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Shotcrete : materials, performance and use
\"This comprehensive textbook covers current state-of-the-art shotcrete technology. It provides an overview of the many and various uses of shotcrete. It includes chapters with case histories on shotcrete materials and mixture proportioning, performance, shotcrete research, equipment and shotcrete application\"-- Provided by publisher.
Book
Reevaluating the roles of histone-modifying enzymes and their associated chromatin modifications in transcriptional regulation
Histone-modifying enzymes are implicated in the control of diverse DNA-templated processes including gene expression. Here, we outline historical and current thinking regarding the functions of histone modifications and their associated enzymes. One current viewpoint, based largely on correlative evidence, posits that histone modifications are instructive for transcriptional regulation and represent an epigenetic ‘code’. Recent studies have challenged this model and suggest that histone marks previously associated with active genes do not directly cause transcriptional activation. Additionally, many histone-modifying proteins possess non-catalytic functions that overshadow their enzymatic activities. Given that much remains unknown regarding the functions of these proteins, the field should be cautious in interpreting loss-of-function phenotypes and must consider both cellular and developmental context. In this Perspective, we focus on recent progress relating to the catalytic and non-catalytic functions of the Trithorax–COMPASS complexes, Polycomb repressive complexes and Clr4/Suv39 histone-modifying machineries.
Recent progress relating to the catalytic and non-catalytic functions of histone modifying complexes warrants a fresh look at the role of histone modifications and the “histone code” model.
Journal Article
Therapeutic targeting of polycomb and BET bromodomain proteins in diffuse intrinsic pontine gliomas
Nucleosomes containing mutant K27M histones in diffuse intrinsic pediatric gliomas (DIPG) exclude PRC2 binding and recruit BET bromodomain proteins; however, residual PRC2-dependent repression of specific loci, is required for DIPG oncogenesis. These results provide a rationale for targeting these epigenetic regulators in patients.
Diffuse intrinsic pontine glioma (DIPG) is a highly aggressive pediatric brainstem tumor characterized by rapid and uniform patient demise
1
. A heterozygous point mutation of histone H3 occurs in more than 80% of these tumors and results in a lysine-to-methionine substitution (H3K27M)
2
,
3
. Expression of this histone mutant is accompanied by a reduction in the levels of polycomb repressive complex 2 (PRC2)-mediated H3K27 trimethylation (H3K27me3), and this is hypothesized to be a driving event of DIPG oncogenesis
4
,
5
. Despite a major loss of H3K27me3, PRC2 activity is still detected in DIPG cells positive for H3K27M
6
,
7
. To investigate the functional roles of H3K27M and PRC2 in DIPG pathogenesis, we profiled the epigenome of H3K27M-mutant DIPG cells and found that H3K27M associates with increased H3K27 acetylation (H3K27ac). In accordance with previous biochemical data
5
, the majority of the heterotypic H3K27M-K27ac nucleosomes colocalize with bromodomain proteins at the loci of actively transcribed genes, whereas PRC2 is excluded from these regions; this suggests that H3K27M does not sequester PRC2 on chromatin. Residual PRC2 activity is required to maintain DIPG proliferative potential, by repressing neuronal differentiation and function. Finally, to examine the therapeutic potential of blocking the recruitment of bromodomain proteins by heterotypic H3K27M-K27ac nucleosomes in DIPG cells, we performed treatments
in vivo
with BET bromodomain inhibitors and demonstrate that they efficiently inhibit tumor progression, thus identifying this class of compounds as potential therapeutics in DIPG.
Journal Article
Histone H3K4 monomethylation catalyzed by Trr and mammalian COMPASS-like proteins at enhancers is dispensable for development and viability
Ali Shilatifard and colleagues generate
Drosophila
lines expressing catalytically deficient Trr, which normally deposits H3K4me1 at enhancers. Trr mutants undergo normal development and show minimal changes in gene expression.
Histone H3 lysine 4 monomethylation (H3K4me1) is an evolutionarily conserved feature of enhancer chromatin catalyzed by the COMPASS-like methyltransferase family, which includes Trr in
Drosophila melanogaster
and MLL3 (encoded by
KMT2C
) and MLL4 (encoded by
KMT2D
) in mammals
1
,
2
,
3
. Here we demonstrate that
Drosophila
embryos expressing catalytically deficient Trr eclose and develop to productive adulthood. Parallel experiments with a
trr
allele that augments enzyme product specificity show that conversion of H3K4me1 at enhancers to H3K4me2 and H3K4me3 is also compatible with life and results in minimal changes in gene expression. Similarly, loss of the catalytic SET domains of MLL3 and MLL4 in mouse embryonic stem cells (mESCs) does not disrupt self-renewal.
Drosophila
embryos with
trr
alleles encoding catalytic mutants manifest subtle developmental abnormalities when subjected to temperature stress or altered cohesin levels. Collectively, our findings suggest that animal development can occur in the context of Trr or mammalian COMPASS-like proteins deficient in H3K4 monomethylation activity and point to a possible role for H3K4me1 on
cis
-regulatory elements in specific settings to fine-tune transcriptional regulation in response to environmental stress.
Journal Article
Uncoupling histone H3K4 trimethylation from developmental gene expression via an equilibrium of COMPASS, Polycomb and DNA methylation
The COMPASS protein family catalyzes histone H3 Lys 4 (H3K4) methylation and its members are essential for regulating gene expression. MLL2/COMPASS methylates H3K4 on many developmental genes and bivalent clusters. To understand MLL2-dependent transcriptional regulation, we performed a CRISPR-based screen with an MLL2-dependent gene as a reporter in mouse embryonic stem cells. We found that MLL2 functions in gene expression by protecting developmental genes from repression via repelling PRC2 and DNA methylation machineries. Accordingly, repression in the absence of MLL2 is relieved by inhibition of PRC2 and DNA methyltransferases. Furthermore, DNA demethylation on such loci leads to reactivation of MLL2-dependent genes not only by removing DNA methylation but also by opening up previously CpG methylated regions for PRC2 recruitment, diluting PRC2 at Polycomb-repressed genes. These findings reveal how the context and function of these three epigenetic modifiers of chromatin can orchestrate transcriptional decisions and demonstrate that prevention of active repression by the context of the enzyme and not H3K4 trimethylation underlies transcriptional regulation on MLL2/COMPASS targets.
A CRISPR screen for MLL2-dependent transcription identifies a compensatory repressive mechanism involving PRC2 and DNA methylation. PRC2 inactivation or DNA demethylation activates gene expression in the absence of H3K4me3.
Journal Article
Resetting the epigenetic balance of Polycomb and COMPASS function at enhancers for cancer therapy
The lysine methyltransferase KMT2C (also known as MLL3), a subunit of the COMPASS complex, implements monomethylation of Lys4 on histone H3 (H3K4) at gene enhancers.
KMT2C
(hereafter referred to as
MLL3
) frequently incurs point mutations across a range of human tumor types, but precisely how these lesions alter MLL3 function and contribute to oncogenesis is unclear. Here we report a cancer mutational hotspot in
MLL3
within the region encoding its plant homeodomain (PHD) repeats and demonstrate that this domain mediates association of MLL3 with the histone H2A deubiquitinase and tumor suppressor BAP1. Cancer-associated mutations in the sequence encoding the MLL3 PHD repeats disrupt the interaction between MLL3 and BAP1 and correlate with poor patient survival. Cancer cells that had PHD-associated
MLL3
mutations or lacked BAP1 showed reduced recruitment of MLL3 and the H3K27 demethylase KDM6A (also known as UTX) to gene enhancers. As a result, inhibition of the H3K27 methyltransferase activity of the Polycomb repressive complex 2 (PRC2) in tumor cells harboring
BAP1
or
MLL3
mutations restored normal gene expression patterns and impaired cell proliferation in vivo. This study provides mechanistic insight into the oncogenic effects of PHD-associated mutations in
MLL3
and suggests that restoration of a balanced state of Polycomb–COMPASS activity may have therapeutic efficacy in tumors that bear mutations in the genes encoding these epigenetic factors.
Interactions between the MLL3 histone methyltransferase and the BAP1–UTX complex set the level of histone H3K27 methylation and suggest a new therapy for MLL3-mutant cancer.
Journal Article
Blimp1/Prdm1 Functions in Opposition to Irf1 to Maintain Neonatal Tolerance during Postnatal Intestinal Maturation
The neonatal intestine is a very complex and dynamic organ that must rapidly adapt and remodel in response to a barrage of environmental stimuli during the first few postnatal weeks. Recent studies demonstrate that the zinc finger transcriptional repressor Blimp1/Prdm1 plays an essential role governing postnatal reprogramming of intestinal enterocytes during this period. Functional loss results in global changes in gene expression patterns, particularly in genes associated with metabolic function. Here we engineered a knock-in allele expressing an eGFP-tagged fusion protein under control of the endogenous regulatory elements and performed genome wide ChIP-seq analysis to identify direct Blimp1 targets and further elucidate the function of Blimp1 in intestinal development. Comparison with published human and mouse datasets revealed a highly conserved core set of genes including interferon-inducible promoters. Here we show that the interferon-inducible transcriptional activator Irf1 is constitutively expressed throughout fetal and postnatal intestinal epithelium development. ChIP-seq demonstrates closely overlapping Blimp1 and Irf1 peaks at key components of the MHC class I pathway in fetal enterocytes. The onset of MHC class I expression coincides with down-regulated Blimp1 expression during the suckling to weaning transition. Collectively, these experiments strongly suggest that in addition to regulating the enterocyte metabolic switch, Blimp1 functions as a gatekeeper in opposition to Irf1 to prevent premature activation of the MHC class I pathway in villus epithelium to maintain tolerance in the neonatal intestine.
Journal Article
The Mll2 branch of the COMPASS family regulates bivalent promoters in mouse embryonic stem cells
Embryonic stem cells (ESCs) possess a unique chromatin landscape in which 'bivalent' domains of trimethylated histone H3 Lys4 (H3K4me3) and Lys27 (H3K27me3) mark key lineage-specific genes. A new study now reports the identification of Mll2 (KMT2b) as the enzyme responsible for implementing H3K4me3 on bivalently marked promoters in ESCs.
Promoters of many developmentally regulated genes, in the embryonic stem cell state, have a bivalent mark of H3K27me3 and H3K4me3, proposed to confer precise temporal activation upon differentiation. Although Polycomb repressive complex 2 is known to implement H3K27 trimethylation, the COMPASS family member responsible for H3K4me3 at bivalently marked promoters was previously unknown. Here, we identify Mll2 (KMT2b) as the enzyme catalyzing H3K4 trimethylation at bivalentlymarked promoters in embryonic stem cells. Although H3K4me3 at bivalent genes is proposed to prime future activation, we detected no substantial defect in rapid transcriptional induction after retinoic acid treatment in Mll2-depleted cells. Our identification of the Mll2 complex as the COMPASS family member responsible for H3K4me3 marking bivalent promoters provides an opportunity to reevaluate and experimentally test models for the function of bivalency in the embryonic stem cell state and in differentiation.
Journal Article
Long-lived unipotent Blimp1-positive luminal stem cells drive mammary gland organogenesis throughout adult life
The hierarchical relationships between various stem and progenitor cell subpopulations driving mammary gland morphogenesis and homoeostasis are poorly understood. Conditional inactivation experiments previously demonstrated that expression of the zinc finger transcriptional repressor Blimp1/PRDM1 is essential for the establishment of epithelial cell polarity and functional maturation of alveolar cells. Here we exploit a
Prdm1.CreERT2-LacZ
reporter allele for lineage tracing experiments. Blimp1 expression marks a rare subpopulation of unipotent luminal stem cells that initially appear in the embryonic mammary gland at around E17.5 coincident with the segregation of the luminal and basal compartments. Fate mapping at multiple time points in combination with whole-mount confocal imaging revealed these long-lived unipotent luminal stem cells survive consecutive involutions and retain their identity throughout adult life. Blimp1
+
luminal stem cells give rise to Blimp1
−
progeny that are invariably Elf5
+
ERα
−
PR
−
. Thus, Blimp1 expression defines a mammary stem cell subpopulation with unique functional characteristics.
The role of stem/progenitor cell populations in mammary gland morphogenesis is not well understood. Here, the authors show that a transcriptional repressor, Blimp1, is expressed in a rare luminal stem cell population, which contribute to duct formation, and survive multiple rounds of pregnancy and involution.
Journal Article
Lactate-dependent transcriptional regulation controls mammalian eye morphogenesis
Mammalian retinal metabolism favors aerobic glycolysis. However, the role of glycolytic metabolism in retinal morphogenesis remains unknown. We report that aerobic glycolysis is necessary for the early stages of retinal development. Taking advantage of an unbiased approach that combines the use of eye organoids and single-cell RNA sequencing, we identify specific glucose transporters and glycolytic genes in retinal progenitors. Next, we determine that the optic vesicle territory of mouse embryos displays elevated levels of glycolytic activity. At the functional level, we show that removal of Glucose transporter 1 and Lactate dehydrogenase A gene activity from developing retinal progenitors arrests eye morphogenesis. Surprisingly, we uncover that lactate-mediated upregulation of key eye-field transcription factors is controlled by the epigenetic modification of histone H3 acetylation through histone deacetylase activity. Our results identify an unexpected bioenergetic independent role of lactate as a signaling molecule necessary for mammalian eye morphogenesis.
Using a combination of eye organoids and mouse models the authors identify a bioenergetic independent role of lactate as a cell signaling molecule required during early stages of eye formation in mice.
Journal Article