderm. Later, mRNA is detected in the neural crest, neural tube, eyes, tailbud and ventral blood islands. In order to test whether xNAP1L has a potential role in gene regulation we overexpressed this protein in animal pole explants and tested the effect on expression of a series of potential target genes. The mRNA encoding the transcription factor GATA-2 was markedly up-regulated by this overexpression. These data support a role for xNAP1L in tissue-restricted gene regulation.

Acetylation of histone H2B mirrors that of H4 and H3 at the chicken beta-globin locus but not at housekeeping genes.

Acetylation of histones H4 and H3 targeted to promoters/enhancers is linked to the activation of transcription, whereas widespread, long range acetylation of the same histones has been linked to the requirement for open chromatin at transcriptionally active loci and regions of V(D)J recombination. Using affinity-purified polyclonal antibodies to tetra/tri-acetylated histone H2B in chromatin immunoprecipitation (ChIP) assays with mononucleosomes from 15-day chicken embryo erythrocytes, a high resolution distribution of H2B acetylation has been determined and compared with that of H4 and H3 at the same genes/loci. At the beta-globin locus, the H2B acetylation is high throughout and in general mirrors that of H3 and H4, consistent with the observation of co-precipitation of hyperacetylated H4 together with the hyperacetylated H2B. In contrast, at the weakly expressed genes glyceraldehyde 3-phosphate dehydrogenase (GAPDH) and Gas41 (housekeeping) and carbonic anhydrase (tissue specific), very little or no hyperacetylated H2B was found despite the presence of acetylated H4 and H3 at their promoters and proximal transcribed sequences. At the inactive lysozyme and ovalbumin genes essentially no acetylation of H2B, H3, or H4 was observed. Acetylation of H2B appears to be principally a feature of only the most actively transcribed genes/loci.

Thyroid hormone-regulated enhancer blocking: cooperation of CTCF and thyroid hormone receptor.

The highly conserved, ubiquitously expressed, zinc finger protein CTCF is involved in enhancer blocking, a mechanism crucial for shielding genes from illegitimate enhancer effects. Interestingly, CTCF-binding sites are often flanked by thyroid hormone response elements (TREs), as at the chicken lysozyme upstream silencer. Here we identify a similar composite site positioned upstream of the human c-myc gene. For both elements, we demonstrate that thyroid hormone abrogates enhancer blocking. Relief of enhancer blocking occurs even though CTCF remains bound to the lysozyme chromatin. Furthermore, chromatin immunoprecipitation analysis of the lysozyme upstream region revealed that histone H4 is acetylated at the CTCF-binding site. Loss of enhancer blocking by the addition of T3 led to increased histone acetylation, not only at the CTCF site, but also at the enhancer and the promoter. Thus, when TREs are adjacent to CTCF-binding sites, thyroid hormone can regulate enhancer blocking, thereby providing a new property for what was previously thought to be constitutive enhancer shielding by CTCF.