TrxG Members Research Articles

Epigenetic factors Polycomb (Pc) and Suppressor of zeste (Su(z)2) negatively regulate longevity in Drosophila melanogaster.

The process of aging is a hallmark of the natural life span of all organisms and individuals within a population show variability in the measures of age related performance. Longevity and the rate of aging are influenced by several factors such as genetics, nutrition, stress, and environment. Many studies have focused on the genes that impact aging and there is increasing evidence that epigenetic factors regulate these genes to control life span. Polycomb (PcG) and trithorax (trxG) protein complexes maintain the expression profiles of developmentally important genes and regulate many cellular processes. Here, we report that mutations of PcG and trxG members affect the process of aging in Drosophila melanogaster, with perturbations mostly associated with retardation in aging. We find that mutations in polycomb repressive complex (PRC1) components Pc and Su(z)2 increase fly survival. Using an inducible UAS-GAL4 system, we show that this effect is tissue-specific; knockdown in fat body, but not in muscle or brain tissues, enhances life span. We hypothesize that these two proteins influence life span via pathways independent of their PRC1 functions, with distinct effects on response to oxidative stress. Our observations highlight the role of global epigenetic regulators in determining life span.

Bivalent complexes of PRC1 with orthologs of BRD4 and MOZ/MORF target developmental genes in Drosophila.

Regulatory decisions in Drosophila require Polycomb group (PcG) proteins to maintain the silent state and Trithorax group (TrxG) proteins to oppose silencing. Since PcG and TrxG are ubiquitous and lack apparent sequence specificity, a long-standing model is that targeting occurs via protein interactions; for instance, between repressors and PcG proteins. Instead, we found that Pc-repressive complex 1 (PRC1) purifies with coactivators Fs(1)h [female sterile (1) homeotic] and Enok/Br140 during embryogenesis. Fs(1)h is a TrxG member and the ortholog of BRD4, a bromodomain protein that binds to acetylated histones and is a key transcriptional coactivator in mammals. Enok and Br140, another bromodomain protein, are orthologous to subunits of a mammalian MOZ/MORF acetyltransferase complex. Here we confirm PRC1-Br140 and PRC1-Fs(1)h interactions and identify their genomic binding sites. PRC1-Br140 bind developmental genes in fly embryos, with analogous co-occupancy of PRC1 and a Br140 ortholog, BRD1, at bivalent loci in human embryonic stem (ES) cells. We propose that identification of PRC1-Br140 "bivalent complexes" in fly embryos supports and extends the bivalency model posited in mammalian cells, in which the coexistence of H3K4me3 and H3K27me3 at developmental promoters represents a poised transcriptional state. We further speculate that local competition between acetylation and deacetylation may play a critical role in the resolution of bivalent protein complexes during development.

Correlation between desiccation stress response and epigenetic modifications of genes in Drosophila melanogaster: An example of environment-epigenome interaction

Animals from different phyla including arthropods tolerate water stress to different extent. This tolerance is accompanied by biochemical changes which in turn are due to transcriptional alteration. The changes in transcription can be an indirect effect on some of the genes, ensuing from the effect of stress on the regulators of transcription including epigenetic regulators. Within this paradigm, we investigated the correlation between stress response and epigenetic modification underlying gene expression modulation during desiccation stress in Canton-S. We report altered resistance of flies in desiccation stress for heterozygote mutants of PcG and TrxG members. Pc/+ mutant shows lower survival, while ash1/+ mutants show higher survival under desiccation stress as compared to Canton-S. We detect expression alteration in stress related genes as well the genes of the Polycomb and trithorax complex in Canton-S subjected to desiccation stress. Concomitant with this, there is an altered enrichment of H3K27me3 and H3K4me3 at the upstream regions of the stress responsive genes. The enrichment of activating mark, H3K4me3, is higher in non-stress condition. H3K27me3, the repressive mark, is more pronounced under stress condition, which in turn, can be correlated with the binding of Pc. Our results show that desiccation stress induces dynamic switching in expression and enrichment of PcG and TrxG in the upstream region of genes, which correlates with histone modifications. We provide evidence that epigenetic modulation could be one of the mechanisms to adapt to the desiccation stress in Drosophila. Thus, our study proposes the interaction of epigenome and environmental factors.

The Trithorax group gene Ash1l regulates quiescence, self-renewal potential and niche occupancy in adult hematopoietic stem cells

Absent, small or homeotic discs 1 (Ash1) encodes a Trithorax group (TrxG) protein first identified in Drosophila. Mammalian Ash1l (Ash1-like) contains a conserved internal SET domain with in vitro H3K36 dimethyltransferase activity. Using a gene trap (GT) model, we discovered an essential role for Ash1l in adult hematopoietic stem cells (HSCs). Ash1lGT/GT mice have a 90% reduction in Ash1l transcripts. Despite preserved HSCs at birth, young adult Ash1l-deficient mice showed profound depletion of long-term HSCs (LT-HSCs) and markedly decreased quiescence (G0 cells). Ash1l-deficient HSCs had decreased expression of p27/p57, two critical regulators of HSC quiescence. In transplantation assays, Ash1l-deficient bone marrow was incapable of long-term reconstitution. Non-myeloablative transplantation of wild-type progenitors into non-irradiated Ash1l-deficient mice led to high engraftment of wild-type LT-HSCs, demonstrating abnormal availability of HSC niches. We assessed if wild-type progenitors engrafting in the non-irradiated Ash1lGT/GT mice retained HSC properties. Donor cells (GFP+) were injected into non-irradiated Ash1lGT/GT mice and allowed to reconstitute for 9 weeks before infusion of wild-type cells CD45.1+. Interestingly, most LT-HSCs in Ash1lGT/GT mice (≥93%) were derived from the initial GFP+ donor and had not been outcompeted by the later infusion of CD45.1+ cells. Thus, wild-type HSCs could stably engraft in the Ash1l-deficient HSC niche, consistent with normal niche function but decreased niche occupancy by Ash1l-deficient HSCs. We next assessed the mechanisms of Ash1l action and its functional interaction with the TrxG member Mixed Lineage Leukemia 1 (Mll1). Ash1l deficiency decreased expression of several posterior Hox genes in HSCs. Loss of both Ash1l and Mll1 led to overt hematopoietic failure, providing the first in vivo evidence that TrxG genes can synergize in mammals. We are currently exploring the molecular mechanisms underlying this cooperativity. Together, our results uncover a Trithorax gene network that controls quiescence, self-renewal and niche occupancy in adult HSCs. Absent, small or homeotic discs 1 (Ash1) encodes a Trithorax group (TrxG) protein first identified in Drosophila. Mammalian Ash1l (Ash1-like) contains a conserved internal SET domain with in vitro H3K36 dimethyltransferase activity. Using a gene trap (GT) model, we discovered an essential role for Ash1l in adult hematopoietic stem cells (HSCs). Ash1lGT/GT mice have a 90% reduction in Ash1l transcripts. Despite preserved HSCs at birth, young adult Ash1l-deficient mice showed profound depletion of long-term HSCs (LT-HSCs) and markedly decreased quiescence (G0 cells). Ash1l-deficient HSCs had decreased expression of p27/p57, two critical regulators of HSC quiescence. In transplantation assays, Ash1l-deficient bone marrow was incapable of long-term reconstitution. Non-myeloablative transplantation of wild-type progenitors into non-irradiated Ash1l-deficient mice led to high engraftment of wild-type LT-HSCs, demonstrating abnormal availability of HSC niches. We assessed if wild-type progenitors engrafting in the non-irradiated Ash1lGT/GT mice retained HSC properties. Donor cells (GFP+) were injected into non-irradiated Ash1lGT/GT mice and allowed to reconstitute for 9 weeks before infusion of wild-type cells CD45.1+. Interestingly, most LT-HSCs in Ash1lGT/GT mice (≥93%) were derived from the initial GFP+ donor and had not been outcompeted by the later infusion of CD45.1+ cells. Thus, wild-type HSCs could stably engraft in the Ash1l-deficient HSC niche, consistent with normal niche function but decreased niche occupancy by Ash1l-deficient HSCs. We next assessed the mechanisms of Ash1l action and its functional interaction with the TrxG member Mixed Lineage Leukemia 1 (Mll1). Ash1l deficiency decreased expression of several posterior Hox genes in HSCs. Loss of both Ash1l and Mll1 led to overt hematopoietic failure, providing the first in vivo evidence that TrxG genes can synergize in mammals. We are currently exploring the molecular mechanisms underlying this cooperativity. Together, our results uncover a Trithorax gene network that controls quiescence, self-renewal and niche occupancy in adult HSCs.

Mll2 controls cardiac lineage differentiation of mouse embryonic stem cells by promoting H3K4me3 deposition at cardiac-specific genes.

Trithorax group (TrxG) proteins play critical roles in transcriptional activation by promoting methylation of histone H3 Lysine 4 (H3K4), but the precise functions of the individual TrxG members during embryonic differentiation are not fully understood. Here we show that Mll2, a TrxG member, is required for proliferation but is dispensable for maintaining the pluripotency of mouse embryonic stem cells (ESCs). In addition, differentiation of ESCs toward mesodermal and endodermal lineages is severely altered and, in particular, the cardiac lineage differentiation of ESCs is completely abolished in the absence of Mll2. Moreover, the expression of core cardiac transcription factors and the levels of H3K4 tri-methylation of these cardiac-specific promoters are significantly decreased by the loss of Mll2. Taken together, our results reveal a critical role for Mll2 in proliferation and cardiac lineage differentiation of mouse ESCs, and provide novel molecular insight into the mechanisms of cardiac development and disease.

SDG2-Mediated H3K4 Methylation Is Required for Proper Arabidopsis Root Growth and Development

Trithorax group (TrxG) proteins are evolutionarily conserved in eukaryotes and play critical roles in transcriptional activation via deposition of histone H3 lysine 4 trimethylation (H3K4me3) in chromatin. Several Arabidopsis TrxG members have been characterized, and among them SET DOMAIN GROUP 2 (SDG2) has been shown to be necessary for global genome-wide H3K4me3 deposition. Although pleiotropic phenotypes have been uncovered in the sdg2 mutants, SDG2 function in the regulation of stem cell activity has remained largely unclear. Here, we investigate the sdg2 mutant root phenotype and demonstrate that SDG2 is required for primary root stem cell niche (SCN) maintenance as well as for lateral root SCN establishment. Loss of SDG2 results in drastically reduced H3K4me3 levels in root SCN and differentiated cells and causes the loss of auxin gradient maximum in the root quiescent centre. Elevated DNA damage is detected in the sdg2 mutant, suggesting that impaired genome integrity may also have challenged the stem cell activity. Genetic interaction analysis reveals that SDG2 and CHROMATIN ASSEMBLY FACTOR-1 act synergistically in root SCN and genome integrity maintenance but not in telomere length maintenance. We conclude that SDG2-mediated H3K4me3 plays a distinctive role in the regulation of chromatin structure and genome integrity, which are key features in pluripotency of stem cells and crucial for root growth and development.

Wdr5 Mediates Self-Renewal and Reprogramming via the Embryonic Stem Cell Core Transcriptional Network

The embryonic stem (ES) cell transcriptional and chromatin-modifying networks are critical for self-renewal maintenance. However, it remains unclear whether these networks functionally interact and, if so, what factors mediate such interactions. Here, we show that WD repeat domain 5 (Wdr5), a core member of the mammalian Trithorax (trxG) complex, positively correlates with the undifferentiated state and is a regulator of ES cell self-renewal. We demonstrate that Wdr5, an "effector" of H3K4 methylation, interacts with the pluripotency transcription factor Oct4. Genome-wide protein localization and transcriptome analyses demonstrate overlapping gene regulatory functions between Oct4 and Wdr5. The Oct4-Sox2-Nanog circuitry and trxG cooperate in activating transcription of key self-renewal regulators, and furthermore, Wdr5 expression is required for the efficient formation of induced pluripotent stem (iPS) cells. We propose an integrated model of transcriptional and epigenetic control, mediated by select trxG members, for the maintenance of ES cell self-renewal and somatic cell reprogramming.

Vertebrate Homologue of Drosophila GAGA Factor

Polycomb group (PcG) and trithorax group (trxG) proteins are chromatin-mediated regulators of a number of developmentally important genes including the homeotic genes. In Drosophila melanogaster, one of the trxG members, Trithorax like ( Trl), encodes the essential multifunctional DNA binding protein called GAGA factor (GAF). While most of the PcG and trxG genes are conserved from flies to humans, a Trl-GAF homologue has been conspicuously missing in vertebrates. Here, we report the first identification of c- Krox/ Th-POK as the vertebrate homologue of GAF on the basis of sequence similarity and comparative structural analysis. The in silico structural analysis of the zinc finger region showed preferential interaction of vertebrate GAF with GAGA sites similar to that of fly GAF. We also show by cross-immunoreactivity studies that both fly and vertebrate GAFs are highly conserved and share a high degree of structural similarity. Electrophoretic mobility shift assays show that vertebrate GAF binds to GAGA sites in vitro. Finally, in vivo studies by chromatin immunoprecipitation confirmed that vertebrate GAF binds to GAGA-rich DNA sequences present in hox clusters. Identification of vertebrate GAF and the presence of its target sites at various developmentally regulated loci, including hox complexes, highlight the evolutionarily conserved components involved in developmental mechanisms across the evolutionary lineage and answer a long-standing question of the presence of vertebrate GAF.

Chromatin remodelling factor Mll1 is essential for neurogenesis from postnatal neural stem cells

Epigenetic mechanisms that maintain neurogenesis throughout adult life remain poorly understood. Trithorax group (trxG) and Polycomb group (PcG) gene products are part of an evolutionarily conserved chromatin remodelling system that activate or silence gene expression, respectively. Although PcG member Bmi1 has been shown to be required for postnatal neural stem cell self-renewal, the role of trxG genes remains unknown. Here we show that the trxG member Mll1 (mixed-lineage leukaemia 1) is required for neurogenesis in the mouse postnatal brain. Mll1-deficient subventricular zone neural stem cells survive, proliferate and efficiently differentiate into glial lineages; however, neuronal differentiation is severely impaired. In Mll1-deficient cells, early proneural Mash1 (also known as Ascl1) and gliogenic Olig2 expression are preserved, but Dlx2, a key downstream regulator of subventricular zone neurogenesis, is not expressed. Overexpression of Dlx2 can rescue neurogenesis in Mll1-deficient cells. Chromatin immunoprecipitation demonstrates that Dlx2 is a direct target of MLL in subventricular zone cells. In differentiating wild-type subventricular zone cells, Mash1, Olig2 and Dlx2 loci have high levels of histone 3 trimethylated at lysine 4 (H3K4me3), consistent with their transcription. In contrast, in Mll1-deficient subventricular zone cells, chromatin at Dlx2 is bivalently marked by both H3K4me3 and histone 3 trimethylated at lysine 27 (H3K27me3), and the Dlx2 gene fails to properly activate. These data support a model in which Mll1 is required to resolve key silenced bivalent loci in postnatal neural precursors to the actively transcribed state for the induction of neurogenesis, but not for gliogenesis.

The multidomain protein Brpf1 binds histones and is required for Hox gene expression and segmental identity

The Trithorax group (TrxG) is composed of diverse, evolutionary conserved proteins that form chromatin-associated complexes accounting for epigenetic transcriptional memory. However, the molecular mechanisms by which particular loci are marked for reactivation after mitosis are only partially understood. Here, based on genetic analyses in zebrafish, we identify the multidomain protein Brpf1 as a novel TrxG member with a central role during development. brpf1 mutants display anterior transformations of pharyngeal arches due to progressive loss of anterior Hox gene expression. Brpf1 functions in association with the histone acetyltransferase Moz (Myst3), an interaction mediated by the N-terminal domain of Brpf1, and promotes histone acetylation in vivo. Brpf1 recruits Moz to distinct sites of active chromatin and remains at chromosomes during mitosis, mediated by direct histone binding of its bromodomain, which has a preference for acetylated histones, and its PWWP domain, which binds histones independently of their acetylation status. This is the first demonstration of histone binding for PWWP domains. Mutant analyses further show that the PWWP domain is absolutely essential for Brpf1 function in vivo. We conclude that Brpf1, coordinated by its particular set of domains, acts by multiple mechanisms to mediate Moz-dependent histone acetylation and to mark Hox genes for maintained expression throughout vertebrate development.

Epigenetic modifications by Trithorax group proteins during early embryogenesis: do members of Trx-G function as maternal effect genes?

Maternal effect genes encode transcripts that are expressed during oogenesis. These gene products are stored in the oocyte and become functional during resumption of meiosis and zygote genome activation, and in embryonic stem cells. To date, a few maternal effect genes have been identified in mammals. Epigenetic modifications have been shown to be important during early embryonic development and involve DNA methylation and post-translational modification of core histones. During development, two families of proteins have been shown to be involved in epigenetic changes: Trithorax group (Trx-G) and Polycomb group (Pc-G) proteins. Trx-G proteins function as transcriptional activators and have been shown to accumulate in the oocyte. Deletion of Trx-G members using conventional knockout technology results in embryonic lethality in the majority of the cases analysed to date. Recent studies using conditional knockout mice have revealed that at least one family member is necessary for zygote genome activation. We propose that other Trx-G members may also regulate embryonic genome activation and that the use of oocyte-specific deletor mouse lines will help clarify their roles in this process.

Trl-GAGA directly interacts with lola like and both are part of the repressive complex of Polycomb group of genes

Epigenetic inheritance to maintain the expression state of the genome is essential during development. In Drosophila, the cis regulatory elements, called the Polycomb Response Elements (PREs) function to mark the epigenetic cellular memory of the corresponding genomic region with the help of PcG and trxG proteins. While the PcG genes code for the repressor proteins, the trxG genes encode activator proteins. The observations that some proteins may function both as PcG and trxG member and that both these group of proteins act upon common cis elements indicate at least a partial functional overlap among these proteins. Trl-GAGA was initially identified as a trxG member but later was shown to be essential for PcG function on several PREs. In order to understand how Trl-GAGA functions in PcG context, we have looked for the interactors of this protein. We identified lola like, aka batman, as a strong interactor of GAGA factor in a yeast two-hybrid screen. lolal also interacts with polyhomeotic and, like Trl, both lolal and ph are needed for iab-7PRE mediated pairing dependent silencing of mini-white transgene. These observations suggest a possible mechanism of how Trl-GAGA plays a role in maintaining the repressed state of target genes involving lolal, which may function as a mediator to recruit PcG complexes.

Mutations in Drosophila heat shock cognate 4 are enhancers of Polycomb.

The homeotic genes controlling segment identity in Drosophila are repressed by the Polycomb group of genes (PcG) and are activated by genes of the trithorax group (trxG). An F(1) screen for dominant enhancers of Polycomb yielded a point mutation in the heat shock cognate gene, hsc4, along with mutations corresponding to several known PcG loci. The new mutation is a more potent enhancer of Polycomb phenotypes than an apparent null allele of hsc4 is, although even the null allele occasionally displays homeotic phenotypes associated with the PcG. Previous biochemical results had suggested that HSC4 might interact with BRAHMA, a trxG member. Further analyses now show that there is no physical or genetic interaction between HSC4 and the Brahma complex. HSC4 might be needed for the proper folding of a component of the Polycomb repression complex, or it may be a functional member of that complex.

Mammalian Trithorax and polycomb-group homologues are antagonistic regulators of homeotic development.

Control of cell identity during development is specified in large part by the unique expression patterns of multiple homeobox-containing (Hox) genes in specific segments of an embryo. Trithorax and Polycomb-group (Trx-G and Pc-G) proteins in Drosophila maintain Hox expression or repression, respectively. Mixed lineage leukemia (MLL) is frequently involved in chromosomal translocations associated with acute leukemia and is the one established mammalian homologue of Trx. Bmi-1 was first identified as a collaborator in c-myc-induced murine lymphomagenesis and is homologous to the Drosophila Pc-G member Posterior sex combs. Here, we note the axial-skeletal transformations and altered Hox expression patterns of Mll-deficient and Bmi-1-deficient mice were normalized when both Mll and Bmi-1 were deleted, demonstrating their antagonistic role in determining segmental identity. Embryonic fibroblasts from Mll-deficient compared with Bmi-1-deficient mice demonstrate reciprocal regulation of Hox genes as well as an integrated Hoxc8-lacZ reporter construct. Reexpression of MLL was able to overcome repression, rescuing expression of Hoxc8-lacZ in Mll-deficient cells. Consistent with this, MLL and BMI-I display discrete subnuclear colocalization. Although Drosophila Pc-G and Trx-G members have been shown to maintain a previously established transcriptional pattern, we demonstrate that MLL can also dynamically regulate a target Hox gene.