Poly(ADP-ribosyl)ation regulates insulator function and intrachromosomal interactions in Drosophila.

Insulators mediate inter- and intrachromosomal contacts to regulate enhancer-promoter interactions and establish chromosome domains. The mechanisms by which insulator activity can be regulated to orchestrate changes in the function and three-dimensional arrangement of the genome remain elusive. Here, we demonstrate that Drosophila insulator proteins are poly(ADP-ribosyl)ated and that mutation of the poly(ADP-ribose) polymerase (Parp) gene impairs their function. This modification is not essential for DNA occupancy of insulator DNA-binding proteins dCTCF and Su(Hw). However, poly(ADP-ribosyl)ation of K566 in CP190 promotes protein-protein interactions with other insulator proteins, association with the nuclear lamina, and insulator activity in vivo. Consistent with these findings, the nuclear clustering of CP190 complexes is disrupted in Parp mutant cells. Importantly, poly(ADP-ribosyl)ation facilitates intrachromosomal interactions between insulator sites measured by 4C. These data suggest that the role of insulators in organizing the three-dimensional architecture of the genome may be modulated by poly(ADP-ribosyl)ation.

Architectural protein subclasses shape 3D organization of genomes during lineage commitment.

Understanding the topological configurations of chromatin may reveal valuable insights into how the genome and epigenome act in concert to control cell fate during development. Here, we generate high-resolution architecture maps across seven genomic loci in embryonic stem cells and neural progenitor cells. We observe a hierarchy of 3D interactions that undergo marked reorganization at the submegabase scale during differentiation. Distinct combinations of CCCTC-binding factor (CTCF), Mediator, and cohesin show widespread enrichment in chromatin interactions at different length scales. CTCF/cohesin anchor long-range constitutive interactions that might form the topological basis for invariant subdomains. Conversely, Mediator/cohesin bridge short-range enhancer-promoter interactions within and between larger subdomains. Knockdown of Smc1 or Med12 in embryonic stem cells results in disruption of spatial architecture and downregulation of genes found in cohesin-mediated interactions. We conclude that cell-type-specific chromatin organization occurs at the submegabase scale and that architectural proteins shape the genome in hierarchical length scales.

Truncated Tau caused by intron retention is enriched in Alzheimer's disease cortex and exhibits altered biochemical properties.

Alzheimer's disease (AD) is characterized by the accumulation of amyloid-ß plaques and Tau tangles in brain tissues. Recent studies indicate that aberrant splicing and increased level of intron retention is linked to AD pathogenesis. Bioinformatic analysis revealed increased retention of intron 11 at the Tau gene in AD female dorsal lateral prefrontal cortex as compared to healthy controls, an observation validated by quantitative polymerase chain reaction using different brain tissues. Retention of intron 11 introduces a premature stop codon, resulting in the production of truncated Tau11i protein. Probing with customized antibodies designed against amino acids encoded by intron 11 showed that Tau11i protein is more enriched in AD hippocampus, amygdala, parietal, temporal, and frontal lobe than in healthy controls. This indicates that Tau messenger RNA with the retained intron is translated in vivo instead of being subjected to nonsense-mediated decay. Compared to full-length Tau441 isoform, ectopically expressed Tau11i forms higher molecular weight species, is enriched in Sarkosyl-insoluble fraction, and exhibits greater protein stability in cycloheximide assay. Stably expressed Tau11i also shows weaker colocalization with α-tubulin of microtubule network in human mature cortical neurons as compared to Tau441. Endogenous Tau11i is enriched in Sarkosyl-insoluble fraction in AD hippocampus and forms aggregates that colocalize weakly with Tau4R fibril-like structure in AD temporal lobe. The elevated level of Tau11i protein in AD brain tissues tested, coupled with biochemical properties resembling pathological Tau species suggest that retention of intron 11 of Tau gene might be an early biomarker of AD pathology.

Widespread rearrangement of 3D chromatin organization underlies polycomb-mediated stress-induced silencing.

Chromosomes of metazoan organisms are partitioned in the interphase nucleus into discrete topologically associating domains (TADs). Borders between TADs are formed in regions containing active genes and clusters of architectural protein binding sites. The transcription of most genes is repressed after temperature stress in Drosophila. Here we show that temperature stress induces relocalization of architectural proteins from TAD borders to inside TADs, and this is accompanied by a dramatic rearrangement in the 3D organization of the nucleus. TAD border strength declines, allowing for an increase in long-distance inter-TAD interactions. Similar but quantitatively weaker effects are observed upon inhibition of transcription or depletion of individual architectural proteins. Heat shock-induced inter-TAD interactions result in increased contacts among enhancers and promoters of silenced genes, which recruit Pc and form Pc bodies in the nucleolus. These results suggest that the TAD organization of metazoan genomes is plastic and can be reconfigured quickly.

Phosphorylation of Tet3 by cdk5 is critical for robust activation of BRN2 during neuronal differentiation.

Tet3 regulates the dynamic balance between 5-methylcyotsine (5mC) and 5-hydroxymethylcytosine (5hmC) in DNA during brain development and homeostasis. However, it remains unclear how its functions are modulated in a context-dependent manner during neuronal differentiation. Here, we show that cyclin-dependent kinase 5 (cdk5) phosphorylates Tet3 at the highly conserved serine 1310 and 1379 residues within its catalytic domain, changing its in vitro dioxygenase activity. Interestingly, when stably expressed in Tet1, 2, 3 triple-knockout mouse embryonic stem cells (ESCs), wild-type Tet3 induces higher level of 5hmC and concomitant expression of genes associated with neurogenesis whereas phosphor-mutant (S1310A/S1379A) Tet3 causes elevated 5hmC and expression of genes that are linked to metabolic processes. Consistent with this observation, Tet3-knockout mouse ESCs rescued with wild-type Tet3 have higher level of 5hmC at the promoter of neuron-specific gene BRN2 when compared to cells that expressed phosphor-mutant Tet3. Wild-type and phosphor-mutant Tet3 also exhibit differential binding affinity to histone variant H2A.Z. The differential 5hmC enrichment and H2A.Z occupancy at BRN2 promoter is correlated with higher gene expression and more efficient neuronal differentiation of ESCs that expressed wild-type Tet3. Taken together, our results suggest that cdk5-mediated phosphorylation of Tet3 is required for robust activation of neuronal differentiation program.

CTCF: an architectural protein bridging genome topology and function.

The eukaryotic genome is organized in the three-dimensional nuclear space in a specific manner that is both a cause and a consequence of its function. This organization is partly established by a special class of architectural proteins, of which CCCTC-binding factor (CTCF) is the best characterized. Although CTCF has been assigned various roles that are often contradictory, new results now help to draw a unifying model to explain the many functions of this protein. CTCF creates boundaries between topologically associating domains in chromosomes and, within these domains, facilitates interactions between transcription regulatory sequences. Thus, CTCF links the architecture of the genome to its function.

Enhancer function: new insights into the regulation of tissue-specific gene expression.

Enhancer function underlies regulatory processes by which cells establish patterns of gene expression. Recent results suggest that many enhancers are specified by particular chromatin marks in pluripotent cells, which may be modified later in development to alter patterns of gene expression and cell differentiation choices. These marks may contribute to the repertoire of epigenetic mechanisms responsible for cellular memory and determine the timing of transcription factor accessibility to the enhancer. Mechanistically, cohesin and non-coding RNAs are emerging as crucial players responsible for facilitating enhancer-promoter interactions at some genes. Surprisingly, these interactions may be required not only to facilitate initiation of transcription but also to activate the release of RNA polymerase II (RNAPII) from promoter-proximal pausing.

Enhancers: emerging roles in cell fate specification.

Enhancers are regulatory DNA elements that dictate the spatial and temporal patterns of gene expression during development. Recent evidence suggests that the distinct chromatin features of enhancer regions provide the permissive landscape required for the differential access of diverse signalling molecules that drive cell-specific gene expression programmes. The epigenetic patterning of enhancers occurs before cell fate decisions, suggesting that the epigenetic information required for subsequent differentiation processes is embedded within the enhancer element. Lineage studies indicate that the patterning of enhancers might be regulated by the intricate interplay between DNA methylation status, the binding of specific transcription factors to enhancers and existing histone modifications. In this review, we present insights into the mechanisms of enhancer function, which might ultimately facilitate cell reprogramming strategies for use in regenerative medicine.

ApoE maintains neuronal integrity via microRNA and H3K27me3-mediated repression.

ApoE regulates neurogenesis, although how it influences genetic programs remains elusive. Cortical neurons induced from isogenic control and ApoE-/- human neural stem cells (NSCs) recapitulated key transcriptomic signatures of in vivo counterparts identified from single-cell human midbrain. Surprisingly, ApoE expression in NSC and neural progenitor cells (NPCs) is not required for differentiation. Instead, ApoE prevents the over-proliferation of non-neuronal cells during extended neuronal culture when it is not expressed. Elevated miR-199a-5p level in ApoE-/- cells lowers the EZH1 protein and the repressive H3K27me3 mark, a phenotype rescued by miR-199a-5p steric inhibitor. Reduced H3K27me3 at genes linked to extracellular matrix organization and angiogenesis in ApoE-/- NPC correlates with their aberrant expression and phenotypes in neurons. Interestingly, the ApoE coding sequence, which contains many predicted miR-199a-5p binding sites, can repress miR-199a-5p without translating into protein. This suggests that ApoE maintains neurons integrity through the target-directed miRNA degradation of miR-199a-5p, imparting the H3K27me3-mediated repression of non-neuronal genes during differentiation.

Altered stability of nuclear lamin-B marks the onset of aging in male Drosophila.

Epigenetic alterations occur during aging, but it remains unclear what epigenetic features are associated with the onset of physiological decline in animals. Nuclear lamin-B forms the filamentous meshwork underneath the nuclear envelope, providing the structural scaffold necessary for genome organization and gene regulation. We found that reduced level of nuclear lamin-B protein coincides with the decline in locomotor activity and stress resistance in young adult male Drosophila. Ubiquitous lamin-B expression improves locomotor activity of the male flies at the expense of lower stress resistance and shorten lifespan. This observation suggests that tissue-specific expression of lamin-B may regulate different aspects of animal physiology during aging. To test this hypothesis, specific GAL-4 lines were used to drive the expression of lamin-B in specific neuronal populations and muscle tissues in male flies. Ectopic expression of lamin-B in the dopaminergic neurons within the protocerebral anterior medial region of the brain improves the locomotor activity of the male flies with little impact on their stress responses and lifespan. Interestingly, age-dependent decrease in the level of lamin-B protein is independent of its mRNA expression. Instead, cellular thermal shift assay showed that lamin-B and CP190 insulator protein undergo significant change in their solubility during aging. This suggests that the increased solubility of lamin-B protein may contribute to its reduced stability and degradation during aging.

E2F and STAT3 provide transcriptional synergy for histone variant H2AZ activation to sustain glioblastoma chromatin accessibility and tumorigenicity.

The histone variant H2AZ is overexpressed in diverse cancer types where it facilitates the accessibility of transcriptional regulators to the promoters of cell cycle genes. However, the molecular basis for its dysregulation in cancer remains unknown. Here, we report that glioblastomas (GBM) and glioma stem cells (GSCs) preferentially overexpress H2AZ for their proliferation, stemness and tumorigenicity. Chromatin accessibility analysis of H2AZ2 depleted GSC revealed that E2F1 occupies the enhancer region within H2AZ2 gene promoter, thereby activating H2AZ2 transcription. Exploration of other H2AZ2 transcriptional activators using a customized "anti-H2AZ2" query signature for connectivity map analysis identified STAT3. Co-targeting E2F and STAT3 synergistically reduced the levels of H2AZ, histone 3 lysine 27 acetylation (H3K27ac) and cell cycle gene transcription, indicating that E2F1 and STAT3 synergize to activate H2AZ gene transcription in GSCs. Remarkably, an E2F/STAT3 inhibitor combination durably suppresses GSC tumorigenicity in an orthotopic GBM xenograft model. In glioma patients, high STAT3 signaling is associated with high E2F1 and H2AZ2 expression. Thus, GBM has uniquely opted the use of E2F1- and STAT3-containing "enhanceosomes" that integrate multiple signaling pathways to achieve H2AZ gene activation, supporting a translational path for the E2F/STAT3 inhibitor combination to be applied in GBM treatment.

NELF-A controls Drosophila healthspan by regulating heat-shock protein-mediated cellular protection and heterochromatin maintenance.

NELF-mediated pausing of RNA polymerase II (RNAPII) constitutes a crucial step in transcription regulation. However, it remains unclear how control release of RNAPII pausing can affect the epigenome and regulate important aspects of animal physiology like aging. We found that NELF-A dosage regulates Drosophila healthspan: Halving NELF-A level in the heterozygous mutants or via neuronal-specific RNAi depletion improves their locomotor activity, stress resistance, and lifespan significantly. Conversely, NELF-A overexpression shortens fly lifespan drastically. Mechanistically, lowering NELF-A level facilitates the release of paused RNAPII for productive transcription of the heat-shock protein (Hsp) genes. The elevated HSPs expression in turn attenuates the accumulation of insoluble protein aggregates, reactive oxidative species, DNA damage and systemic inflammation in the brains of aging NELF-A depleted flies as compared to their control siblings. This pro-longevity effect is unique to NELF-A due to its higher expression level and more efficient pausing of RNAPII than other NELF subunits. Importantly, enhanced resistance to oxidative stress in NELF-A heterozygous mutants is highly conserved such that knocking down its level in human SH-SY5Y cells attenuates hydrogen peroxide-induced DNA damage and apoptosis. Depleting NELF-A reconfigures the epigenome through the maintenance of H3K9me2-enriched heterochromatin during aging, leading to the repression of specific retrotransposons like Gypsy-1 in the brains of NELF-A mutants. Taken together, we showed that the dosage of neuronal NELF-A affects multiple aspects of aging in Drosophila by regulating transcription of Hsp genes in the brains, suggesting that targeting transcription elongation might be a viable therapeutic strategy against age-onset diseases like neurodegeneration.

Increased intron retention is a post-transcriptional signature associated with progressive aging and Alzheimer's disease.

Intron retention (IR) by alternative splicing is a conserved regulatory mechanism that can affect gene expression and protein function during adult development and age-onset diseases. However, it remains unclear whether IR undergoes spatial or temporal changes during different stages of aging or neurodegeneration like Alzheimer's disease (AD). By profiling the transcriptome of Drosophila head cells at different ages, we observed a significant increase in IR events for many genes during aging. Differential IR affects distinct biological functions at different ages and occurs at several AD-associated genes in older adults. The increased nucleosome occupancy at the differentially retained introns in young animals suggests that it may regulate the level of IR during aging. Notably, an increase in the number of IR events was also observed in healthy older mouse and human brain tissues, as well as in the cerebellum and frontal cortex from independent AD cohorts. Genes with differential IR shared many common features, including shorter intron length, no perturbation in their mRNA level, and enrichment for biological functions that are associated with mRNA processing and proteostasis. The differentially retained introns identified in AD frontal cortex have higher GC content, with many of their mRNA transcripts showing an altered level of protein expression compared to control samples. Taken together, our results suggest that an increased IR is an conserved signature that is associated with aging. By affecting pathways involved in mRNA and protein homeostasis, changes of IR pattern during aging may regulate the transition from healthy to pathological state in late-onset sporadic AD.

Poly(ADP-ribosyl)ation of OVOL2 regulates aneuploidy and cell death in cancer cells.

Poly(ADP-ribosyl)ation (PARylation) is a post-translational modification by which poly ADP-ribose (PAR) polymers are covalently added to proteins through a PAR polymerase (PARP). Here, using proteomic approach, we identify the transcriptional regulator, OVOL2, is a novel substrate of PARP1 and can be PARylated at residues Lysine 145, Lysine 176, and Lysine 212 within its C2H2 zinc finger domains. Overexpression of PARylated OVOL2 alters cell morphology and induces lagging chromosomes and aneuploidy. To define the underlying molecular mechanism by which OVOL2 induces abnormal cell cycle and centrosome amplification, we uncover that the OVOL2 elevates the protein levels of Cyclin E by enhancing its stability. Furthermore, we identify Skp2, the E3 ubiquitin ligase of Cyclin E, as a direct target of PARylated OVOL2. Using ChIP assay, the OVOL2 binding site on the promoter region of Skp2 is mapped. To further explore the physiological effect, we show that PARylated OVOL2 can induce cell death. Furthermore, to investigate PARylated OVOL2 function in vivo, we further develop a null-mice xenograft model and generate MMTV-PyVT transgenic mice and monitor the effect of wild-type OVOL2 and non-PARylated OVOL2-3K/A mutants on tumor progression. Consistently, overexpression of wild-type OVOL2 in both null-mice xenograft and MMTV-PyVT transgenic mice displays significantly reduction of tumor progression, respectively, further indicating that the function of OVOL2 as a tumor suppressor in vivo is highly regulated by PARylation. Taken together, our study sheds new light on PARP1-induced PARylation as a critical event in the OVOL2-mediated regulation of chromosomal integrity and suppression of cancer cells growth.

Membrane targeting and asymmetric localization of Drosophila partner of inscuteable are discrete steps controlled by distinct regions of the protein.

Asymmetric division of neural progenitors is a key mechanism by which neuronal diversity in the Drosophila central nervous system is generated. The distinct fates of the daughter cells derived from these divisions are achieved through preferential segregation of the cell fate determinants Prospero and Numb to one of the two daughters. This is achieved by coordinating apical and basal mitotic spindle orientation with the basal cortical localization of the cell fate determinants during mitosis. A complex of apically localized proteins, including Inscuteable (Insc), Partner of Inscuteable (Pins), Bazooka (Baz), DmPar-6, DaPKC, and G alpha i, is required to mediate and coordinate basal protein localization with mitotic spindle orientation. Pins, a molecule which directly interacts with Insc, is a key component required for the integrity of this complex; in the absence of Pins, other components become mislocalized or destabilized, and basal protein localization and mitotic spindle orientation are defective. Here we define the functional domains of Pins. We show that the C-terminal region containing the G alpha i binding GoLoco motifs is necessary and sufficient for targeting to the neuroblast cortex, which appears to be a prerequisite for apical localization of Pins. The N-terminal tetratricopeptide repeat-containing region of Pins is required for two processes; TPR repeats 1 to 3 plus the C-terminal region are required for apical localization but are insufficient to recruit Insc to the apical cortex, whereas TPR repeats 1 to 7 plus C-terminal Pins can perform both functions. Hence, the abilities of Pins to cortically localize, to apically localize, and to restore Insc apical localization are all separable, and all three capabilities are necessary to mediate asymmetric division. Moreover, the need for N-terminal Pins can be obviated by fusing a minimal Insc functional domain with the C-terminal region of Pins; this chimeric molecule is apically localized and can fulfill the functions of both Insc and Pins.

Insulator function and topological domain border strength scale with architectural protein occupancy.

BACKGROUND: Chromosome conformation capture studies suggest that eukaryotic genomes are organized into structures called topologically associating domains. The borders of these domains are highly enriched for architectural proteins with characterized roles in insulator function. However, a majority of architectural protein binding sites localize within topological domains, suggesting sites associated with domain borders represent a functionally different subclass of these regulatory elements. How topologically associating domains are established and what differentiates border-associated from non-border architectural protein binding sites remain unanswered questions. RESULTS: By mapping the genome-wide target sites for several Drosophila architectural proteins, including previously uncharacterized profiles for TFIIIC and SMC-containing condensin complexes, we uncover an extensive pattern of colocalization in which architectural proteins establish dense clusters at the borders of topological domains. Reporter-based enhancer-blocking insulator activity as well as endogenous domain border strength scale with the occupancy level of architectural protein binding sites, suggesting co-binding by architectural proteins underlies the functional potential of these loci. Analyses in mouse and human stem cells suggest that clustering of architectural proteins is a general feature of genome organization, and conserved architectural protein binding sites may underlie the tissue-invariant nature of topologically associating domains observed in mammals. CONCLUSIONS: We identify a spectrum of architectural protein occupancy that scales with the topological structure of chromosomes and the regulatory potential of these elements. Whereas high occupancy architectural protein binding sites associate with robust partitioning of topologically associating domains and robust insulator function, low occupancy sites appear reserved for gene-specific regulation within topological domains.